Rational design, synthesis and biological evaluation of 1,3,4-oxadiazole pyrimidine derivatives as novel pyruvate dehydrogenase complex E1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.03.011

On the basis of previous study on 2-methylpyrimidine-4-ylamine derivatives I, further synthetic optimization was done to find potent PDHc-E1 inhibitors with antibacterial activity. Three series of novel pyrimidine derivatives 6, 11 and 14 were designed and synthesized as potential Escherichia coli PDHc-E1 inhibitors by introducing 1,3,4-oxadiazole-thioether, 2,4-disubstituted-1,3-thiazole or 1,2,4-triazol-4-amine-thioether moiety into lead structure I, respectively. Most of 6, 11 and 14 exhibited good inhibitory activity against E. coli PHDc-E1 (IC₅₀ 0.97-19.21 μM) and obvious inhibitory activity against cyanobacteria (EC₅₀ 0.83-9.86 μM). Their inhibitory activities were much higher than that of lead structure I. 11 showed more potent inhibitory activity against both E. coli PDHc-E1 (IC₅₀ < 6.62 μM) and cyanobacteria (EC₅₀ < 1.63 μM) than that of 6, 14 or lead compound I. The most effective compound 11d with good enzyme-selectivity exhibited most powerful inhibitory potency against E. coli PDHc-E1 (IC₅₀ = 0.97 μM) and cyanobacteria (EC₅₀ = 0.83 μM). The possible interactions of the important residues of PDHc-E1 with title compounds were studied by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that 11d had more potent inhibitory activity than that of 14d or I due to its 1,3,4-oxadiazole moiety with more binding position and stronger interaction with Lsy392 and His106 at active site of E. coli PDHc-E1.

Full text of DOI:10.1016/j.bmc.2016.03.011

Accepted Manuscript
Rational design, synthesis and biological evaluation of 1,3,4-oxadiazole pyri-
midine derivatives as novel pyruvate dehydrogenase complex E1 inhibitors
Haifeng He, Wei Wang, Yuan Zhou, Qin Xia, Yanliang Ren, Jiangtao Feng,
Hao Peng, Hongwu He, Lingling Feng
PII:
S0968-0896(16)30160-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.011
BMC 12860
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
2 February 2016
4 March 2016
Please cite this article as: He, H., Wang, W., Zhou, Y., Xia, Q., Ren, Y., Feng, J., Peng, H., He, H., Feng, L., Rational
design, synthesis and biological evaluation of 1,3,4-oxadiazole pyrimidine derivatives as novel pyruvate
dehydrogenase complex E1 inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/
j.bmc.2016.03.011
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Rational design, synthesis and biological evaluation of 1,3,4-oxadiazole
pyrimidine derivatives as novel pyruvate dehydrogenase complex E1 inhibitors
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Haifeng Hea1, Wei Wangb1, Yuan Zhoua, Qin Xiaa, Yanliang Rena, Jiangtao Fenga, Hao Penga,
Hongwu He*a and Lingling Feng*a
5
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
bXi'an Modern Chemistry Research Institute, Xi'an 710065, PR China,
* To whom correspondence should be addressed. Tel./fax: +86 (0)27 67867960
E-mail addresses: he1208@mail.ccnu.edu.cn (H. He), fll708@mail.ccnu.edu.cn (L. Feng)
1 These authors equally contributed to this work.
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Abstract
On the basis of previous study on 2-methylpyrimidine-4-ylamine derivatives I, further synthetic
optimization was done to find potent PDHc-E1 inhibitors with antibacterial activity. Three series
of novel pyrimidine derivatives 6, 11 and 14 were designed and synthesized as potential E. coli
PDHc-E1 inhibitors by introducing 1,3,4-Oxadiazole-thioether, 2,4-disubstituted-1,3-thiazole or
1,2,4-triazol-4-amine-thioether moiety into lead structure I, respectively. Most of 6, 11 and 14
exhibited good inhibitory activity against E. coli PHDc-E1 (IC50 0.97 to 19.21 µM) and obvious
inhibitory activity against cyanobacteria (EC50 0.83 to 9.86 µM). Their inhibitory activities were
much higher than that of lead structure I. 11 showed more potent inhibitory activity against both E.
coli PDHc-E1 (IC50 < 6.62 ꢀM) and cyanobacteria (EC50 < 1.63 ꢀM) than that of 6, 14 or lead
compound I. The most effective compound 11d with good enzyme-selectivity exhibited most
powerful inhibitory potency against E. coli PDHc-E1 (IC50 = 0.97 µM) and cyanobacteria (EC50
=
0.83 µM). The possible interactions of the important residues of PDHc-E1 with title compounds
were studied by molecular docking, site-directed mutagenesis, and enzymatic assays. The results
indicated that 11d had more potent inhibitory activity than that of 14d or I due to its
1,3,4-Oxadiazole moiety with more binding position and stronger interaction with Lsy392 and
His106 at active site of E.coli PDHc-E1.
Keywords antibacterial activity, PDHc-E1 inhibitor, cyanobacteria, 1,3,4-Oxadiazole
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1. Introduction
The frequent application of bactericides with same or limited mode of action has led to the
wide spread evolution of resistance.1-3 It is, therefore, necessary to develop efficient bactericides
with novel structure or mode of action to overcome microbial disease.
The pyruvate dehydrogenase complex (PDHc) plays a pivotal role in cellular metabolism
catalyzing the oxidative decarboxylation of pyruvate and the subsequent acetylation of coenzyme
A (CoA) to acetyl-CoA.4-5 The overall reaction of oxidative decarboxylation can be simply
exhibited in Fig. 1.
Fig.1 Insert Here
The complex (PDHc) is comprised of three different enzyme components (E1, E2 and E3) and
a number of cofactors.6 Pyruvate dehydrogenase complex E1 component (PDHc-E1, EC1.2.4.1) is
the initial member of PDHc, which catalyzes the first step of multistep process, under condition of
using thiamine diphosphate (ThDP) and Mg2+ as cofactors.7–9 Particularly PDHc-E1 catalyzes the
first irreversible step among multistep process which is catalyzed by PDHc. Hence blocking the
activity of PDHc-E1 is the best way to inactivate the PDHc. ThDP plays an important role in the
enzyme reaction and the catalysis mechanism.10 Therefore, we selected E. coli PDHc-E1 as the
target pattern of bacterium to design new cofactor ThDP analogs as inhibitors of PDHc-E1 with
bactericidal activity.
