A new and efficient ZnCl2-catalyzed synthesis and biological evaluation of novel 2-amino-3,5-dicyano-4-aryl-6-aryl-aminopyridines as potent antibacterial agents against Helicobacter pylori (HP)

Article abstract of DOI:10.1016/j.tet.2015.09.018

Here a new and efficient method via ZnCl₂-catalyzed direct cyclization of diverse benzylidenemalononitriles and arylamines has been developed. With this method, a variety of novel 2-amino-3,5-dicyano-4-aryl-6-aryl-aminopyridines (2a-2v) could be easily prepared under the mild conditions with board substrate/functional group tolerance and decent product yields. The biological activities of the selected compounds (2c, 2e, 2g, 2i, 2k, 2m, 2n and 2o) has also been evaluated as new antibacterial agents. The results demonstrated that almost all of the compounds had more potent antibacterial activities against HP than that of clinically used drugs, Ornidazole, Metronidazole, Nitrimidazine and Clarithromycin, suggesting that further development of such compounds might be of great interest.

Full text of DOI:10.1016/j.tet.2015.09.018

Accepted Manuscript
A new and efficient ZnCl -catalyzed synthesis and biological evaluation of novel 2-
2
amino-3,5-dicyano-4-aryl-6-aryl-aminopyridines as potent antibacterial agents against
Helicobacter Pylori (HP)
Jijun Huang, Jie Zhou, Senchuan Song, Huacan Song, Zhiyong Chen, Wei Yi
PII:
S0040-4020(15)30047-8
10.1016/j.tet.2015.09.018
TET 27115
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 27 June 2015
Revised Date: 2 September 2015
Accepted Date: 7 September 2015
Please cite this article as: Huang J, Zhou J, Song S, Song H, Chen Z, Yi W, A new and efficient ZnCl -
2
catalyzed synthesis and biological evaluation of novel 2-amino-3,5-dicyano-4-aryl-6-aryl-aminopyridines
as potent antibacterial agents against Helicobacter Pylori (HP), Tetrahedron (2015), doi: 10.1016/
j.tet.2015.09.018.
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ACCEPTED MANUSCRIPT
Graphical Abstract
R1
Developed methodology
CN
Biological application
Br
CN
R1
NC
HN
CN
NC
ZnCl2
CN
+
o
EtOH, 80 C
N
NH2
HN
N
2i
NH2
2a-v
H N
2
R2
2
2 examples
up to 88% yield
MIC50 = 8 µg/mL
MIC90 = 8 µg/mL
R2
Cl
Herein a new and efficient method via ZnCl -catalyzed direct cyclization of diverse
2
benzylidenemalononitriles and arylamines for one-pot synthesis of novel
2
-amino-3,5-dicyano-4-aryl-6-aryl-aminopyridines as potent antibacterial agents is
described.

ACCEPTED MANUSCRIPT
A new and efficient ZnCl -catalyzed synthesis and biological evaluation of novel
2
2
-amino-3,5-dicyano-4-aryl-6-aryl-aminopyridines as potent antibacterial agents
against Helicobacter Pylori (HP)
a
a
a
a*
b
c*
Jijun Huang , Jie Zhou , Senchuan Song , Huacan Song ,Zhiyong Chen , Wei Yi
a
School of Chemistry and Chemical Engineering, Sun yat-Sen University,Guangzhou 510275, P.R.
China
b
Guangdong Provincial Public Laboratory of Analysis and Testing Technology, China National
Analytical Center (Guangzhou), Building 34, 100 Xianlie Middle Road, Guangzhou 510070, P.R.
China
c
VARI/SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of
Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, P.R. China
Corresponding authors. Tel.: +86 20 84110918; fax: +86 20 84112245.
E-mail addresses: yjhxhc@mail.sysu.edu.cn (H. C. Song) and yiwei2@mail2.sysu.edu.cn (W. Yi).
1

ACCEPTED MANUSCRIPT
Abstract
Here a new and efficient method via ZnCl -catalyzed direct cyclization of diverse
2
benzylidenemalononitriles and arylamines has been developed. With this method, a
variety of novel 2-amino-3,5-dicyano-4-aryl-6-aryl-aminopyridines (2a-2v) could be
easily prepared under the mild conditions with board substrate/functional group
tolerance and decent product yields. The biological activities of the selected
compounds (2c, 2e, 2g, 2i, 2k, 2m, 2n and 2o) has also been evaluated as new
antibacterial agents. The results demonstrated that almost all of the compounds had
more potent antibacterial activities against HP than that of clinically used drugs,
Ornidazole, Metronidazole, Nitrimidazine and Clarithromycin, suggesting that further
development of such compounds might be of great interest.
Keywords: ZnCl -catalyzed cyclization; 2,6-diamino-3,5-dicyano-4-arylpyridines;
2
antibacterial activity; Helicobacter Pylori (HP).
2

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1
. Introduction
Polysubstituted pyridine system has attracted considerable attentions since this
class of compounds demonstrated many kinds of interesting biological activities. For
example, 2-amino(or chloro)-3,5-dicyano-4-alkyl(or aryl)-6-substituted amino(or
alkoxy, alkylthio)pyridine compounds could be used not only for the treatment of
1
,2
Alzheimer and neuronal vascular diseases (I, II in Fig. 1), but also for the treatment
3
,4
of HIV-1 integrase (III in Fig. 1).
Besides, 2-Amino-3-cyano-4-substituted
5
pyridines (IV in Fig. 1) were identified as potent adenosine receptor antagonists
,
6
protection against edema and inhibition of plasma PGE2. Moreover, they are key and
useful synthons for rapid and efficient construction of other biologically important
7
-9
pharmacophores such as pyridopyrimidinone. Driven by their potential in biological
and chemical application, to date, a large number of polysubstituted pyridines have
been successfully constructed by using a variety of developed methods.
Figure 1. Chemical structures of representative polysubstituted pyridine compounds.
OC H5
R1
2
R
NC
X
CN
Br
O
NC
R
CN
NC
N
I
NH2
NC
CN
Br
N
II
NH2
H N
2
N
R2
H N
N
S
2
R = H or Ph
X = alkoxy, cycloalkylamino,
alkylamino or alkylthio
IV
O
O
III
R = alkyl, halogen or OH
R = furan-2-yl or phenyl
1
R = Cl or NMe2
2
Among
these
polysubstituted
pyridine
compounds,
2
-amino-3,5-dicyano-4-aryl-6-substituted aminopyridine compounds occupied a
prevalent position because of their broad ranges of fascinating biological
1
,2,10
properties.
However, to the best of our knowledge, there are only a few reported
2
,11-14
methods about their synthesis
(Scheme 1). The first method was the substitution
reaction of 2-amino-6-chloro-4-phenyl-pyridine-3,5-dicarbonitrile with the
3

