Synthesis and Activity of 1,2,3-Triazole Aminopyrimidines against Cyanobacteria as PDHc-E1 Competitive Inhibitors

Article abstract of DOI:10.1021/acs.jafc.9b02878

Cyanobacteria harmful algal blooms are of global concern, but all currently available algicides in the market are nonselective and have potential side effects on nontarget species. In the present work, two series of compounds (4 and 6) comprising 16 novel

Full text of DOI:10.1021/acs.jafc.9b02878

Subscriber access provided by UNIV OF WESTERN ONTARIO
Functional Structure/Activity Relationships
Synthesis and Activity of 1,2,3-Triazole Aminopyrimidines
against Cyanobacteria as PDHc-E1 Competitive Inhibitors
Yuan Zhou, Jiangtao Feng, Lingling Feng, Dan Xie, Hao Peng, Meng Cai, and Hongwu He
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02878 • Publication Date (Web): 22 Oct 2019
Downloaded from pubs.acs.org on October 23, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 34
Journal of Agricultural and Food Chemistry
1
2
Synthesis and Activity of 1,2,3-Triazole Aminopyrimidines against Cyanobacteria as
PDHc-E1 Competitive Inhibitors
3
Yuan Zhou1, Jiangtao Feng1, Lingling Feng, Dan Xie, Hao Peng, Meng Cai*, Hongwu He*
4
5
Key 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
6
*CORRESPONDING AUTHOR FOOTNOTE
7
8
To whom correspondence should be addressed. Tel./fax: +86 (0)27 67867960, E-mail
addresses: he1208@mail.ccnu.edu.cn,cm122587@163.com.
9
1 These authors equally contributed to this work.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 34
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Abstract
Cyanobacteria harmful algal blooms are of a global concern, but all currently available algicides
in market are non-selective and have potential side effects on non-target species. In the present
work, two series of compounds (4 and 6) comprising 16 novel 1,2,3-triazole aminopyrimidines
were rational designed and synthesized as control agent for cyanobacteria. Our design focus was
the inhibiting cyanobacteria by inhibition against pyruvate dehydrogenase complex E1
(PDHc-E1). Compounds 4 and 6 showed potent inhibition against Escherichia coli PDHc-E1 (IC50
= 4.13-23.76 μM) and also strong algicidal activities against Synechocystis sp. PCC 6803 (EC50
=
1.7-8.1 μM) and Microcystis sp. FACHB905 (EC50 = 2.1-11.8 μM). In particular, the algicidal
activities of 6d against four algal species were not only higher than that of prometryn; they were
also comparable to or higher than that of copper sulfate. The analogs 4c, 4d, 6d, and 6e displayed
potent algicidal activities and inhibition of E. coli PDHc-E1 but exhibited negligible inhibition of
porcine PDHc-E1. As revealed by molecular docking, site-directed mutagenesis, enzymatic
assays, and an inhibition kinetic analysis, 4c and 6d inhibited PDHc-E1 in a competitive manner.
Our results suggest that highly selective, effective algicides can be developed by rationally
designing competitive PDHc-E1 inhibitors.
Keywords: synthesis; PDHc-E1 inhibitor; anti-cyanobacteria; selectivity; molecular docking
ACS Paragon Plus Environment

Page 3 of 34
Journal of Agricultural and Food Chemistry
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
1 Introduction
Along with increasing eutrophication and global warming, cyanobacteria harmful algal
blooms (Cyano-HABs) have become a worldwide concern.1-3 Cyano-HABs can destroy aquatic
ecosystems by depleting oxygen levels, with cyanobacteria themselves producing cyanotoxins that
threaten aquatic organism survival and human health.4, 5 An urgent need to control Cyano-HABs
therefore exists. One of the most convenient control methods is the application of chemicals.6, 7
Applied chemicals include inorganic copper fungicides (e.g., copper sulfate, copper oxychloride,
and copper citrate), herbicides (e.g., prometryne, diquat, paraquat, atrazine, and simazine), and
chemical oxidants (e.g., chlorine dioxide, hydrogen peroxide, potassium permanganate, and
calcium peroxide).8 Unfortunately, all currently available algicides in market are non-selective and
have potential side effects on non-target species, including human beings. This non-selectivity has
served as the impetus for designing chemicals that target unique enzymes of cyanobacteria.
Pyruvate dehydrogenase E1 (PDHc-E1), a member of the pyruvate dehydrogenase complex,
catalyzes the only irreversible step of a multistep process using thiamine diphosphate (ThDP) and
Mg2+ as cofactors.9 PDHc-E1 is therefore a feasible target for the evaluation of novel algicides.
Given the indispensability of ThDP in pyruvate metabolism in microbe,10, 11 the design and
synthesis of ThDP analogs as PDHc-E1 inhibitors may be a good strategy for the discovery of
novel algicides.
We thus aimed to design an algicide that can selectively inhibit PDHc-E1. Cyanobacteria and
Escherichia coli, which are both prokaryotes, have nuclei without nuclear membranes and lack
membrane-bound plastids. Because the crystal structure of cyanobacterial PDHc-E1 has not been
reported, we were only able to design new inhibitors against PDHc-E1 from E. coli (PDB code:
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 34
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
1L8A).12 We first synthesized a series of 1,2,3-triazole aminopyrimidines I (Fig. 1) and
demonstrated their effectiveness as inhibitors against E. coli PDHc-E1.13 However, these
compounds displayed poor algicidal activity. Compounds II, III and IV (Fig. 1) were then
synthesized by modifying the phenoxy moiety of I. Most of these new compounds showed potent
inhibition against both E. coli PDHc-E1 and cyanobacteria that was significantly superior to I.14
Particularly, compound IV-21l (R1= I, R = 6-Br) (Fig.1) has the best inhibition against the
cyanobacteria strain Synechocystis sp. PCC6803 with an EC50 of 0.7 μM.14 However, the synthesis
process of this compound is complicated, and its water- and fat-solubility is poor because of its
three different nitro-containing heterocycles. These findings encouraged us to design more potent
PDHc-E1 inhibitors by modifying I with the aid of E. coli PDHc-E1 structure-based molecular
docking methods. The molecular docking analysis indicated that the “linker portion” between the
1,2,3-triazole and benzene ring of I could not form hydrogen bonds with any amino acid residues
in the active site of E. coli PDHc-E1. We hypothesized that the formation of a hydrogen bond
between the “linker portion” and the amino acid residues in the active site should be greatly
beneficial for enhancing inhibition against E. coli PDHc-E1. For this purpose, we considered an
imine or amide group containing a NH or C=O moiety, which could be used as a hydrogen donor
(NH) or receptor (C=O). Our recent work has therefore focused on using an imine or amide group
as the
“linker portion”
to
obtain
a
novel
series
of 5-((4-((substituted
(4) and
amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-
2-methylpyrimidin-4-amine
1-((4-amino-2-methylpyrimidin-5-yl)methyl)-N-substituted-1H-1,2,3-triazole-4-carboxamide (6)
compounds (Fig. 2).
Here, we report the chemical synthesis of the 2 series of compounds (4 and 6) and their
ACS Paragon Plus Environment

