Design, synthesis, biological evaluation and molecular docking of amide and sulfamide derivatives as Escherichia coli pyruvate dehydrogenase complex E1 inhibitors

Article abstract of DOI:10.1039/c5ra22573f

In this study, a series of novel amide derivatives and sulfamide derivatives as potential E. coli PDHc E1 inhibitors were designed and synthesized by optimizing the linker between triazole and benzene ring moieties based on the structure of lead compound

Full text of DOI:10.1039/c5ra22573f

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ARTICLE
Design, synthesis, biological evaluation and molecular
docking of amide and sulfamide derivatives as Escherichia
coli pyruvate dehydrogenase complex E1 inhibitors
Received 00th January 20xx,
Accepted 00th January 20xx
Haifeng Hea1, Jiangtao Fenga1, Junbo Heb, Qin Xiaa, Yanliang Rena, Fang Wanga, Hao Penga, Hongwu
He*a and Lingling Feng*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this study, a series of novel amide derivatives and sulfamide derivatives as potential E. coli PDHc E1 inhibitors were
designed and synthesized by optimizing the linker between triazole and benzene ring moieties based on the structure of
lead compound I as thiamin diphosphate (ThDP) analogs. Their inhibitory activities against E. coli PDHc E1 were examined
in vitro and their inhibitory activities against microbial diseases were further evaluated. Most of these compounds exhibit
good inhibitory activity against E. coli PHDc E1 (IC50 1.99 to 25.66 µM) and obvious antibacterial activity. 5a, 5c and 9i
showed 90-100% antibacterial activity against Xanthimonas oryzae pv Oryzae (Xoo), Acidovorax avenae subsp. Avenae
(Aaa) and cyanobacteria. Sulfamide derivatives 9 showed more potent inhibitory activity against E.coli PDHc E1 (IC50 <14
µM) than that of amide derivatives 5 or lead compound I. Especially 9d (IC50 = 2.95 µM) and 9k (IC50 = 1.99 µM) exhibited
not only most powerful inhibitory potency against E. coli PDHc E1, but also 9k showed 99% antibacterial activity against
Aaa at 500 µg/mL and almost best inhibition 97% against cyanobacteria at 20 µg/mL. Furthermore, the binding mode of 5d
and 9d to E.coli PDHc E1 was analyzed by molecular docking method. The possible interactions of 9d with the important
residues of E. coli PDHc E1 were further verified via site-directed mutagenesis enzymatic assays, and fluorescence spectral
analysis. Both theoretical and experimental results revealed that 9d could display more powerful interaction than that of
5d or I by forming hydrogen bond between sulfamide linkage and residue Lsy392, Tyr599 and His106 at active site of E.coli
PDHc E1. 9k, 9d and 9i with both potent enzyme inhibition and significant antibacterial activity, could be used as novel
lead compounds for further optimization. These results proved that a series of compounds with potential antibacterial
activity could be obtained by the biorational design of E. coli PDHc E1 inhibitors.
Introduction
modes of action to overcome microbial disease and bactericides
resistance.
Bactericides play important role in modern agriculture due to the
harm of bacterium in agriculture. Although there are many
The pyruvate dehydrogenase complex (PDHc) plays
a key
bactericides can control these bacterium infections, the repeated regulatory role in cellular metabolism catalyzing the oxidative
use of the same bactericides or repeated treatment with decarboxylation of pyruvate and the subsequent acetylation of
bactericides having the same mode of action, has resulted in the coenzyme A (CoA) to acetyl-CoA3-4. The overall reaction of oxidative
wide spread evolution of resistance1,2. It is essential to develop decarboxylation can be simply shown in Fig. 1.
efficient microbicides or bactericides with novel structures or
a Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College
of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079,
Fig.1 Oxidative decarboxylation catalyzed by PDHc.
P. R. China
bCollege of Food Science & Engineering, Wuhan Polytechnic University, Wuhan
430023, China
The complex is comprised of three different enzymes
1Those authors contributed equally to this work
components
including
pyruvate
dehydrogenase
(E1),
E-mail: he1208@mail.ccnu.edu.cn; fll708@mail.ccnu.edu.cn
Fax: 86-27-67867960; 86-27-67867960
dihydrolipoamide
acetyltransferase (E2),
dihydrolipoamide
Electronic Supplementary Information (ESI) available: [Assay of E.coli PDHc E1 (in
vitro) and site-directed mutagenesis of PDHc E1; Molecular docking; Fluorescence
spectral analyses; Inhibitory bacterial activity and fungal activity evaluation of
compounds; characterization datas mentioned in the paper]. See
DOI: 10.1039/x0xx00000x
dehydrogenase (E3) and
a
number of cofactors5. Pyruvate
dehydrogenase complex E1 component (PDHc E1, EC 1.2.4.1) is the
initial member of PDHc, which catalyzes the first step of multistep
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process, using thiamine diphosphate (ThDP) and Mg2+ as cofactors fungicidal activity. In fact some reported ThDP analogs are
6–8. Especially this PDHc E1 catalyzed process is the rate limiting step unsuitable for the usage as agricultural chemicals due to their
among multistep process which is catalyzed by PDHc. Therefore, complex structure with highly charged pyrophosphate and poor
blocking the activity of PDHc E1 will be the best way to inactivate bioavailability. Aiming at the above-mentioned problems, both the
the PDHc. In our study, E. coli PDHc E1 was selected as the target, thiazolium ring and the pyrophosphate moiety in ThDP were
which should be an interesting site of action for bactericide design. replaced by 1,2,3-triazole ring and substituted benzene ring,
Some ThDP analogs as inhibitors of PDHc E1 in E. coli have been respectively. It has been verified that some hit compounds with
studied and reported (such as triazole ThDP in Fig. 2) because of the aminopyrimidine, triazole and benzene ring moiety could be
important role of ThDP in oxidative decarboxylation catalyzed by effective in occupying the ThDP-binding pocket of PDHc E1 by using
PDHc 9-13. However there was little report about their bactericidal or structure-based molecular docking14
.
Fig.2 ThDP analogue and design of new amide and sulfamide derivatives as E.coli PDHc E1 inhibitors.
In order to obtain potential fungicide or bactericide by designing ((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1, 2, 3- triazol-4-
PHDc E1 inhibitor, series of 2-methylpyrimidine-4-ylamine yl)methyl)-substituted-benzenesulfonamide hydrochloride 9.
derivatives I containing 1,2,3-triazole ring and substituted benzene
Herein, we report the design and synthesis of series of new
ring (Fig. 2) as ThDP analogs had been firstly chemically synthesized
amide and sulfamide derivatives 5a–e and 9a-k by incorporating the
and demonstrated to be effective inhibitors against E. coli PDHc E1
15-16. The structure skeleton of I avoided the high charge of the
active amide or sulfamide pharmacophore as a “linker” to form
novel ThDP analog as potential inhibitors against E. coli PDHc E1
pyrophosphate moiety and some of these compounds exhibited
(Fig.2). Some amide and sulfamide derivatives have attracted our
attention due to their excellent antibacterial activity17-18. Therefore,
antifungal activity. These preliminary progresses encouraged us to
choose
I as lead compound for further optimization. As a
these title compounds are expected to be good E.coli PDHc E1
inhibitors with bactericidal activity. In order to make sure the effect
of benzene ring in parent structure on inhibitory activity, N-((1-((4-
amino-2-methylpyrimidin-5-yl) methyl)-1H-1,2,3-triazol-4-yl)methyl)
methanesulfonamide hydrochloride 13 also was designed and
synthesized.
distinguishing feature, ether (C-O-C) group was used as a linker to
connect the triazole and benzene ring in lead compound I. The
result of molecular docking showed that there was no interaction
between oxygen atom of ether moiety and amino acid residue in
the active site of PHDc-E1 16. It was thought that the formation of
hydrogen bond between the structural part of linker and amino acid
residues in the active site should be much beneficial for enhancing
inhibition against E. coli PDHc E1. In order to increase the inhibitory
potency against E. coli PDHc E1, the scaffold of 2-methylpyrimidine-
4-ylamine derivatives containing 1,2,3-triazole and benzene ring
were kept, further optimization focused on “linker part” between
the triazole and benzene ring. Considering amide or sulfamide
group containing NH, C=O or O=S=O structure unit, which could be
used as hydrogen donor (NH) or hydrogen receptor (C=O, O=S=O).