Certain ThDP analogs have been reported as efficient inhibitors of PDHc-E1 (such as ThTDP,
ThTTDP, and triazole-ThDP in Fig. 2).11-15 However there were few reports about their
bactericidal or fungicidal activity, due to their complex structure with highly charged
pyrophosphate, poor bioavailability16 and poor enzyme-selective inhibition between
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microorganisms and mammals.12
Aiming at the aforementioned problems, a series of 2-methylpyrimidine-4-ylamine derivatives
containing 1,2,3-triazole ring and substituted benzene ring I, had been firstly chemically
synthesized in our laboratory. II, III, IV and V were further synthesized as ThDP analogs by the
modification of I (Fig. 2).17–21 Some of them were demonstrated to be effective inhibitors of E.
coli PDHc-E1 with moderate antifungal and antibacterial activity. These 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 Insert Here
The high charge of the pyrophosphate moiety in ThDP has been replaced by the low charge of
the substituted phenoxy group in I. The analysis of molecular docking indicated that the
substituted phenoxy (“A and B part”) moiety could occupy the binding site of pyrophosphate in
active site of E. coli PDHc-E1. However, unliked the interaction of oxygen atom of pyrophosphate
with amino acid residues, the oxygen atom of ether bond (“A part”) in I could not form hydrogen
bond with any amino acid residue in active site of E. coli PDHc-E1.17 It was thought that
inhibitory potency against E. coli PDHc-E1 should be increased by enhancing the interaction of
the “A or B part” with PDHc-E1. Therefore, the scaffold of I was kept, further optimization
focused on “A or B part”. In this work, 1,3,4-Oxadiazole-thioether moiety as “A part” was
introduced
into
I
to
replace
phenoxy-ether
bond
and
produce
novel
2-methyl-4-amino-5-((4-(((5-substituted-pheny-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-
triazol-1-yl)methyl)pyrimidine 6. Moreover, 2,4-disubstituted-1,3-thiazole group as “B part”
was further introduced into the parent structure 6 to replace substituted benzene ring and form
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novel
2-methyl-4-amino-5-((4-(((5-(2,4-disubstituted-thiazol-5-yl)-1,3,4-oxadiazol-2-yl)thio)
methyl)-1H-1,2,3-triazol-1-yl)methyl)pyrimidine 11 (Fig. 3). On the other hand, considering that
1,3,4-Oxadiazole-thioether or 1, 3-thiazole moiety could be as hydrogen bond receptor which
would benefit the interaction of the “A and B part” with PDHc-E1, some 1,3,4-Oxadiazole and
1,3-thiazole derivatives also showed excellent antibacterial activity.22-25 Therefore these novel
ThDP analog, 1,3,4-Oxadiazole pyrimidine derivatives 6 and 11 are expected to be good E.coli
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PDHc-E1 inhibitors with bactericidal activity.
Fig. 3 Insert Here
In order to examine the effect of the structure of “A part” on inhibitory activity,
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1,3,4-Oxadiazole-thioether moiety in pyrimidine
derivatives 11 was replaced with
1,2,4-triazol-thioether moiety to give novel 2-methyl-4-amine-5-((4-(((5-(2,4-disubstituted-thiazol)
-4H-1,2,4-triazol-4-amine-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)methy l)pyrimidine derivatives
14 ( Fig. 3).
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Herein, we report the synthesis of new 1,3,4-Oxadiazole pyrimidine derivatives 6, 11 and
1,2,4-triazolyl pyrimidine derivatives 14. Their enzyme inhibition, antibacterial and antifungal
activity were examined. The interaction mode of some title compounds with E.coli PDHc-E1 was
studied by molecular docking method, probable inhibition mechanism was discussed. The
enzyme-selectivity of representative compounds between pig heart and E. coli PDHc-E1 was also
examined.
2. Chemistry
The synthetic route of 6a-l, 11a-d and 14a-d is depicted in Scheme 1.
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Scheme 1. Insert Here
Vitamin B1 or thiamine hydrochloride as starting material was used to prepare
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5-azido-methyl-2-methylpyrimidine-4-ylamine 1, according to the literature method.26 1 is the key
intermediate for the preparation of title compounds 6a-l, 11a-d and 14a-d. The title compounds
could be synthesized by a five-step sequence starting from starting material. Various substituted
ethyl benzoate 2 reacted with hydrazine hydrate in ethanol to produce corresponding hydrazide 3.
Under same condition, 2,4-disubstituted-1,3-thiazole-5-ethyl formate 7 could be converted into
corresponding hydrazide 8.
The preparation of 5-substituted-phenyl-1,3,4-Oxadiazole-2-thiol
4
was
achieved by
the reaction of hydrazide derivatives 3 with carbon disufide under strong basic conditions
followed by acidification with dilute hydrochloric acid. Under same condition, 5-(2,4-disubstituted
-thiazol-5-yl)-1,3,4-Oxadiazole-2-thiol 927 could be obtained from hydrazide derivatives 8 using
carbon disulfide. 4-Amino-5-(2,4-disubstituted-thiazol-5-yl)-4H-1,2,4-triazole-3-thiol 12 was
prepared by the cyclization of hydrazide derivatives 8 , in which 8 was firstly converted into the
corresponding potassium dithiocarbamate and further cyclized with hydrazine hydrate. The key
intermediate, 5, 10 or 13 with terminal alkynes, was prepared via reaction of 3-bromopropyne
with corresponding substituted thiol 4, 9 or 12 respectively in refluxing acetone with K2CO3 as
base. The 1,2,3-triazol ring in the skeleton of title compounds 6, 11 and 14 could be constructed
by applying ‘click chemistry’. In our present work, 6a-l, 11a-d and 14a-d were synthesized by the
1,3-dipolar cycloaddition of 1 with substituted-prop-2-yn-1-thioethers 5, 10, or 13 respectively
using CuI as catalyst in the presence of Et3N and THF. All synthesized compounds were
characterized by 1H NMR, 13C NMR and mass spectrometry (HR-EIMS).
3. Results and discussion
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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
modified by replacing “A part” with 1,3,4-Oxadiazole-thioether moiety (Fig.3). Several
1,3,4-Oxadiazole pyrimidine derivatives 6a-e were firstly synthesized and their inhibitory
potency against E. coli PDHc-E1 were evaluated. The IC50 values of 6a-e and Ia-e17 are
summarized in Table 1. All 6a-e displayed higher inhibitory activity than that of Ia-e with same R
on the benzene ring. Especially the inhibitory activity of 6e was 7-folds higher than that of Ie. The
results showed that the inhibitory potency could be significantly improved by replacing ether bond
at “A part” in I with 1,3,4-Oxadiazole-thioether moiety. This suggested that the introduction of
1,3,4-Oxadiazole-thioether moiety at “A part” should be much beneficial for enhancing inhibition
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against E. coli PDHc-E1.