Page 5 of 34
Journal of Agricultural and Food Chemistry
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
biological activities against E. coli PDHc-E1 and five algal species (Synechocystis sp. PCC6803,
Microcystis sp. FACHB905, Anabaena sp. FACHB82, Aphanizomenon sp. FACHB1395, and
Nostoc sp. FACHB713).
2 Material and Methods
2.1 Chemistry
All melting points (m.p.) were determined with a digital model X-5 apparatus (uncorrected).
1H and 19F nuclear magnetic resonance (NMR) spectra were recorded on a Bruker spectrometer at
400 MHz and 376 MHz with tetramethylsilane as the internal standard. Chemical shifts were
reported in δ (ppm) (parts per million) values. A MicroMass GCT CA 055 instrument was used to
acquire high-resolution electron impact mass spectra (HR-EIMS) under electron impact (70 eV)
conditions. Elemental analyses were performed on a Vario ELIIICHNSO element analyzer, and
crystal structures were obtained with Bruker AEPEX DUO CCD diffractometer. All chemicals
and reagents used for syntheses were purchased from commercial sources; they were of AR grade
and used without further purification.
2.2 Procedure for the preparation of 5-azidomethyl-2-methylpyrimidine-4-yl-amine 1.
As described by Erixon,15 a stirring suspension of 30.6 mmol thiamine chloride (Vitamin B1),
o
75.4 mmol sodium azide and 3.0 mmol sodium sulfite in water (100mL) was heated at 65 C.
After 5 h, 100 mmol citric acid (pH ≈ 4) was added into the aqueous solution. The reaction
mixture was stirred at room temperature. After 30 min, potassium carbonate was added until the
pH value reached 8.0. A white precipitation of the product began forming immediately. The
suspension was thoroughly stirred and filtered. The filter cake was washed with saturated
potassium carbonate solution and dried to afford a white solid (Yield: 65%, m.p. 150-152 oC).
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 34
94
95
2.3 General procedure for preparation of N-(prop-2-yn-1-yl)anilines 3.
According to the procedure described by Majumdar,16 to a stirred suspension of an
appropriate substituted aniline 2 (12 mmol) and potassium carbonate (10 mmol) in dry DMF (15
mL) was added propargyl bromide (10 mmol). The reaction mixture was stirred at room
temperature and monitored by thin layer chromatography (TLC). After the reaction finished, the
reaction mixture was washed with water (15 mL) and extracted with EtOAc (60 mL). The
combined organic extracts were then washed with brine (20 mL), dried over Na2SO4, and
concentrated in vacuo. The crude products was further purified by a silica gel column using a 1:20
(v/v) acetate/petroleum ether mixture as the eluent, which afforded the corresponding
N-(prop-2-yn-1-yl)aniline 3. A series of 3 could be obtained by this procedure in the yields of
72-85 %.
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
2.4 General procedure for preparation of N-phenylpropiolamides 5.
Compounds 5 were synthesized via the method repoted by Ramachandran.17 To a stirred
suspension of dicyclohexylcarbodiimide (DCC) (12 mmol) in dichloromethane (DCM) (50 mL),
o
o
o
propiolic acid was added slowly at -5 C ~ 0 C. After 1 h of stirring at 0 C, a solution of
substituted aniline (10 mmol) in DCM (20 mL) was added to the reaction mixture and then stirred
at room temperature for 24 h. After the reaction finished, the reaction mixture was filtered and the
filtrate was evaporated in vacuo. The residue was purified by a silica gel column using a 1:50 (v/v)
ethyl acetate/petroleum ether as the eluent, which afforded the corresponding
N-phenylpropiolamide 5. A series of 5 could be obtained by this procedure in the yields of
53-79%.
2.5 General procedure for the synthesis of 1,2,3-triazole aminopyrimidines 4 and 6.
ACS Paragon Plus Environment

Page 7 of 34
Journal of Agricultural and Food Chemistry
116
To a stirring suspension of 1 (2 mmol) and an appropriate intermediate 3 or 5 (2 mmol) in
THF, CuI (0.2 mmol) and Et3N (2.4 mmol) were added sequentially. The reaction mixture was
stirred at room temperature for overnight prior the addition of water (30 mL). The product
suspension was filtered and dried. The filter cake was further purified by recrystallization
(methanol/dichloromethane) to give corresponding title compound 4 or 6. Two series of 4 and 6
could be obtained by same procedure.
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
2.5.1 2-methyl-5-((4-((phenylamino)methyl)-1H-1,2,3-triazol-1-yl)methyl)pyrimidin-4-amine
(4a)
Yellow solid; Yield 57%; m.p.188-189 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.29 (s,
3H, CH3), 4.24 (d, J = 5.4 Hz, 2H, CH2), 5.37 (s, 2H, CH2), 6.03 (s, 1H, Ar-H), 6.51 (t, J = 7.0 Hz,
1H, Ar-H), 6.58 (d, J = 7.8 Hz, 2H, Ar-H), 6.91 (s, 2H, Ar-H, 1,2,3-trizole), 7.03 (t, J = 7.4 Hz,
2H, NH, pyrimidine-H), 7.93 (s, 2H, NH2); HRMS (ESI): calcd. for C15H17N7 [M+H]+ 296.16182,
found: 296.16210; Elemental Anal. Calcd for C15H17N7: C, 61.00; H, 5.80; N, 33.20. Found: C,
60.87; H, 5.91; N, 32.75.
2.5.2
5-((4-(((2-chlorophenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-methylpyrimidin-4-a
mine (4b)
Yellow solid; Yield 60%; m.p.136-138 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.29 (s,
3H, CH3), 4.39 (d, J = 5.5 Hz, 2H, CH2), 5.36 (s, 2H, CH2), 5.90 (t, J = 5.6 Hz, 1H, Ar-H), 6.55 (t,
J = 7.1 Hz, 1H, Ar-H), 6.71 (d, J = 8.3 Hz, 1H, Ar-H), 6.91 (s, 2H, Ar-H, 1,2,3-triazole-H), 7.05
(t, J = 7.4 Hz, 1H, NH), 7.21 (d, J = 7.6 Hz, 1H, pyrimidine-H), 7.92 (s, 2H, NH2); HRMS (ESI):
calcd. for C15H16ClN7 [M+H]+ 330.12285, found: 330.12283; Elemental Anal. Calcd for
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 34
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
C15H16ClN7: C, 54.63; H, 4.89; N, 29.73. Found: C, 54.71; H, 4.95; N, 29.64.
2.5.3
5-((4-(((3-chlorophenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-methylpyrimidin-4-a
mine (4c)
Yellow solid; Yield 67%; m.p.149-151 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.32 (s,
3H, CH3), 4.30 (d, J = 5.7 Hz, 2H, CH2), 5.44 (s, 2H, CH2), 6.46 (t, J = 5.6 Hz, 1H, Ar-H),
6.51-6.63 (m, 2H, Ar-H), 6.66 (s, 1H, Ar-H), 6.97 (s, 2H, 1,2,3-triazole-H, pyrimidine-H), 7.08 (t,
J = 8.0 Hz, 1H, NH), 8.01 (s, 2H, NH2); HRMS (ESI): calcd. for C15H16ClN7 [M+H]+ 330.12285,
found: 330.12276; Elemental Anal. Calcd for C15H16ClN7: C, 54.63; H, 4.89; N, 29.73. Found: C,
54.55; H, 4.91; N, 29.81.
2.5.4
5-((4-(((4-chlorophenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-methylpyrimidin-4-a
mine (4d)
Yellow solid; Yield 72%; m.p.174-176 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.32 (s,
3H, CH3), 4.26 (d, J = 3.9 Hz, 2H, CH2), 5.45 (s, 2H, CH2), 6.29 (s, 1H, Ar-H), 6.62 (d, J = 8.6
Hz, 2H, Ar-H), 6.97 (s, 2H, Ar-H, 1,2,3-triazole-H), 7.08 (d, J = 8.6 Hz, 2H, pyrimidine-H, NH),
7.98 (s, 2H, NH2); HRMS (ESI): calcd. for C15H16ClN7 [M+H]+ 330.12285, found: 330.12296;
Elemental Anal. Calcd for C15H16ClN7: C, 54.63; H, 4.89; N, 29.73. Found: C, 54.56; H, 4.77; N,
29.75.
2.5.5
5-((4-(((3-bromophenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2-methylpyrimidin-4-a
mine (4e)
ACS Paragon Plus Environment