Therefore, amide or sulfamide group as a “linker part” was
introduced into the parent structure respectively to design novel
series of N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1, 2, 3-
triazol-4-yl)methyl)-substituted-benzamide hydrochloride 5, and N-
In this work, both inhibitory activity against E.coli PDHc E1 and
antibacterial activity of title compounds 5, 9 and 13 were examined.
The interaction mode of the important residues of E.coli PDHc E1
with some title compounds was also studied by molecular docking
method and site-directed mutagenesis, probable inhibition
mechanism was discussed.
Results and discussion
Synthesis
The synthetic route of title compounds 5a–e, 9a-k and 13 is
depicted in Scheme 1. Vitamin B or thiamine hydrochloride as
starting material was used to prepare 5-azido-methyl-2-
methylpyrimidine-4-ylamine 1, according to the literature
2 | J. Name., 2012, 00, 1-3
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method19. 1 is the key intermediate for the preparation of title
The 1,2,3-triazol ring in the parent skeleton of title compounds
compounds 5, 9 and 13. The title compounds 5a–e, 9a-k and 13 could be constructed by a synthetic method of ‘click chemistry’.
could be synthesized by a four or five-step sequence starting from Important intermediates, 4a-e, 8a-k or 12 with key skeleton
substituted benzoic acid, substituted sulfonyl chloride or methyl containing 1,2,3-triazol ring were prepared by the Cu-catalyzed 1,3-
sulfonyl chloride respectively. Various substituted benzoic acid dipolar cycloaddition of 1 with N-substituted-prop-2-yn-1-amines
reacted with oxalyl chloride in the presence of DMF to produce including 3a–e, 7a-k, or 11 respectively using CuI and Et3N in the
substituted benzoyl chlorides 2a–e, which reacted with presence of THF.
propargylamine to form N-(prop-2-yn-1-yl)-sub-benzamide 3a–e
In order to increases the solubility of compounds, 4a-e, 8a-k and
using Et3N as base. Under same condition, substituted sulfonyl
12 were further converted into corresponding hydrochloride 5a–e,
chloride 6a-k or methyl sulfonyl chloride 10 could be converted into
9a-k and 13 by using hydrochloric acid. All title compounds, 5a–e,
9a-k and 13 were characterized by 1H NMR, 13C NMR, mass
corresponding N-(prop-2-yn-1-yl)-sub-benzenesulfonamide 7a-k or
N-(prop-2-yn-1-yl)methanesulfonamide 11, respectively.
spectrometry (MS), and confirmed by elementary analysis.
NH2
NH2
CH3
S
a
N
N3
N
N
OH
H3C
N
H3C
N
1
Vitamin B1
R
NH2
N
R
R
R
HN
N
N
Cl
O
OH
O
c
d
1
b
N
HN
N
H3C
R
O
O
4a-e
3a-3e
2a-2e
NH2HCl
N
N
N
e
HN
N
H3C
N
O
5a-e
NH2
R
R
R
Cl
HN
O
S
c
N
N
S
O
S
O
d
1
N
H
N
N
O
O
O
N
H3C
6a-k
7a-k
8a-k
NH2HCl
R
O
S
N
N
e
N
H
N
N
O
N
H3C
9a-k
NH2
NH2HCl
O
O
S
Cl
HN
N
N
N
N
e
c
d
1
N
H
S
S
O
N
S
O
H
N
N
N
N
O
O
O
O
N
N
H3C
H3C
10
12
13
11
Scheme 1. Reagents and conditions (a) NaN3, Na2SO3, H2O, 60-65 oC 56%; (b) C2O2Cl2 DCM/DMF, 0 oC; (c) CHCCH2NH2, Et3N, DCM 0 oC 70-
85%; (d) CuI, Et3N, THF, rt, 10-15 h; (e) HCl.
Inhibitory potency against E.coli PDHc E1
results showed that the inhibitory activity of 5a–e against E.coli
PDHc E1 could be improved by introducing amide bond into the
“linker part”. Some compounds 5 exhibited better inhibitory activity
than that of lead compound I, such as inhibitory activity, 5a > Ia; 5e
> Ie, Especially 5e showed 7-folds activity higher than that of Ie.
However 5c showed lower inhibitory activity than that of Ic. On the
In order to enhance the inhibitory potency against E. coli PDHc E1,
lead structure I was modified by replacing ether bond (C-O-C) in the
structure skeleton I with amide linkage.
Several amide derivatives 5a–e were firstly synthesized and
evaluated for their inhibitory activities against E.coli PDHc E1. The
IC50 values of 5a–e and some I16 were summarized in Table 1. The
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basis of this work, the structure skeleton of 5 was further optimized
by introducing sulfamide bond as a linker to form a series of 9a-k.
inhibitory potency against E. coli PDHc E1. Compared with ether
bond or amide bond, the sulfamide bond as linker was much
beneficial to inhibitory activity. These results suggest that the
structure skeleton of 9 is better than both 5 and lead structure I for
finding more powerful PDHc E1 inhibitor.
It was found that the inhibitory activity was further enhanced by
changing amide into sulfamide linkage. When R kept same, 9a, 9c,
and 9d (IC50 = 4.01±0.17, 5.99±0.37, and 2.95±0.14 µM) with
sulfamide linkage displayed higher inhibitory activity than that of
corresponding 5a, 5c, and 5d (IC50 = 25.66±1.34 , 24.27±1.84, and
13.52±1.48 µM) with amide linkage. As an example, 9a (R=H)
showed 6-folds activity higher than that of 5a (R=H). Although 9e
showed weaker inhibitory activity than that of 5e, it was still higher
As shown in Table 1, R on the benzene ring also had great
influence on the inhibitory activity base on the structure skeleton of
9. The inhibitory activity of 9 could be greatly enhanced by
optimizing R on the benzene ring. The effects of R on inhibitory
activity can be shown by following activity sequence, 9b (R=4-CH3,
IC50 = 12.93 µM) < 9g (R=4-Br, IC50 = 11.64 µM) < 9e (R=4-Cl, IC50
=
than that of Ie. It was also observed that 9a, 9c, 5e, 9f (IC50
4.01±0.17, 5.99±0.37, 3.16±0.46 and 7.53±0.31 µM) showed higher
inhibitory activity than that of corresponding compounds ( IC50
=
10.53 µM) < 9h (R=4-F, IC50 = 9.11 µM) < 9f (R=4-OCH3, IC50 = 7.53
µM) < 9i (R=2,4,6-Me3, IC50 = 6.60 µM) < 9c (R=4-NO2, IC50 = 5.99
µM) < 9j (R=2-NO2, IC50 = 5.31 µM) < 9a (R=H, IC50 = 4.01 µM) < 9d
(R=3-NO2, IC50 = 2.95 µM) < 9k (R=3-NO2-4-Cl, IC50 = 1.99 µM). These
result showed all compounds 9 with NO2 as R, such as 9c, 9j, 9d, 9k
exhibited better inhibitory activity than that of compounds 9 with
=
75.5±0.02, 6.7±0.48, 6.9±1.19 and 30.2±3.45 µM) , which had been
reported20 with oxime ether linkage.