Table 1 Insert Here
Further optimization was focused on R of 6. As shown in Table 1, R at 4-position on the
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benzene ring was favorable to inhibitory activity. Inhibitory activity against E. coli PDHc-E1
decreased in the following order: 6a (R=4-NO2, IC50 = 7.59 ꢀM) > 6c (R=2-NO2, IC50 = 9.84
ꢀM)> 6i (R=3-NO2, IC50 = 10.38 ꢀM); 6b (R=4-Cl, IC50 = 7.08 ꢀM) > 6l (R=2-Cl, IC50=12.39
ꢀM); 6f (R=4-F, IC50 = 9.11 ꢀM) > 6j (R=2-F, IC50 = 19.21 ꢀM). Small size group as R at
4-position of benzene ring seemed to be good for inhibitory activity. For example, 6f with 4-F as
R showed better inhibitory activity (IC50 = 9.11 ꢀM) than that of 6j with 4-CF3 as R (IC50 = 15.87
ꢀM). In the other hand, single substituent was favorable for inhibitory activity, such as 6b (R=4-Cl,
IC50= 7.08 ꢀM) > 6l (R=2-Cl-4-Cl, IC50= 13.18 ꢀM); 6a (R=4-NO2, IC50= 7.59 ꢀM) > 6c
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(R=2-Cl-4-NO2, IC50 = 12.80 ꢀM). Above results indicated that the inhibitory activity of 6 was
also dependent upon the structure, position and size of R on the benzene ring.
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6
was
further
modified
by replacing
substituted
benzene
ring
with
2,4-disubstituted-1,3-thiazole moiety at “B part” to form a series of 1,3,4-Oxadiazole pyrimidine
derivatives 11. Compared with 6, the inhibitory activity of 11 was further enhanced. As shown in
Table 1 and 2, 11a-d (IC50 = 0.97-6.62 µM) displayed better inhibitory activity than 6a-l (IC50
=
7.08-19.21 µM). Among 11a-d, compounds with CF3 as R1 displayed higher inhibitory activity
than that of compounds with CH3 as R1, irrespective of NH2 or CH3NH as R2. For example, the
inhibitory activity of 11d (R1=CF3) was 6-folds higher than that of 11b (R1=CH3). The inhibitory
potency against E. coli PDHc-E1 could be remarkably enhanced by introducing both
1,3,4-Oxadiazole-thioether and 2,4-disubstituted-1,3 -thiazole moiety to replace both ether bond
and benzene ring at “A and B part” in I.
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Table 2 Insert Here
In order to examine the effect of “A part” on inhibitory activity against E.coli PDHc-E1, 11
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was modified to produce 14 by replacing 1,3,4-Oxadiazole-thioether moiety in 11 with
1,2,4-triazol-4-amine-thioether moiety as “A part”. The inhibitory activity of 14 was shown in
Table 2. All 14a-d displayed weaker inhibitory activity than that of corresponding 11a-d with
same R1 and R2. Especially 14d showed inhibitory activity 19-folds lower than that of 11d. The
result showed that the loss of 1,3,4-Oxadiazole moiety led to a decrease in inhibitory activity
against E.coli PDHc-E1. It indicated that the structure of “A part” had a significant effect on
inhibitory activity against E.coli PDHc-E1.
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3.2. Antibacterial activity
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Most reported ThDP analogs as E. coli PDHc-E1 inhibitors had no useful antibacterial or
antifungal activity. 6, 11, and 14 as E. coli PDHc-E1 inhibitors were evaluated for their
antifungal and antibacterial activity. Interestingly all tested compounds displayed good
antibacterial activity, but very weak fungicidal activity. As shown in Fig. 4. 6, 11, and 14 exhibited
obvious inhibitory potency against cyanobacteria at 10 µM. Especially 6b, 6c, 6e, 6f, 6g, 6h, 6i,
11a-d, and 14a-c could 90-99% control cyanobacteria, but I only showed <30% inhibitory
potency.
Fig.4 Insert Here
EC50 values of 6, 11, 14 and I were further evaluated their inhibitory potency against
cyanobacteria. The structure and EC50 of 6, 11, 14 and I are listed in Tables 1 and 2. 6, 11, and 14
exhibited obvious inhibitory potency against cyanobacteria with EC50 ranging from 0.83 to 9.86
µM. However I with EC50 > 50 µM. According to the data in Tables 1 and 2, we see that the
inhibitory potency of some tested compounds against cyanobacteria is related to their inhibition
against E. coli PDHc-E1. 6, 11, and 14 with the IC50 values ranging from 0.97 to 19.21 µM against
E. coli PHDc-E1 could exhibit moderate to good antibacterial activity against cyanobacteria. I
with much weak inhibitory potency against E. coli PHDc-E1 also showed much weak activity
against cyanobacteria.
As shown in Tables 1 and 2, most 6, 11 and 14 exhibited much higher inhibitory potency
against both E. coli PDHc-E1 and cyanobacteria than that of I. Especially 1,3,4-Oxadiazole
pyrimidine derivatives 11 were much favorable to both enzyme inhibition and antibacterial activity.
It could be noticed that 14a-d containing 1,2,4-triazole moiety as “A part” showed weaker
inhibition against both E. coli PDHc-E1 and cyanobacteria than that of 11a-d containing
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1,3,4-Oxadiazole moiety as “A part”. 1,3,4-Oxadiazole pyrimidine derivative 11d was found to be
the most effective compound against E. coli PDHc-E1 (IC50 = 0.97 µM), and against
cyanobacteria with EC50 value of 0.83 µM. However 1,2,4-triazole pyrimidine derivative 14d
showed poor activity against E. coli PDHc-E1 (IC50 = 15.67 µM), and against cyanobacteria with
EC50 value of 9.86 µM.
Above observation showed that both enzyme inhibition and antibacterial activity could be
greatly
enhanced
by
introducing
both
1,3,4-Oxadiazole-thioether
and
2,4-disubstituted-1,3-thiazole moiety at “A and B part” to replace both ether bond and benzene
ring in I. These results suggested that 1,3,4-Oxadiazole-thioether and 1,3-thiazole moiety as “A
and B part” in structure 11 would be much beneficial for enhancing the interaction of 11 with E.
coli PDHc-E1 by forming hydrogen bond. Although most 6, 11 and 14 exhibited obvious
antibacterial activity against cyanobacteria, they had no significant fungicidal activity against
fungus. As shown in Table 3, most 6, 11, and 14 showed < 60% inhibitory potency against A.
solani, R. solani, B. cinerea and C. lagenarium. It showed that most 6, 11, and 14 could
selectively inhibit bacterium due to their good inhibition against PDHc-E1 from E. coli.
Table 3 Insert Here
Table 4 Insert Here
Some reported ThDP analogs as PDHc-E1 inhibitors not only had powerful inhibition potency
against E. coli PDHc-E1, but also had powerful inhibition potency against human PDHc-E1,
because they were had poor enzyme-selective inhibition between mammals and bacteria.28 In this
work, 11a-d with good inhibition against E. coli PDHc-E1 (IC50 = 0.97-6.62 µM) were selected to
test their inhibition against Pig heart PDHc-E1. As shown in Table 4, 11a-d showed <30%
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inhibitory potency against pig heart PDHc-E1. These findings showed that 11a-d had better
enzyme-selective inhibition between microorganism and mammal, possibly acting as specific
inhibitors against bacteria.