Page 9 of 34
Journal of Agricultural and Food Chemistry
160
Yellow solid; Yield 65%; m.p.143-145 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.29 (s,
3H, CH3), 4.25 (d, J = 4.4 Hz, 2H, CH2), 5.39 (s, 2H, CH2), 6.31-6.46 (m, 1H, Ar-H), 6.61 (dd, J =
25.3, 7.1 Hz, 2H, Ar-H), 6.76 (s, 1H, Ar-H), 6.95 (dd, J = 19.2, 11.0 Hz, 3H, NH, 1,2,3-trizole-H,
pyrimidine-H), 7.95 (s, 2H, NH2); HRMS (ESI): calcd. for C15H16BrN7 [M+H]+ 374.07233, found:
374.07211; Elemental Anal. Calcd for C15H16BrN7: C, 48.14; H, 4.31; N, 26.20. Found: C, 48.01;
H, 4.42; N, 26.43.
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
2.5.6
2-methyl-5-((4-(((3-(trifluoromethyl)phenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)pyri
midin-4-amine (4f)
Yellow solid; Yield 62%; m.p.144-146 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.32 (s,
3H, CH3), 4.36 (s, 2H, CH2), 5.43 (s, 2H, CH2), 6.63 (s, 1H, Ar-H), 6.77-7.13 (m, 5H, Ar-H,
1,2,3-triazole-H, pyrimidine-H), 7.29 (s, 1H, NH), 8.02 (s, 2H, NH2); 19F NMR (376 MHz,
DMSO-d6) δ(ppm): -61.25; HRMS (ESI): calcd. for C16H16F3N7 [M+H]+ 364.1492, found:
364.14954; Elemental Anal. Calcd for C16H16F3N7: C, 52.89; H, 4.44; N, 26.98. Found: C, 53.05;
H, 4.51; N, 27.04.
2.5.7
2-methyl-5-((4-(((4-(trifluoromethyl)phenyl)amino)methyl)-1H-1,2,3-triazol-1-yl)methyl)pyri
midin-4-amine (4g)
Yellow solid; Yield 69%; m.p.150-152 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.28 (s,
3H, CH3), 4.31 (s, 2H, CH2), 5.39 (s, 2H, CH2), 6.58 (s, 1H, Ar-H), 6.84 (d, J = 28.3 Hz, 5H,
Ar-H, 1,2,3-triazole-H, pyrimidine-H), 7.24 (s, 1H, NH), 7.97 (s, 2H, NH2); 19F NMR (376 MHz,
DMSO-d6) δ(ppm): -61.24; HRMS (ESI): calcd. for C16H16F3N7 [M+H]+ 364.1492, found:
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 34
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
364.14949; Elemental Anal. Calcd for C16H16F3N7: C, 52.89; H, 4.44; N, 26.98. Found: C, 52.71;
H, 4.52; N, 26.84.
2.5.8 2-methyl-5-((4-((p-tolylamino)methyl)-1H-1,2,3-triazol-1-yl)methyl)pyrimidin-4-amine
(4h)
o
1
Yellow solid; Yield 72%; m.p.75-77 C; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.32 (s,
6H, 2CH3), 4.44 (s, 2H, CH2), 5.52 (s, 2H, CH2), 7.01 (s, 1H, Ar-H), 7.21 (d, J = 28.3 Hz, 5H,
Ar-H, 1,2,3-triazole-H, pyrimidine-H), 7.67 (s, 1H, NH), 8.68 (s, 2H, NH2); HRMS (ESI): calcd.
for C16H19N7 [M+H]+ 310.17747, found: 310.17727; Elemental Anal. Calcd for C16H19N7: C,
62.12; H, 6.19; N, 31.69. Found: C, 59.98; H, 6.05; N, 31.64.
2.5.9
1-((4-amino-2-methylpyrimidin-5-yl)methyl)-N-phenyl-1H-1,2,3-triazole-4-carboxamide (6a)
Yellow solid; Yield 65%; m.p.249-251 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.33 (s,
3H, CH3), 5.54 (s, 2H, CH2), 7.06 (d, J = 32.0 Hz, 3H, Ar-H), 7.33 (s, 2H, Ar-H), 7.82 (s, 2H,
NH2), 8.10 (s, 1H, 1,2,3-triazole-H), 8.71 (s, 1H, pyrimidine-H), 10.45 (s, 1H, NH); HRMS (ESI):
calcd. for C15H15N7O [M+H]+ 310.14108, found: 310.14102; Elemental Anal. Calcd for
C15H15N7O: C, 58.24; H, 4.89; N, 31.70. Found: C, 58.38; H, 4.77; N, 31.58.
2.5.10
1-((4-amino-2-methylpyrimidin-5-yl)methyl)-N-(3-chlorophenyl)-1H-1,2,3-triazole-4-carboxa
mide (6b)
Yellow solid; Yield 78%; m.p.>260 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.31 (s, 3H,
CH3), 5.53 (s, 2H, CH2), 6.98 (s, 2H, Ar-H), 7.13 (d, J = 7.4 Hz, 1H, Ar-H), 7.33 (d, J = 7.5 Hz,
1H, Ar-H), 7.73 (d, J = 8.2 Hz, 1H, 1,2,3-triazole-H), 7.97 (s, 2H, NH2), 8.71 (s, 1H,
ACS Paragon Plus Environment