ACCEPTED MANUSCRIPT
2
corresponding amine (Scheme 1a). However, it needed harsh conditions and gave the
desired product in low yields. The second method was one-pot three-component
condensation reaction of substituted benzaldehydes, malononitrile and
1
1-13
14
alkylamines
(Scheme 1b) or ammonia (Scheme 1c). The latter is easier to
implement in a more step-/atom- economic way than the former, while this reaction
was not compatible for arylamines. Obviously, the development of a new and highly
efficient procedure to improve the current limited scope of substrates and to obtain
new analogues of 2-amino-3,5-dicyano-4-aryl-6-substituted aminopyridine for
immediate drug screening is still high desired.
Scheme 1. The reported methods for synthesizing 2-amino-3,5-dicyano-4-aryl-6-substituted
aminopyridine compounds
a) Samadi group, 2011
R
N
R
N
CN
CN
NC
Cl
CN
NC
CN
R C(OC H ) +
Nucleophile
2
5 3
Me N
NH2
NH2
2
R =H or Ph
b) Tu group, 2005; Ramakrishnan group, 2003; Khan group, 2014
NC
NH2
CN
R1
N
R1
CHO +
+
NH(R )
2 2
CN
NC
N(R2)2
R = alkyl or halogen
1
R = alkyl
2
c) Shi group, 2015
NC
NH2
N
CN
CN
R1
R1
CHO +
+ NH -H O
3 2
NC
NH2
R = alkyl, halogen or Ar
1
On the other hand, the increasing incidence of bacterial infection is a growing
global health problem. Nowadays, infectious diseases caused by various bacteria
especially constantly emerging antibiotic resistant bacteria are still a major concern
and account for almost 13 50,000 deaths worldwide daily. Accordingly, to overcome
4

ACCEPTED MANUSCRIPT
the threats, modern medicine has an eager quest for new antibacterial drugs. Based on
these, so far many efforts have been spent in this field, and a large number of
1
5-23
naturally occurring and synthetic antibiotics have been reported.
However, most
of them are not potent enough to be put into practical use due to their weak individual
activities or safety concerns. Undoubtedly, there is ongoing demand for developing
novel antibacterial drugs with more promising potency including better activities and
lower side effects.
Taking advantage of aforementioned information, here we report for the first time a
mild and efficient ZnCl -catalyzed cyclization of diverse benzylidenemalononitriles
2
and
arylamines
for
direct
construction
of
novel
2
-amino-3,5-dicyano-4-aryl-6-aryl-aminopyridines. Moreover, in continuing our
2
4,25
program aimed to search for potent drugs for bacterial infections,
the antibacterial
activities against helicobacter pylori (HP) of the selected compounds were evaluated
and the structure-activity relationships (SARs) were discussed. The nice data from
biological evaluation suggested that further development of such compounds for
antibacterial drug discovery might be of great interest.
2
2
. Results and discussion
.1. Chemistry
Scheme 2. The hoped three-component condensation reaction of substituted benzaldehyde,
malononitrile and substituted phenylamine.
NC
NH2
N
R1
CN
CN
R1
CHO +
+ R2
NH2
X
Glycol
NC HN
R = R = H, methyl or methoxy
R2
1
2
As reported (Scheme 1b), alkylamines were employed not only as the reagents,
more importantly, but also as the basic catalysts to be involved in the
5

ACCEPTED MANUSCRIPT
three-component condensation reaction. However, as illustrated in Scheme 2, we
found that the weakly basic aniline could not catalyze this reaction. With this in mind,
at the outset of this study, we chose benzaldehyde (1.0 eq.), malononitrile (2.1 eq.)
and aniline (2.1 eq.) as a model reaction in the presence of catalyst amount of
o
pyrrolidine at 75 C in glycol for 24 h. Unfortunately, no desired product was
observed (Scheme 3). Inferior results were also obtained in other selected basic
catalysts such as piperidine, triethylamine, morpholine or pyridine.
Scheme 3. Reaction of benzaldehyde, malononitrile and aniline with using pyrrolidine,
piperidine, triethylamine, morpholine and pyridine as catalysts, respectively.
NC
NH2
basic catalyst
X
CN
CN
N
CHO +
+
NH
2
NC HN
Scheme 4. Reaction of benzaldehyde, malononitrile and aniline with pyrrolidine as catalyst.
Piperidine
CN
CN
(catalyst)
+
H N
2
CHO
+
+
NH2
CN
CN
NC
-Benzylidene-
malononitrile
2
X
NC
NC
CN
CN
2-Phenylpropane-
NC
1,1,3,3-tetracarbonitrile
To our surprise, 2-benzylidenemalononitrile was detected as the main product in
the above reaction system, although the molecular ratio of benzaldehyde and
malononitrile in the reaction mixture was 1:2.1 (Scheme 4). This result revealed that
the designed reaction might follow a quite different mechanism compared with the
previously reported alkylamine-mediated reaction, which prompted us to explore an
alternative reaction condition for starting this reaction, such as the well-known Lewis
acid-catalyzed reaction system.
6

ACCEPTED MANUSCRIPT
Table 1 Optimization of reaction conditions.
NC
NC
NH2
N
CN
CN
cat. [Lewis acid]
Solvent
+
H N
2
HN
o
d
Entry
Catalyst
TsOH
Solvent
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
DMF
T ( C)
75
75
75
75
75
75
75
75
75
75
75
75
60
50
80
80
80
80
80
80
T (h)
Yield (%)
1
2
3
4
5
6
7
8
9
12
12
12
12
12
12
12
12
12
12
12
12
12
12
2.0
3.5
5.0
5.0
5.0
5.0
0
0
AlCl
BO
COOH
Zn(OCOCH
FeCl
3
H
3
0
3
CF
0
3
)
0
3
2
0
3
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
ZnCl
81
10
0
2
2
2
2
2
2
2
2
2
2
2
2
2
2
DCE
1
0
1
DMSO
THF
0
1
21
0
1
1
1
1
1
1
2
3
4
5
6
7
Toluene
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
77
65
63
79
86
52
38
83
a
b
c
18
19
20
Reaction conditions: benzylidenemalononitrile (6.0 mmol), aniline (6.9 mmol), Lewis acid-catalyst (9.0 mmol),
a
b
c
d
solvent (20 mL). 6.0 mmol ZnCl was used. 3.0 mmol ZnCl was used. Performed on a gram-scale. Isolated
2
2
yields.
In order to confirm above conjecture, next we selected the
benzylidenemalononitrile and aniline as the model substrates for the optimization of
Lewis acid-catalyzed reaction system (Table 1). After extensive screening the
common Lewis acids as catalysts (Table 1, entries 1-7), we were pleased to find that
o
the reaction of benzylidenemalononitrile with aniline at 75 C in EtOH using ZnCl as
2
the catalyst gave the desired product 2a in good yield (81%) along with the formation
2
6
of N-benzylideneaniline as the by-porduct (Table 1, entry 7). Changing EtOH to
other solvents obviously inhibited the process (Table 1, entries 7-12). Further
7

ACCEPTED MANUSCRIPT
investigation showed that the reaction time and temperature also obviously influenced
the reaction efficiencies (Table 1, entries 13-17). The yield was raised to 86% with a
shorter reaction time (5.0 h vs 12 h) when the reaction temperature was increased to
o
8
0 C (Table 1, entry 17). Moreover, an attempt to decrease the catalyst loading cut
down the yield sharply (Table 1, entries 18-19). Finally, we were pleased to find that
the reaction could conveniently be scaled up to a gram level without a decrease in
isolated yield (Table 1, entry 20). In summary, the optimal conditions in ethanol
o
included ZnCl (150 mol %) at 80 C for 5.0 h under air.
2
With the optimized conditions in hand, we next examined the influence exerted by
substituents at the benzylidenemalononitrile moiety. The results were shown in
Scheme 5. In general, the reaction proceeded smoothly to give the desired products in
high yields and also showed good compatibility with many valuable functional groups
such as methoxy and bromo substituents. It was noteworthy that the hydroxyl group
remains intact after reaction, providing easy handles for further synthetic elaborations.
Subsequently, we also explored the versatility of the optimized system by testing a
representative set of arylamines. As shown in Scheme 5, both electro-donating and
-withdrawing groups on 4-position of arylamine are all well tolerated affording the
expected products in good yields. Tolerance to the chloro and bromo functional
groups was especially noteworthy since they are useful for subsequent cross-coupling
reactions.
8