Above observation showed the “linker part” between the triazole
and benzene ring in parent structure played a very important role in
Table 1 Structures and inhibitory activity (IC50) of novel amide derivatives 5a-e and sulfamide derivatives 9a–k and 13 against E.coli PDHcE1
IC50a(µM)
55.15±4.65
25.66±2.34
4.01±0.17
13.69±1.56
12.93±0.30
8.8±0.35
IC50a(µM)
3.16±0.46
10.53±0.70
81.62±5.85
7.53±0.31
11.64±0.39
9.11±0.40
6.60±0.16
5.31±0.31
1.99±0.08
7.40±0.64
Compd.
R
Compd.
R
4-Cl
Ia
5a
9a
H
5e
9e
If
H
4-Cl
H
4-OCH3
4-OCH3
4-Br
4-Me
4-Me
4-NO2
4-NO2
4-NO2
3-NO2
3-NO2
4-Cl
9f
5b
9b
Ic
9g
9h
9i
4-F
24.27±1.84
2,4,6-Me3
2-NO2
3-NO2-4-Cl
5c
9c
5d
9d
Ie
5.99±0.37
9j
13.52±1.48
9k
13
2.95±0.14
26.44±1.68
aIC50 (µM) value is defined as the micromolar concentration required for 50% inhibition on PDHc E1 from E. coli in vitro.
other substituents, and NO2 at 3-position on the benzene ring was found to be most effective compound against E.coli PDHc E1. It
seems to be favorable to inhibitory activity. Compound with exhibited 6-folds activity higher than that of 9b with 4-CH3 as R.
electron-withdrawing group as R was promotive for inhibitory Above results indicated that the inhibitory activity of 9 was also
activity against E.coli PDHc E1. Especially, 9k with 3-NO2, 4-Cl as R
4 | J. Name., 2012, 00, 1-3
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dependent upon the structure and position of R on the benzene Antibacterial activity
ring.
Most reported ThDP analogs as E. coli PDHc E1 inhibitors did not
In order to examine the effect of benzene ring in structure 9 on show antibacterial activity or antifungal activity. In order to find
inhibitory activity, the benzene ring was replaced with a methyl useful PDHc E1 inhibitors with antibacterial activity or antifungal
group to prepare compound 13 and its inhibitory activity against activity, amide derivatives 6a–e, sulfamide derivatives 9a-k and 13
E.coli PDHc E1 was tested. The result showed that the lost of as new E. coli PDHc E1 inhibitors were evaluated for their antifungal
benzene ring led to a decrease in inhibitory activity against E.coli activity against Gibberella zeae (G. zeae), Rhizoctonia solani (R.
PDHc E1 compared with 13 (IC50 = 7.40 µM) with 9a (R=H , IC50
=
solani), Botrytis cinerea (B.cinerea), Alternaria solani (A. solani), and
Oryzae (Xoo)
4.01 µM) or 9k (R=3-NO2-4-Cl, IC50 = 1.99 µM). It indicated that antibacterial activity against Xanthimonas oryzae pv
．
substituted benzene ring also an important pharmacophore for Acidovorax avenae subsp. Avenae (Aaa) and cyanobacteria.
structure 9.
Table 2 Antibacterial activity of compounds 5, 9 and 13
Compd
R
Inhibitory potencyb Inhibitory potencyb
(%) 20 µg/mL
Compd
R
Inhibitory potencyb
(%) 20 µg/mL
Inhibitory potencyb
(%) 500 µg/mL
(%) 500 µg/mL
cyanobacteria
Aaaa
<10
<10
<10
<10
99
Xooa
<10
<10
<10
<10
95
cyanobacteria
Aaaa
84
80
84
60
80
90
79
99
89
Xooa
76
Ia
Ic
H
4-NO2
4-Cl
<20
<20
<20
<20
92
9d
9e
9f
3-NO2
4-Cl
97
94
94
77
96
99
94
97
99
95
100
100
81
Ie
4-OCH3
4-Br
If
4-OCH3
H
9g
9h
9i
5a
5b
5c
5d
5e
9a
9b
9c
4-F
75
4-Me
4-NO2
3-NO2
4-Cl
90
60
93
2,4,6-Me3
2-NO2
100
99
94
99
90
9j
76
39
96
9k
13
3-NO2-4-Cl
28
59
71
95
85
H
95
50
99
CuSO4
Streptomycin Sulfate
4-Me
4-NO2
94
78
74
97
98
97
90
80
aXoo, Xanthimonas oryzae pv
．
Oryzae； Aaa, Acidovorax avenae subsp. Avenae; bInhibitory potency (%) against the growth of
pathogenic fungi at 100 mg/mL, 0 (no effect), 100% (completely kill).
It is very interesting to find all the title compounds displayed 9g. Moreover 5a, 5c, 9c, 9i, and 9k could 90-99% control Aaa, and
good antibacterial activity, but very weak fungicidal activity. As 5a-5e, 9a, 9e, 9f, 9i and 9j could 90-100% control Xoo at 500 µg/mL,
shown in Table 2, all the title compounds 5 and 9 exhibited obvious but compounds I only showed <20% inhibitory potency against
antibacterial activity against cyanobacteria, Xoo or Aaa. All 5 and 9 cyanobacteria, Xoo and Aaa. The effect of these compounds against
could 90-99% control cyanobacteria at 20 µg/mL, except 5d, 5e and cyanobacteria, Xoo and Aaa were comparable to commercial
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bactericide, CuSO4 or streptomycin sulfate as a positive control. Table 3 Antifungal activity of compounds 5, 9 and 13
Inhibitory potencyb (%)
Especially 5a, 5c and 9i showed 90-100% antibacterial activity
Compd.
against cyanobacteria, Xoo and Aaa.
G. zeaea
R.solania B.cinereaa A.solania
According to the data in Table 1 and 2, all title compounds 5 and
9 with the IC50 values ranging from 1.99 to 25.66 µM against E. coli
PHDc E1 could exhibit moderate to good antibacterial activity
against Xoo and Aaa at 500 µg/mL, and showed obviously inhibition
against cyanobacteria at 20 µg/mL. However I with much weaker
inhibitory potency against E. coli PHDc E1 showed much weaker or
no inhibition against cyanobacteria, Xoo and Aaa. When the ether
(C-O-C) group as a linker in lead structure I was replaced by an
amide or sulfamide group, both enzyme inhibitory potency and
antibacterial activity could be greatly enhanced. Most title
compounds 5 and 9 with amide or sulfamide linkage exhibited
higher inhibitory potency and antibacterial activity than that of lead
compounds I with ether linkage. And all of the reported compounds
5a
5b
5c
5d
5e
9a
0
0
0
0
33
20
26
64
39
0
0
0
0
0
23
0
43
39
19
26
22
19
7
46
7
18
9
with oxime ether linkage had no antibacterial activity reported20
.
9b
9c
9d
9e
9f
14
23
14
23
28
3
18
3
When R was kept same, 9d, or 9e with sulfamide linkage seemed to
exhibit better inhibition against cyanobacteria than that of
corresponding amide derivatives 5d or 5e. The results showed
that R on the benzene ring also had significant influence on the
inhibition against cyanobacteria, Xoo and Aaa, but there was not
clear regularity to establish the relationship of the structure and
position of R on antibacterial activity.
