Design, synthesis and biological evaluation of novel inhibitors against cyanobacterial pyruvate dehydrogenase multienzyme complex E1

Article abstract of DOI:10.1016/j.bmc.2019.01.021

Cyanobacterial pyruvate dehydrogenase multienzyme complex E1 (PDHc E1)is a potential target enzyme for finding inhibitors to control harmful cyanobacterial blooms. In this study, a series of novel triazole thiamin diphosphate (ThDP)analogs were designed a

Full text of DOI:10.1016/j.bmc.2019.01.021

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel inhibitors against
cyanobacterial pyruvate dehydrogenase multienzyme complex E1
Jiangtao Fenga,c, Haifeng Hea,b,c, Yuan Zhoua, Xiaoliang Guoa, Honglin Liua, Meng Caia,
⁎
⁎
Fang Wanga, Lingling Fenga, , Hongwu Hea,
a Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China
b School of Chemistry and Chemical Engineering, Jiangxi Science and Technology Normal University, Nanchang 330013, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cyanobacterial pyruvate dehydrogenase multienzyme complex E1 (PDHc E1) is a potential target enzyme for
finding inhibitors to control harmful cyanobacterial blooms. In this study, a series of novel triazole thiamin
diphosphate (ThDP) analogs were designed and synthesized by modifying the substituent group of triazole ring
and optimizing triazole-benzene linker as potential cyanobacterial PDHc E1 (Cy-PDHc E1) inhibitors. Their
inhibitory activities against Cy-PDHc E1 in vitro and algicide activities in vivo were further examined. Most of
these compounds exhibited prominent inhibitory activities against Cy-PDHc E1 (IC50 1.48–4.48 μM) and good
algicide activities against Synechocystis PCC6803 (EC50 0.84–2.44 µM) and Microcystis aeruginosa FACHB905
(EC50 0.74–1.77 µM). Especially, compound 8d showed not only the highest inhibitory activity against Cy-PDHc
E1 (IC50 1.48 μM), but also the most powerful inhibitory selectivity between Cy-PDHc E1 (inhibitory rate
98.90%) and porcine PDHc E1 (inhibitory rate only 9.54%). Furthermore, the potential interaction between
compound 8d and Cy-PDHc E1 was analyzed by a molecular docking method and site-directed mutagenesis and
enzymatic analysis and fluorescence spectral analysis. These results indicated that compound 8d could be used
as a hit compound for further optimization and might have potential to be developed as a new algicide.
Pyruvate dehydrogenase multienzyme
component E1
Inhibitor
Cyanobacterial blooms
Thiamin diphosphate (ThDP) analogs
Algicide
1. Introduction
Pyruvate dehydrogenase multienzyme complex (PDHc) catalyzes
the oxidative decarboxylation of pyruvate, and subsequently acetylates
With the increasing eutrophication and global warming, harmful
cyanobacterial blooms (HCB) have become a major threat to human
health and ecological system because HCB have increased all over the
world during recent decades and they are possibly expanding further in
the recent future1. Several approaches have been developed to control
HCB, including (1) physical methods, such as nutrient management by
phosphorus-binding clays2, high-frequency sonication for disrupting
cell membranes and retarding photosynthetic activity3; (2) biological
methods, such as viruses, bacteria or fungi4; (3) chemical methods, such
as herbicides (e.g. diuron) and copper-based compounds (e.g. copper
sulfate)5,6. Among these approaches, chemical methods could rapidly
eradicate HCB and have been used for many decades. However, these
chemical compounds are no longer recommended because of their
lengthy environmental persistence and toxic effect on both cyano-
bacteria and other non-target aquatic organisms5,6. Therefore, it is very
necessary and urgent to develop novel selective and safe algicides tar-
geting cyanobacteria.
coenzyme A (CoA) to acetyl-CoA during the tricarboxylic acid meta-
bolic pathway using thiamine diphosphate (ThDP) and Mg2+ as co-
factors7. PDHc poses a key role in cyanobacteria cellular metabolism.
Pyruvate dehydrogenase complex E1 [PDHc E1, EC1.2.4.1] is the first
and most important enzyme during the multienzyme complex com-
posed of three enzymes. The PDHc E1 catalyzes the first step of thiamin
diphosphate-dependent oxidative decarboxylation of pyruvate, which is
the rate limiting step in the overall reaction catalyzed by the PDHc. It
had been reported that PDHc-E1 enzyme was taken as the potent target
of antibacterial and antifungal and herbicide for a long time8,9. These
reports showed that cyanobacterial PDHc E1 (Cy-PDHc E1) possibly
could be a potential candidate enzymatic target for searching specific
algicide to control HCBs.
So far, few studies reported about the inhibitors or algicides tar-
geted to the Cy-PDHc E1. It has been reported that cofactor ThDP plays
an important role in the oxidative decarboxylation catalyzed by PDHc
E1. Currently, based on the structure of ThDP, some of its analogs had
⁎ Corresponding authors.
E-mail addresses: fll708@mail.ccnu.edu.cn (L. Feng), he1208@mail.ccnu.edu.cn (H. He).
c These authors equally contributed to this work.
https://doi.org/10.1016/j.bmc.2019.01.021
Received 10 December 2018; Received in revised form 18 January 2019; Accepted 21 January 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Feng,J.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.01.021

J. Feng et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 1. Structures of known potential ThDP analog
inhibitors against PDHc E1.
been reported as effective inhibitors against plant PDHc E1 or
Escherichia coli (E. coli) PDHc E1 in vitro and against herbicide or an-
tifungal or antibacterial activities in vivo (Fig. 1)9–13. Compound I is an
effective inhibitor of Escherichia coli (E. coli) PDHc E1 in vitro in our
previous study16, but we found that it was almost not effective against
Cy-PDHc E1.
compounds 6a-6g and 8a-8g were synthesized by thiamine hydro-
chloride (Vitamin B1) as starting materials16,20,21
.
The 3a-3g compounds were synthesized by the reaction of various
substituted benzoic acid with oxalyl chloride using dichloromethane
(DCM) as solvent. 3a-3g compounds were reacted with propynol to
prepare substituted benzoate 4a-4g using triethylamine (Et3N) as base.
Then the substituted benzoate 4a-4g was used to prepare 5a-5g in the
presence of CuI, Et3N and THF. Compounds 5a-5g were further con-
verted into corresponding hydrochloride 6a-6g by using hydrochloric
acid for increasing the water solubility. Substituted benzoate 4a-4g
reacted with N-iodomorpholine to produce compounds 7a-7g in the
presence of catalyzer CuI and solvent tetrahydrofuran (THF). 8a-8g
were synthesized using intermediates 7a-7g as materials in the same
methods.