ACCEPTED MANUSCRIPT
Scheme 5. Substrate scope.
NC
NH2
N
CN
R1
+
H N
2
R2
ZnCl2
CN
R1
o
EtOH, 80 C
NC HN
a-v
R2
2
NC
NH2
N
NC
NH2
N
NC
NH
N
2
NC HN
a, 86%
NC HN
2b, 84%
CH3
NC HN
2c, 85%
OCH
3
2
NC
NH2
NC
NH2
N
NC
NH2
N
N
Br
Br
NC HN
2e, 74%
NC HN
d, 73%
Cl
NC HN
2
2f, 82%
NC
NC
NH2
NC
NH2
N
NH2
N
Br
N
Br
Br
NC HN
2h, 83%
OCH3
NC HN
g, 85%
CH3
NC HN
Cl
2
2i, 67%
NC
NC
NH2
NC
NH2
N
NH2
N
H CO
N
Br
3
H CO
3
NC HN
2k, 83%
NC HN
j, 78%
Br
NC HN
CH3
2
2l, 85%
NC
NC
NH2
NC
NH2
N
NH2
N
H CO
N
H CO
3
3
H CO
3
NC HN
2n, 73%
Cl
NC HN
OCH3
NC HN
2o, 85%
NC
NH2
Br
2
m, 88%
NH2
O
O
NC
H
3
CO
NC
NH2
H
CO
3
N
H CO
3
N
HO
N
H CO
NC HN
R = H, 2p, 83%
R
3
NC HN
R
NC HN
2v, 83%
R = CH , 2t, 84%
3
CH , 2q, 83%
3
OCH , 2u, 84%
3
OCH , 2r, 84%
3
Br, 2s, 82%
Reaction conditions: Cat. ZnCl (9.0 mmol), the corresponding benzylidenemalononitriles (6.0 mmol) and anilines
2
o
(6.9 mmol) in EtOH (20 mL) at 80 C for 5.0 h under air. Isolated yields.
Encouraged by the above results, we finally expanded the scope of functional
groups attached at both benzylidenemalononitrile and arylamine moieties. Thus,
several typical substituents such as methoxy, bromo, methyl and chloro were selected
9

ACCEPTED MANUSCRIPT
as the effectors for the investigation (Scheme 5). As expected, the corresponding
double-substituted or poly-substituted products were obtained in good yields.
Furthermore, we were pleased to find that benzylidenemalononitrile moiety, bearing a
bulkier substituent at benzene ring such as 3,4-methylenedioxy or 3,4,5-trimethoxy,
also smoothly reacted with 4-functionized arylamines to deliver the corresponding
products under the optimized conditions without significant decrease in the product
yields, which further illustrated the remarkable robustness of our developed ZnCl2
catalytic system.
Scheme 6. Proposed mechanism.
CN
CN
CN
CN
C N: ZnCl
ZnCl2
R2
2
C N:
H N
2
..
ZnCl2
H N
2
H N
2
(I)
CNH
C N:
C N: ZnCl
H N
..
H
2
ZnCl2
C N:
ZnCl2
NC
CN
C N: ZnCl2
2
H N
2
CN
C N: ZnCl2
H N
2
CN
(II)
H
N
H
HN
C
NC
H HN
HN
N
NC
NC
N
Elimination
N
C
N
Air
Oxidized
NH
III)
CN
NC
NH2
NC
NH
2
H
N
(
(IV)
Based on these observations and literature precedents, we used 2-benzylidene-
malononitrile and aniline as model reaction to propose a possible mechanism
illustrated in Scheme 6. First, 2-benzylidenemalononitrile reacted with aniline in the
presence of ZnCl2 forming intermediate I which reacted further with
2
-benzylidenemalononitrile generating intermediate II. Catalyzing by ZnCl again, II
2
1
0

ACCEPTED MANUSCRIPT
reacted with aniline and followed by an intramolecular ring-closing reaction forming
intermediate III. Sequentially, III was changed into IV by eliminating
N-benzylidenebenzenamine, and finally, IV was oxidized by air and aromatized into
2
-amino-3,5-dicyano-4-phenyl-6-phenylaminopyridine.
In addition, from Scheme 6 we found that ZnCl took part in four reaction steps:
2
(1) the addition reaction of 2-benzylidenemalononitrile and aniline, (2) the addition
reaction of 2-benzylidenemalononitrile and I, (3) the addition reaction of II and
aniline, and (4) the intramolecular ring-closing reaction forming III. Taken together,
ZnCl played a vital role in the outcome of this reaction.
2
2
.2. Biology
With these synthesized 2-amino-3,5-dicyano-4-aryl-6-arylaminopyridines in hand,
their biological activities of selected samples against eleven HP strains (ATCC
Hp11637 and clinically separated HP strains, Hp05-5, Hp05-6, Hp05-7, Hp05-8,
Hp05-9, Hp05-10, Hp05-11, Hp05-12, Hp05-13, Hp05-14) were evaluated by usual
2
7-31
procedure,
and the results were presented in Tables 2-4. Here clinically used
antibacterial drugs, Ornidazole (D ), Metronidazole (D ), Nitrimidazine (D ) and
1
2
3
Clarithromycin (D ), were employed as standard references.
4
11

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Table 2 The MIC values of synthesized compounds against HP-strains in comparison with four
6
4
drug references, D , D , D and D respectively in 10 and 10 of colony forming units of
1
2
3
4
HP strains.
Bacterial
strains
Colony forming
MIC(ꢀg/mL)
units /cfu/mL
2c 2e
32 16
32 16
32 16
2g
16
16
16
2i
8
2k
16
32
16
64
16
16
32
32
16
16
16
2m
2n
4
2o
32
D1 D2
64 64
D3
64
D4
64
6
11637
10
16
32
16
64
16
16
32
32
32
16
16
HP05-5
HP05-6
HP05-7
HP05-8
HP05-9
HP05-10
HP05-11
HP05-12
HP05-13
HP05-14
8
8
32
128
64
128 64
128 32
128 128
128 16
128 64
128 64
128 64
128 64
128 64
128 64
64 128
64 128
64 128
64 128
64 128
64 128
64 128
64 128
64 128
64 128
8
4
64 128 128
64
8
64
4
32 16
32 16
64 32
64 32
32 16
32 16
32 16
16
16
16
16
16
16
16
32
8
4
32
8
8
64
8
32
4
64
8
32
8
8
32
8
8
32
4
1
1637
10
32 16
32 16
32 16
16
16
16
8
8
16
32
16
64
16
16
32
32
16
16
32
16
64
16
16
32
32
32
4
8
32
32
64
64
64
64
HP05-5
HP05-6
HP05-7
HP05-8
HP05-9
HP05-10
HP05-11
HP05-12
128 64
64 128
64 128
64 128
64 128
64 128
64 128
64 128
64 128
8
4
128 128 32
64 128 128
64
8
64
4
64
32
32
64
64
32
128 128
128 16
128 64
128 64
128 64
128 64
32 16
32 16
64 32
64 32
32 16
16
16
16
16
16
8
4
8
8
8
32
4
8
First, in order to evaluate the anti-HP activities of the target compounds and
investigate the effects of the concentrations of colony forming units of bacteria on the
6
4
activity, two concentrations (10 and 10 units /cfu/mL) of colony forming units of HP
strains was used to determine the MIC values of samples (Table 2). As shown in
Table 2, the results showed that the similar MIC value for each sample were obtained
in two different concentrations, suggesting that the antibacterial activity against HP of
the tested sample was not related with the concentration of colony forming units of
bacteria in the inoculums.
1
2