3.3. Analyses of the interaction between inhibitors and E.coil PDHc-E1
In order to understand the inhibition of 11 and 14 against E. coli PDHc-E1, the interaction
mode of 11 and 14 with active site of E.coli PDHc-E1 was further explored. The molecular
docking simulation of several compounds was carried out by using the SURFLEX module of
SYBYL package.16 1,3,4-Oxadiazole pyrimidine derivative 11d as the best PDHc-E1 inhibitor
and its corresponding 1,2,4-triazolyl pyrimidine derivative 14d were selected to analyse their
interaction with E. coil PDHc-E1 by molecular docking.
The binding mode of 11d or 14d with amino acid residues is shown in Fig. 5 A and B,
respectively. As shown in Fig. 5, 11d or 14d with a ‘V’ conformation can occupy the
ThDP-binding pocket and bind in the active site of E.coli PDHc-E1. On the right side of the‘V’
conformation, the 4-aminopyrimidine ring of 11d or 14d displays a π–π stacking with the
side chain ring of Phe602，also forms hydrogen bonds with Met194, Glu571 and Val192, which is
similar to the interactions of ThDP or lead structure I with corresponding amino acid residues. For
the left side of the ‘V’ conformation of 11d or 14d, trifluoromethyl group as R1 on the 1,3-thiazole
ring of 11d or 14d has an interaction with Lys392 in the active site of E.coli PDHc-E1 by forming
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hydrogen bond.
Fig. 5 Insert Here
In order to confirm the prediction of molecular docking, site-directed mutagenesis and
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enzymatic assays were further performed. As shown in Fig. 6, the IC50 value of 11d against
mutants M194A (19.05 ꢀM), E571A (5.02 ꢀM), V192A (33.17 ꢀM), K392A (20.44 ꢀM) or F602A
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(5.08 ꢀM) was about 18 times, 4 times, 33 times, 20 times or 4 times higher than its value against
wild-type PDHc-E1 (0.97 ꢀM), respectively. These results suggest that the interaction between
11d and Met194, Glu571, Val192, Lys392 or Phe602 by forming hydrogen bond plays an
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important role in the binding of 11d with E.coli PDHc-E1.
Fig. 6 Insert Here
It was very interesting to explore the different of the binding mode of I, 11 and 14 by
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comparing with “A part” in their parent structures. No hydrogen bond between any amino acid
residues and the oxygen atom of ether bond as “A part” at the middle of the ‘V’ conformation of I
was observed by molecular docking in our previous study.15 However the two nitrogen atoms
(N-N) and oxygen atom (C-O-C) of 1,3,4-Oxadiazole moiety as “A part” at the middle of the ‘V’
conformation of compound 11d could form two strong hydrogen bond with Lsy392 and a strong
hydrogen bond with His106 , respectively (Fig. 5A). As shown in Fig. 5B, the two nitrogen atoms
(N-N) of 1,2,4-triazolyl moiety in pyrimidine derivative 14d is turned 180 degree comparing
with that of 1,3,4-Oxadiazole moiety in pyrimidine derivative 11d. In this case, the two nitrogen
atoms (N-N) of 1,2,4-triazolyl moiety in 14d only form a hydrogen bond with His106, but no
other hydrogen bond between 1,2,4-triazolyl moiety and Lsy392 or any amino acid residues was
observed.
Further site-directed mutagenesis and enzymatic assays showed that the IC50 values of 11d
against the mutants K392A (20.44 ꢀM) or H106A (11.61 ꢀM) was about 20 times or 11 times
higher than its value against wild-type PDHc-E1 enzyme (0.97 ꢀM) (Fig. 6). These results also
revealed that the interaction between 11d and Lsy392 or His106 by forming hydrogen bond had a
significant contribution for its inhibitory activity against E.coli PDHc-E1.
Above observation indicated that 1,3,4-Oxadiazole moiety as “A part” in 11 was most
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favorable to enzyme inhibition against E. coli PDHc-E1 due to more binding position and stronger
interaction with the active site of E.coli PDHc-E1. However 1,2,4-triazolyl moiety or ether bond
as “A part” had fewer binding position or no interaction with amino acid residues, which led to
weak enzyme inhibition of 14 or I. These results provided us a reasonable explanation for why
1,3,4-Oxadiazole pyrimidine derivatives 11 had more potent inhibitory activity against E.coli
PDHc-E1 than that of 1,2,4-triazolyl pyrimidine derivatives 14 or I.
4. Conclusion
On the basis of lead structure I, 1,3,4-Oxadiazole pyrimidine derivatives 6a-l, 11a-d and
1,2,4-triazolyl pyrimidine derivatives 14a-d as potential E. coli PDHc-E1 inhibitors were designed
and synthesized. As novel ThDP analogs, all 6a-l, 11a-d and 14a-d with the IC50 values ranging
from 0.97 to 19.21 µM against E. coli PHDc E1 could exhibit moderate to good inhibitory potency
against cyanobacteria (EC50 = 0.83-9.86 µM). However I with much weaker inhibition against E.
coli PHDc-E1 showed no inhibitory activity against cyanobacteria. The inhibitory potency of
compounds against both E. coli PDHc-E1 and cyanobacteria could be greatly increased by
replacing the ether bond of I with 1,3,4-Oxadiazole-thioether, 2,4-disubstituted-1,3-thiazole or
1,2,4-triazol-4-amine-thioether moiety as “A and B part”. 11 with 1,3,4-Oxadiazole-thioether and
2,4-disubstituted-1,3-thiazole moiety as “A and B part” showed more potent inhibitory activity
against both E. coli PDHc-E1 and cyanobacteria than that of 6, 14 or I. The most effective
compound 11d (IC50 = 0.97 µM) exhibited not only much stronger inhibition against E. coli
PDHc-E1 than that of corresponding 1,2,4-triazolyl pyrimidine 14d (IC50 = 15.67 µM), but also
displayed much higher inhibitory potency (EC50 = 0.83 µM) against cyanobacteria than that of
14d (EC50 = 9.86 µM). The above findings showed that there was some correlation between
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enzyme inhibition and antibacterial activity.
Binding mode analysis revealed that 11d displayed much powerful interaction by forming
hydrogen bond between 1,3,4-Oxadiazole moiety and Lsy392, His106 at active site of E.coli
PDHc-E1. The site-directed mutagenesis and enzymatic assays further proved that the interaction
between 1,3,4-Oxadiazole moiety in 11d with Lsy392 or His106 had a significant contribution for
its inhibitory activity against E.coli PDHc-E1. It suggested that 11d had more potent inhibitory
activity against E.coli PDHc-E1 and bacterium than that of 14d or lead compound I due to
1,3,4-Oxadiazole moiety as a “A part” with more binding position and stronger interaction with
important amino acid residues than that of 14d or I. These results elucidated that
1,3,4-Oxadiazole-thioether and 2,4-disubstituted-1,3-thiazole moiety as “A and B part” were much
beneficial than substituted ether bond in the parent structure I for enhancing both inhibition
against E. coli PDHc-E1 and cyanobacteria. 11a-d with weak inhibition against pig PDHc-E1
acted as special inhibitor selectively against E. coli PDHc-E1 and bacterium. Therefore, the
skeleton of 1,3,4-Oxadiazole pyrimidine derivatives 11 could be as the novel lead structure of
bactericide 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
Mercury-Plus 400 spectrometer and chemical shifts were recorded in parts per million (ppm) with
TMS as the internal reference. High-resolution electron impact mass spectra (HR-EIMS) were
recorded under electron impact (70 eV) condition using a MicroMass GCT CA 055 instrument.