In order to find the inhibitors of Cy-PDHc E1, structurally diversi-
fied inhibitors should be investigated. Based on the structure of the
triazole-ThDP analogs and compound I, firstly, considering that benzoic
acid and benzoate derivatives have received much attention especially
for their structures and biological activities, such as anticancer, anti-
microbial activities17–19, we remained the 2-methylpyrimidine-4-yla-
mine moiety of triazole ThDP in our new compounds. Then, because the
triazole ring moiety has wider biological activities and plays an im-
portant role in bio-conjugation due to easily form powerful pharma-
cophores, we tried to remain the triazole ring from triazole-ThDP
analogs in our new compounds. Furthermore, the highly charged pyr-
ophosphate moiety of ThDP analogs leads a bad bioavailability and
negligible anti-microbial activity. Our previous studies have been de-
monstrated that these highly charged pyrophosphate moiety could be
replaced with substituted benzene ring to solve the problem and some
All title compounds 6a-6g and 8a-8g were characterized by 1H
NMR, 13C NMR, mass spectrometry (MS), and confirmed by elementary
analysis.
2.2. Inhibitory activity and selectivity of the new ThDP analogs against Cy-
PDHc E1 and Porcine PDHc E1 in vitro
herbicide and antifungal and antibacterial candidates were found12–15
.
As shown in Table 1, the compounds Ia-Id, 6a-6g and 8a-8g were
evaluated for their inhibitory activities against Cy-PDHc E1 in vitro. The
lead compounds Ia-Id showed strong inhibitory activity against E. coli
PDHc E1 in our previous study16. However, when we determined the
inhibitory activities of lead compounds Ia-Id against Cy-PDHc E1, the
results showed that compounds Ia-Id had very slight inhibitory activity
against Cy-PDHc E1 (Table 1). Then compounds 5a-5g were designed
and synthesized by introducing ester group (OeC]O) as a linker to
connect the 1, 2, 3-triazole ring with benzene ring. But due to their poor
water solubility, compounds 5a-5g were further converted into corre-
sponding hydrochloride 6a-6g by using hydrochloric acid to increase
their solubility. The most exciting part to us was that all the compounds
6a-6g had much better inhibitory activity against Cy-PDHc E1 (IC50
3.37–4.48 µM) than that of lead compounds Ia-Id.
So we used benzene ring to substitute the pyrophosphate group and
introduced ester group (OeC]O) as a functional linker to connect the
1, 2, 3-triazole ring with benzene ring. It is very interesting that the
optimized compounds showed higher selective inhibition activity
against Cy-PDHc E1. These results encouraged us to further design and
synthesize a series of ThDP analogs as potential inhibitors of Cy-PDHc
E1.
In this study, we designed and synthesized a series of (1-((4-amino-
2-methyl pyrimidin-5-yl) methyl)-1H-1,2,3-triazol-4-yl) methyl sub-
stituted benzoate by remaining the 2- methylpyrimidine-4-ylamine
moiety and the triazole ring and using the ester group (OeC]O) as a
functional linker between 1,2,3-triazole ring and substituted benzene
ring (Fig. 2). These compounds not only exhibited higher inhibitory
activities against Cy-PDHc E1 in vitro, but also had good algicide ac-
tivities against Synechocystis PCC6803 and Microcystis aeruginosa
FACHB905 than that of triazole-ThDP or compound I in viro. Especially
compound 8d showed the highest inhibitory activity against Cy-PDHc
E1 and the most powerful inhibitory selectivity between Cy-PDHc E1
and porcine PDHc E1. Furthermore, the potential interaction between
compound 8d and Cy-PDHc E1 was analyzed by a molecular docking
method and site-directed mutagenesis and enzymatic analysis and
fluorescence spectral analysis.
Subsequently, compounds 8a-8g were synthesized in order to in-
vestigate the effect of introduction of iodine on the inhibitory activity
against Cy-PDHc E1. As shown in Table 1, compounds 8a-8g with io-
dine (I) showed higher inhibitory activity (IC50 = 1.48–2.35 µM)
against Cy-PDHc E1 than that of compounds 6a-6g (IC50
3.37–4.48 µM). Especially compound 8d exhibited highest inhibitory
activity (IC50 = 1.48 µM).
Based on these results, it can be speculated that the ester group of
compounds 6a-6g and 8a-8g was the most important factor for their
inhibitory activities against Cy-PDHc E1, at the same time, the in-
troduction of iodine group to the 5-position of 1, 2, 3-triazole is to the
benefit for increasing the inhibitory activity of compounds 8a-8g
against Cy-PDHc E1.
2. Results and discussion
2.1. Design and synthesis of the new ThDP analogs 8a-8g
Furthermore, in order to examine the selectivity of these com-
pounds, the inhibitory activities of compounds 6a-6g and 8a-8g against
Porcine PDHc E1 were determined. As showed in Table 1, our results
As shown in Scheme 1, the (1-((4-amino-2-methyl pyrimidin-5-yl)
methyl)-1H- 1,2,3- triazol −4-yl) methyl substituted benzoate
Fig. 2. Design of novel ThDP analog inhibitors
against cyanobacterial PDHc E1.
2

J. Feng et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Scheme 1. Reagents and conditions (a) NaN3, Na2SO3, H2O, 60–65 °C, 5 h; (b) C2O2Cl2, DCM/DMF, rt, 2–3 h; (c) propynol, Et3N, DCM, rt; (d) CuI, Et3N, THF, rt, 12 h;
(e) HCl, CH3OH,rt; (f) N-iodomorpholine, CuI, THF, rt, 1 h; (g) CuI, Et3N, THF, rt, 12 h.
Table 1
Structures and activities against Cy-PDHc E1 or Porcine PDHc-E1 of compounds 6a-6g and 8a-8g.
a
b
c
Compd.
R1
Cy-PDHc-E1 IC50 (µM)
Inhibitory rate (%)
EC50 (μM)
Cy-PDHc-E1
Porcine PDHc-E1
PCC6803d
FACHB905d
Ia
H
> 100
> 100
> 100
> 100
19.0
8.64
16.2
29.6
95.5
95.7
99.5
97.8
94.2
93.2
95.3
96.5
99.2
95.4
98.9
95.6
98.7
97.1
–
–
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
Ib
4-NO2
4-F
–
Ic
–
Id
4-Cl
H
–
6a
6b
6c
6d
6e
6f
3.58
3.57
3.88
3.37
3.55
3.73
4.48
1.94
1.69
1.61
1.48
2.35
1.79
1.88
–
0.25
0.14
0.05
0.16
0.12
0.19
0.27
0.19
0.06
0.19
0.05
0.16
0.10
0.08
15.9
13.5
17.8
7.31
–
1.66
1.64
2.11
2.44
2.25
1.81
1.43
1.44
1.09
0.84
1.17
0.90
1.16
1.14
1.47
0.11
1.17
1.33
1.22
1.43
1.11
1.29
1.20
0.99
0.95
0.78
0.90
1.06
0.74
0.85
1.77
0.06
0.06
0.05
0.11
0.05
0.21
0.13
0.08
0.02
0.05
0.07
0.07
0.08
0.03
0.14
4-NO2
4-F
0.38
0.20
0.13
0.24
0.56
0.11
0.18
0.05
0.15
0.11
0.24
0.15
0.06
0.08
2-Cl
4-Cl
3-Cl
4-CH3
H
9.62
–
6g
8a
8b
8c
8d
8e
8f
21.0
10.4
15.3
9.50
–
4-NO2
4-F
2-Cl
4-Cl
3-Cl
4-CH3
–
5.13
–
8g
CuSO4
–
a
IC50 (µM) is defined as the concentration required for 50% inhibition against Cy-PDHc E1 in vitro and were estimated statistically by origin7.5 software. Values
are means
SD (n = 3).
b
c
d
Inhibitory rate (%) of compounds against Cy-PDHc E1 or Porcine PDHc E1 in vitro at 100 µM.
