Design, synthesis and evaluation of phenylthiazole and phenylthiophene pyrimidindiamine derivatives targeting the bacterial membrane

Article abstract of DOI:10.1016/j.ejmech.2020.112141

As the continuous rise in the incidence of antibiotic resistance, it is urgent to develop novel chemical scaffolds with antibacterial activities to control the spread of resistance to conventional antibiotics. In this study, a series of phenylthiazole and

Full text of DOI:10.1016/j.ejmech.2020.112141

Journal Pre-proof
Design, synthesis and evaluation of phenylthiazole and phenylthiophene
pyrimidindiamine derivatives targeting the bacterial membrane
Tingting Fan, Weikai Guo, Ting Shao, Wenbo Zhou, Pan Hu, Mingyao Liu, Yihua
Chen, Zhengfang Yi
PII:
S0223-5234(20)30108-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112141
EJMECH 112141
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 December 2019
Revised Date: 10 February 2020
Accepted Date: 10 February 2020
Please cite this article as: T. Fan, W. Guo, T. Shao, W. Zhou, P. Hu, M. Liu, Y. Chen, Z. Yi, Design,
synthesis and evaluation of phenylthiazole and phenylthiophene pyrimidindiamine derivatives targeting
the bacterial membrane, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112141.
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14g
Con.
in vivo
in vitro
E. coli.
1
2
3
4
5
6
7
8
9
10 11 12
A
B
C
D
E
F
G
H
S. aureus.
***
*
**
Destruction of cytoplasmic membrane
Elimination of viable pathogens

Design, synthesis and evaluation of
phenylthiazole and phenylthiophene
pyrimidindiamine derivatives targeting the
bacterial membrane
Tingting Fana,b,1, Weikai Guoa,1, Ting Shaoa, Wenbo Zhoua, Pan Hua, Mingyao Liua,
Yihua Chena* and Zhengfang Yia*
aEast China Normal University and Shanghai Fengxian District Central Hospital
Joint Center for Translational Medicine, Shanghai Key Laboratory of Regulatory
Biology, Institute of Biomedical Sciences and School of Life Sciences, East China
Normal University, 200241 Shanghai, China
bChangzhi Medical College, Changzhi 046000, Shanxi, China
*Corresponding authors.
*Z.Y.: Phone: +86-21-54345016. Fax: +86-21-54344922. E-mail: zfyi@bio.ecnu.edu.cn
*Y.C.: Phone: +86-21-24206647. Fax: +86-21-54344922. E-mail: yhchen@bio.ecnu.edu.cn
1These authors contributed equally to this work.
Keywords: Antibacterial activity, Thiazole, Thiophene, Membrane damage

ABSTRACT
As the continuous rise in the incidence of antibiotic resistance, it is urgent to develop
novel chemical scaffolds with antibacterial activities to control the spread of
resistance to conventional antibiotics. In this study, a series of phenylthiazole and
phenylthiophene pyrimidindiamine derivatives were designed and synthesized by
modifying the hit compound (N2-isobutyl-N4-((4-methyl-2-phenylthiazol-5-yl)methyl)
pyrimidine-2,4-diamine) and their antibacterial activities were evaluated both in vitro
and
in
vivo.
Among
the
tested
compounds,
compound
14g
(N4-((5-(3-bromophenyl)thiophen-2-yl)methyl)-N2-isobutylpyrimidine-2,4-diamine)
displayed the best antibacterial activities, which was not only capable of inhibiting E.
coli and S. aureus growth at concentrations as low as 2 and 3 µg/mL in vitro, but also
efficacious in a mice model of bacteremia in vivo. Unlike conventional antibiotics,
compound 14g was elucidated to mainly destroy the bacterial cell membrane, with the
dissipation of membrane potential and leakage of contents, ultimately leading to cell
death. The destruction of cell structure is challenging to induce bacterial resistance,
which suggested that compound 14g may be a kind of promising alternatives to
antibiotics against bacteria.
1. Introduction
Antibiotics have been essential life-saving drugs to revolutionize medicine
radically [1]. Indeed, since the discovery of penicillin in 1928, several highly effective
antibiotics have been developed for clinical treatments of bacterial infections [2]. It is
estimated that the widespread use of antibiotics has extended human life expectancy

by about 30 years in the developed countries [3]. However, the extensive availability
of antibiotics has also caused many uncontrollable adverse effects [4], the most
serious of which is the induction of bacterial resistance [5, 6]. The efficacies of drugs
will be severely reduced or even invalidated once bacterial resistance occurs [7].
Numerous researches pointed out that bacterial resistance to antibiotics has reached
alarming levels worldwide [8-12]. It was suggested that the number of deaths due to
bacterial resistance might increase from the current estimated 700,000 to 10,000,000
annually by 2050 [13]. There is little way to stop bacteria from developing adaptation
to the environment and antibiotics [14, 15]. The continuous development of new
drugs to keep pace with, or even exceed the evolution and spread of bacterial
resistance is widely recognized as one of the effective strategies to prevent the dawn
of the post-antibiotic era. However, there are few new drugs currently in the discovery
and clinical pipeline, as natural antibiotics are difficult and expensive to synthesize or
isolate [16]. What’s worse, most of the current drug developments are based on the
modification of existing antibiotic structures, which are easy to induce resistance
again with a high probability of cross-resistance [7]. Therefore, designing and
discovering more novel antibacterial agents with different scaffolds are urgent to
alleviate this situation.
In this study, to discover novel antibacterial candidates with different skeletons
from traditional antibiotics, an in-house library of more than 200 newly synthesized
compounds was screened based on their antibacterial effects at lower dilutions. As a
result, the hit compound N2-isobutyl-N4-((4-methyl-2-phenylthiazol-5-yl)methyl)