Page 11 of 34
Journal of Agricultural and Food Chemistry
204
pyrimidine-H), 10.63 (s, 1H, NH); HRMS (ESI): calcd. for C15H14ClN7O [M+H]+ 344.10211,
found: 344.10201; Elemental Anal. Calcd for C15H14ClN7O: C, 52.41; H, 4.10; N, 28.52. Found:
C, 52.53; H, 4.05; N, 28.64.
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
2.5.11
1-((4-amino-2-methylpyrimidin-5-yl)methyl)-N-(4-chlorophenyl)-1H-1,2,3-triazole-4-carboxa
mide (6c)
Yellow solid; Yield 82%; m.p.>260 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.31 (s, 3H,
CH3), 5.54 (s, 2H, CH2), 6.99 (s, 2H, Ar-H), 7.36 (d, J = 5.9 Hz, 2H, Ar-H), 7.82 (d, J = 6.2 Hz,
3H, NH2, 1,2,3-triazole-H), 8.69 (s, 1H, pyrimidine-H), 10.59 (s, 1H, NH); HRMS (ESI): calcd.
for C15H14ClN7O [M+H]+ 344.10211, found: 344.10215; Elemental Anal. Calcd for C15H14ClN7O:
C, 52.41; H, 4.10; N, 28.52. Found: C, 52.26; H, 3.98; N, 28.44.
2.5.12
1-((4-amino-2-methylpyrimidin-5-yl)methyl)-N-(3-bromophenyl)-1H-1,2,3-triazole-4-carbox
amide (6d)
White solid; Yield 77%; m.p.>260 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.34 (s, 3H,
CH3), 5.57 (s, 2H, CH2), 7.04 (s, 2H, Ar-H), 7.32 (s, 2H, Ar-H), 7.83 (s, 1H, NH2), 8.17 (s, 1H,
1,2,3-triazole-H), 8.76 (s, 1H, pyrimidine-H), 10.68 (s, 1H, NH); HRMS (ESI): calcd. for
C15H14BrN7O [M+H]+ 388.0516, found: 388.05113; Elemental Anal. Calcd for C15H14BrN7O: C,
46.41; H, 3.63; N, 25.26. Found: C, 46.53; H, 3.55; N, 25.40.
2.5.13
1-((4-amino-2-methylpyrimidin-5-yl)methyl)-N-(4-bromophenyl)-1H-1,2,3-triazole-4-carbox
amide (6e)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 34
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
White solid; Yield 84%; m.p.>260 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.34 (s, 3H,
CH3), 5.56 (s, 2H, CH2), 7.03 (s, 2H, Ar-H), 7.54 (d, J = 6.8 Hz, 2H, Ar-H), 7.83 (d, J = 7.1 Hz,
2H, NH2), 8.12 (s, 1H, 1,2,3-triazole-H), 8.75 (s, 1H, pyrimidine-H), 10.65 (s, 1H, NH); HRMS
(ESI): calcd. for C15H14BrN7O [M+H]+ 388.0516, found: 388.05100; Elemental Anal. Calcd for
C15H14BrN7O: C, 46.41; H, 3.63; N, 25.26. Found: C, 46.27; H, 3.72; N, 25.14.
2.5.14
1-((4-amino-2-methylpyrimidin-5-yl)methyl)-N-(4-fluorophenyl)-1H-1,2,3-triazole-4-carboxa
mide (6f)
Yellow solid; Yield 71%; m.p.>260 oC; 1H NMR (400 MHz, DMSO-d6) δ(ppm): 2.32 (s, 3H,
CH3), 5.56 (s, 2H, CH2), 7.02 (s, 2H, Ar-H), 7.18 (t, J = 7.7 Hz, 3H, Ar-H, , 1,2,3-triazole-H), 7.83
(s, 2H, NH2), 8.71 (s, 1H, pyrimidine-H), 10.56 (s, 1H, NH); 19F NMR (376 MHz, DMSO-d6)
δ(ppm): -118.62; HRMS (ESI): calcd. for C15H14FN7O [M+H]+ 328.13166, found: 328.13175;
Elemental Anal. Calcd for C15H14FN7O: C, 55.04; H, 4.31; N, 29.95. Found: C, 54.87; H, 4.51; N,
30.08.
2.5.15
1-((4-amino-2-methylpyrimidin-5-yl)methyl)-N-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazole
-4-carboxamide (6g)
o
1
White solid; Yield 76%; m.p.193-195 C; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.31 (s,
3H, CH3), 5.53 (s, 2H, CH2), 6.99 (s, 2H, Ar-H), 7.43 (d, J = 7.0 Hz, 1H, Ar-H), 7.50-7.67 (m, 1H,
Ar-H), 8.06 (d, J = 7.6 Hz, 2H, NH2), 8.28 (s, 1H, 1,2,3-triazole-H), 8.73 (s, 1H, pyrimidine-H),
10.80 (s, 1H, NH); 19F NMR (376 MHz, DMSO-d6) δ(ppm): -61.19; HRMS (ESI): calcd. for
C16H14F3N7O [M+H]+ 378.12847, found: 378.12864; Elemental Anal. Calcd for C16H14F3N7O: C,
ACS Paragon Plus Environment

Page 13 of 34
Journal of Agricultural and Food Chemistry
248
50.93; H, 3.74; N, 25.98. Found: C, 51.07; H, 3.55; N, 26.13.
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
2.5.16
1-((4-amino-2-methylpyrimidin-5-yl)methyl)-N-(p-tolyl)-1H-1,2,3-triazole-4-carboxamide
(6h)
o
1
White solid; Yield 77%; m.p.245-247 C; H NMR (400 MHz, DMSO-d6) δ(ppm): 2.31 (s,
3H, CH3), 3.63 (s, 3H, CH3), 5.61 (s, 2H, CH2), 7.12 (d, J = 34.8 Hz, 4H, Ar-H), 7.72 (s, 3H, NH2,
1,2,3-triazole-H), 8.73 (s, 1H, pyrimidine-H), 10.40 (s, 1H, NH); HRMS (ESI): calcd. for
C16H17N7O [M+H]+ 324.15673, found: 324.15644; Elemental Anal. Calcd for C16H17N7O: C,
59.43; H, 5.30; N, 30.32. Found: C, 59.57; H, 5.55; N, 29.90.
2.6 Crystallographic study
A colorless single crystal of 4b suitable for X-ray analysis was cultured from a mixture of
methanol and dichloromethane at room temperature. Unit cell determination and data collection
were performed using Mo Kα radiation ( = 0.71073 Å) on a Bruker AEPEX DUO CCD
diffractometer in - scan mode at 298(2) K. The structure was solved directly using the
SHELXS program of the SHELXTL package and refined by full-matrix least-squares methods
with SHELXL. The position of all hydrogen atoms were determined from difference Fourier
maps. All non-hydrogen atoms of 4b were refined with anisotropic thermal parameters.
2.7 Enzyme inhibition assay for PDHc-E1
The preparation of PDHc-E1 from E. coli and the measurement of enzymatic activities were
essentially as previously reported.18 The half maximal inhibitory concentration (IC50) was
determined in
a reaction mixture containing 50 mM K3PO4 (pH 6.4), 0.4 mM
2,6-dichlorophenolindophenol (DCPIP), 50 μM sodium pyruvate as the substrate, 5 μg PDHc-E1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 34
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
purified enzyme, and 50 μM ThDP. Title compounds with different concentration gradients were
incubated for 5 min with PDHc-E1 at 37 oC prior to addition of the pyruvate substrate. IC50 values
were obtained from a nonlinear least-squares fitting of the data using the Hill kinetic equations
under the Growth/Sigmoidal model in Origin 7.0 software as described previously.19
Site-directed mutations were introduced to PDHc-E1 using the Fast Mutagenesis System
(TransGen Biotech, Beijing, China).The pMAL-c2X-PDHc-E1 plasmid constructed by Feng19 was
used as a wild-type template. All operations were performed according to the manufacturer’s
protocol (TransGen Biotech). The accuracy of the desired mutations was confirmed by DNA
sequencing. Mutant PDHc-E1 was expressed and purified in the same manner as that used for the
wild type.
2.8 Fluorescence spectroscopic analysis
Fluorescence spectroscopic measurements were carried out on a FluoreMax-P fluorescence
spectrophotometer (Horiba Jobin Yvon, Longjumeau, France) according to a previously described
protocol.20 Fluorescence quenching of wild-type and mutant PDHc-E1 by different concentrations
of the title compounds was measured at an emission of 305-500 nm in a 1-cm cuvette. The
excitation wavelength was at 290 nm. To correct for background interference, wild-type or mutant
PDHc-E1 in buffer was used as a control. The binding constant Ka was calculated using the
following formula: ln [(F0 – F) / F] = ln Ka + n ln [Q], where F0 and F are the fluorescence
intensities of protein in the absence and presence of the title compounds, respectively, and [Q] is
the concentration of the quencher. A plot of ln [(F0 – F) / F] vs. ln [Q] gave a straight line using
least squares analysis. The intercept on the Y-axis was equal to the log value of Ka.
2.9 Determination of inhibition types
ACS Paragon Plus Environment