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Unless otherwise noted, reagents were purchased from commercial suppliers and used without
further purification. Intermediate 5-(azidomethyl)-2-methylpyrimidine -4-amine 1 was synthesized
according to the existing methods.26
5.2 General procedure for preparation of 3a-l and 8a-d.
A mixture of substituted ethyl benzoate 2 (10 mmol) and hydrazine hydrate (20 mmol) in ethanol
was stirred at room temperature for 3 h and then filtered. The crude product recrystallized from
absolute alcohol to give 3a-l, which were used directly for the next step. Under this same
condition, the intermediate compounds 8a-d were also prepared.
5.3 General procedure for preparation of 4a-l and 9a-d.
Substituted benzohydrazide 3 (5 mmol) was dissolved in a solution of potassium hydroxide (6
mmol) in water (4 mL) and ethanol (20 mL). Carbon disulfide (10 mmol) was then added while
stirring and the reaction mixture was heated under reflux for 8 h. The solvents were removed
under reduced pressure; the residue was treated with water and then filtered. The filtrate was
cooled, and then neutralized to pH 3 using dilute hydrochloric acid and the separated product thiol
4a-l was filtered, washed with water, dried and recrystallised from benzene as yellow crystals,
which were used directly for the next step. Under this same condition, the intermediate
compounds 9a-d were also prepared.
5.4 General procedure for preparation of 12a-d.
To
a solution of potassium hydroxide (6 mmol) in absolute ethanol (30 mL),
2,4-disubstituted-1,3-thiazole-5-carbohydrazide 8 (5 mmol) and carbon disulphide (10 mmol)
were added and the mixture was stired for 16 h. The precipitated potassium dithiocarbazinate was
collected by filtering, washed with ethyl acetate and dried under vacuum. The potassium salt was
15

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used in the next step without further purification. A suspension of the potassium salt, water (4.0
mL) and hydrazine hydrate (3 mL) were heated under reflux for 5 h, and then the mixture was
diluted with 50 mL water and subsequent acidification with dilute acetic acid gave a white
precipitate which was filtered, washed with water and recrystallized from aqueous DMF and
obtained 12a-d as yellow crystals in good yield.
5.5 General procedure for preparation of 5a-l, 10a-d and 13a-d.
A solution of substituted thiol 4 (5 mmol), 3-bromopyropyne (0.71 g, 6 mmol) and K2CO3 (1.38 g,
10 mmol) in acetone (20 mL) was heated under reflux until the reaction was complete based on
TLC monitoring. Then residue was dissolved in water (20 mL) and then filtered. The crude
product recrystallized from absolute alcohol to give 5a-l, which were used directly for the next
step. Under this same condition, the intermediate compounds 10a-d and 13a-d were also prepared.
5.6 General procedure for preparation of target compounds 6a-l, 11a-d and 14a-d.
CuI (0.04g, 0.2 mmol) was added to a stirred solution of 5-azidomethyl-2-methylpyrimidine-4-yla
mine 1 (0.33g, 2 mmol) and 2-substituted-phenyl-5-(prop-2-yn-1-ylthio)-1,3,4-oxadiazole 5 (2
mmol) in THF (10mL) followed by Et3N (0.24g, 2.4 mmol). After overnight stirring at room
temperature, the reaction mixture was poured into water (20 mL) and then filtered. The crude
products were purified by column chromatography on silica gel and elution with
petroleumether/acetone (2:1, v/v) to give the corresponding pure title compounds 6a-l. Under this
same condition, the intermediate compounds 11a-d and 14a-d were also prepared.
5.6.1.
2-methyl-4-amine-5-((4-(((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-triaz
ol-1-yl)methyl)pyrimidine (6a)
16

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Yellow solid; Yield 90%; m.p. 143-145 oC; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.33 (s, 3H,
CH3), 4.67 (s, 2H, SCH2), 5.45 (s, 2H, CH2), 7.06 (s, 2H, NH2), 8.16 (s, 1H, pyrimidine-5-yl-H),
8.18 (s, 2H, Ar-H), 8.37 (d, 2H, Ar-H, J = 6.3 Hz); 13C NMR (100 MHz, DMSO-d6) ꢁ(ppm): 25.2,
27.0, 46.9, 108.2, 124.0, 124.7, 127.8, 128.6, 142.3, 149.2, 156.0, 161.5, 164.1, 164.6, 167.3;
HR-EIMS (EI) m/z 426.1102 (M+H)+, calcd. for C17H16N9O3S m/z = 426.1097.
5.6.2.
2-methyl-4-amine-5-((4-(((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-tria
zol-1-yl)methyl)pyrimidine (6b)
o
1
White solid; Yield 93%; m.p. 189-191 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.35 (s, 3H,
CH3), 4.64 (s, 2H, -SCH2-), 5.45 (s, 2H, CH2), 7.06 (s, 2H, NH2), 7.63 (s, 2H, Ar-H), 7.92 (s, 2H,
Ar-H), 8.14 (s, 1H, pyrimidine-5-yl-H); 13C NMR (100 MHz, DMSO-d6) ꢁ(ppm): 25.0, 26.7, 46.9,
107.5, 118.0, 120.3, 124.6, 124.9, 134.6, 147.9, 149.0, 155.0, 156.7, 162.9, 165.2; HR-EIMS (EI)
m/z 415.0890 (M+H)+, calcd. for C17H16ClN8OS m/z = 415.0856.
5.6.3.
2-methyl-4-amine-5-((4-(((5-(2-nitrophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-triaz
ol-1-yl)methyl)pyrimidine (6c)
1
Yellow solid; Yield 90%; m.p. 171-173 oC; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.31 (s, 3H,
CH3), 4.67 (s, 2H, -SCH2-), 5.48 (s, 2H, CH2), 7.22 (s, 2H, NH2), 7.89 (s, 2H, Ar-H), 7.98 (s, 1H,
pyrimidine-5-yl-H), 8.16 (s, 2H, Ar-H); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 24.5, 26.2,
46.2, 107.5, 120.1, 120.6, 123.4, 127.5, 131.3, 133.4, 147.2, 150.2, 150.0, 151.1, 158.8, 165.0,
167.3; HR-EIMS (EI) m/z 426.1102 (M+H)+, calcd. for C17H16ClN8OS m/z = 426.1097.