ACCEPTED MANUSCRIPT
Table 3 The MIC , MIC and MIC range values of compounds 2a-2h against HP-strains in
5
0
90
comparison with four drug references, D , D , D and D
1
2
3
4.
MIC value (ꢀg/mL)
Samples
MIC value (ꢀg/mL)
Sample
MIC50
MIC90
MIC-range
MIC50
MIC90
MIC-range
2
2
2
c
e
g
32
16
16
8
64
32
16
8
32-64
16-128
16-128
8-64
2n
2o
D1
D2
D3
D4
8
32
64
4-64
32-128
64-128
16-128
64-128
64
32
128
64
128
64
2
i
2
k
16
16
32
32
16-64
16-64
128
64
128
64
2
m
Therefore, next the minimum inhibitory concentrations at the inhibitory
percentages of 50%, 90% (named MIC50 and MIC , respectively) and minimum
90
inhibitory concentration range (MIC-range) of samples against HP-strains were
determined under the condition of single concentration (Table 3). The results
demonstrated that most of target compounds performed more potent antibacterial
activities against eleven HP-strains than D , D , D and D . Especially, compound 2i
1
2
3
4
was found to be the most potent antibacterial agent with the same MIC and MIC
90
5
0
value (8 ꢀg/mL).
From the antibacterial activities of the synthesized 2-amino-3,5-dicyano-4-aryl-6-
arylaminopyridine compounds, the following SAR results could be derived:
(1) Compared with the reference drugs, all the selected compounds showed potent
anti-HP activities, suggesting that 2-amino-3,5-dicyano-4-aryl-6-arylaminopyridine
moiety could serve as a privileged structural core motif for new and potent
antibacterial drug discovery.
(2) In general, at 4-position of phenyl rings, the compounds bearing electro-
withdrawing halogen groups showed better antibacterial activities than that bearing
electro-donating methyl or methoxy group (2e vs 2c, 2i vs 2g, and 2n vs 2m). The
results
suggested
that
the
antibacterial
activities
of
1
3

ACCEPTED MANUSCRIPT
2
-amino-3,5-dicyano-4-aryl-6-arylaminopyridines might be associated with the
electronic character of the substituent on the 4-position of benzene. In the present
investigation, the halogen substitution for improving the antibacterial activity strength
followed the order: Cl >Br (2n vs 2o).
(3) Double-substituted compounds 2g, 2i and 2m-n (except 2o) have more potent
anti-HP activities than that bearing mono-substituted group (2c and 2e), suggesting
that the introduction of an additional functional group might be efficacious for the
anti-HP activity. Furthermore, compared with the most active compounds 2i and 2n, it
showed that bromo group was more favorable than methoxy group for improving the
potent antibacterial activities.
Table 4 The statistical data of anti-HP activity of synthesized compounds in comparison with
four drug references.
a
Sample N
Mean
Standard
deviation
Standard 5%confidence interval for mean
Min-
Max-
error
Lower bound
Upper bound
imum imum
2
2
2
c
e
g
11
11
11
11
11
11
11
11
11
11
1.587249
1.340952
1.286219
0.985189
1.340952
1.368318
0.903090
1.641982
2.079844
1.751447
0.1406109 0.0423958
0.2812218 0.0847916
0.2722919 0.0820991
0.2722919 0.0820991
0.2069737 0.0624049
0.2069737 0.0624049
0.4038741 0.1217726
0.2069737 0.0624049
0.0907640 0.0273664
0.2260004 0.0681417
1.492785
1.152024
1.103291
0.802261
1.201905
1.229271
0.631764
1.502935
2.018868
1.599618
1.681713
1.529879
1.469147
1.168117
1.479999
1.507365
1.174416
1.781029
2.140820
1.903276
1.5051 1.8062
1.2041 2.1072
1.2041 2.1072
0.9031 1.8062
1.2041 1.8062
1.2041 1.8062
0.6021 1.8062
1.5051 2.1072
1.8062 2.1072
1.2041 2.1072
2
i
2
k
2
m
2
n
o
2
D1
D2
D3
11
11
2.079844
0.0907640 0.0273664
2.018868
2.140820
1.8062 2.1072
D4
1.806180
1.513199
0.0000000 0.0000000
0.4474078 0.0327177
1.806180
1.448653
1.806180
1.577744
1.8062 1.8062
0.3010 2.1072
Total 132
a
number of strains used in this test
Finally, The proceeded MIC test data were treated by using SPSS statistical
software, and the results including mean, standard deviation, standard error, 5%
confidence interval for mean, minimum and maximum values were listed in Table 4.
As shown in Table 4, we found that 2i and 2n had the highest anti-HP activities in a
1
4

ACCEPTED MANUSCRIPT
similar order of magnitude (P > 0.05), which was significantly better than that of the
other tested compounds and selected four drug references (P < 0.05). This further
confirmed our obtained results shown as above.
3
. Conclusion
In summary, we have developed for the first time a ZnCl -catalyzed direct
2
cyclization of diverse benzylidenemalononitriles and arylamines. With this method, a
wide range of novel 2-amino-3,5-dicyano-4-aryl-6-aryl-aminopyridines were
conveniently synthesized in high yields with board substrate/functional group
tolerance. Moreover, the application of the synthesized compounds was demonstrated,
the results showed that most of target compounds have more potent antibacterial
activities against HP strains (including ATCC Hp11637 and eleven kinds of clinically
separated HP strains) than currently used four antibacterial drugs D , D , D and D .
1
2
3
4
In particular, compound 2i was found to be the most potent antibacterial agent with
the same MIC50 and MIC90 value (8 ꢀg/mL). SAR analysis showed: (1)
2
-amino-3,5-dicyano-4-aryl-6-aryl-aminopyridine moiety could serve as a privileged
structural core motif for new and potent antibacterial drug discovery; and (2) the
electronic character and number of the substituent on the 4-position of benzene played
a key role in determining the antibacterial activity, suggesting that further
development of such compounds might be of great interest.
4
4
. Experimental section
.1. General
Melting points were determined on a WRS-1B digital instrument without
correction. NMR spectra were recorded on a Varian Mercury-Plus 300 spectrometer
in DMSO-d . All chemical shifts (δ) were quoted in parts per million and coupling
6
constants (J) were given in Hertz. Mass spectra were obtained from VG ZAB-HS,
1
5