EC50 (μM) is defined as the concentration required for 50% inhibition against PCC6803 or FACHB905. Values are means
PCC6803, Synechocystis sp. PCC6803; FACHB905, Microcystis aeruginosa FACHB905.
SD (n = 3).
3

J. Feng et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
showed that compounds 6a-6g and 8a-8g had obviously selective in-
hibition between Cy-PDHc E1 and Porcine PDHc-E1. All the compounds
6a-6g and 8a-8g had stronger inhibition against Cy-PDHc E1 (in-
hibitory rate > 90%) than that of their inhibition against porcine PDHc
E1 (inhibitory rate only 7.31–21.0%) in vitro at the same concentration
(100 µM). It indicated that compounds 6a-6g and 8a-8g showed lower
toxicity and owed more eco-friendly characterizations as potential al-
gicide.
2.3. Algicide activities of the new ThDP analog inhibitors against
cyanobacteria in vivo
Most known ThDP analogs showed antibacterial activity or anti-
fungal or herbicide activity8,9,12–16, but few researches reported about
ThDP analogs with algicide activities. In order to examine the practi-
cality of compounds 6a-6g and 8a-8g, we further evaluated their al-
gicide activities against two model cyanobacteria strains Synechocystis
sp. PCC6803 (PCC6803) and Microcystis aeruginosa FACHB905
(FACHB905). As shown in Table 1, compounds Ia-Id did not display
algicide activities (EC50 > 100 µM), conversely the corresponding
compounds 6a-6g and 8a-8g exhibited dramatically high algicide ac-
tivities (EC50 = 0.74–2.44 µM). Especially compounds 8a-8g showed
higher algicide activities than that of CuSO4, a kind of widely used
algicide. These results indicated that the algicide activity of these ThDP
analogs also could be significantly enhanced by introducing ester group
(OeC]O) as a linker to connect the 1, 2, 3-triazole ring with benzene
ring and introducing iodine group to the 5-position of 1, 2, 3-triazole.
Fig. 3. Binding modes of compound 8d target into active site of Cy-PDHc E1,
PDHc E1 is shown in cartoon, compound 8d is shown in stick, some key re-
sidues are shown in lines and hydrogen bonds are shown in dotted lines.
2.4. The interaction between the novel ThDP analog inhibitors and Cy-
PDHc E1
Because it showed the highest inhibitory activity (IC50 = 1.48 µM)
against Cy-PDHc E1 and higher inhibitory activity against
Synechocystis sp. PCC6803 (PCC6803) and Microcystis aeruginosa
FACHB905 (FACHB905), compound 8d was selected as a hit compound
for the study of the interaction mode of the novel ThDP analogs (8a-8g)
with the potential target Cy-PDHc E1. Firstly, the modeling 3D struc-
ture of Cy-PDHc E1 was generated on SwissModel server (https://
swissmodel.expasy.org/)22 by homology modeling using the crystal
structure of Geobacillus stearothermophilus PDHc E1 (Geo-PDHc E1)
(PDB: 1 W88) as a template which has 38.4% identity with Cy-PDHc
E123. Then the reliability of the 3D model structure of Cy-PDHc E1 was
further confirmed by Ramachandran plot analyses of PROCHECK and
QMEAN24–26. Molecular docking simulation analysis of compound 8d
with the active site of Cy-PDHc E1 was performed by using the SUR-
FLEX module of SYBYL package.
Fig. 4. Binding constants (Kb) determined by fluorescence spectral analyses for
the binding of compound 8d to the wild type (WT) and its mutants. Values are
means
SD (n = 3).
with compound 8d.
3. Conclusion
Based on the structure of ThDP, in this study, a series of novel ThDP
analogs 6a-6g and 8a-8g were designed by optimizing triazole-benzene
linker and modifying the substituent group of triazole ring. Then they
were synthesized as potential inhibitors of Cy-PDHc E1 by using click
chemistry. All the compounds 6a-6g (IC50 3.37–4.48 µM) had much
better inhibitory activity against Cy-PDHc E1 than that of lead com-
pounds Ia-Id. Compounds 8a-8g with iodine (I) in the 5-position of 1,
2, 3-triazole showed higher inhibitory activity (IC50 = 1.48–2.35 µM)
against Cy-PDHc E1 than that of compounds 6a-6g. Especially com-
pound 8d exhibited highest inhibitory activity (IC50 = 1.48 µM). These
results showed that the ester group of compounds 6a-6g and 8a-8g was
the most important factor for their inhibitory activities against Cy-PDHc
E1, at the same time, the introduction of iodine group to the 5-position
of 1, 2, 3-triazole was to the benefit for increasing the inhibitory ac-
tivity of compounds 8a-8g against Cy-PDHc E1.
The binding mode of compound 8d is shown in Fig. 3. It can be seen
that 8d could occupy the ThDP-binding pocket of Cy-PDHc E1, which
has the ‘V’conformation27,28. The aminopyrimidine ring of compound
8d has similar interactions with amino acid residues to ThDP or ThTDP
or compound I in the ‘V’ conformation, which can form backbone hy-
drogen bonds with the amino acid residues Ala129 and Ile131. More-
over, strong π-π stacking can be formed between aminopyrimidine ring
and amino acid residue Phe85. It was extraordinary that the ester group
of compound 8d obviously owned hydrogen bond interaction with
amino acid residues Tyr80 and Arg81 in the active site of Cy-PDHc E1.
This is the possibly important reason that the inhibition activity of
compound 8d with ester group against Cy-PDHc E1 was higher than
that of compound I. To validate the predictive interaction between
compounds 8d and the amino acids binding with ester group, we fur-
ther performed site-directed mutagenesis and fluorescence spectral
analyses as shown in Fig. 4. The binding constant (Kb) against the
Y80A, R81A and F85A were investigated using fluorescence spectral
analysis. In line with our theoretical prediction, Kbs of the three mu-
tants are obviously lower than the Kb value of the wild type enzyme
(Fig. 4), suggesting that Y80, R81 and F85 have stronger interaction
It is more interesting that all the compounds 6a-6g and 8a-8g had
stronger inhibition against Cy-PDHc E1 (inhibitory rate > 90%) than
that of their inhibition against porcine PDHc E1 (inhibitory rate only
7.31–21.0%) in vitro at the same concentration (100 µM), indicating
that they owned possibly lower toxicity and more eco-friendly char-
acterizations as potential algicide. Furthermore, the corresponding
4

J. Feng et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
compounds 6a-6g and 8a-8g exhibited dramatically high algicide ac-
tivities (EC50 = 0.74–2.44 µM). Especially compounds 8a-8g showed
better algicide activities even than that of CuSO4.