Page 15 of 34
Journal of Agricultural and Food Chemistry
292
A series of experiments was performed to determine the inhibition kinetics of 4c and 6d. The
inhibitor concentrations of 4c were 0, 10, and 20 μM and those of 6d were 0, 5, and 10 μM.
Substrate (ThDP) concentrations were 0, 0.1, 0.2, 0.4, 0.8, 2, 4, 10, 16, 20, 30, 50, and 100 μM in
all kinetic studies. Pre-incubation and measurement times were the same as described in section
2.7. DMSO was used as a blank control for background correction. The reaction velocity was
calculated using the following formula: V = ΔOD600 / (K × 5), where ΔOD600 is the absorbance
difference at 600 nm over 5 min, K is the slope of the DCPIP standard curve, and 5 is the reaction
time of the kinetic experiments. A similar set of experiments in the absence of inhibitors 4c and 6d
was also performed under the same conditions. The kinetic parameters Vmax and Km were obtained
by curve fitting according to the classical Michaelis-Menten equation.
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
2.10 Molecular docking
The crystal structure of the PDHc-E1 (PDB code: 1L8A) was obtained from the PDB data
base (Protein Data Bank, https://www.rcsb.org/). Hydrogen atoms were first added to the protein
using the PDB2PQR server.21 ThDP and hit compounds were constructed and optimized with
Gasteiger charges using SYBYL7.0 and then docked into the active pocket of the prepared
PDHc-E1 using Surflex-Dock.14 AutoDock 4.222 was used to investigate the binding modes of the
newly developed compounds.
2.11 In vivo anti-algal assay
Synechocystis sp. PCC6803, Microcystis sp. FACHB905, Anabaena sp. FACHB82,
Aphanizomenon sp. FACHB1395, and Nostoc sp. FACHB713 were cultured in 96-well
microplates according to a previously described method.2 The inhibition rate of the title
compounds against the five cyanobacteria species was estimated using the following formula:
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 34
314
315
316
317
318
319
320
321
322
323
324
325
inhibition (%) = {1➖ [∆ODt,680➖ ∆ODi,680] / ∆ODo,680}×100, where, ∆ODo,680 and ∆ODt,680 are
absorbance differences at 680 nm of the cyanobacteria in the blank control and title compounds
amended cultures, respectively, and ∆ODi,680 is the absorbance difference at 680 nm of the best
compound in each assay. Half maximal effective concentrations (EC50) were estimated with
logistic fitting. DMSO was used as a blank control. The commonly used algicides prometryn and
copper sulfate, and compound IV-21l (R1 = I, R = 6-Br) (Fig.1), which displayed the best
inhibition against PCC6803 (EC50 = 0.7 μM) in our previous study,14 served as positive controls.
3 Results and Discussion
3.1 Chemistry
3.1.1 Synthesis
Sixteen novel ThDP analogs in the series of 4 and 6 were designed and synthesized (Scheme
1). The synthetic routes are rather simple and convenient.
NH2
NH2
N
N
N
N3
NaN3, Na2SO3
OH
S
H2O, citric acid, K2CO3
N
N
1
Vitamin B1
NH2
N
H
N
NH2
Br
1
N
N
N
N
R
R
R
R
R
CuI, THF, Et3N
K2CO3, DMF
HN
N
2
4a-h
3
O
NH2
N
H
N
O
NH2
HO
1
N
N
R
CuI, THF, Et3N
O
DCC, DCM
HN
N
2
6a-h
5
326
327
Scheme 1. Synthesis of the title compounds 4 and 6
In this synthetic route, compound 1, which was synthesized from thiamine hydrochloride,
328
329
was in turn an important intermediate in the synthesis of 4 and 6. The skeleton of title compounds
ACS Paragon Plus Environment

Page 17 of 34
Journal of Agricultural and Food Chemistry
330
4 and 6 contains a 1,2,3-triazole ring, which can be readily constructed via “click chemistry”, a
concept introduced by Noble laureate K. Barry Sharpless.23, 24 In the work presented here, series 4
and 6 were synthesized from 1 via a 1,3-dipolar cycloaddition reaction that employed copper(I)
iodide and triethylamine in conjunction with terminal alkynes 3 and 5, respectively. Copper(I)
iodide acts as a catalyst in this process, in which Cu+ catalyzes the reaction of terminal alkynes
and azide groups to form 1,2,3-triazole ring.25, 26 Compounds 3 were prepared by the reaction of
propargyl bromide with the appropriate substituted anilines in DMF with K2CO3 as a base, while
compounds 5 were obtained by the condensation of substituted anilines with propiolic acid in the
presence of DCC.
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
Sixteen novel title compounds were smoothly synthesized by the above-mentioned synthetic
method, which had the advantages of being simple, mild, easy and efficient. The generated
1
compounds were fully characterized by H NMR, 19F NMR, and HRMS and confirmed by
elemental analysis.
3.1.2 Crystal structure of compound 4b
Crystal data for 4b are presented in Table 1, while Fig. 3 provides a perspective view based
on the atomic labeling system (CCDC-1536365; these data are available free of charge from the
Cambridge Crystallographic Data Centre). According to the structural analysis, 4b has a U shaped
conformation, which is close to its binding pose.
3.2 Enzyme inhibitory activity against E. coli PDHc-E1
The inhibitory activity of title compounds 4a-h against E. coli PDHc-E1 was evaluated. The
resulting IC50 values, which are summarized in Table 2, suggested that the inhibition of 4a-h
could be improved by introducing an imine group into the linker portion. The structural skeleton
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 34
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
of 4 was accordingly further optimized by introducing an amide group as a linker to generate the
series 6a-h.
We found that the inhibitory activity was further enhanced by changing the imine group into
an amide group. All compounds in series 6 had higher inhibitory activities than those of series 4
and lead compound I. For a given R group, compounds with an amide linkage, i.e., 6a (R = H,
IC50 = 14.70 ± 0.14 μM), 6b (R = 3-Cl, IC50 = 9.48 ± 0.25 μM), 6c (R = 4-Cl, IC50 = 7.12 ± 0.08
μM), 6d (R = 3-Br, IC50 = 4.13 ± 0.26 μM), 6g (R = 3-CF3, IC50 = 7.41 ± 0.01 μM), and 6h (R =
4-CH3, IC50 = 10.06 ± 0.17 μM), had higher inhibitory activities than the corresponding
compounds with an imine group, i.e., 4a (R = H, IC50 = 46.32 ± 0.24 μM), 4c (R = 3-Cl, IC50
=
11.53 ± 0.33 μM), 4d (R = 4-Cl, IC50 = 15.54 ± 0.01 μM), 4e (R = 3-Br, IC50 = 10.91 ± 0.65 μM),
4f (R = 3-CF3, IC50 = 15.74 ± 0.14 μM), and 4h (R = 4-CH3, IC50 = 23.76 ± 0.01 μM). We
observed that compounds in both series 4 and 6 with an electron-withdrawing R group, i.e., Br, F,
Cl, or CF3, displayed higher inhibition than their counterparts containing the electron-donating R
group CH3. Compounds with no substitution on the benzene ring, such as 4a and 6a, showed the
weakest inhibition in both series 4 and 6. The highest inhibitory activity, an IC50 value of 4.13
μM, was exhibited by 6d, which had 3-Br as the R group. According to our results, the linker
between the 1,2,3-triazole and benzene ring in the parent structure played a critical role in the
inhibition against E. coli PDHc-E1. Compared with an ether group, the use of an imine group or
amide group as a linker was much more beneficial to the inhibitory activity.
3.3 Inhibitory mechanisms of 4 and 6
To determine the inhibitory mechanisms of series 4 and 6, compounds 4c and 6d were chosen
and their kinetic inhibitions on E. coli PDHc-E1 were studied. We determined the maximum
ACS Paragon Plus Environment