18
18
18
7
9
14
28
The bioassay showed that 9d and 9k with sulfamide linkage were
the most two effective compounds (IC50 = 2.95 and 1.99 µM)
against E. coli PDHc E1. 9d and 9k with 3-NO2 and 3-NO2-4-Cl as R
exhibited much higher inhibitory potency against E. coli PDHc E1
than that of corresponding 5d (R= 3-NO2, IC50 = 13.52 µM). It was
found that 9d and 9k with 97% inhibitory potency also displayed
higher inhibitory activity against cyanobacteria than that of 5d with
76 % inhibitory potency at 20 µg/mL. 9k, displaying the most potent
inhibitory activity against E. coli PDHc E1, showed almost best
inhibitory activity against cyanobacteria at 20 µg/mL and Aaa at 500
µg/mL. It was noted that 9i with potent inhibitory potency against E.
coli PDHc E1 displayed 90-100% inhibition against cyanobacteria,
Xoo and Aaa at the test concentration.
10
17
7
27
9
9g
9h
9i
19
9
9
31
12
21
25
15
18
9
23
23
19
28
32
35
7
9j
9
9k
13
27
18
14
Above observation showed that the degree of antibacterial
activity of most tested compounds positively correlated with that of
their inhibition against E. coli PDHc E1. Both amide and sulfamide
group, especial sulfamide linkage is much favorable to both enzyme
inhibition against E. coli PDHc E1 and antibacterial activity against
Xoo, Aaa and cyanobacteria. Although 5a-e and 9a-e exhibited
obvious antibacterial activity, they had no significant fungicidal
activity against tested fungus. As shown in Table 3, all amide
derivatives 5a-e and sulfamide derivatives 9a-e only showed <50 %
(0 - 43 %) inhibitory potency against G. zeae, R. solani, B. cinerea
and A. solani. According to our previous results, Ia, Ic, Ie also had
similar weak inhibitory potency (0-73%) against G. zeae, R. solani,
aG. zeae, Gibberella zeae; R. solani, Rhizoctonia solani;
B.cinerea, Botrytis cinerea; A. solani, Alternaria solani
bInhibitory potency (%) against the growth of pathogenic
fungi at 100 mg/mL, 0 (no effect), 100% (completely kill).
Analyses of the interaction between inhibitors and E.coil PDHc E1
In order to understand the mechanism of antibacterial activity, the
interaction mode of amide derivative 5 or sulfamide derivative 9
with active site of E.coli PDHc E1 was explored. Several molecular
docking simulation studies were carried out by using the SURFLEX
module of SYBYL package 14. In this study, both 5d and 9d with 3-
NO2 as R displaying significant inhibitory activity against E.coli PDHc
E1, were selected as hit compounds for molecular docking.
15
B. cinerea and A. solani . These results suggested that these title
compounds 5 and 9 could selectively inhibit bacterium due to their
good inhibition against PDHc E1 from E. coli. Their inhibition against
cyanobacteria is worth further examination.
6 | J. Name., 2012, 00, 1-3
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E1. Above observation showed that the binding mode of 4-
aminopyrimidine and substituted benzene ring in 5d and 9d with
amino acid residues in the active site of E.coli PDHc E1were very
similar to the binding mode of lead compound I.
It was very interesting to explore the different of the binding
mode of I, 5 and 9 by comparing the linker in their parent
structures. No hydrogen bond between any amino acid residues
and the oxygen atom of ether bond (C-O-C) as a linker at the middle
of the ‘V’ conformation of structure I was observed according to
previous molecular docking study on structure I14-16. However the
oxygen atom of amide (C=O) bond as a linker at the middle of the
‘V’ conformation of compound 5d could form a strong hydrogen
bond with residue Lsy392 (Fig. 3A), which was very important for
stabilizing the bound of 5d with the enzyme. As shown in Fig. 3B,
one of the oxygen atoms of sulfamide linkage in 9d can form two
strong hydrogen bonds with Lsy392, the other oxygen atom of
sulfamide linkage can form a strong hydrogen bond with His106.
Meanwhile, the 1,2,3-triazole moiety of 9d also form a hydrogen
bong with Tyr599. It showed that the binding mode of 9d with
sulfamide linkage displayed much powerful interaction than that of
5d with amide linkage or lead compound I. Site-directed
mutagenesis and enzymatic assays showed that the IC50 values of
compound 9d against the mutants K392A (22.19 µM), H106A (21.14
µM) and Y599A (9.61 µM) were about 7.5-fold, 6-fold and 2.3-fold
higher than its value against wild-type PDHc E1 enzyme (2.95 µM)
(Fig. 4). This suggests that the interaction between 9d and residue
Fig. 3 Binding modes of compound 5d (A) and 9d (B) target into
active site of E.coli PDHc E1, in which PDHc E1 is shown in ribbon,
ligands and some key residues are shown in stick, both coordination
bonds and hydrogen bonds are shown in dashed lines
Lsy392, Tyr599 or His106 by forming hydrogen bond has
a
significant contribution for its inhibitory activity against E.coli PDHc
E1.
The binding mode of 5d and 9d is shown in Fig. 3 A and B,
respectively. 5d and 9d with the ‘V’ conformation occupies the
ThDP-binding pocket and bind in the active site of E.coli PDHc E1.
On the right side of the ‘V’ conformation, the aminopyrimidine ring
of 5d and 9d can form hydrogen bonds with amino acid residues
Met194, Glu571 and Val192 (Fig. 3 A and B), which is similar to the
interactions of ThDP or lead structure I with amino acid residues.
The nitryl group as R on the benzene ring of 5d or 9d respectively
has an interaction with Asn260 and Ser109 in the active site of E.coli
PDHc E1 by forming hydrogen bonds. In order to validate the
prediction of molecular docking, site-directed mutagenesis and
enzymatic assays were performed. These results showed the IC50
values of 9d against mutants M194A (20.2 µM), G571A (21.67 µM),
V192A (42.13 µM) and S109A (13.78 µM) were about 5.8-fold, 6.3-
fold, 13.3-fold and 3.7-fold higher than its value against wild-type
PDHc E1 enzyme (2.95 µM), respectively (Fig. 4). These results
suggest that the hydrogen bonding interaction between 9d and
amino acid residues Met194, Glu571, Val192 or Ser109 plays an
important role in the binding of 9d with E.coli PDHc E1.
It showed that the prediction of molecular docking correlated
well to the results of site-directed mutagenesis. These results
provided us a reasonable explanation for why sulfamide derivative
had more potent inhibitory activity against E.coli PDHc E1 than that
of amide derivative 5d or lead compound I. It indicated that
sulfamide linkage was most favorable to enzyme inhibition against
E. coli PDHc E1 due to more binding position and stronger
interaction with the active site of E.coli PDHc E1 then that of 5d or I.
It is not possible to confirm the interaction between 9d and
Asn260 through the enzymatic assay of the N260A mutant directly
due to this mutant exhibits much less enzymatic activity. Therefore
the binding constant (Kb) values of N260A were investigated using
fluorescence spectral. As shown in Fig. 5, the Kb value of wild type
enzyme (3100 M−1) is over 9-fold higher than the Kb value of N260A
(330 M−1), suggesting that there is a stronger interaction between
9d and Asn260. These results further confirm and explain the
binding-mode between the inhibitors and the active site of PDHc
Fig. 4 The IC50 values of compound 9d against the wild type (WT)
and mutants of E. coli PDHc E1
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ARTICLE
Journal Name
9d had more potent inhibitory activity against E.coli PDHc E1 or
bacterium than that of 5d or lead compound I due to sulfamide
group as a “linker part” with more binding position and stronger
interaction in the active site of E.coli PDHc E1 than 5d or I. These
results proved that sulfamide group as a “linker part” of triazole and
benzene ring in the parent structure was much favorable to both
inhibition against E. coli PDHc E1 and bacterium. To the best of our
knowledge, 9k, 9d and 9i seem to be the first ThDP analogs as PDHc
E1 inhibitors with both potent enzyme inhibition and significant
antibacterial activity, and they could be used as lead compound for
further optimization. These results proved that antibacterial activity
compounds could be obtained by the biorational design of E. coli
PDHc E1 inhibitors.