5.6.4.
17

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2-methyl-4-amine-5-((4-(((5-(2,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-
triazol-1-yl)methyl)pyrimidine (6d)
o
1
White solid; Yield 91%; m.p. 173-175 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.34 (s, 3H,
CH3), 4.65 (s, 2H, -SCH2-), 5.46 (s, 2H, CH2), 7.05 (s, 2H, NH2), 7.62 (s, 1H, Ar-H), 7.88 (s, 1H,
Ar-H), 7.93 (s, 1H, Ar-H), 8.15 (s, 1H, pyrimidine-5-yl-H); 13C NMR (100 MHz, DMSO-d6)
ꢁ(ppm): 25.5, 27.2, 45.1, 105.5, 120.0, 122.2, 125.6, 127.4, 128.4, 133.4, 133.9, 149.9, 151.0,
151.4, 158.7, 164.9, 167.2; HR-EIMS (EI) m/z 471. 0320 (M+Na)+, calcd. for C17H14Cl2N8OSNa
m/z = 471. 0286.
5.6.5.
2-methyl-4-amine-5-((4-(((5-phenyl-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)m
ethyl)pyrimidine (6e)
o
1
White solid; Yield 88%; m.p. 138-140 C ; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.34 (s, 3H,
CH3), 4.67 (s, 2H, -SCH2-), 5.48 (s, 2H, CH2), 7.18 (s, 2H, NH2), 7.58 (s, 3H, Ar-H), 7.93 (s, 2H,
Ar-H), 8.18 (s, 1H, pyrimidine-5-yl-H); 13C NMR (100 MHz, DMSO-d6) ꢁ(ppm): 23.5, 25.2, 45.2,
106.5, 116.9, 120.0, 122.0, 123.9, 128.2, 146.8, 147.9, 153.8, 155.6, 161.8, 164.1; HR-EIMS (EI)
m/z 381.1236 (M+H)+, calcd. for C17H17N8OS m/z = 381.1246.
5.6.6.
2-methyl-4-amine-5-((4-(((5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-tria
zol-1-yl)methyl)pyrimidine (6f)
o
1
White solid; Yield 89%; m.p 132-134 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.34 (s, 3H,
CH3), 4.64 (s, 2H, -SCH2-), 5.45 (s, 2H, CH2), 7.05 (s, 2H, NH2), 7.41 (d, 2H, Ar-H, J = 6.6 Hz),
7.98 (s, 2H, Ar-H), 8.14 (s, 1H, pyrimidine-5-yl-H); 13C NMR (100 MHz, DMSO-d6) ꢁ(ppm):
18

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420
25.5, 27.2, 45.2, 108.2, 116.0, 119.9, 120.2, 129.4, 129.5, 149.8, 150.9, 156.8, 158.6, 164.8, 166.1,
167.1; HR-EIMS (EI) m/z 399.1151 (M+H)+, calcd. for C17H16FN8OS m/z = 399.1152.
5.6.7.
2-methyl-4-amine-5-((4-(((5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1
H-1,2,3-triazol-1-yl)methyl)pyrimidine (6g)
o
1
White solid; Yield 89%; m.p 132-134 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.30 (s, 3H,
CH3), 4.66 (s, 2H, -SCH2-), 5.43 (s, 2H, CH2), 7.00 (s, 2H, NH2), 7.94 (s, 2H, Ar-H), 8.14 (s, 2H,
Ar-H), 8.14 (s, 1H, pyrimidine-5-yl-H); 13C NMR (100 MHz, DMSO-d6) ꢁ(ppm): 25.2, 27.0, 47.5,
108.2, 118.2, 119.9, 124.1, 124.3, 126.6, 129.5, 129.8, 130.0, 130.3, 149.8, 150.9, 156.8, 158.6,
164.8, 167.1; HR-EIMS (EI) m/z 449.1133 (M+H)+, calcd. for C18H16F3N8OS m/z = 449.1120.
5.6.8.
2-methyl-4-amine-5-((4-(((5-(3,5-dinitrophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-tr
iazol-1-yl)methyl)-2-methylpyrimidine (6h)
o
1
Yellow solid; Yield 87%; m.p 163-165 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.37 (s, 3H,
CH3), 4.66 (s, 2H, -SCH2-), 5.48 (s, 2H, CH2), 7.10 (s, 2H, NH2), 7.60 (s, 2H, Ar-H), 7.60 (s, 1H,
1,2,3-triazol-1-yl-H), 7.94 (s, 1H, pyrimidine-5-yl-H), 7.94 (s, 1H, Ar-H); 13C NMR (100 MHz,
DMSO-d6) ꢁ(ppm): 25.5, 27.8, 46.5, 108.5, 118.6, 119.7, 122.6, 127.4, 142.3, 149.2, 151.0, 157.5,
158.9, 164.9, 167.5; HR-EIMS (EI) m/z 471.0950 (M+H)+, calcd. for C17H15N10O5S m/z =
471.0948.
5.6.9.
2-methyl-4-amine-5-((4-(((5-(3-nitrophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-triaz
ol-1-yl)methyl)pyrimidine (6i)
19

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Yellow solid; Yield 90%; m.p 171-173 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.28 (s, 3H,
CH3), 4.72 (s, 2H, -SCH2-), 5.51 (s, 2H, CH2), 7.18 (s, 2H, NH2), 7.88 (s, 1H, Ar-H), 8.20 (s, 1H,
pyrimidine-5-yl-H), 8.36 (s, 1H, Ar-H), 8.43 (s, 1H, Ar-H), 8.62 (s, 1H, Ar-H); 13C NMR (100
MHz, DMSO-d6) ꢁ(ppm): 25.1, 27.2, 46.1, 107.6, 119.1, 120.9, 122.1, 127.1, 127.6, 130.1, 145.1,
149.1, 150.0, 155.5, 158.5, 164.2, 167.1; HR-EIMS (EI) m/z 426. 1069 (M+H)+, calcd. for
C17H16N9O3S m/z = 426. 1097.
5.6.10.
2-methyl-4-amine-5-((4-(((5-(2-fluorophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-tria
zol-1-yl)methyl)pyrimidine (6j)
o
White solid; Yield 90%; m.p 165-167 C; 1H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.33 (s, 3H,
CH3), 4.64 (s, 2H, -SCH2-), 5.45 (s, 2H, CH2), 7.04 (s, 2H, NH2), 7.42 (d, 2H, Ar-H, J = 21.1 Hz),
7.65 (s, 1H, Ar-H), 7.96 (s, 1H, Ar-H), 8.15 (s, 1H, pyrimidine-5-yl-H); 13C NMR (100 MHz,
DMSO-d6) ꢁ(ppm): 25.1, 27.5, 46.1, 107.3, 116.5, 119.6, 123.2, 125.4, 129.5, 131.5, 149.2, 150.8,
151.4, 157.4, 158.6, 159.4, 164.2, 167.1; HR-EIMS (EI) m/z 399.1143 (M+H)+, calcd. for
C17H16FN8OS m/z = 399.1152.