OCH2), 7.47 (s, 2H, NH2), 7.61 (s, 1H, Ar-H), 7.88 (s, 2H, Ar-H), 7.95 (s,
1H, 1,2,3-triazol-4-yl-H), 8.40 (s, 2H, Ar-H), 8.67 (s, 1H, pyrimidin-6-
yl-H), 14.07 (s, 1H, HCl); ESI-MS m/z: 325.2 [M + 1]+; HRMS (ESI):
The results of molecular docking together with site-directed muta-
genesis and fluorescence spectral analysis showed that triazole-benzene
linker and the substituent group of triazole ring are the possibly im-
portant reasons for the higher inhibition activity of compound 8d.
These results indicated that compound 8d could be used as a hit com-
pound for further optimization and might have potential to be devel-
oped as a new algicide.
calcd. for
C
16H16N6O2 [M + H]+ 325.14075, found: 325.14118;
Elemental Anal. Calcd for C16H17ClN6O2: C, 53.26; H, 4.75; N, 23.29.
Found: C, 53.55; H, 4.99; N, 23.54.
4.1.3.1. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl-4-nitrobenzoate hydrochloride 6b. Yellow solid; Yield 90%; m.p
140–142 °C; 1H NMR (400 MHz, DMSO‑d6) δ(ppm): 2.50 (s, 3H, CH3),
5.44 (s, 2H, CH2), 5.81 (s, 2H, OCH2), 8.13 (s, 2H, NH2), 7.91 (s, 1H,
1,2,3-triazol-4-yl-H), 8.13 (s, 1H, Ar-H), 8.29 (s, 1H, pyrimidin -6-yl-H),
8.29 (s, 2H, Ar-H), 8.60 (s, 1H, Ar-H); 13C NMR (100 MHz, DMSO‑d6)
δ(ppm): 25.3, 47.0, 58.2, 108.6, 125.5, 127.5, 129.0, 132.0, 138.5,
141.4, 156.8, 160.9, 164.0, 167.6; ESI-MS m/z: 370.2 [M + 1]+; HRMS
(ESI): calcd. for C16H15N7O4 [M + H]+ 370.12583, found: 370.12679;
Elemental Anal. Calcd for C16H16ClN7O4: C, 47.36; H, 3.97; N, 24.16.
Found: C, 47.19; H, 4.03; N, 24.55.
4. Experimental procedures
4.1. Chemical synthesis procedures
All chemical reagents were commercially available and treated with
standard methods. Melting points (mp) were measured on an electro-
thermal melting point apparatus and were uncorrected. 1H and 13C
NMR spectra were recorded at 400 MHz, in DMSO‑d6 solution on a
Varian Mercury-Plus 400 MHz 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. Elemental analysis (EA) was measured on a Vario ELIII
CHNSO elemental analyzer. Intermediate 5-(azidomethyl)-2-methyl-
4.1.3.2. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl-4-fluorobenzoate hydrochloride 6c. Yellow solid; Yield 88%;
m.p 126–128 °C; 1H NMR (400 MHz, DMSO‑d6) δ(ppm): 2.50 (s, 3H,
CH3), 5.37 (s, 2H, CH2), 5.52 (s, 2H, OCH2), 7.13 (s, 2H, NH2), 7.32 (s,
2H, Ar-H), 7.98 (s, 2H, Ar-H), 8.01 (s, 1H, 1,2,3-triazol-4-yl-H), 8.26 (s,
1H, pyrimidin-5-yl-H); 13C NMR (100 MHz, DMSO‑d6) δ(ppm): 24.3,
46.4, 57.3, 107.2, 114.8, 115.3, 124.1, 125.0, 131.4, 141.7, 155.2,
160.7, 163.1, 163.9, 165.7, 166.4; ESI-MS m/z: 343.3 [M + 1]+; HRMS
(ESI): calcd. for C16H15FN6O2 [M + H]+ 343.13133, found: 343.13204;
Elemental Anal. Calcd for C16H16ClFN6O2: C, 50.73; H, 4.26; N, 22.19.
Found: C, 50.88; H, 4.45; N, 22.34.
pyrimidine-4-amine
1 was synthesized according to the existing
methods29
.
4.1.1. General procedure for preparation of substituted prop-2-yn-1-yl
substituted benzoate 4a-4g
Intermediates 3a-3g were synthesized respectively by the reaction
of corresponding substituted benzoic acid (5 mmol) with oxalyl
chloride (10 mmol) in the presence of dichloromethane (10 ml). The
solution of the substituted benzoyl chloride 3a-3g was added dropwise
to the solution of propynol (6 mmol), trimethylamine (6 mmol) and
dichloromethane (20 ml) at 0 °C. Then the reaction mixture was stirred
at room temperature until the reaction was completed based on the TLC
monitoring. The reaction mixture was washed with water (15 ml) and
extracted three times with dichloromethane. Then the organic phases
were washed sequentially with 10% NaOH solution, brine, dried with
MgSO4 and evaporated under reduced pressure to get crude products.
The crude products were purified by column chromatography (silica
gel, ethyl acetate: petroleum ether = 1:3 as eluent) to obtain 4a-4g.
4.1.3.3. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl-2-chlorobenzoate hydrochloride 6d. Yellow solid; Yield 89%;
m.p 136–138 °C; 1H NMR (400 MHz, DMSO‑d6) δ(ppm): 2.50 (s, 3H,
CH3), 5.38 (s, 2H, CH2), 5.59 (s, 2H, OCH2), 7.17 (s, 2H, NH2), 7.43 (s,
1H, Ar-H), 7.55 (s, 2H, Ar-H), 7.75 (s, 1H, Ar-H), 8.00 (s, 1H, 1,2,3-
triazol-4-yl-H), 8.26 (s, 1H, pyrimidin-6-yl-H); 13C NMR (100 MHz,
DMSO‑d6) δ (ppm): 26.0, 46.5, 58.1, 108.8 125.2, 127.8, 129.0, 130.6,
133.3, 139.0, 140.1, 141.9, 156.7, 162.1, 164.7, 167.5; ESI-MS m/z:
359.1 [M + 1]+; HRMS (ESI): calcd. for C16H15ClN6O2 [M + H]+
359.10178, found: 359.10356; Elemental Anal. Calcd for
C
16H16Cl2N6O2: C, 48.62; H, 4.08; N, 21.26. Found: C, 48.89; H,
4.44; N, 21.54.