Page 19 of 34
Journal of Agricultural and Food Chemistry
374
velocity (Vmax) and Michaelis constant (Km) in the presence and absence of either 4c or 6d.
Michaelis-Menten equation curves are shown in Fig. 4, and Vmax and Km are listed in Table 3.
As indicated by Table 3, Vmax remained nearly constant whether 4c or 6d was in the presence or
absence, while Km increased as the concentration of either 4c or 6d was increased. On the basis of
these observations, we conclude that 4c and 6d act as competitive inhibitors of ThDP against E.
coli PDHc-E1.
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
3.4 Selectivity
Many ThDP analogs with poor enzyme selectivity, such as thiamine thiothiazolone
pyrophosphate have been reported to have high inhibitory activity against both E. coli PDHc-E1
(Ki = 64 nM) and human PDHc-E1 (Ki = 74 nM).27 Determining the inhibition selectivity of our
candidates between mammals and E. coli PDHc-E1 is thus important. We selected porcine
PDHc-E1 as a mammalian target because its sequence is 98.5% similar to that of human
PDHc-E1. Compounds 4c, 4e, 6d, and 6e, which had good inhibitory activity against both E. coli
PDHc-E1 and cyanobacteria, were chosen for the enzyme-selectivity examination. We found that
these compounds exhibited 100% inhibition against E. coli PDHc-E1 at 100 μM, but their
inhibition was negligible (<19%) against porcine PDHc-E1. The excellent selectivity of these new
ThDP analogs indicates they may have low mammalian toxicity (Table 4).
3.5 Analyses of interactions
To identify the possible binding mode of the title compounds, compounds 4e and 6d were
docked into the active pocket of PDHc-E1. As shown in Fig. 5, compounds 4e and 6d occupy the
ThDP-binding pocket in a ‘V’ conformation. On the right side of the ‘V’ conformation, the
4-aminopyrimidine ring of 4e and 6d forms hydrogen bonds with residues V192, M194, and
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 34
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
E571, which is similar to the interactions between ThDP and the corresponding residues.12 On the
left side of the ‘V’ conformation, the terminal benzene ring of 4e and 6d can form cation-π and
hydrophobic interaction with K392 and L264, respectively.
It was found that 4e (IC50 = 10.91 ± 0.65 μM) and 6d (IC50 = 4.13 ± 0.26 μM) displayed
significantly higher inhibition against E. coli PDHc-E1 than did Ib (IC50 = 26.44 ± 1.68 μM). We
next investigated the difference in binding when the ether group (the linker) was modified to an
imine or amide group. As shown in Fig. 5, the benzene ring of 4e and 6d can participate in
hydrophobic interactions with L264, which is obviously different from compound I. As illustrated
in Fig. 5B, a new hydrogen bond between H640 and the N atom of 1,2,3-triazole is additionally
formed as a result of the introduction of the amide group, which changes of the structure of 6 and
contributes to its better inhibition against PDHc-E1 (IC50 = 4.13 μM). In contrast, only a weak
intermolecular interaction takes place between 4e and H640 according to molecular docking
analysis.
To further confirm the predicted interactions of the title compounds with E. coli PDHc-E1, 4e
and 6d were studied by site-directed mutagenesis and enzymatic assays. The IC50 values of 4e and
6d against mutants V192A (73.03 and 41.75 μM, respectively), M194A (87.45 and 33.13 μM,
respectively), L264A (105.41 and 44.73 μM, respectively), and H640A (25.52 and 22.79 μM,
respectively) were much higher than the IC50 values against the wild-type PDHc-E1 (10.91 and
4.13 μM, respectively) (Fig. 6). According to the results, the residues, V192, M194, L264 and
H640 play an important role in the binding of 4e or 6d with E. coli PDHc-E1. PDHc-E1 with an
E571A or K392A mutation is completely inactive, however, which prevented the detection of
enzyme inhibitory activity. The binding affinity of the title compounds to mutants E571A and
ACS Paragon Plus Environment