ACCEPTED MANUSCRIPT
LCMS-2010A or LCQ DECA XP spectrometer. All commercially available reagents
and solvents were used without further purification. Ornidazole (D , lot number
1
1
0336-0001) and Metronidazole (D , 100191-200305) were purchased from National
2
Institutes for Food and Drug Control; Nitrimidazine (D ,) was provided by the
3
Industry of Sichuan antibiotics; Clarithromycin (D , lot number 040908) was
4
purchased from Zhejiang Huayi Medicines Co. Dimethyl sulfoxide was used as
solvent to dissolve all the reference drugs. HP strains (including ATCC Hp11637 and
eleven kinds of clinically separated HP strains, Hp05-5, Hp05-6, Hp05-7, Hp05-8,
Hp05-9, Hp05-10, Hp05-11, Hp05-12, Hp05-13, Hp05-14) used in these studies were
provided by professor Fulian Hu (Peking University).
4
.2. Chemistry
General procedure for the synthesis of 2-amino-3,5-dicyano-4-aryl-6-arylamino-
pyridines 2a-2v: Substituted benzylidenemalononitrile compounds were prepared by
the condensation reaction of appropriate substituted benzaldehydes and malononitrile
in the presence of piperidine or pyrrolidine in ethanol.
To the solution of substituted benzylidenemalononitrile (6.0 mmol) in anhydrous
ethanol (20 mL) were added appropriate substituted aniline (6.9 mmol), cat. ZnCl2
o
(
9.0 mmol), the mixture was stirred at 80 C for 5.0 h. Then, the mixture was cooled
to room temperature and slowly poured into 150 mL of ice-water by stirring. The
precipitate solid was filtered, washed, recrystallized in the mixture of acetone and
methanol to afford pure target compounds 2a-2v.
4
.2.1. 2-Phenylamino-6-amino-4-phenylpyridine-3,5-dicarbonitrile (2a)
o
1
Colorless powder solid, m.p. 255-256 C. H NMR (DMSO-d , 300MHz) δ: 7.10
6
(
t, J = 7.4 Hz, 1H), 7.32 (t, J = 7.8 Hz, 2H), 7.52-7.55 (m, 7H, ArH + NH ), 7.66 (d, J
2
1
3
=
9.0 Hz, 2H), 9.12 (s, 1H, NH). C NMR (DMSO-d , 75MHz) δ: 81.9, 82.7, 116.3,
6
1
6

ACCEPTED MANUSCRIPT
116.6, 123.2, 124.4, 128.9, 129.2, 130.6, 135.6, 139.4, 149.1, 157.9, 161.2. IR (KBr) ν:
3
621, 3328, 3220, 3127, 3032, 2218, 1623, 1575, 1551, 1491, 822, 751, 680, 616.
+
FAB-MS m/z (%): 312 [(M + H) , 10].
4
.2.2. 2-(p-Tolylamino)-6-amino-4-phenylpyridine-3, 5-dicarbonitrile (2b)
o
1
Colorless powder solid, m.p. 275 C (dec.). H NMR (DMSO-d , 300MHz) δ:
6
2
.26 (s, 3H), 7.09 (d, J = 8.1 Hz, 2H), 7.45-7.51 (m, 9H, ArH + NH ), 9.01 (s, 1H,
2
1
3
NH). C NMR (DMSO-d , 75MHz) δ: 21.4, 81.8, 82.6, 112.8, 116.8, 123.6, 129.0,
6
1
1
29.3, 129.5, 130.6, 133.6, 135.7, 136.8, 158.0, 161.2. IR (KBr) ν: 3325, 3225, 2211,
+
627, 1551, 1514, 822, 779. FAB-MS m/z (%):326 [(M + H) , 25]. Anal. calcd. for
C H N : C 73.83, H 4.65, N 21.52; found C 73.26, H 4.84, N 21.35.
20
15
5
4
.2.3. 2-(4-Methoxyphenylamino)-6-amino-4-phenylpyridine-3,5-dicarbonitrile (2c)
o
1
Colorless powder solid, m.p. 241 C (dec.). H NMR (DMSO-d , 300MHz) δ:
6
3
8
1
3
.75 (s, 3H), 6.87 (d, J = 9.0 Hz, 2H), 7.33-7.42 (br s, 2H, NH ), 7.45-7.57 (m, 7H),
2
1
3
.98 (s, 1H, NH). C NMR (DMSO-d , 75MHz) δ: 56.1, 81.6, 82.4, 114.4, 116.9,
6
17.0, 125.7, 129.2, 129.5, 130.8, 132.5, 136.0, 156.9, 158.4, 161.5. IR (KBr) ν: 3483,
314, 3223, 2211, 1636, 1558, 1509, 1420, 1242, 822, 696. FAB-MS m/z: 342[(M +
+
H) , 4]. Anal. calcd. for C H N : C 70.37, H 4.43, N 20.52; found C 69.92, H 4.50,
20
15
5
N 20.46.
4
.2.4. 2-(4-Chlorophenylamino)-6-amino-4-phenylpyridine-3, 5-dicarbonitrile (2d)
o
1
Colorless powder solid, m.p. 255 C (dec.). H NMR (DMSO-d , 300MHz) δ:
6
7
9
1
3
.34 (d, J = 8.7 Hz, 2H), 7.50-7.54 (m, 7H, ArH + NH ), 7.69 (d, J = 8.7 Hz, 2H),
2
1
3
.27 (s, 1H, NH). C NMR (DMSO-d , 75MHz) δ: 82.2, 82.8, 116.6, 124.8, 128.1,
6
28.7, 128.9, 129.2, 130.6, 135.5, 138.4, 157.6, 160.9, 161.1. IR(KBr) ν: 3478, 3326,
218, 2210, 1632, 1590, 1550, 1490, 818, 740, 697, 602. FAB-MS m/z (%): 345 [(M
1
7

ACCEPTED MANUSCRIPT
+
+
H) , 5]. Anal. calcd. for C H ClN : C 66.00, H 3.50, N 20.25; found C 65.97, H
19 12 5
3
.58, N 20.23.
.2.5. 2-(4-Bromophenylamino)-6-amino-4-phenylpyridine-3, 5-dicarbonitrile (2e)
4
o
1
Colorless powder solid, m.p. 275 C (dec.). H NMR (DMSO-d , 300MHz) δ:
6
1
3
7
.44-7.54 (m, 9H), 7.64 (d, J = 9.0Hz, 2H), 9.25 (s, 1H, NH). C NMR (DMSO-d ,
6
7
5MHz) δ: 82.2, 82.8, 116.2, 116.5, 125.2, 128.9, 129.2, 130.6, 131.7, 135.5, 138.9,
57.6, 160.9, 161.1. IR (KBr) ν: 3469, 3337, 3225, 2212, 1643, 1591, 1549, 1489,
1
+
8
16, 737, 696, 601. FAB-MS m/z (%): 391 [(M + H) , 8]. Anal. calcd. for C H BrN :
19
12
5
C 58.48, H 3.10, N 17.95; found C 58.34, H 3.21, N 17.81.
4
.2.6. 2-Phenylamino-6-amino-4-(4-bromophenyl)pyridine-3,5-dicarbonitrile (2f)
o
1
Colorless powder solid, m.p. 271 C (dec.). H NMR (DMSO-d , 300MHz) δ: 7.08
6
(t, J = 7.4 Hz, 1H), 7.30 (t, J = 7.8 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.55 (br s, 2H,
1
3
NH ), 7.63 (d, J = 7.8 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 9.15 (s, 1H, NH). C NMR
2
(
DMSO-d , 75MHz) δ: 81.7, 82.5, 116.5, 123.3, 124.2, 124.5, 129.0, 131.1, 132.3,
6
1
34.8, 139.3, 157.7, 159.7, 161.0. IR (KBr) ν: 3335, 3225, 2210, 1652, 1506, 1303,
+
1
137, 1020, 810. FAB-MS m/z (%): 389 [(M + H) , 10].
4
.2.7. 2-(p-Tolylamino)-6-amino-4-(4-bromophenyl)pyridine-3,5-dicarbonitrile (2g)
o
1
Colorless powder solid, m.p. 270 C (dec.). H NMR (DMSO-d , 300MHz) δ: 2.30
6
(
s, 3H), 7.13 (d, J = 8.4 Hz, 2H), 7.47-7.52 (m, 6H, ArH + NH ), 7.78 (d, J = 8.4 Hz,
2
1
3
2
1
3
H), 9.09 (s, 1H, NH). C NMR (DMSO-d , 75MHz) δ: 21.1, 81.4, 82.3, 116.6,
6
23.6, 124.2, 129.4, 131.1, 132.3, 133.7, 134.9, 136.7, 157.9, 159.6, 161.1. IR (KBr) ν:
500, 3324, 3225, 2211, 1628, 1531, 1513, 1425, 822, 779, 669. FAB-MS m/z: 404
+
[
(M + H) , 20]. Anal. calcd. for C H BrN : C 59.42, H 3.49, N 17.32; found C 58.99,
2
0
14
5
H 3.68, N 17.88.
4
.2.8. 2-(4-Methoxyphenylamino)-6-amino-4-(4-bromophenyl)pyridine-
1
8