Fig. 5 Binding constants (Kb) determined by fluorescence spectral
analyses for the binding of compound 9d to the wild type (WT) and
mutants of E. coli PDHc E1.
Experimental procedures
General procedures
Melting points (mp) were measured on an electrothermal melting
point apparatus and were uncorrected.1H NMR and 13C NMR
spectra were recorded at 400 MHz, in DMSO-d6 solution on a
Varian Mercury-Plus 400 spectrometer and chemical shifts were
recorded in parts per million (ppm) with TMS as the internal
reference. 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. Unless otherwise noted,
reagents were purchased from commercial suppliers and used
without further purification. Intermediate 5-(azidomethyl)-2-
Conclusions
In summary, two series of amide and sulfamide derivatives 5a-e, 9a-
k and 13 were synthesized as potential E. coli PDHc E1 inhibitors.
Their inhibition against both E. coli PDHc E1 and microbial disease
were evaluated. SAR analyses indicated that the inhibitory potency
of compounds against both E. coli PDHc E1 and bacterium could be
greatly increased by replacing ether bond (C-O-C) linkage in lead
structure I with amide or sulfamide linkage. All 5a-e or 9a-k and 13
with the IC50 values ranging from 1.99 to 25.66 µM against E. coli
PHDc E1 could exhibit moderate to good antibacterial activity
against cyanobacteria，Xoo and Aaa. However I with much weaker
inhibitory potency against E. coli PHDc E1 showed no antibacterial
activity against cyanobacteria，Xoo and Aaa. Sulfamide derivatives
9 showed more potent inhibitory activity against E.coli PDHc E1 (IC50
<14 µM) than that of amide derivatives 5 or lead compound I. 9d
(R= 3-NO2, IC50 = 2.95 µM) and 9k (R= 3-NO2-4-Cl IC50 = 1.99 µM)
with sulfamide linkage exhibited much higher inhibitory potency
against E. coli PDHc E1 than that of corresponding 5d (R= 3-NO2 IC50
= 13.52 µM). Meanwhlie, 9d and 9k also displayed 97% inhibition
against cyanobacteria at 20 µg/mL, much higher than that of 5d
(with 76% inhibitory potency). Especially, 9k, displaying the most
potent inhibitory activity against E. coli PDHc E1, showed almost
best inhibitory activity against cyanobacteria or Aaa at 20 µg/mL or
500 µg/mL respectively. The above findings showed that there was
some correlation between enzymatic inhibition and antibacterial
activity.
methylpyrimidin-4-amine
existing methods19
1 was synthesized according to the
.
General procedure for preparation of N-(prop-2-yn-1-yl)-
(substituent)benzamide (3a-e)
The reaction of substituted benzoic acid (10 mmol) with oxalyl
chloride (1.89 g, 15 mmol) gave substituted benzoyl chloride. A
solution of the substituted benzoyl chloride was added dropwise to
the solution of prop-2-yn-1-amine (0.55 g, 10 mmol) and
trimethylamine (1.21 g, 12 mmol) in dichloromethane (15mL) at 0
oC. Then the reaction mixture was stirred for 3.5 h at room
temperature. Dichloromethane (20 mL) was added to the mixture
and the mixture was washed with water, a solution of sodium
hydroxide (1 M), a solution of diluted hydrochloric acid (1 M) and
brine, dried over sodiumsulfate and filtered. The solvent was
evaporated under reduced pressure to get crude products. The
crude products were recrystallized with dichloromethane to give
the pure compounds 3a-e, which were used directly for the next
step.
The interaction mode of amide derivatives 5 or sulfamide
derivatives 9 with active site of E.coli PDHc E1 was explored by
molecular docking to understand the mechanism of antibacterial
activity. Binding mode analysis revealed that 9d displayed much
powerful interaction by forming hydrogen bond between sulfamide
linkage and residue Lsy392, Tyr599 and His106 at active site of E.coli
PDHc E1.
General procedure for preparation of N-(prop-2-yn-1-yl))-
(substituent)benzenesulfonamide (7a-k) and
yl)methanesulfonamide 11
N-(prop-2-yn-1-
A solution of the substituted sulfonyl chloride (10 mmol) was added
dropwise to the solution of prop-2-yn-1-amine (0.55 g, 10 mmol)
and trimethylamine (1.21 g, 12 mmol) in dichloromethane (15mL)
at 0 oC. Then the reaction mixture was stirred for 3.5 h at room
These possible binding modes of 9d with important residues of
PDHc E1 were further verified via site-directed mutagenesis,
enzymatic assays, and fluorescence spectral analysis. It suggested
8 | J. Name., 2012, 00, 1-3
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temperature. Dichloromethane (20 mL) was added to the mixture 109.4, 124.3, 128.6, 133.8, 139.2, 143.5, 148.6, 150.9, 160.9, 162.6,
and the mixture was washed with water, a solution of sodium 164.1; ESI-MS m/z: 369.3 (M-Cl)+; Elemental Anal. Calcd for
hydroxide (1 M), a solution of diluted hydrochloric acid (1 M) and C16H17ClN8O3: C, 47.47; H, 4.23; N, 27.68. Found: C, 47.60; H, 4.48;
brine, dried over sodiumsulfate and filtered. The solvent was N, 27.35.
evaporated under reduced pressure to get crude products. The
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
crude products were recrystallized with dichloromethane to give
yl)methyl)-3-nitrobenzamide hydrochloride (5d)
the pure compounds 7a-k and 11, which were used directly for the
o
1
White solid; Yield 63%; m.p. 188-189 C; H NMR (400 MHz, DMSO-
d6) δ(ppm): 2.52 (s, 3H, CH3), 4.57 (s, 2H, CH2), 5.65 (s, 2H, CH2),
7.80 (s, 1H, Ar-H), 8.32 (s, 2H, NH2), 8.38 (s, 2H, Ar-H), 8.73 (s, 1H,
1,2,3-triazol-4-yl-H), 8.88 (s, 1H, Ar-H), 9.22 (s, 1H, pyrimidin-5-yl-H),
9.59 (s, 1H, NH), 14.90 (s, 1H, HCl); 13C NMR (100 MHz, DMSO-d6)
δ(ppm): 25.4, 35.4, 46.6, 108.5, 127.2, 127.4, 128.8, 130.4, 133.3,
148.2, 151.7, 154.5, 160.9, 165.7, 167.6; ESI-MS m/z: 369.4 (M-Cl)+;
Elemental Anal. Calcd for C16H17ClN8O3: C, 47.47; H, 4.23; N, 27.68.
Found: C, 47.48; H, 4.55; N, 27.54.
next step.
General procedure for preparation of N-((1-((4-amino-2-
methylpyrimidin-5-yl)methyl)-
1H-1,2,3-triazol-4-yl)methyl)-
(substituent)benzamide hydrochloride (5a-e); N-((1- ((4-amino-2-
methylpyr-imidin-5-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-
(substituent)benzenesulfonamide hydrochloride (9a-k) and N-((1-
((4-amino-2-methylpyrimidin-5-yl)
methyl)-1H-1,2,3-triazol-4-
yl)methl)meth-anesulfonamide hydrochloride 13
We added CuI (0.04g, 0.2 mmol) to a stirred solution of 5-
azidomethyl-2-methylpyrimidine-4-yla mine 1 (0.33g, 2 mmol) and
3a–e, 7a-k or 11 (2 mmol) in THF (10mL) followed by Et3N (0.24g,
2.4 mmol). After over night stirring at room temperature, the
reaction mixture was poured into water (50 mL), and the
precipitate was collected by filtration and dried under atmospheric
pressure obtained 4a–e, 8a-k and 12. Then they reacted with 36%
hydrogen chloride afforded title compounds 5a–e, 9a-k and 13.