4.1.2. General procedure for preparation of compounds 6a-6g and 8a-8g
4.1.3.4. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl-4-chlorobenzoate hydrochloride 6e. White solid; Yield 54%;
m.p. 188–189 °C; 1H NMR (400 MHz, DMSO‑d6) δ (ppm): 2.29 (s, 3H,
CH3), 5.37 (s, 2H, CH2), 5.44 (s, 2H, OCH2), 6.96 (s, 2H, NH2), 7.59 (d,
2H, J = 7.2 Hz, Ar-H), 7.94 (d, 2H, J = 7.2 Hz, Ar-H), 8.02 (s, 1H,
1,2,3-triazol-4-yl-H), 8.25 (s, 1H, pyrimidin-5-yl-H); 13C NMR
(100 MHz, DMSO‑d6) δ (ppm): 25.3, 46.8, 59.2, 108.4, 124.3, 127.8,
129.0, 131.5, 138.1, 140.9, 142.0, 156.8, 161.4, 164.5, 167.3; HRMS
Intermediate 5-azidomethyl-2-methylpyrimidine-4-yla mine
1
(1.5 mmol) was reacted with 4a-4g respectively in the presence of THF
(10 ml), CuI (0.075 mmol) and Et3N (3 mmol)20. The reaction was
stirred at room temperature until the reaction was complete based on
TLC monitoring and then poured into water to form precipitate. The
precipitate was collected by filtration, dried under atmospheric pres-
sure and washed with DCM (10 ml) to obtained 5a–5g, which were
used directly to react with 36% hydrogen chloride for preparing com-
pounds 6a-6g.
(ESI): calcd. for
C
16H15ClN6O2 [M + H]+ 359.10178, found:
359.10257; Elemental Anal. Calcd for C16H15ClN6O2 (358.10): C,
53.56; H, 4.21; N, 23.42. Found: C, 53.93; H, 4.15; N, 22.98.
Intermediates 4a-4g (2 mmol) were dissolved in THF (10 ml) and
reacted with N-iodomorpholine (3 mmol) by using CuI (0.1 mmol) as
catalyzer. The reaction mixture was stirred at room temperature for 1 h
until white precipitate formed. Then the white precipitate was washed
three times with THF (10 ml) to prepare 7a-7g. Then compounds 8a-8g
were synthesized by 7a-7g respectively reacting with compound 1 in
the same way which was used to synthesize 5a–5g.
4.1.3.5. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl-3-chlorobenzoate hydrochloride 6f. Yellow solid; Yield 90%;
m.p 135–137 °C; 1H NMR (400 MHz, DMSO‑d6) δ(ppm): 2.50 (s, 3H,
CH3), 5.39 (s, 2H, OCH2), 5.61 (s, 2H, CH2), 7.23 (s, 2H, NH2), 8.02 (s,
1H, 1,2,3-triazol-4-yl-H), 7.53 (s, 1H, Ar-H), 7.72 (s, 1H, Ar-H), 7.87 (s,
2H, Ar-H), 8.28 (s, 1H, pyrimidin-6-yl-H); 13C NMR (100 MHz,
DMSO‑d6) δ (ppm): 26.2, 47.4, 58.3, 109.5, 124.9, 128.3, 129.2,
131.7, 132.5, 138.9, 140.4, 141.9, 156.3, 161.6, 164.4, 167.5; ESI-MS
m/z: 359.2 [M + 1]+; HRMS (ESI): calcd. for C16H15ClN6O2 [M + H]+
359.10178, found: 359.10229; Elemental Anal. Calcd for
4.1.3. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl) methyl-benzoate hydrochloride 6a
Yellow solid: Yield 96%; m.p 172–174 °C; 1H NMR (400 MHz,
DMSO‑d6) δ (ppm): 2.50 (s, 3H, CH3), 5.35 (s, 2H, CH2), 5.61 (s, 2H,
5

J. Feng et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
C16H16Cl2N6O2: C, 48.62; H, 4.08; N, 21.26. Found: C, 48.55; H, 4.22;
N, 21.40.
131.3, 136.2, 139.2, 142.5, 157.2, 161.4, 166.0, 168.2; HRMS (ESI):
calcd. for C16H14ClIN6O2 [M + 1]+ 484.99842, found: 484.99794;
Elemental Anal. Calcd for C16H14ClN6O2(483.99): C, 39.65; H, 2.91;
N, 17.34. Found: C, 39.38; H, 2.73; N, 17.23.
4.1.3.6. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl-4-methylbenzoate hydrochloride 6g. Yellow solid; Yield 89%;
m.p 132–134 °C; 1H NMR (400 MHz, DMSO‑d6) δ(ppm): 2.50 (s, 3H,
CH3), 2.65 (s, 3H, CH3), 5.50 (s, 2H, CH2), 5.75 (s, 2H, OCH2), 7.44 (s,
2H, NH2), 7.44 (s, 2H, Ar-H), 7.96 (s, 2H, Ar-H), 7.96 (s, 1H, 1,2,3-
triazol-4-yl-H), 8.44 (s, 1H, pyrimidin-6-yl-H); 13C NMR (100 MHz,
DMSO‑d6) δ(ppm): 22.6, 25.0, 47.3, 58.2, 108.2, 125.5, 127.5, 129.2,
131.8, 138.7, 141.8, 156.7, 161.6, 164.8, 167.5; ESI-MS m/z: 339.2
[M + 1]+; HRMS (ESI): calcd. for C17H18N6O2 [M + H]+ 339.15640,
found: 339.15706; Elemental Anal. Calcd for C17H19ClN6O2: C, 54.47;
H, 5.11; N, 22.42. Found: C, 54.66; H, 5.34; N, 22.56.
4.1.3.12. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-3-chlorobenzoate 8f. Celadon solid; Yield 88%; m.p.
185–186 °C; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.50(s, 3H, CH3),
5.39(s, 2H, CH2), 5.51(s, 2H, -CH2O), 6.95(s, 2H, NH2), 7.57(s, 1H, Ar-
H), 7.75(s, 1H, Ar-H), 7.90(s, 2H, Ar-H), 13C NMR(100 MHz, DMSO‑d6)
δ(ppm): 25.7, 47.0, 58.1, 108.5, 124.7, 127.9, 128.8, 131.1, 138.3,
142.3, 143.0, 156.4, 161.9, 165.6, 167.7; HRMS (ESI): calcd. for
C
16H14ClIN6O2 [M + 1]+ 484.99842, found: 484.99568; Elemental
Anal. Calcd for C16H14ClN6O2 (483.99): C, 39.65; H, 2.91; N, 17.34.
Found: C, 39.74; H, 2.86; N, 17.15.
4.1.3.7. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)-methylbenzoate 8a. Green solid; Yield 91%; m.p.
198–199 °C; 1H NMR (400 MHz, DMSO‑d6) δ (ppm): 2.33 (s, 3H,
CH3), 5.36 (s, 2H, CH2), 5.48 (s, 2H, -CH2O), 6.95 (s, 2H, NH2), 7.52
4.1.3.13. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-4-methoxybenzoate 8g. Green solid; Yield 75%; m.p.