5.6.11.
2-methyl-4-amine-5-((4-(((5-(2-chloro-4-nitrophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,
2,3-triazol-1-yl)methyl)pyrimidine (6k)
o
1
Yellow solid; Yield 93%; m.p 162-164 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.28 (s, 3H,
CH3), 4.67 (s, 2H, -SCH2-), 5.41 (s, 2H, CH2), 6.91 (s, 2H, NH2), 8.14 (s, 1H, pyrimidine-5-yl-H),
8.20 (s, 1H, Ar-H), 8.23 (s, 1H, 1,2,3-triazol-1-yl-H), 8.40 (s, 1H, Ar-H), 8.43 (s, 1H, Ar-H); 13C
NMR (100 MHz, DMSO-d6) ꢁ(ppm): 25.4, 27.8, 47.1, 108.2, 121.5, 124.4, 126.2, 127.1, 130.1,
20

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132.7, 144.4, 149.2, 158.5, 165.5, 164.1, 164.7, 167.5; HR-EIMS (EI) m/z 460.0710 (M+H)+,
calcd. for C17H15ClN9O3S m/z = 460.0707.
5.6.12.
2-methyl-4-amine-5-((4-(((5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-tria
zol-1-yl)methyl)pyrimidine (6l)
o
1
White solid; Yield 86%; m.p 126-128 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.33 (s, 3H,
CH3), 4.65 (s, 2H, -SCH2-), 5.45 (s, 2H, CH2), 7.04 (s, 2H, NH2), 7.53-7.67 (m, 3H, Ar-H), 7.92 (s,
1H, Ar-H), 8.15 (s, 1H, pyrimidine-5-yl-H); 13C NMR (100 MHz, DMSO-d6) ꢁ(ppm): 25.1, 27.5,
46.1, 107.3, 119.5, 125.3, 126.4, 128.1, 129.7, 131.8, 149.2, 150.8, 150.4, 151.3, 158.3, 164.1,
167.4; HR-EIMS (EI) m/z 415.0831 (M+H)+, calcd. for C17H16ClN8OS m/z = 415.0856.
5.6.13.
2-methyl-4-amine-5-((4-(((5-(2-amine-4-methylthiazol)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-
1,2,3-triazol-1-yl)methyl)pyrimidine (11a)
o
1
Gray solid; Yield 85%; m.p 229-230 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.30 (s, 3H,
CH3), 2.36 (s, 3H, thiazol-CH3), 4.54 (s, 2H, -SCH2-), 5.42 (s, 2H, CH2), 6.93 (s, 2H, pyrimidine
-4-NH2), 7.76 (s, 2H, thiazol-2-NH2), 8.10 (s, 1H, pyrimidine-5-yl-H); 13C NMR (100 MHz,
DMSO-d6) ꢁ(ppm): 14.7, 23.6, 27.8, 46.3, 105.2, 118.3, 119.9, 125.6, 146.1, 149.9, 150.6, 158.6,
164.7, 167.2, 167.8; HR-EIMS (EI) m/z 417.1031 (M+H)+, calcd. for C15H17N10OS2 m/z =
417.1028.
5.6.14.
2-methyl-4-amine-5-((4-(((5-(2-amine-N,4-dimethylthiazol)-1,3,4-oxadiazol-2-yl)thio)methyl)
-1H-1,2,3-triazol-1-yl)methyl)pyrimidine (11b)
21

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486
Gray solid; Yield 86%; m.p 189-191 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.31 (s, 3H,
CH3), 2.40 (s, 3H, thiazol-CH3), 2.87 (s, 3H, NCH3), 4.54 (s, 2H, -SCH2-), 5.43 (s, 2H, CH2), 6.95
(s, 2H, pyrimidine-4-NH2), 8.10 (s, 1H, pyrimidine-5-yl-H), 8.28 (s, 1H, NH); 13C NMR (100
MHz, DMSO-d6) ꢁ(ppm): 14.7, 18.5, 22.5, 27.0, 45.2, 105.8, 118.2, 119.9, 125.1, 146.5, 149.8,
150.9, 158.6, 164.5, 164.8, 167.8; HR-EIMS (EI) m/z 431.1190 (M+H)+, calcd. for C16H19N10OS2
m/z = 431.1185.
5.6.15.
2-methyl-4-amine-5-((4-(((5-(2-amine-4-trifluoromethylthiazol)-1,3,4-oxadiazol-2-yl)thio)met
hyl)-1H-1,2,3-triazol-1-yl)methyl)pyrimidine (11c)
o
1
Gray solid; Yield 81%; m.p 201-202 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.32 (s, 3H,
CH3), 4.58 (s, 2H, -SCH2-), 5.47 (s, 2H, CH2), 7.02 (s, 2H, pyrimidine-4-NH2), 8.12 (s, 1H,
pyrimidine-5-yl-H), 8.29 (s, 2H, thiazol-2-NH2); 13C NMR (100 MHz, DMSO-d6) ꢁ(ppm): 14.5,
23.9, 28.5, 47.1, 106.5, 118.9, 119.9, 125.6, 128.7, 128.4, 128.2, 127.9, 146.1, 148.2, 149.9, 158.9,
164.2, 164.7, 167.9; HR-EIMS (EI) m/z 471.0750 (M+H)+, calcd. for C15H14F3N10OS2 m/z =
471.0746.
5.6.16.
2-methyl-4-amine-5-((4-(((5-(2-amine-N-methyl-4-trifluoromethylthiazol)-1,3,4-oxadiazol-2-y
l)thio)methyl)-1H-1,2,3-triazol-1-yl)methyl)pyrimidine (11d)
o
1
Gray solid; Yield 79%; m.p 197-198 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.30 (s, 3H,
CH3), 2.91 (s, 3H, NCH3), 4.57 (s, 2H, -SCH2-), 5.43 (s, 2H, CH2), 6.93 (s, 2H, pyrimidine
-4-NH2), 8.10 (s, 1H, pyrimidine-5-yl-H), 8.76 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6)
ꢁ(ppm): 14.7, 20.5, 22.5, 27.0, 45.2, 104.9, 119.3, 120.3, 126.7, 127.8, 128.1, 128.4, 128.6, 148.6,
22

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149.8, 150.9, 158.9, 160.4, 164.8, 167.8; HR-EIMS (EI) m/z 485.0910 (M+H)+, calcd. for
C16H16F3N10OS2 m/z = 485.0902.
5.6.17.