Page 21 of 34
Journal of Agricultural and Food Chemistry
418
K392A was therefore assessed using fluorescence spectroscopy. The association constant (Ka) of
4e to mutants E571A and K392A was found to be decreased more than 30 fold, compared with
that of 4e to the wild-type PDHc-E1 (Table 5). The binding affinity of 6d to both mutants was
also decreased more than 24 fold (Table 5). The above observations indicate that the predictions
based on molecular docking were in accord with the experimental results of site-directed
mutagenesis.
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
3.6 Inhibition against cyanobacteria
To examine the practicality of title compounds 4 and 6, several compounds with acceptable
inhibition against E. coli PDHc-E1 were selected and evaluated for their anti-cyanobacteria
activity. Five algal species (Synechocystis sp. PCC6803, Microcystis sp. FACHB905, Anabaena
sp. FACHB82, Aphanizomenon sp. FACHB1395, and Nostoc sp. FACHB713) with agricultural
and environmental significance were used for this assay. Prometryn (a commercial herbicide) and
copper sulfate, two algicides currently in common use, were used as positive controls. Compound
IV-21l (R1= I, R = 6-Br) (Fig.1), which displayed the best inhibition against PCC6803 (EC50 = 0.7
μM) in our previously published study,14 was also selected as a positive control.
As shown in Table 6, most compounds in series 4 and 6 had poor inhibition against
FACHB713, but all title compounds except 4a showed a broad spectrum of algicidal activity
against four of the algal species (PCC6803, FACHB905, FACHB82, and FACHB1395).
Compounds 4b-4h and 6a-6d had better potency against PCC6803 and FACHB905 than did
prometryn. For example, 4c, 4e, 4g, 4h, 6d, 6e, and 6g, with EC50 < 3 μM, showed stronger
inhibition against both algal species than did prometryn (PCC6803, EC50 = 13.1±0.7; FACHB905,
14.7 ± 0.8 μM). The algicidal activity of 4c (EC50 = 1.7 ± 0.2 μM) against PCC6803 was also
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 34
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
comparable to that of copper sulfate (EC50 = 1.8 ± 0.1 μM). Moreover, 6d not only exhibited
much higher algicidal activity (PCC6803, EC50 = 1.6 ± 0.3 μM; FACHB905, EC50 = 2.2 ± 0.2 μM;
FACHB82, EC50 = 1.2 ± 0.1 μM; FACHB1395, EC50 = 1.3 ± 0.3 μM) than prometryn but also had
activity comparable to or higher than that of copper sulfate (PCC6803, EC50 = 1.8 ± 0.1 μM;
FACHB905, 1.5 ± 0.1 μM; FACHB82, EC50 = 2.8 ± 0.3 μM; FACHB1395, EC50 = 1.6 ± 0.1 μM).
Although the previously reported compound IV-21l showed excellent inhibition against PCC6803
(EC50 = 0.7 ± 0.1 μM) and FACHB713 (EC50 = 7.8 ± 2.2 μM), its inhibitory activity against
FACHB905 (EC50 = 4.4 ± 0.2 μM), FACHB82 (EC50 = 9.2 ± 1.1 μM) and FACHB1395 (EC50
=
4.5 ± 0.2 μM) was significantly lower than that of compound 6d. It’s worth noting that compound
IV-21l should be synthesized by a six-step sequence starting from 2-amino-5-bromobenzoic acid.
However, it takes only three steps to synthesize compound 6d. The synthesis of 6d has the
advantage of being simple and easy. In addition, IV-21l has poor water- and fat-solubility because
of its three different nitro-containing heterocycles. Overall, 6d is a better candidate for further
study than IV-21l.
The inhibitory potency of the title compounds against cyanobacteria seems to be positively
well correlated with their inhibition against E. coli PDHc-E1. Compounds with higher levels of E.
coli PDHc-E1 inhibition displayed stronger potency against the different cyanobacterial species.
For instance, 4b-h and 6a-h exhibited higher levels of inhibition against E. coli PDHc-E1 (IC50
=
4.13-23.76 μM) and also exhibited good potency against PCC6803 (EC50 = 1.6-4.4 μM) and
FACHB905 (EC50 = 2.1-8.5 μM). In particular, 6d, which had the best inhibition against E. coli
PDHc-E1, also displayed potent inhibition against PCC6803, FACHB905, FACHB82, and
FACHB1395. The reverse was true for compounds I and 4a, which had lower levels of enzyme
ACS Paragon Plus Environment

Page 23 of 34
Journal of Agricultural and Food Chemistry
462
inhibition (IC50 > 26 μM) and lower algicidal activities (EC50 > 50 μM). These results indicate that
an effective algicide can be developed by designing a potent E. coli PDHc-E1 inhibitor.
Acknowledgments
463
464
465
466
467
468
469
The work was supported in part by the National Research and Development Plan
(2017YFD0200506), the National Natural Science Foundation of China (21877047, 31701820,
21472062, and 21907035), the 111 Project B17019, the Natural Science Foundation of Hubei
Province of China (2017CFB232), the National Key Research Development Program of China
(2018YFD0200100).
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 34
REFERENCES
(1) Yu, Y.; Zeng, Y. D.; Li, J.; Yang, C. Y.; Zhang, X. H.; Luo, F.; Dai, X. Z. An algicidal
Streptomyces amritsarensis strain against Microcystis aeruginosa strongly inhibits microcystin
synthesis simultaneously. Sci. Total Environ. 2019, 650, 34-43.
(2) Li, D.; Han, X. Y.; Tu, Q. D.; Feng, L. L.; Wu, D.; Sun, Y.; Chen, H. F.; Li, Y. J.; Ren, Y. L.;
Wan, J. Structure-Based Design and Synthesis of Novel Dual-Target Inhibitors against
Cyanobacteria Fructose-1,6-Bisphosphate Aldolase and Fructose-1,6-Bisphosphatase. J. Agric.
Food Chem. 2013, 61, 7453-7461.
(3) Li, H.; Pan, G. Simultaneous Removal of Harmful Algal Blooms and Microcystins Using
Microorganism- and Chitosan-Modified Local Soil. Environ. Sci. Technol. 2015, 49, 6249-6256.
(4) Dalu, T.; Wasserman, R. J. Cyanobacteria dynamics in a small tropical reservoir:
Understanding spatio-temporal variability and influence of environmental variables. Sci. Total
Environ. 2018, 643, 835-841.
(5) Saraf, S. R.; Frenkel, A.; Harke, M. J.; Jankowiak, J. G.; Gobler, C. J.; McElroy, A. E. Effects
of Microcystis on development of early life stage Japanese medaka (Oryzias latipes): Comparative
toxicity of natural blooms, cultured Microcystis and microcystin-LR. Aquat. Toxicol. 2018, 194,
18-26.
(6) Sun, Y.; Zhang, R.; Li, D.; Feng, L. L.; Wu, D.; Feng, L. N.; Huang, P. P.; Ren, Y. L.; Feng, J.
T.; Xiao, S.; Wan, J. Pharmacophore-Based Virtual Screening and Experimental Validation of
Novel Inhibitors against Cyanobacterial Fructose-1,6-/Sedoheptulose-1,7-bisphosphatase. J.
Chem. Inf. Model. 2014, 54, 894-901.
(7) Han, X. Y.; Zhu, X. Y.; Zhu, S. H.; Wei, L.; Hong, Z. Q.; Guo, L.; Chen, H. F.; Chi, B.; Liu,
ACS Paragon Plus Environment