200-201 °C ; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.29(s, 3H, CH3),
2.36(s, 3H, -CH3), 5.35(s, 2H, CH2), 5.44(s, 2H, -CH2O), 6.95(s, 2H,
NH2), 7.32(d, 2H, J = 8.0 Hz, Ar-H), 7.83(d, 2H, J = 8.0 Hz, Ar-H),
8.01(s, 1H, pyrimidin-5-yl-H); 13C NMR(100 MHz, DMSO‑d6) δ(ppm):
25.7, 29.7, 46.9, 59.2, 109.1, 124.8, 127.6, 129.0, 131.1, 139.0, 143.3,
155.8, 163.3, 165.1, 167.5; HRMS (ESI): calcd. for C17H17IN6O2 [M
+1]+ 465.05304, found: 465.05161; Elemental Anal. Calcd for
C17H17IN6O3(464.05): C, 42.51; H, 3.57; N, 17.50. Found: C, 42.44;
H, 3.58; N, 17.67.
(s, 3H, Ar-H), 7.94(s, 2H, Ar-H), 7.66 (s, H, pyrimidin-5-yl-H Ar-H); 13
C
NMR (100 MHz, DMSO‑d6) δ(ppm): 25.9, 46.9, 57.7, 108.8, 128.5,
129.4, 131.5, 133.5, 138.5, 142.6, 156.2, 160.2, 164.8, 167.6; HRMS
(ESI): calcd. for C16H15IN6O2 [M + 1]+ 451.03739, found: 451.03703;
Elemental Anal. Calcd for C16H15IN6O2(450.03): C, 42.68; H, 3.36; N,
18.61. Found: C, 42.64; H, 3.43; N, 19.67.
4.1.3.8. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-4- nitrobenzoate 8b. Yellow solid; Yield 86%; m.p.
192–193 °C; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.39(s, 3H, CH3),
5.36(s, 2H, CH2), 5.55(s, 2H, -CH2O), 6.98(s, 2H, NH2), 7.36(s, 2H, Ar-
H), 8.01(s, 2H, Ar-H), 8.01(s, 1H, pyrimidin-5-yl-H); 13C NMR
(100 MHz, DMSO -d6) δ(ppm): 25.7, 47.9, 57.6, 108.4, 124.9, 127.9,
129.2, 131.3, 138.7, 141.8, 156.6, 161.6, 165.6, 166.7; HRMS (ESI):
4.2. Expression and purification of Cy-PDHc E1 and its activity
measurements
calcd. for
C
16H14IN7O4 [M + 1]+ 496.02247, found: 496.02199;
The genes encoding Cy-PDHc-E1 (Gene Bank Number: BA000022.2)
were directly amplified from genomic DNA of Synechocystis PCC6803
and then were inserted into the pETDuet-1 vector (Novagen). Positive
transformants were verified by sequencing and then they were co-
transformed into Escherichia coli BL-21 (DE3) together with plasmid
pGro7 for over-expressing chaperonins GroEL and GroES. For expres-
sion of the enzyme, the recombination stain was inoculated in 2xYT
medium with 100 µg/ml ampicillin and 20 µg/ml chloramphenicol at
37 °C until reaching a cell density to A600 of 0.5–0.6. The culture was
induced with a final concentration of 0.5 mM IPTG and 0.5 mg/mL L-
Arabinose for 24 h at 22 °C before harvesting. The harvested pellets
were resuspended in buffer (300 mM NaCl, 50 mM potassium phos-
phate buffer, 1% glycerol and 1 mM DTT, 1 mM MgCl2, pH 7.2) and
sonicated, subsequently the lysate supernatant containing soluble cel-
lular materials was loaded on a Ni2+-NTA resin (Novagen) in a stan-
dard procedure. Proteins were eluted in two steps with 300 mM NaCl,
50 mM potassium phosphate buffer, and 1% glycerol and 1 mM DTT,
1 mM MgCl2, pH 7.2, 75 mM imidazole and 250 mM imidazole. The
isolated proteins were dialyzed against 300 mM NaCl, 50 mM potassium
phosphate buffer, and 1% glycerol and 1 mM DTT, 1 mM MgCl2, pH 7.2.
Purified protein was stored in 50% (v/v) glycerol at −20 °C. The con-
centrations of purified proteins were determined by the Bradford
Elemental Anal. Calcd for C16H14IN7O4(495.02): C, 38.80; H, 2.85; N,
19.80. Found: C, 38.88; H, 3.01; N, 20.05.
4.1.3.9. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-4-fluorobenzoate 8c. Yellow solid; Yield 73%; m.p.
207–209 °C ; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.30(s, 3H, CH3),
4.50(s, 2H, CH2), 5.42(s, 2H, -CH2O), 6.91(s, 2H, NH2), 7.53(d, 2H,
J = 7.4 Hz, Ar-H), 7.88(d, 2H, J = 7.5 Hz, Ar-H), 8.45(s, 1H, pyrimidin-
5-yl-H); 13C NMR(100 MHz, DMSO‑d6) δ(ppm): 24.7, 47.9, 57.6, 108.6,
115.1, 124.2, 127.5, 131.6, 142.5, 157.4, 161.6, 164.2, 165.3, 166.9,
167.9; HRMS (ESI): calcd. for C16H14FIN6O2 [M + 1]+ 469.02797,
found: 469.02847; Elemental Anal. Calcd for C16H14FIN6O2 (468.02):
C, 41.04; H, 3.01; N, 17.95. Found: C, 40.87; H, 3.42; N, 18.01.
4.1.3.10. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-2-chlorobenzoate 8d. Celadon solid; Yield 86%; m.p.
185–187 °C; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.36(s, 3H, CH3),
4.94(s, 2H, CH2), 5.50(s, 2H, -CH2O), 7.00(s, 2H, NH2), 7.55–7.59(m,
2H, Ar-H), 7.76(d, 2H, J = 4.0 Hz, Ar-H), 8.18 (s, 1H, pyrimidin-5-yl-
H); 13C NMR(100 MHz, DMSO‑d6) δ(ppm): 26.1, 48.1, 58.3, 108.6,
125.0, 128.3, 129.0, 130.8, 132.0, 138.7, 141.7, 142.6, 155.8, 161.2,
164.0, 167.5; HRMS (ESI): calcd. for
C
16H14ClIN6O2 [M + 1]+
method19
.
484.99842, found: 484.99809; Elemental Anal. Calcd for
Cy-PDHc E1 activity was determined by monitoring the reduction of
2, 6 – DiChlorophenol IndoPhenol (2,6-DCIP) at 600 nm using a mi-
croplate reader (Bioteck Synergy2, USA)20. Cofactor ThDP and sub-
strate pyruvate were purchased from Sigma. A standard reaction mix-
ture containing 50 mM K3PO4 (pH 7.2), 0.375 mM 2, 6-
DiChlorophenolIndoPhenol (DCIP), 0.8 mM sodium pyruvate as sub-
strate, 0.1 mg/ml purified Cy-PDHc E1 enzyme. The reaction mixtures
were incubated for 3 min at 37 °C, then added different concentrations
of ThDP (ranging from 0 to 200 µM). One unit of activity is defined as
the amount of 2, 6-DCIP reduced.