2-methyl-4-amine-5-((4-(((5-(2-amine-4-methylthiazol)-4H-1,2,4-triazol-4-amine-2-yl)thio)me
thyl)-1H-1,2,3-triazol-1-yl)methyl)pyrimidine (14a)
o
1
Gray solid; Yield 86%; m.p 219-220 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.31 (s, 3H,
CH3), 2.37 (s, 3H, thiazol-CH3), 4.46 (s, 2H, -SCH2-), 5.42 (s, 2H, CH2), 5.99 (s, 2H,
1,2,4-triazol-NH2), 6.96 (s, 2H, pyrimidine-4-NH2), 7.23 (s, 2H, thiazol-2-NH2), 8.10 (s, 1H,
pyrimidine-5-yl-H); 13C NMR (100 MHz, DMSO-d6) ꢁ(ppm): 15.3, 24.3, 27.9, 46.9, 106.3, 118.7,
119.9, 135.5, 149.9, 150.9, 151.3, 155.4, 158.7, 164.2, 167.6; HR-EIMS (EI) m/z 431.1300
(M+H)+, calcd. for C15H19N12S2 m/z = 431.1297.
5.6.18.
2-methyl-4-amine-5-((4-(((5-(2-amine-N,4-dimethylthiazol)-4H-1,2,4-triazol-4-amine-2-yl)thi
o)methyl)-1H-1,2,3-triazol-1-yl)methyl)pyrimidine (14b)
o
1
Yellow solid; Yield 76%; m.p 188-189 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.31 (s, 3H,
CH3), 2.41 (s, 3H, thiazol-CH3), 2.83 (s, 3H, NCH3), 4.45 (s, 2H, -SCH2-), 5.41 (s, 2H, CH2), 6.00
(s, 2H, 1,2,4-triazol-NH2), 6.96 (s, 2H, pyrimidine-4-NH2), 7.75 (s, 1H, 1,2,3-triazol-H), 8.08 (s,
1H, pyramidine-5-yl-H), 8.29 (s, 1H, NH); 13C NMR(100 MHz, DMSO-d6) ꢁ(ppm): 12.7, 13.5,
21.0, 25.1, 44.8, 105.8, 116.2, 117.9, 131.5, 147.8, 148.9, 149.3, 153.2, 156.6, 162.5, 162.8;
HR-EIMS (EI) m/z 445.1450 (M+H)+, calcd. for C16H21N12S2 m/z = 445.1454.
5.6.19.
2-methyl-4-amine-5-((4-(((5-(2-amine-4-trifluoromethylthiazol)-4H-1,2,4-triazol-4-amine-2-y
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l)thio)methyl)-1H-1,2,3-triazol-1-yl)methyl)pyrimidine (14c)
o
1
Brown solid; Yield 77%; m.p 169-171 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.32 (s, 3H,
CH3), 4.52 (s, 2H, -SCH2-), 5.46 (s, 2H, CH2), 6.02 (s, 2H, 1,2,4-triazol-NH2), 7.01 (s, 2H,
pyrimidine-4-NH2), 7.82 (s, 2H, thiazol-2-NH2), 8.13 (s, 1H, pyrimidine-5-yl-H),; 13C NMR (100
MHz, DMSO-d6) ꢁ(ppm): 14.7, 23.4, 28.9, 47.8, 109.5, 118.9, 116.9, 125.1, 128.5, 128.9, 129.6,
130.3, 151.1, 152.6, 157.2, 160.6, 164.2, 166.7; HR-EIMS (EI) m/z 485.1012 (M+H)+, calcd. for
C15H16F3N12S2 m/z = 485.1014.
5.6.20.
2-methyl-4-amine-5-((4-(((5-(2-amine-N-methyl-4-trifluoromethylthiazol)-1,3,4-oxadiazol-2-y
l)thio)methyl)-4H-1,2,4-triazol-4-amine-2-yl)methyl)pyrimidine (14d)
o
1
Yellow solid; Yield 75%; m.p 156-157 C; H NMR (400 MHz, DMSO-d6) ꢁ(ppm): 2.31 (s, 3H,
CH3), 2.89 (s, 3H, NCH3), 4.48 (s, 2H, -SCH2-), 5.45 (s, 2H, CH2), 6.01 (s, 2H, 1,2,4-triazol-NH2),
6.96 (s, 2H, pyrimidine-4-NH2), 8.10 (s, 1H, pyrimidine-5-yl-H), 8.32 (s, 1H, NH); 13C NMR (100
MHz, DMSO-d6) ꢁ(ppm): 15.3, 22.5, 23.5, 27.6, 47.2, 110.1, 121.3, 126.3, 129.9, 131.9, 132.1,
133.4, 133.6, 150.9, 155.8, 157.3, 159.6, 160.4, 166.6; HR-EIMS (EI) m/z 499.1174 (M+H)+,
calcd. for C16H18F3N12S2 m/z = 499.1171.
5.7. Assay of E.coli PDHc-E1(in vitro) and site-directed mutagenesis of PDHc-E1
The expressing plasmid pMal-C2X-PDHc E1 was transformed into E. coli strain TB1 and
inoculated in Luria–Bertani broth containing 2% glucose and 30 mg/ml ampicillin at 37 ℃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℃ 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
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Sciences). The concentrations of purified proteins were determined by the method of Bradford29
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℃.
The inhibitory activities of synthesized compounds were measured by the enzymatic assay.
PDHc-E1 activity was assayed by a modified method of N. Nemeria30 and measured by
monitoring the reduction of 2,6-DichloroPhenolindophenol (2,6-DCPIP) at 600 nm using a
microplate reader (BioTek Synergy 2, USA). The total volume of 100 ꢀL reaction mixture
contained 50 mM K3PO4 , pH 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 reaction mixtures were incubated for 3
min at 37 ℃, then different concentrations of ThDP (ranging from 0 to 200 ꢀM) were added 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-(V0-V∞)/((IC50/I)n + 1).31 Where V,
V0 , and V∞ are the 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 (ꢀmol/min/mg of PDHc-E1).
Site-directed mutagenesis of PDHc E1 was accomplished by the introduction of specific base
changes into a double-stranded DNA plasmid, as described previously. DNA encoding 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
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mutagenizing primer. The methylated plasmid was digested with DpnI, and 4 µL of each reaction
was used to transform the DH5ꢂ competent cells. All mutations were confirmed 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.
5.8. Molecular docking
For docking purposes, the crystallographic coordinates of the PDHc-E1 with bound ThDP from E.
coli (PDB code: 1L8A)32 were obtained from Brookhaven Data Bank. Hydrogen atoms were
added to the structure allowing for appropriate ionization at physiological pH. The protonated
state of several important residues, such as His142, Tyr177, Glu751, His640 and Met 194, 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 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. All calculations were performed on a CCNU Grid-based
computational
environment
(CCNUGrid
website
http://www.202.114.32.71:8090/ccnu/chem/platform.xml).
572
Acknowledgments
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The work was supported in part by the National Basic Research Program of China (No.
2010CB126100); National Natural Science Foundation of China (No. 21172090 and 21472062);
Natural Science Key Foundation of Hubei Province (No. 2014CFA111); Excellent doctorial
dissertation cultivation grant from Central China Normal University.
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