Page 25 of 34
Journal of Agricultural and Food Chemistry
Y.; Feng, L. L.; Ren, Y. L.; Wan, J. A Rational Design, Synthesis, Biological Evaluation and
Structure-Activity Relationship Study of Novel Inhibitors against Cyanobacterial
Fructose-1,6-bisphosphate Aldolase. J. Chem. Inf. Model. 2016, 56, 73-81.
(8) Matthijs, H. C. P.; Jančula, D.; Visser, P. M.; Maršálek, B. Existing and emerging cyanocidal
compounds: new perspectives for cyanobacterial bloom mitigation. Aquat. Ecol. 2016, 50,
443-460.
(9) Nemeria, N.; Baykal, A.; Joseph, E.; Zhang, S.; Yan, Y.; Furey, W.; Jordan, F. 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, 6565-6575.
(10) Patel, M. S.; Korotchkina, L. G. The Biochemistry of the Pyruvate Dehydrogenase Complex.
Biochem. Mol. Biol. Educ. 2003, 31, 5-15.
(11) Balakrishnan, A.; Nemeria, N. S.; Chakraborty, S.; Kakalis, L.; Jordan, F. Determination of
Pre-Steady-State Rate Constants on the Escherichia coli Pyruvate Dehydrogenase Complex
Reveals That Loop Movement Controls the Rate-Limiting Step. J. Am. Chem. Soc. 2012, 134,
18644-18655.
(12) Arjunan, P.; Nemeria, N.; Brunskill, A.; Chandrasekhar, K.; Sax, M.; Yan, Y.; Jordan, F.;
Guest, J. R.; Furey, W. Structure of the Pyruvate Dehydrogenase Multienzyme Complex E1
Component from Escherichia coli at 1.85Å Resolution. Biochemistry 2002, 41, 5213-5221.
(13) He, J. B.; Feng, L. L.; Li, J.; Tao, R. J.; Wang, F.; Liao, X.; Sun, Q. S.; Long, Q. W.; Ren, Y.
L.; Wan, J.; He, H. W. Design, synthesis and biological evaluation of novel
2-methylpyrimidine-4-ylamine derivatives as inhibitors of Escherichia coli pyruvate
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 34
dehydrogenase complex E1. Bioorg. Med. Chem. 2012, 20, 1665-1670.
(14) Zhou, Y.; Feng, J. T.; He, H. W.; Hou, L. F.; Jiang, W.; Xie, D.; Feng, L. L.; Cai, M.; Peng,
H. Design, Synthesis, and Potency of Pyruvate Dehydrogenase Complex E1 Inhibitors against
Cyanobacteria. Biochemistry 2017, 56, 6491-6502.
(15) Erixon, K. M.; Dabalos, C. L.; Leeper, F. J. Synthesis and biological evaluation of
pyrophosphate mimics of thiamine pyrophosphate based on a triazole scaffold. Org. Biomol.
Chem. 2008, 6, 3561-3572.
(16) Majumdar, K. C.; Ganai, S. An expedient approach to substituted triazole[1,5-α][1,4]
benzodiazepines via Cu-catalyzed tandem Ullmann C-N coupling/azide-alkyne cycloaddition.
Tetrahedron Lett. 2013, 54, 6192-6195.
(17) Ramachandran, P. V.; Rudd, M. T.; Reddy, M. V. R. Stereoselective synthesis of
hex-2-(E)-en-4-yn-1,6-dioates and E,Z-muconic acid diesters via organo-catalyzed self-coupling
of propiolates. Tetrahedron Lett. 2005, 46, 2547-2549.
(18) Ren, Y. L.; He, J. B.; Feng, L. L.; Liao, X.; Jin, J.; Li, Y. J.; Cao, Y.; Wan, J.; He, H. W.
Structure-based rational design of novel hit compounds for pyruvate dehydrogenase multienzyme
complex E1 components from Escherichia coli. Bioorg. Med. Chem. 2011, 19, 7501-7506.
(19) Feng, L. L.; Sun, Y.; Deng, H.; Li, D.; Wan, J.; Wang, X. F.; Wang, W. W.; Liao, X.; Ren, Y.
L.;
Hu,
X.
P.
Structural
and
biochemical
characterization
of
fructose-1,6/sedoheptulose-1,7-bisphosphatase from the cyanobacterium Synechocystis strain
6803. FEBS J. 2014, 281, 916-926.
(20) Feng, L. L.; He, J. B.; He, H. F.; Zhao, L. L.; Deng, L. F.; Zhang, L.; Zhang, L.; Ren, Y. L.;
Wan, J.; He, H. W. The design, synthesis and biological evaluation of novel thiamin diphosphate
ACS Paragon Plus Environment

Page 27 of 34
Journal of Agricultural and Food Chemistry
analog inhibitors against the pyruvate dehydrogenase multienzyme complex E1 from Escherichia
coli. Org. Biomol. Chem. 2014, 12, 8911-8918.
(21) Dolinsky, T. J.; Nielsen, J. E.; McCammon, J. A.; Baker, N. A. PDB2PQR: an automated
pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 2004,
32, 665-667.
(22) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.;
Olson, A. J. AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor
Flexibility. J. Comput. Chem. 2009, 30, 2785-2791.
(23) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function from
a Few Good Reactions. Angew. Chem. Int. Ed. 2001, 40, 2004-2021.
(24) Wu, P.; Malkoch, M.; Hunt, J. N.; Vestberg, R.; Kaltgrad, E.; Finn, M. G.; Fokin, V. V.;
Sharpless, K. B.; Hawker, C. J. Multivalent, bifunctional dendrimers prepared by click chemistry.
Chem. Commun. 2005, 37, 5775-5777.
(25) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A Stepwise Huisgen
Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal
Alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596-2599.
(26) Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.; Sharpless, K. B.; Fokin,
V. V. Copper(I)-Catalyzed Synthesis of Azoles. DFT Study Predicts Unprecedented Reactivity
and Intermediates. J. Am. Chem. Soc. 2005, 127, 210-216.
(27) Nemeria, N.; Yan, Y.; Zhang, Z.; Brown, A. M.; Arjunan, P.; Furey, W.; Guest, J. R.; Jordan,
F. Inhibition of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Subunit and Its
Tyrosine 177 Variants by Thiamin 2-Thiazolone and Thiamin 2-Thiothiazolone Diphosphates. J.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 34
Biol. Chem. 2001, 276, 45969-45978.
ACS Paragon Plus Environment

Page 29 of 34
Journal of Agricultural and Food Chemistry
Tables
Table 1. Crystallographic data of compound 4b.
Parameters
Data
chemical formula
formula weight
crystal system
space group
crystal color
a (Å)
C15H16ClN7
329.80
monoclinic
P2(1)/c
colorless
14.488(4)
9.884(2)
12.319(3)
90
b (Å)
c (Å)
 (deg)
115.098(4)
90
 (deg)
 (deg)
V (Å)
1597.6(7)
4
Z
Dcalc (Mgm-3)
 range (deg)
hkl range
1.371
2.58-26.00
-17  h  16; -12  k  12; -15  l  15
F (0 0 0)
688
no. collected refl.
no. ind. Refl. (Rint)
data/ restraints/ parameters
Absorption coefficient (mm-1)
R1; wR2 [I > 2(I)]
R1; wR2 (all data)
GOOF
14090
3126 (0.0908)
3126 / 0 / 209
0.250
0.0500; 0.1225
0.0776; 0.1434
1.015
Table 2. Structure and inhibitory activity of I, 4, and 6
NH2
N
NH2
NH2
O
R
R
R
N
N
N
N
N
N
N
N
N
O
HN
HN
N
N
N
N
N
L
4
6
IC50 (μM) Inhibitory
IC50 (μM) Inhibitory
Compd.
R
H
Compd.
R
against E. coli PDHc-E1
55.15 ± 4.65
against E. coli PDHc-E1
23.76 ± 0.01
14.70±0.14
L1
L2
L3
4a
4b
4c
4h
6a
6b
6c
6d
6e
6f
4-CH3
H
4-Cl
26.44 ± 1.68
3-CF3
H
32.73 ± 0.17
3-Cl
4-Cl
3-Br
4-Br
4-F
9.48 ± 0.25
46.32 ± 0.24
7.12 ± 0.08
2-Cl
3-Cl
4-Cl
13.41 ± 0.82
4.13 ± 0.26
11.53 ± 0.33
5.65 ± 0.01
4d
15.54 ± 0.01
5.92 ± 0.02
ACS Paragon Plus Environment