C
16H14ClN6O2(483.99): C, 39.65; H, 2.91; N, 17.34. Found: C, 39.28;
H, 3.16; N, 17.79.
4.1.3.11. (1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-
triazol-4-yl)methyl-4-chlorobenzoate 8e. Grass green solid; Yield 84%;
m.p. 192–193 °C; 1H NMR(400 MHz, DMSO‑d6) δ(ppm): 2.33(s, 3H,
CH3), 5.36(s, 2H, CH2), 5.49(s, 2H, CH2O), 6.94(s, 2H, NH2), 7.59(s,
2H, Ar-H), 7.94(s, 2H, Ar-H), 7.59(s, 1H, pyrimidin-5-yl-H); 13C NMR
(100 MHz, DMSO‑d6) δ(ppm): 25.9, 46.7, 58.1, 108.2, 124.7, 128.4,
6

J. Feng et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
4.3. Evaluation of inhibitory activity of compounds against Cy-PDHc E1
and porcine PDHc E1 in vitro
Acknowledgments
The work was supported by National Research and Development
Plan (No. 2017YFD0200506), the Natural Science Foundation of China
(No. 21877047, 21472062, 21877046, 21867011, 21472061 and
31701820), and the Project of the Science Fund of Jiangxi Education
Office (GJJ170672), the Project of Hubei Natural Science Foundation
The inhibitory activities of the compounds were measured by
evaluating the Cy-PDHc E1 activity in vitro. A standard reaction mixture
for the inhibition rate (%) of compounds against Cy-PDHc E1 is that
contains 50 mM K3PO4 (pH 7.2), 0.375 mM 2, 6-DCIP, 0.1 mg/ml pur-
ified PDHc E1 enzyme and 200 µM ThDP and the 100 μM compounds.
After the mixture was incubated for 5 min at 37 °C, the saturating
substrate pyruvate was added for triggering the reaction. The standard
reaction mixture for half maximal inhibitory concentration (IC50) de-
termination is as same as the above except for the compounds ranging
from 0 to 100 μM. All the kinetic data were fit to Growth/Sigmoidal
model from origin 7.0 software.
(2017CFB232),
China
postdoctoral
science
foundation
(2017M622486). The work was also financially supported by self-de-
termined research funds of CCNU from the colleges’ basic research and
operation of MOE (CCNU18ZDPY02) and the Program of Introducing
Talents of Discipline to Universities of China (111 program, B17019).
References
Porcine PDHc-E1 was purchased from Sigma. Porcine E1 activity
was determined by monitoring the reduction of 3-(4,5- dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 566 nm using a mi-
croplate reader (Bioteck Synergy2, USA). The inhibition rate (%) of
compounds against porcine PDHc-E1 was assayed at the final con-
centration (100 μM). A standard reaction mixture containing 1.0 mM
MgCl2, 0.2 mM ThDP, 0.5 mM MTT, 6.5 mM phenazine methosulfate
(PMS), 50 mM K3PO4, pH 7.0, 0.75 mg/mL enzyme and 100 μM com-
pounds. The mixture was incubated for 3 min at 37 °C, and then 2 mM
sodium pyruvate was added to initiate the reaction.
1. Huisman Jef, Codd Geoffrey A, Paerl Hans W, Ibelings Bas W, Verspagen Jolanda
MH, Visser Petra M. Cyanobacterial blooms. Nat Rev Microbiol. 2018;16:471–483.
2. Lürling Miquel, Faassen Elisabeth J. Controlling toxic cyanobacteria: effects of
dredging and phosphorus - binding clay on cyanobacteria and microcystins. Water
Res. 2012;46(5):1447–1459.
3. Rajasekhar Pradeep, Fan Linhua, Nguyen Thang, Roddick Felicity A. Impact of so-
nication at 20kHz on Microcystis aeruginosa, Anabaena circinalis, and Chlorella sp.
Water Res. 2012;46(5):1473–1481.
4. Gerphagnon Mélanie, Macarthur Deborah J, Latour Claire Delphine, Gachon MM,
Van Ogtrop Floris, Gleason Frank H. Télesphore Sime-Ngando. Microbial players
involved in the decline of filamentous and colonial cyanobacterial blooms with a
focus on fungal parasitism. Environ Microbiol. 2015;17(8):2573–2587.
5. Matthijs Hans CP, Jančula Daniel, Visser Petra M. Blahoslav Maršálek. Existing and
emerging cyanocidal compounds: new perspectives for cyanobacterial bloom miti-
gation. Aquat Ecol. 2016;50(3):443–460.
4.4. Inhibitory activity evaluation of compounds against Synechocystis
PCC6803 and Microcystis aeruginosa FACHB905 in vivo
6. Huo Xiangchen, Chang DeWei, Tseng Michael JingHua, Burch D, Lin TsairFuh.
Exposure of microcystis aeruginosa to hydrogen peroxide under light: kinetic modeling
of cell rupture and simultaneous microcystin degradation. Environ Sci Technol.
2015;49(9):5502–5510.
Inhibitory activities of compounds and half maximal effective in-
7. Harris Robert A, BowkerKinley Melissa M, Huang Boli, Pengfei Wu. Regulation of the
activity of the pyruvate dehydrogenase complex. Adv Enzyme Regul.
2015;42:249–259.
hibitory concentration (EC50
) against Synechocystis PCC6803 and
Microcystis aeruginosa FACHB905 was determined in vivo. Synechocystis
PCC6803 and Microcystis aeruginosa FACHB905 were cultured in BG11
medium at 28 °C for 7 days at a 12 h ligh/12 h dark cycle until the
logarithmic growth phase. The 1% cultured cells were then transferred
to a fresh medium containing different compounds. After culturing for
7 days, the OD680 were determined, and the inhibition rate and half
maximal effective concentration (EC50) were calculated as described
8. Birkenstock Timo, Liebeke Manuel, Winstel Volker, et al. Exometabolome analysis
identifies pyruvate dehydrogenase as a target for the antibiotic triphe-
nylbismuthdichloride in multiresistant bacterial pathogens. J Biol Chem.
2012;287(4):2887–2895.
9. He HongWu, Yuan JunLin, Peng Hao, Chen Ting, Shen Ping. ShuQing Wan, Yanjun
Li, HongLiang Tan, YaHui He, JunBo He, Yan Li. Studies of o, o-dimethyl α-(2,4-
dichloro- phenoxyacetoxy) ethylphosphonate (hw02) as a new herbicide. 1. Synthesis
and herbicidal activity of hw02 and analogues as novel inhibitors of pyruvate de-
hydrogenase complex. J Agric Food Chem. 2011;59(9):480–4813.
previously29
.
10. Palaniappa Arjunan, Krishnamoorthy Chandrasekhar, Martin Sax, et al. Structural
determinants of enzyme binding affinity: the E1 component of pyruvate dehy-
drogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone
diphosphate. Biochemistry. 2004;43(9):2405–2411.