ACCEPTED MANUSCRIPT
3
,5-dicarbonitrile (2h)
o
1
Colorless powder solid, m.p. 260 C (dec.). H NMR (DMSO-d , 300MHz) δ:
6
3
.73 (s, 3H), 6.87 (d, J = 9.0 Hz, 2H), 7.44-7.49 (m, 6H, ArH + NH ), 7.75 (d, J = 8.4
2
1
3
Hz, 2H), 9.04 (s, 1H, NH). C NMR (DMSO-d , 75MHz) δ: 55.8, 81.2, 81.9, 114.1,
6
115.5, 116.6, 124.2, 125.5, 131.1, 132.1, 132.3, 134.9, 156.6, 159.6, 161.1. IR (KBr) ν:
3
500, 3369, 3297, 3168, 2214, 1655, 1552, 1509, 1421, 827, 775, 666, 617. EI-MS
+
+
+
+
m/z (%): 420 [M , 100], 405 [M -CH , 30], 324 [M -CH -Br-H, 30], 296 [M
2
3
-
CH OPhNH, 12]. Anal. calcd. for C H BrN O: C 57.16, H 3.36, N 16.66; found C
3
20 14
5
5
4
7.34, H 3.59, N 16.35.
.2.9. 2-(4-Chlorophenylamino)-6-amino-4-(4-bromophenyl)pyridine-
,5- dicarbonitrile (2i)
Colorless powder solid, m.p. 260-261 C. H NMR (DMSO-d , 300MHz) δ: 7.33
3
o
1
6
(
d, J = 8.7 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.60 (br s, 2H, NH ), 7.68 (d, J = 9.0 Hz,
2
1
3
2
8
1
H), 7.76 (d, J = 8.4 Hz, 2H), 9.29 (s, 1H, NH). C NMR (DMSO-d , 75MHz) δ:
6
2.0, 82.6, 116.4, 124.3 124.9, 128.1, 128.7, 131.1, 132.3, 134.7, 138.4, 157.6, 159.8,
61.0. IR (KBr) ν: 3383, 3285, 3239, 2212, 1581, 1489, 1424, 1072, 881, 820, 667.
+
FAB-MS m/z: 426 [(M + H) , 6].
4
.2.10. 2-(4-Bromophenylamino)-6-amino-4-(4-bromophenyl)pyridine-
,5-dicarbonitrile (2j)
Colorless powder solid, m.p. 285 C (dec.). H NMR (DMSO-d , 300MHz) δ: 7.44
3
o
1
6
(
d, J = 7.5 Hz, 4H), 7.61 (d, J = 8.7 Hz, 4H, 2ArH + NH ), 7.74 (d, J = 8.4 Hz, 2H),
2
9
.27 (s, 1H, NH). IR (KBr) ν: 3449, 3331, 3230, 2912, 2216, 1638, 1572, 1505, 1445,
+
1
034, 816, 742, 607. FAB-MS m/z (%): 470 [(M + H) , 5].
4
.2.11. 2-Phenylamino-6-amino-4-(4-methxoyphenyl)pyridine-3,5-dicarbonitrile (2k)
o
1
Colorless powder solid, m.p. 235-237 C. H NMR (DMSO-d , 300MHz) δ: 3.83
6
1
9

ACCEPTED MANUSCRIPT
(s, 3H), 7.04-7.10 (m, 3H), 7.29 (t, J = 7.8 Hz, 2H), 7.45 (d, J = 8.7 Hz, 4H, ArH +
NH ), 7.63 (d, J = 7.8 Hz, 2H), 9.00 (s, 1H, NH). IR(KBr) ν: 3838, 3760, 3320, 2981,
2
-
2
213, 1581, 1420, 1240, 1162, 825, 743, 694. ESI-MS m/z: 340 [M - H] .
4
.2.12. 2-(p-Tolylamino)-6-amino-4-(4-methoxyphenyl)pyridine-
3,5-dicarbonitrile (2l)
o
1
Colorless powder solid, m.p. 270-272 C. H NMR (DMSO-d , 300MHz) δ: 2.27
6
(
s, 3H), 3.83 (s, 3H), 7.07-711 (m, 4H), 7.39 (br s, 2H, NH ), 7.43-7.49 (m, 4H), 8.96
2
(s, 1H, NH). IR (KBr) ν: 3435, 3370, 3210, 2200, 1640, 1546, 1458, 1256, 1175, 846,
+
7
80, 650. EI-MS m/z (%): 355 [M , 100].
4
.2.13. 2-(4-Methoxyphenylamino)-6-amino-4-(4-methoxyphenyl)pyridine-
3,5-dicarbonitrile(2m)
o
1
Colorless powder solid, m.p. 248-249 C. H NMR (DMSO-d , 300MHz) δ: 3.75
6
(s, 3H), 3.84 (s, 3H), 6.89 (d, J = 9.0 Hz, 2H), 7.11 (d, J = 9.0 Hz, 2H), 7.34 (br s, 2H,
1
3
NH ), 7.46 (d, J = 8.5 Hz, 2H), 7.15 (d, J = 9.0 Hz, 2H), 8.93 (s, 1H, NH). C NMR
2
(
DMSO-d , 75MHz) δ: 55.2, 55.3, 80.7, 81.5, 113.5, 113.9, 116.4, 124.7, 127.0, 130.0,
6
1
31.6, 156.0, 157.6, 160.0, 160.5, 160.6. IR (KBr) ν: 3478, 3431, 3372, 3211, 2198,
-
1
638, 1544, 1459, 1225, 1180, 840, 779, 651. EI-MS m/z: 370 [M-H] . Anal. calcd.
for C H N O : C 67.91, H 4.61, N 18.86; found C 65.04, H 5.19, N 18.94.
2
1
17
5
2
4
.2.14. 2-(4-Cholorophenylamino)-6-amino-4-(4-methoxyphenyl)pyridine-
,5- dicarbonitrile (2n)
Colorless powder solid, m.p. 256-257 C. H NMR (DMSO-d , 300MHz) δ: 3.85
3
o
1
6
(s, 3H), 7.10 (d, J = 8.4, Hz 2H), 7.33 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H),
7
.51 (br s, 2H, NH ), 7.69 (d, J = 8.7 Hz, 2H), 9.21 (s, 1H, NH). IR (KBr) ν: 3469,
2
3
348, 3210, 2212, 1624, 1548, 1502, 1243, 1151, 1014, 887, 816. ESI-MS m/z: 374
-
[
M-H] .
2
0