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)-4-chlorobenzamide hydrochloride (5e)
White solid; Yield 84%; m.p. 165-167 oC ; 1H NMR (400 MHz, DMSO-
d6) δ(ppm): 2.51 (s, 3H, CH3), 4.52 (s, 2H, CH2), 5.62 (s, 2H, CH2),
7.55 (d, 2H, NH2, J = 4.6Hz), 7.93 (d, 2H, Ar-H, J = 4.8Hz), 8.25 (s, 1H,
1,2,3-triazol-4-yl-H), 8.32 (s, 1H, Ar-H), 8.85 (s, 1H, Ar-H), 9.22 (s,
1H, pyrimidin-5-yl-H), 9.25 (s, 1H, NH), 14.83 (s, 1H, HCl); 13C NMR
(100 MHz, DMSO-d6) δ(ppm): 25.4, 35.0, 46.8, 108.6, 123.3, 128.2,
132.6, 135.5, 139.7, 144.6, 156.2, 161.9, 164.7, 167.4; ESI-MS m/z:
358.3 (M-Cl)+; Elemental Anal. Calcd for C16H17Cl2N7O: C, 48.74; H,
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)benzamide hydrochloride (5a)
o
Green solid; Yield 78%; m.p. 197-199 C; 1H NMR (400 MHz, DMSO- 4.35; N, 24.87. Found: C, 48.37; H, 4. 78; N, 24.72.
d6) δ(ppm): 2.53 (s, 3H, CH3), 4.57 (s, 2H, CH2), 5.69 (s, 2H, CH2),
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)benzenesulfonamide hydrochloride (9a)
7.49 (s, 2H, NH2), 7.55 (s, 1H, Ar-H), 7.94 (s, 2H, Ar-H), 8.34 (s, 1H,
Ar-H), 8.40 (s, 1H, 1,2,3-triazol-4-yl-H), 8.87 (s, 1H, Ar-H), 9.17 (s,
1H, pyrimidin-5-yl-H), 9.23 (s, 1H, NH); 14.83 (s, 1H, HCl); 13C
NMR(100 MHz, DMSO-d6) δ(ppm): 20.6, 33.9, 45.0, 108.8, 122.0,
126.4, 127.5, 130.5, 132.9, 143.0, 152.5, 160.2, 162.0, 165.1; ESI-MS
m/z: 324.3 (M-Cl)+; Elemental Anal. Calcd for C16H18ClN7O: C,
53.41; H, 5.04; N, 27.25. Found: C, 53.19; H, 5.25; N, 27.75.
o
1
Yellow solid; Yield 86%; m.p 142-143 C; H NMR (400 MHz, DMSO-
d6) δ(ppm): 2.54 (s, 3H, CH3), 4.05 (s, 2H, CH2), 5.62 (s, 2H, CH2),
7.58 (d, 2H, NH2, J = 6.7Hz), 7.61 (d, 1H, Ar-H, J = 6.4Hz), 7.79 (d, 2H,
Ar-H, J = 6.8Hz), 8.18 (s, 1H, 1,2,3-triazol-4-yl-H), 8.29 (s, 2H, Ar-H),
8.91 (s, 1H, pyrimidin-5-yl-H), 9.24 (s, 1H, NH); 14.97 (s, 1H, HCl); 13C
NMR (100 MHz, DMSO-d6) δ(ppm): 20.8, 37.0, 44.9, 108.7, 122.3,
125.6, 128.4, 131.7, 133.7, 139.1, 143.1, 160.2, 161.8; ESI-MS m/z:
360.3 (M-Cl)+; Elemental Anal. Calcd for C15H18ClN7O2S: C, 45.51; H,
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)-4-methylbenzamide hydrochloride (5b)
o
1
White solid; Yield 71%; m.p. 132-133 C; H NMR (400 MHz, DMSO- 4.58; N, 24.77. Found: C, 45.65; H, 4.76; N, 24.89.
d6) δ(ppm): 2.33 (s, 3H, CH3), 2.53 (s, 3H, CH3), 4.50 (s, 2H, CH2),
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)-4-methylbenzenesulfonamide hydrochloride (9b)
5.64 (s, 2H, CH2), 7.25 (d, 2H, NH2, J = 6.9Hz), 7.80 (d, 2H, Ar-H, J =
6.9Hz), 8.29 (s, 2H, Ar-H), 8.87 (s, 1H, 1,2,3-triazol-4-yl-H), 9.05 (s,
1H, pyrimidin-5-yl-H), 9.20 (s, 1H, NH), 14.92 (s, 1H, HCl); 13C NMR
(100 MHz, DMSO-d6) δ(ppm): 20.2, 25.6, 35.1, 48.0, 108.5, 127.2,
128.2, 130.4, 133.7, 148.8, 154.5, 160.3, 164.6, 166.6; ESI-MS m/z:
338.4 (M-Cl)+; Elemental Anal. Calcd for C17H20ClN7O: C, 54.62; H,
5.39; N, 26.23. Found: C, 54.49; H, 5.66; N, 26.51.
o
1
Yellow solid; Yield 80%; m.p 156-158 C; H NMR (400 MHz, DMSO-
d6) δ(ppm): 2.39 (s, 3H, CH3), 2.53 (s, 3H, CH3), 4.00 (s, 2H, CH2),
5.64 (s, 2H, CH2), 7.39 (d, 2H, NH2, J = 7.5Hz), 7.69 (d, 2H, Ar-H, J =
7.4Hz), 8.18 (s, 1H, 1,2,3-triazol-4-yl-H), 8.24 (s, 1H, Ar-H), 8.31 (s,
1H, Ar-H), 8.92 (s, 1H, pyrimidin-5-yl-H), 9.24 (s, 1H, NH), 14.94 (s,
1H, HCl); 13C NMR (100 MHz, DMSO-d6) δ(ppm): 20.4, 20.7, 36.8,
44.9, 108.4, 125.5, 128.7, 135.7, 135.8, 140.9, 141.4, 142.9, 160.0,
161.6; ESI-MS m/z: 374.4 (M-Cl)+; Elemental Anal. Calcd for
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)-4-nitrobenzamide hydrochloride (5c)
Green solid; Yield 93%; m.p. 239-240 oC; 1H NMR (400 MHz, DMSO - C16H20ClN7O2S: C, 46.88; H, 4.92; N, 23.92. Found: C, 46.66; H, 4.55;
d6) δ(ppm): 2.52 (s, 3H, CH3), 4.56 (s, 2H, CH2), 5.65 (s, 2H, CH2), N, 23.88.
8.15 (d, 2H, NH2, J = 6.9Hz), 8.33 (s, 4H, Ar-H), 8.88 (s, 1H, 1,2,3-
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
triazol-4-yl-H), 9.22 (s, 1H, pyrimidin-5-yl-H), 9.54 (s, 1H, NH), 14.80
(s, 1H, HCl); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 21.0, 34.6, 45.4,
yl)methyl)-4-nitrobenzenesulfonamide hydrochloride (9c)
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o
Yellow solid; Yield 65%; m.p 125-126 C; 1H NMR (400 MHz, DMSO- (M-Cl)+; Elemental Anal. Calcd for C15H17BrClN7O2S: C, 37.95; H,
d6) δ(ppm): 2.52 (s, 3H, CH3), 4.13 (s, 2H, CH2), 5.55 (s, 2H, CH2), 3.61; N, 20.65. Found: C, 37.89; H, 3.57; N, 20.54.