4.5. Structure-based docking
11. Arjunan Palaniappa, Sax Martin, Brunskill Andrew, et al. A thiamin-bound, pre-
decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1
subunit induces large scale disorder-to-order transformations in the enzyme and
reveals novel structural features in the covalently bound adduct. J Biol Chem.
2006;281(22):15296–15303.
The amino acid sequence of Cy-PDHc E1 obtained from the NCBI
database (NCBI Reference Sequence: NC_000911.1) shared 38.4%
identity with Geobacillus Stearothermophilus PDHc E1 (Geo-PDHc E1,
PDB ID: 1W88)23. The modeling 3D structure of cyanobacteria PDHc E1
was built by using SWISSMODEL server and taking Geo-PDHc E1 crystal
structure as templated22. Cofactors ThDP and Mg2+ were derived from
the template crystal structure. The model 3D structure of Cy-PDHc E1
was further validated by Procheck25 and Q-mean program26. The re-
sults of Ramachandran plot were that 89.9% of the residues were dis-
tributed in the favored regions, 8.0% in the additionally allowed re-
gions, 1.5% in the generously allowed regions, only 0.6% of the
residues were in the disallowed regions. The model 3D structure with
cofactors of Cy-PDHc E1 was used for molecular docking analyses. The
structure of compound 8d was prepared with SYBYL7.0 and hydrogen
atoms were added to the structure. Then the SURFLEX module of
SYBYL7.0 package was performed to explore the interaction model of
Cy-PDHc E1 with compound 8d. All atoms located within 6.5 Å away
from the cofactor ThDP were selected into the active site, and the
corresponding amino acid residue was involved in the active site if only
one of its atoms was selected. Other parameters were set to default. All
calculations were performed on the CCNU Grid website http://www.
202.114.32.71:8090/ccnu/chem/platform.xml.
12. He JunBo, Ren YanLiang, Sun QiuShuang, et al. Design, synthesis and molecular
docking of amide and urea derivatives as Escherichia coli PDHC-E1 inhibitors. Bioorg
Med Chem. 2014;22(12):3180–3186.
13. Zhou Yuan, Zhang Shasha, He Hongwu, et al. Design and synthesis of highly selective
pyruvate dehydrogenase complex E1 inhibitors as bactericides. Bioorg Med Chem.
2018;26:84–95.
14. He JunBo, HaiFeng He Lu.Lu, Zhao Li Zhang, et al. Synthesis and antifungal activity
of 5-iodo-1,4-disubstituted-1,2,3-triazole derivatives as pyruvate dehydrogenase
complex E1 inhibitors. Bioorg Med Chem. 2015;23(7):1395–1401.
15. He Haifeng, Wang Wei, Zhou Yuan, et al. Rational design, synthesis and biological
evaluation of 1, 3, 4- oxadiazole pyrimidine derivatives as novel pyruvate dehy-
drogenase complex E1 inhibitors. Bioorg Med Chem. 2016;24(8):1879–1888.
16. He Junbo, Feng Lingling, Li Jing, et al. Design, synthesis and biological evaluation of
novel 2-methylpyrimidine-4-ylamine derivatives as inhibitors of Escherichia coli
pyruvate dehydrogenase complex E1. Bioorg Med Chem. 2012;20(5):1665–1670.
17. Ohno Hiroaki, Minamiguchi Daiki, Nakamura Shinya, Shu Keito, Okazaki Shiho,
Honda Maho. Ryosuke Misu, Hirotomo Moriwaki, Shinsuke Nakanishi, Shinya Oishi,
Takayoshi Kinoshita, Isao Nakanishi, NobutakaFujii. Structure - activity relationship
study of 4 - (thiazol -5 - yl) benzoic acid derivatives as potent protein kinase CK2
inhibitors. Bioorganic Medicinal Chemistry. 2016;24(5):1136–1141.
18. Nihei Ken-ichi. Atsuko Nihei and Isao Kubo*Rational Design of Antimicrobial Agents:
Antifungal Activity of Alk(en)yl Dihydroxybenzoates and Dihydroxyphenyl
Alkanoates. Bioorg Med Chem Lett. 2003;13(22):3993–3996.
19. Kratky Martin, Bosze Szilvia, Baranyai Zsuzsa, et al. Synthesis and in vitro biological
evaluation of 2- (phenyl carbamoyl) phenyl 4-substituted benzoates. Bioorg Med
Chem. 2015;23(4):868–875.
7

J. Feng et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
20. Erixon Karl M, Dabalos Chester L, Leeper Finian J. Synthesis and biological eva-
luation of pyrophosphate mimics of thiamine pyrophosphate based on a triazole
scaffold. Org Biomol Chem. 2008;6(19):3561–3572.
25. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to
check the stereochemical quality of protein structures. J Appl Crystallogr.
1993;26(2):283–291.
21. Natalia Nemeria, Ahmet Baykal, Ebenezer Joseph, et al. Tetrahedral intermediates in
thiamin diphosphate-dependent decarboxylations exist as a 1', 4'-imino tautomeric
form of the coenzyme, unlike the michaelis complex or the free coenzyme.
Biochemistry. 2004;43(21):6565–6575.
26. Benkert Pascal, Biasini Marco, Schwede Torsten. Toward the estimation of the ab-
solute quality of individual protein structure models. Bioinformatics.
2011;27(3):343–350.
27. Hawkins Christopher F, Borges Adolfo, Perham Richard N. Cloning and sequence
analysis of the genes encoding the α and β subunits of the E1 component of the
pyruvate dehydrogenase multienzyme complex of bacillus stearothermophilus. Eur J
Biochem. 2010;191(2):337–346.
22. Waterhouse Andrew, Bertoni Martino, Bienert Stefan, Studer Gabriel, Tauriello
Gerardo, Gumienny Rafal, Heer Florian T, de Beer Tjaart AP. Christine Rempfer,
Lorenza Bordoli, Rosalba Lepore, and Torsten Schwede. Swiss-model: homology
modelling of protein structures and complexes. Nucleic Acids Res. 2018;46(Web
Server issue):W296–W303.
28. Lessard IA, Perham RN. Expression in Escherichia coli of genes encoding the E1 alpha
and E1 beta subunits of the pyruvate dehydrogenase complex of bacillus stear-
othermophilus and assembly of a functional E1 component (alpha 2 beta 2) in vitro. J
Biol Chem. 1994;269(14):10378–10383.
23. Frank René AW, Titman Christopher M, Venkatesh Pratap J, Luisi Ben F, Perham
Richard N. A molecular switch and proton wire synchronize the active sites in
thiamine enzymes. Science. 2004;306(5697):872–876.
29. Zhou Yuan, Feng Jiangtao, He Hongwu, et al. Design, synthesis and potency of
pyruvate dehydrogenase complex e1 inhibitors against cyanobacteria. Biochemistry.
2017;56(49):6491–6502.
24. Martí-Renom Marc A, Stuart Ashley C, Fiser András, Sánchez Roberto, Melo
Francisco, Šali Andrej. Comparative protein structure modeling of genes and gen-
omes. Ann Rev Biophys Biomol Struct. 2000;29(1):291–325.
8