ACCEPTED MANUSCRIPT
4
.2.15. 2-(4-Bromophenylamino)-6-amino-4-(4-methoxyphenyl)pyridine-
3,5- dicarbonitrile (2o)
o
1
Colorless powder solid, m.p. 260-261 C. H NMR (DMSO-d , 300MHz) δ: 3.83
6
(
s, 3H), 7.09 (d, J = 8.7 Hz, 2H), 7.43- 7.50 (m, 6H, 4ArH + NH ), 7.62 (d, J = 9.0 Hz,
2
2
1
4
H), 9.19 (s, 1H, NH). IR (KBr) ν: 3478, 3376, 3312, 3194, 2926, 2205, 1616, 1558,
-
507, 1260, 1179, 1027, 889, 824,772. ESI-MS m/z: 418 [M-H] .
.2.16. 2-Phenylamino-6-amino-4-(benzo[d][1,3]dioxol-5-yl)pyridine-
3,5- dicarbonitrile (2p)
o
1
Colorless powder solid, m.p. 266 C (dec.). H NMR (DMSO-d , 300MHz) δ:
6
6
2
2
3
4
.13 (s, 2H), 6.97 (dd, J = 8.1, 1.5Hz, 1H), 7.04-7.09 (m, 3H), 7.29 (m, 2H), 7.46 (br s,
H, NH ), 7.63 (d, J = 7.5Hz, 2H), 9.05 (s, 1H, NH). IR (KBr)ν: 3446, 3337, 3227,
2
913, 2212, 1624, 1559, 1497, 1451, 1277, 1032, 926, 853, 814, 739. ESI-MS m/z:
-
54 [M – H] .
.2.17. 2-(p-Tolylamino)-6-amino-4-(benzo[d][1,3]dioxol-5-yl)pyridine-
3,5- dicarbonitrile (2q)
o
1
Colorless powder solid, m.p. >300 C. H NMR (DMSO-d , 300MHz) δ: 2.27 (s,
6
3
7
1
4
H), 6.12 (s, 2H), 6.97 (dd, J = 8.1, 1.5Hz, 1H), 7.08 (m, 4H), 7.41(br s, 2H, NH2),
.47 (d, J = 8.4Hz, 2H), 8.98 (s, 1H, NH). IR (KBr) ν: 3856, 3745, 3613, 2929, 2208,
-
646, 1560, 1418, 1246, 1032, 881, 668. ESI-MS (m/z): 368 [M - H] .
.2.18. 2-(4-Methoxyphenylamino)-6-amino-4-(benzo[d][1,3]dioxol-5-yl)pyridine-
3,5-dicarbonitrile (2r)
o
1
Colorless powder solid, m.p. 250-251 C. H NMR (DMSO-d , 300MHz) δ: 3.73
6
(s, 3H), 6.12 (s, 2H), 6.87 (d, J = 9.0 Hz, 2H), 6.95 (dd, J = 7.8, 1.5Hz, 1H), 7.07 (d, J
=
7.8 Hz, 2H), 7.35 (brs, 2H, NH ), 7.45 (d, J = 8.7 Hz, 2H), 8.95 (s, 1H, NH). IR
2
2
1

ACCEPTED MANUSCRIPT
(KBr) ν: 3445, 3336, 3225, 2915, 2213, 1626, 1559, 1495, 1450, 1347, 1283, 1259,
-
1
032, 926, 856, 815, 735. ESI-MS m/z: 384 [M - H] .
4
.2.19. 2-(4-Bromophenylamino)-6-amino-4-(benzo[d][1,3]dioxol-5-yl)pyridine-
3,5-dicarbonitrile (2s)
o
1
Colorless powder solid, m.p. 258-260 C. H NMR (DMSO-d , 300MHz) δ: 6.13
6
(s, 2H), 6.98 (d, J = 8.1, 1H), 7.08 (d, J = 7.5, 2H), 7.44 (d, J = 8.7, 2H), 7.52 (br s,
2
2
H, NH ), 7.63 (d, J = 9.0Hz, 2H), 8.98 (s, 1H, NH). IR (KBr) ν: 3484, 3324, 3225,
2
937, 2834, 2208, 1624, 1561, 1246, 1125, 1030, 883, 826, 721. ESI-MS m/z: 433
-
[
M - H] .
4
.2.20. 2-(p-Tolylamino)-6-amino-4-(3,4,5-trimethoxyphenyl)pyridine-
,5- dicarbonitrile (2t)
3
o
1
Colorless powder solid, m.p. 214 C (dec.). H NMR (DMSO-d , 300MHz) δ:
6
2
2
1
4
.20 (s, 3H), 3.66 (s, 3H), 3.73 (s, 6H), 6.76 (s, 2H), 7.02 (d, J = 8.1, 2H), 7.34 (brs,
H, NH ), 7.39 (d, J = 8.4, 2H), 8.91 (s, 1H, NH). IR (KBr) ν: 3336, 2930, 2209, 1613,
2
-
555, 1511, 1427, 1246, 1179, 1034, 880, 832. ESI-MS m/z: 414 [M - H] .
.2.21. 2-(4-Methoxyphenylamino)-6-amino-4-(3,4,5-trimethoxyphenyl) pyridine-
3,5-dicarbonitrile (2u)
o
1
Colorless powder solid, m.p. 212-213 C. H NMR (DMSO-d , 300MHz) δ: 3.74
6
(
s, 6H), 3.82 (s, 6H), 6.85-6.89 (m, 4H), 7.38 (brs, 2H, NH ), 7.48 (d, J = 8.7, 2H),
2
8
1
4
.98 (s, 1H, NH). IR (KBr) ν: 3325, 2937, 2834, 2208, 1624, 1561, 1513, 1314, 1246,
-
125, 1031, 826. ESI-MS (m/z): 430 [M - H] .
.2.22. 2-Phenylamino-6-amino-4-(3-hydroxy-4-methoxyphenyl)pyridine-
3,5-dicarbonitrile (2v)
o
1
Colorless powder solid, m.p. 274 C (dec.). H NMR (DMSO-d , 300MHz) δ: 3.72
6
(s, 3H), 6.80-6.88 (m, 2H), 6.96-7.01 (m, 2H), 7.21 (t, J = 7.8, 2H), 7.34 (brs, 2H,
2
2

ACCEPTED MANUSCRIPT
NH ), 7.55 (d, J = 7.5, 2H), 8.93 (s, 1H, NH), 9.46 (s, 1H, OH). IR (KBr) ν: 3438,
2
3
266, 3221, 3128, 3044, 2927, 2209, 1605, 1571, 1495, 1322, 1106, 896, 752, 687.
-
ESI-MS m/z: 356 [M - H] .
4
.3. Biology
The antibacterial activities in vitro of all the target compounds were tested against HP
strains (including ATCC Hp11637 and eleven kinds of clinically separated HP strains,
Hp05-5, Hp05-6, Hp05-7, Hp05-8, Hp05-9, Hp05-10, Hp05-11, Hp05-12, Hp05-13,
Hp05-14), using Ornidazole (D ), Metronidazole (D ), Nitrimidazine (D ) and
1
2
3
2
7-31
Clarithromycin (D ) as references, according to the method reported by other
and
4
2
4,25
our
groups with some slight modifications. Briefly, all of the selected compounds
were evaluated for their antibacterial activity using conventional agar-dilution method.
Twofold serial dilutions of the compounds and reference drugs were prepared in
Mueller–Hinton agar. Drugs were dissolved in dimethylsulfoxide (DMSO, 1 mL) and
the solution was diluted with water (9 mL) without precipitation. Further progressive
double dilution with melted Mueller–Hinton agar was performed to obtain the
required concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1 ꢀg/mL. Petri dishes
4
6
were incubated with 1x10 or 1x10 colony forming units (cfu) and incubated at 37 °C
for 18 h. The minimal inhibitory concentration (MIC) was the lowest concentration of
the test compound, which resulted in no visible growth on the plate. MIC and MIC
5
0
90
values represents a concentration giving 50% and 90% inhibition of the corresponding
HP activity, respectively. To ensure that the solvent had no effect on bacterial growth,
a control test was performed with test medium supplemented with DMSO at the same
dilutions as used in the experiment.
Acknowledgements
We thank the Chinese Postdoctoral Science Foundation (2012M511158 and
2
3