8.02 (d, 2H, NH2, J = 7.6Hz), 8.20 (s, 1H, 1,2,3-triazol-4-yl-H), 8.37 (d,
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
3H, Ar-H, J = 7.7Hz), 8.67 (s, 1H, Ar-H), 8.77 (s, 1H, pyrimidin-5-yl-H),
yl)methyl)-4-fluorobenzenesulfonamide hydrochloride (9h)
9.23 (s, 1H, NH), 14.52 (s, 1H, HCl); 13C NMR (100 MHz, DMSO-d6)
o
1
Yellow solid; Yield 88%; m.p 157-159 C; H NMR (400 MHz, DMSO-
d6) δ(ppm): 2.53 (s, 3H, CH3), 4.06 (s, 2H, CH2), 5.61 (s, 2H, CH2),
7.66 (s, 2H, NH2), 7.79 (s, 2H, Ar-H), 8.24 (s, 1H, 1,2,3-triazol-4-yl-H),
8.30 (s, 1H, Ar-H), 8.40 (s, 1H, Ar-H), 8.89 (s, 1H, pyrimidin-5-yl-H),
9.24 (s, 1H, NH), 14.85 (s, 1H, HCl); 13C NMR (100 MHz, DMSO-d6)
δ(ppm): 20.4, 36.5, 44.4, 108.0, 125.0, 127.2, 128.0, 132.2, 135.5,
137.4, 142.6, 159.6, 161.2; ESI-MS m/z: 405.3 (M-Cl)+; Elemental
δ(ppm): 21.0, 38.9, 46.6, 109.0, 124.2, 127.9, 141.6, 145.5, 146.6,
149.1, 160.8, 162.4; ESI-MS m/z: 405.3 (M-Cl)+; Elemental Anal.
Calcd for C15H17ClN8O4S: C, 40.87; H, 3.89; N, 25.42. Found: C, 40.77;
H, 3.55; N, 25.66.
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)-3-nitrobenzenesulfonamide hydrochloride (9d)
o
Yellow solid; Yield 89%; m.p 81-83 C; 1H NMR (400 MHz, DMSO-d6) Anal. Calcd for C15H17ClFN7O2S: C, 43.53; H, 4.14; N, 23.69. Found: C,
δ(ppm): 2.52 (s, 3H, CH3), 4.14 (s, 2H, CH2), 5.54 (s, 2H, CH2), 7.86 (t, 43.66; H, 4.43; N, 23.55.
1H, Ar-H, J = 7.9Hz), 8.13-8.18 (m, 2H, NH2), 8.28 (s, 1H, 1,2,3-
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
triazol-4-yl-H), 8.43-8.47 (m, 2H, Ar-H), 8.69 (s, 1H, Ar-H), 8.83 (s,
1H, pyrimidin-5-yl-H), 9.22 (s, 1H, NH), 14.77 (s, 1H, HCl); 13C NMR
yl)methyl)-2,4,6-trimethylbenzenesulfonamide hydrochloride (9i)
o
1
Yellow solid; Yield 76%; m.p 106-108 C; H NMR (400 MHz, DMSO-
d6) δ(ppm): 2.24 (s, 3H, CH3), 2.53 (s, 9H, 3CH3), 4.03 (s, 2H, CH2),
5.56 (s, 2H, CH2), 6.98 (d, 2H, NH2, J = 8.8Hz), 8.02 (d, 1H, Ar-H, J =
8.0Hz), 8.11 (s, 1H, 1,2,3-triazol-4-yl-H), 8.27 (s, 1H, Ar-H), 8.84 (s,
1H, pyrimidin-5-yl-H), 9.24 (s, 1H, NH), 14.82 (s, 1H, HCl); 13C NMR
(100 MHz, DMSO-d6) δ(ppm): 19.2, 20.3, 21.2, 35.4, 44.4, 108.0,
125.6, 130.0, 132.5, 135.3, 136.4, 139.5, 142.3, 159.5, 161.1; ESI-MS
(100 MHz, DMSO-d6) δ(ppm): 21.0, 37.7, 45.2, 109.5, 120.7, 121.2,
126.8, 131.0, 132.4, 133.5, 141.9, 143.7, 147.4, 161.1, 162.7; ESI-MS
m/z: 405.4 (M-Cl)+; Elemental Anal. Calcd for C15H17ClN8O4S: C,
40.87; H, 3.89; N, 25.42. Found: C, 40.86; H, 3.99; N, 25.44.
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)-4-chlorobenzenesulfonamide hydrochloride (9e)
o
Yellow solid; Yield 86%; m.p 163-164 C; 1H NMR (400 MHz, DMSO- m/z: 402.4 (M-Cl)+; Elemental Anal. Calcd for C18H24ClN7O2S: C,
d6) δ(ppm): 2.53 (s, 3H, CH3), 4.06 (s, 2H, CH2), 5.62 (s, 2H, CH2), 49.37; H, 5.52; N, 22.39;. Found: C, 49.55; H, 5.66; N, 22.66.
7.64 (d, 2H, NH2, J = 6.6Hz), 7.78 (s, 2H, Ar-H), 8.24 (s, 1H, 1,2,3-
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
triazol-4-yl-H), 8.29 (s, 1H, Ar-H), 8.40 (s, 1H, Ar-H), 8.89 (s, 1H,
pyrimidin-5-yl-H), 9.23 (s, 1H, NH), 14.85 (s, 1H, HCl); 13C NMR (100
yl)methyl)-2-nitrobenzenesulfonamide hydrochloride (9j)
o
1
Yellow solid; Yield 89%; m.p 103-105 C; H NMR (400 MHz, DMSO-
d6) δ(ppm): 2.53 (s, 3H, CH3), 4.23 (s, 2H, CH2), 5.57 (s, 2H, CH2),
7.82 (d, 2H, NH2, J = 7.2Hz), 7.94 (d, 2H, Ar-H, J = 7.3Hz), 8.14 (s, 1H,
1,2,3-triazol-4-yl-H), 8.29 (s, 1H, Ar-H), 8.77 (s, 1H, pyrimidin-5-yl-H),
8.85 (s, 1H, Ar-H), 9.22 (s, 1H, NH), 14.84 (s, 1H, HCl); 13C NMR (100
MHz, DMSO-d6) δ(ppm): 20.8, 37.2, 44.9, 108.8, 123.7, 124.6, 128.8,
131.9, 132.1, 133.4, 140.0, 143.2, 146.5, 160.3, 162.0 ; ESI-MS m/z:
MHz, DMSO-d6) δ(ppm): 20.7, 37.1, 44.9, 108.8, 124.8, 127.7, 128.5,
132.5, 136.2, 138.1, 143.1, 160.3, 161.9; ESI-MS m/z: 394.3 (M-Cl)+;
Elemental Anal. Calcd for C15H17Cl2N7O2S: C, 41.87; H, 3.98; N,
22.79. Found: C, 41.68; H, 3.69; N, 22.66.
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)-4-methoxybenzenesulfonamide hydrochloride (9f)
o
Yellow solid; Yield 69%; m.p 134-135 C; 1H NMR (400 MHz, DMSO- 405.3 (M-Cl)+; Elemental Anal. Calcd for C15H17ClN8O4S: C, 40.87; H,
d6) δ(ppm): 2.54 (s, 3H, CH3), 3.86 (s, 3H, CH3), 4.00 (s, 2H, CH2), 3.89; N, 25.42;. Found: C, 40.80; H, 3.99; N, 25.49.