ACCEPTED MANUSCRIPT
2
013T60477) and National Natural Science Foundation of China (81125023) for
financial support on this study. We also thank Professor Fulian Hu (Peking University)
for providing HP strains (including ATCC Hp11637 and clinically separated HP
strains, Hp05-5, Hp05-6, Hp05-7, Hp05-8, Hp05-9, Hp05-10, Hp05-11, Hp05-12,
Hp05-13, Hp05-14) used in these studies.
References and notes
1
. (a) Samadi, A.; Marco-Contelles, J.; Soriano, E.; Álvarez-Pérez, M.; Chioua, M.; Romero, A.;
González-Lafuente, L.; Gandía, L.; Roda, J. M.; López, M. G.; Villarroya, M.; García, A. G.;
Ríos, C. d. L. Bioorg. Med. Chem. 2010, 18, 5861.
2
3
4
. Samadi, A.; Silva, D.; Chioua, M.; Carreiras, M. d. C.; Marco, J. C. Synth. Commun. 2011, 41,
2
859.
. Deng, J.; Sanchez, T.; Al-Mawsawi, Q. L.; Dayam, R.; Yunes, A. R.; Garofalo, A.; Bolger, B.
M.; Neamati, N. Bioorg. Med. Chem. 2007, 15, 4985.
. Murata, T.; Shimada, M.; Sakakibara, S.; Yoshino, T.; Kadono, H.; Masuda, T.; Shimazaki,
M.; Shintani, T.; Fuchikami, K.; Sakai, K.; Inbe, H.; Takeshita, K.; Niki, T.; Umeda, M.;
Bacon, K. B.; Ziegelbauer K. B.; Lowinger, T. B. Bioorg. Med. Chem. Lett. 2003, 13, 913.
. Mantri, M.; Link, R.; de Vries, H.; Beukers, M. W.; Brussee, J.; Ijzerman, A. P. J. Med. Chem.
5
2
008, 51, 4449.
6
7
8
9
. Said, S. A. Monatsh Chem. 2009, 140, 573.
. Ibrahim, A. D.; Ismail, M. N. S. Eur. J. Med. Chem. 2011, 46, 5825.
. Mosaad, M. S.; Samir, A. M.; Amira, S. I. Molecules 2010, 15, 1882.
. Yang, T.; He, H.; Ang, W.; Yang, Y.-H.; Yang, J.-Z.; Lin, Y.-N.; Yang, H.-C.; Pi, W.-Y.; Li,
Z.-C.; Zhao, Y.-L.; Luo, Y.-F.; Wei, Y.-Q. Molecules 2012, 17, 2351.
0. Piper, J. R.; McCaleb, G. S.; Montgomery, J. A.; Kisliuk, R. L.; Gaumont, Y.; Sirotnaks, F. M.;
J. Med. Chem. 1986, 29, 1080.
1
1
1. Tu, S.-J.; Li, T.-J.; Zhu, S.-L.; HZou, X.; Wang, Q. Acta Crystallogr. E 2005, E61, o1883.
2. Raghukumar, V.; Thirumalai, D.; Ramakrishnan, T. V.; Karunakarac, V.; Ramamurthy, P.;
1
2
4

ACCEPTED MANUSCRIPT
Tetrahedron 2003, 59, 3761.
1
3. Sarkar, S.; Das, D. K.; Khan, A. T. RSC Adv. 2014, 4, 53752.
1
4. Yang, J.-J.; Li, J.-R.; Hao, P. F.; Qiu, F.-D.; Liu, M.-X.; Zhang, Q.; Shi, D.-X. Dyes Pigments
2
015, 116, 97.
1
5. Pugachev, M. V.; Shtyrlin, N. V.; Sysoeva, L. P.; Nikitina, E. V.; Abdullin, T. I.; Iksanova, A.
G.; Ilaeva, A. A.; Musin, R. Z.; Berdnikov, E. A.; Shtyrlin, Y. G. Bioorg. Med. Chem. 2013, 21,
4
388.
6. Liu, Z.; Cheng, W.; Liu, D.; van Ofwegen, L.; Proksch, P.; Lin, W. Tetrahedron 2014, 70,
703.
1
1
8
7. Fardeau, S.; Dassonville-Klimpt, A.; Audic, N.; Sasaki, A.; Pillon, M.; Baudrin, E.; Mullié, C.;
Sonnet, P. Bioorg. Med. Chem. 2014, 22, 4049.
1
8. Li, S.; Shah, N. P. Food Chem. 2014, 165, 262.
1
9. Roy, S.; Bauza, A.; Banik, R.; Biswas, S. C.; Frontera, A.; Das, S. Tetrahedron 2014, 70,
6
931.
2
2
2
2
2
2
2
0. Li, P.; Shi, L.; Gao, M.-N.; Yang, X.; Xue, W.; Jin, L.-H.; Hu, D.-Y.; Song, B.-A. Bioorg. Med.
Chem. Lett. 2015, 25, 481.
1. Barniol-Xicota, M.; Escandell, A.; Valverde, E.; Julián, E.; Torrents, E.; Vázquez, S. Bioorg.
Med. Chem. 2015, 23, 290.
2. Li, X.; Sheng, J.; Huang, G.; Ma, R.; Yin, F.; Song, D.; Zhao, C.; Ma, S. Eur. J. Med. Chem.
2
015, 97, 32.
3. Qin, X.-J.; Zhao, Y.-L.; Lunga,P.-K.; Yang, X.-Q.; Song, C.-W.; Cheng, G.-G.; Liu, L.; Chen,
Y.-Y.; Liu, Y.-P.; Luo, X.-D. Tetrahedron 2015, 71, 4372.
4. Liu, J.-B.; Yi, W.; Hu, J.-M.; Wu, F.-Y.; Zhao, L.-Z.; Song, H.-C.; Wang, Z.-H. Chem. Pharm.
Bull. 2010, 58, 1127.
5. Liu, J.-B.; Cao, R.-H.; Wang, Z.-H.; Peng, W.-L.; Song, H.-C. Eur. J. Med. Chem. 2009, 44,
1
737.
6. The reaction was carried out for 5 h under the conditions of table 1-entry 7, and the obtained
mixture was monitored by ESI-HRMS analysis (see below). The results showed that the
desired product 2a and the compound N-benzylideneaniline were generated.
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ACCEPTED MANUSCRIPT
2
2
2
7. Bae, E. A.; Han, J. M.; Kim, D. H. Planta Med. 1999, 65, 442.
8. Matu, E. N.; van Staden, J. J. Ethnopharmacol. 2003, 87, 35.
9. National Committee for Clinical Laboratory Standards, “Methods for Dilution Antimicrobial
Susceptability Tests for Bacteria that Grow Aerobically-Third Edition; approved Standard;
NCCLS Document M7-A3 (ISBN 1-56238-209-8),” NCCLS, Villanova, PA 1993.
0. Si, Y.-C. J. Pharm. Pract. 2001, 19, 80.
3
3
1. Ma, X.-R.; Su, D.-M. Drug microbiology testing handbook, Beijing, Science Press, 2000, 210.
2
6