5.66 (s, 2H, CH2), 7.11 (s, 2H, NH2), 7.74 (d, 2H, Ar-H, J = 4.5Hz), 8.09
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
(s, 1H, 1,2,3-triazol-4-yl-H), 8.29 (s, 2H, Ar-H), 8.90 (s, 1H, pyrimidin-
5-yl-H), 9.24 (s, 1H, NH), 14.85 (s, 1H, HCl); 13C NMR (100 MHz,
yl)methyl)-4-chloro-3–nitrobenzenesulfonamide hydrochloride (9k)
o
1
Yellow solid; Yield 80%; m.p 103-105 C; H NMR (400 MHz, DMSO-
d6) δ(ppm): 2.52 (s, 3H, CH3), 4.15 (s, 2H, CH2), 5.58 (s, 2H, CH2),
8.02 (d, 2H, NH2, J = 10.7Hz), 8.23 (s, 1H, Ar-H), 8.31 (s, 1H, 1,2,3-
triazol-4-yl-H), 8.41 (s, 1H, Ar-H), 8.70 (s, 1H, pyrimidin-5-yl-H), 8.82
(s, 1H, Ar-H), 9.22 (s, 1H, NH), 14.66 (s, 1H, HCl); 13C NMR (100 MHz,
DMSO-d6) δ(ppm): 20.5, 36.9, 44.6, 108.5, 121.0, 123.0, 128.1,
130.5, 135.1, 132.2, 139.5, 143.0, 146.0, 160.2, 161.8; ESI-MS m/z:
DMSO-d6) δ(ppm): 20.2, 36.2, 44.4, 55.0, 107.7, 112.8, 125.9, 127.1,
129.7, 131.6, 142.4, 159.3, 160.0, 161.1 ; ESI-MS m/z: 390.4 (M-Cl)+;
Elemental Anal. Calcd for C16H20ClN7O3S: C, 45.12; H, 4.73; N, 23.02.
Found: C, 45.55; H, 4.92; N, 23.43.
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)-4-bromobenzenesulfonamide hydrochloride (9g)
o
Yellow solid; Yield 79%; m.p 178-179 C; 1H NMR (400 MHz, DMSO- 439.2 (M-Cl)+; Elemental Anal. Calcd for C15H16Cl2N8O4S: C, 37.90; H,
d6) δ(ppm): 2.61 (s, 3H, CH3), 4.14 (s, 2H, CH2), 5.70 (s, 2H, CH2), 3.39; N, 23.57;. Found: C, 37.88; H, 3.42; N, 23.55.
7.81 (d, 2H, NH2, J = 7.9Hz), 7.88 (d, 2H, Ar-H, J = 8.0Hz), 8.32 (s, 1H,
N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-1H-1,2,3-triazol-4-
1,2,3-triazol-4-yl-H), 8.38 (s, 1H, Ar-H), 8.51 (s, 1H, Ar-H), 9.00 (s,
yl)methyl)methanesulfonamide hydrochloride (13)
1H, pyrimidin-5-yl-H), 9.33 (s, 1H, NH), 15.01 (s, 1H, HCl); 13C NMR
Yellow solid; Yield 66%; m.p 29-30 oC; 1H NMR (400 MHz, DMSO-d6)
(100 MHz, DMSO-d6) δ(ppm): 20.9, 37.4, 45.1, 109.1, 124.9, 125.6,
δ(ppm): 2.57 (s, 3H, CH3), 2.91 (s, 3H, CH3), 4.24 (s, 2H, CH2), 5.63 (s,
127.0, 128.1, 131.7, 138.8, 143.3, 160.5, 162.2; ESI-MS m/z: 440.2
2H, CH2), 8.35 (s, 2H, NH2), 7.59 (s, 1H, 1,2,3-triazol-4-yl-H), 8.77 (s,
10 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5RA22573F
1H, pyrimidin-5-yl-H), 9.22 (s, 1H, NH), 14.49 (s, 1H, HCl); 13C NMR
(100 MHz, DMSO-d6) δ(ppm): 18.0, 33.6, 36.7, 42.5, 104.8, 125.1,
134.5, 140.1, 156.7, 158.2; ESI-MS m/z: 298.4 (M-Cl)+; Elemental
Anal. Calcd for C10H16ClN7O2S: C, 35.98.71; H, 4.83; N, 29.37. Found:
C, 35.78; H, 4.66; N, 29.23.
11. P. Arjunan, K. Chandrasekhar, M. Sax, A. Brunskill, N.
Nemeria, F. Jordan, W. Furey, Biochemistry., 2004, 43
,
2405-2411.
12. P. Arjunan, M. Sax, A. Brunskill, K. Chandrasekhar, N.
Nemeria, S. Zhang, F. Jordan, W. Furey, J. Biol. Chem., 2006,
281, 15296-15303.
Molecular docking
13. K. M. Erixon, C. L. Dabalos, F. J. Leeper, Org. Biomol. Chem.,
For docking purposes, the crystallographic coordinates of the PDHc-
E1 with bound ThDP from E. coli (PDB code: 1L8A) were obtained
from Brookhaven Data Bank. Hydrogen atoms were added
2008, 6, 3561-3572.
14. Y. L. Ren, J. B. He, L. L. Feng, X. Liao, J. Jin, Y. J. Li, Y. Cao, J.
Wan and He, H. W. Bioorg. Med. Chem., 2011, 19, 7501-
7506.
to the structure allowing for appropriate ionization at physiological
pH. The protonated state of several important residues, such as
His142, Tyr177, Glu751, His640 and Met 194, were adjusted by
using SYBYL7.3 (Tripos, St. Louis, USA) in favor of forming
reasonable hydrogen bond with the ligand. Molecular docking
analysis was carried out by the SURFLEX module of SYBYL package
to explore the interaction model for the active site of PDHc-E1 with
its ligand. All atoms located within the range of 6.5 Å from any atom
of the cofactor ThDP were selected into the active site, and the
corresponding amino acid residue was, therefore, involved into the
active site if only one of its atoms was selected. Other default
parameters were adopted in the SURFLEX-docking calculations. All
calculations were performed on a CCNU Grid-based computational
environment(CCNU Grid website
15. J. B. He, L. L.Feng, J. Li, R. J. Tao, F. Wang, X. Liao, Q. S. Sun, Q.
W. Long, Y. L.Ren, J. Wan and H. W. He, Bioorg. Med.
Chem., 2012, 20, 1665-1670.
16. J. B. He, H. F. He, L. L. Zhao, L. Zhang, G. Y. You, L. L. Feng, J.
Wan and H. W. He, Bioorg. Med. Chem., 2015, 23, 1395-
1401
17. M. T. Labro, Bioorg. Clinical. Microbiology. Reviews., 2000, 13
,
615-650.
18. S. SS. Printsevskaya Svetlana, E. SE. Solovieva Svetlana, N. EN.
Olsufyeva Eugenia, P. EP. Mirchink Elena, B. EB. Isakova
Elena, E. D. Clercq Erik, J. Balzarini Jan, N. MN.
Preobrazhenskaya Maria, J. Med. Chem., 2005, 48, 3885-
3890
19. K. M. Erixon, C. L. Dabalos, F. J. Leeper, Org. Biomol. Chem.,
2008, 6, 3561-3572.
http://www.202.114.32.71:8090/ccnu/chem/platform.xml).
20. L. L. Feng, J. B. He, H. F. He, L. L. Zhao, L. F. Deng, L. Zhang, L.
Zhang, Y. L. Ren, J. Wan and H. W. He, Org. Biomol. Chem.,
2014, 12, 8911-8918
Acknowledgements
The work was supported in part by the National Basic Research
Program of China (No. 2010CB126100); the National Natural
Science Foundation of China (Nos. 20772042, 21172090, 21272089,
21472061 and 21472062); Excellent doctorial dissertation
cultivation grant from Central China Normal University.
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DOI: 10.1039/C5RA22573F
Graphic abstract for
Design, synthesis, biological evaluation and molecular docking of amide and sulfamide
derivatives as Escherichia coli pyruvate dehydrogenase complex E1 inhibitors
Optimal binding mode of novel E. coli PDHc E1 inhibitor 9d
9d IC50 = 2.95µM