pyrimidine-2,4-diamine displayed mild antibacterial activities against Escherichia coli
(E. coli) and Staphylococcus aureus (S. aureus) with minimum inhibitory
concentration (MIC) of 16 µg/mL (Fig. 1). As been known, the 2,4-pyrimidindiamine
scaffold has existed in several antibacterials, such as trimethoprim a (TMP) [17],
Mycobacterial dihydrofolate reductase inhibitor b (SRI-20094) [18], and Bacillus
anthracis dihydrofolate reductase inhibitors c [19] and d [20] (Fig. 1). Besides, a
series of hybrid of tetrahydrocarbazole with 2,4-diaminopyrimidine scaffold has also
shown antibacterial activities in our previous studies [21]. To further optimize the
antibacterial activities and explore the structure-activity relationship, a series of
phenylthiazole and phenylthiophene pyrimidindiamine derivatives were designed and
synthesized based on the hit compound. Firstly, it was investigated for the positions of
the diaminopyrimidinyl group linking to the thiazole ring as well as their antibacterial
activities. Secondly, we replaced the thiazole ring with the thiophene ring and
modified the substitutions in pyrimidine ring at 2-position and that in phenyl ring at
the left part. Among them, compound 14g showed the most antibacterial effects with
MIC value of 2 and 3 µg/mL for E. coli and S. aureus, respectively.

Fig 1. The structure of the hit compound as well as several known antimicrobial
agents with diaminopyrimidine skeleton.
The conventional antibiotics primarily exert antibacterial effect by entering cells
and interfering with the physiological activities in the cells, such as inhibiting cell
wall synthesis, DNA replication and protein synthesis, thus giving bacteria chance to
get tolerance by changing drug targets, preventing drugs from entering, and degrading
drugs [22]. In contrast, compound 14g was found to achieve antibacterial effects
mainly by destroying the bacterial cell membrane structure. The cell membranes are
essential material guarantees for intracellular physiological activities, increasing
studies have focused on the development of membrane-active compounds with a
broad-spectrum activity [23, 24]. The normal physiological function of incomplete
membranes will be interrupted because there are many important proteins distributed
on the cell membrane and the increased membrane permeability will promote the

entry of other drugs to produce a synergistic effect [25]. These may contribute to the
antibacterial efficacy of compound 14g. Furthermore, the membrane-active
compounds can overcome non-inherited antibiotic resistance since both actively
dividing cells and non-dividing cells need intact membranes to remain viable [26],
and there is little chance for inactive bacterial cells to develop resistance [24, 27, 28].
Hence, compound 14g with a novel structure and acting by destroying the cell
membrane can minimize the emergence of resistance. This study is meaningful for the
development of new antibacterial drugs in the future.
2. Chemistry
The synthetic route of compounds 6 and 7 was shown in Scheme 1.
Thiobenzamide 1 was condensed with ethyl bromopyruvate to give the critical
intermediate ethyl 2-phenylthiazole-4-carboxylate 2. Compound 3 was obtained by
reducing 2 with LiAlH4, and then it was brominated with phosphorus tribromide to
afford 4, which was subsequently converted to the relevant amine 5 via Gabriel
reaction. Compound 5 was coupled with 2,4-dichloropyrimidine to yield target
compound 6, which was underwent a nucleophilic substitution reaction with
isobutylamine to produce 7.
Synthesis of the compounds 13a-k and 14a-h followed the general procedures
illustrated in Scheme 2. Different boronic acids were undergone Suzuki coupling
reactions with bromothiophene derivatives to afford compound 9. Subsequently, the
treatment of compound 9 with hydroxylamine hydrochloride generated compound 10,
which was further reduced to obtain compound 11. Compound 12 was prepared

through coupling 11 with 2,4-dichloropyrimidine, which was underwent similar
nucleophilic substitution reactions with different amines to produce compounds 13a-k
and 14a-h.
Scheme 1. Synthesis of compounds 6 and 7. Reagents and conditions: (a) ethyl
bromopyruvate, EtOH, reflux; (b) LiAlH4, Et2O, 0 °C then to r.t; (c) PBr3, DCM, r.t;
.
(d) potassium phthalimide, DMF, 60 °C; (e) NH2NH2 H2O, reflux; (f)
2,4-dichloropyrimidine, DIEA, EtOH, 40 °C; (g) isobutylamine, DIEA, n-BuOH,
reflux.
OH
OH
R2
R2
R2
b
a
B
N
S
OH
S
CHO
8
10
9
c
R2
R2
d
H
R2
e
H
N
S
N
N
R1
S
12
N
NH2
Cl
S
N
N
13a-k and 14a-h
11
R1 = i-Bu-NH- or Et-NH- or n-Pr-NH- or n-Bu-NH-
or (n-Pr)2N- or (n-Bu)2N- or (i-Bu)2N- or pyrrolidinyl
or piperazinyl or morpholinyl
R2 = -H or -OBn or -OMe or -t-Bu or -F or -Cl or -Br
Scheme 2. Synthesis of compounds 13a-k and 14a-h. Reagents and conditions: (a)
5-bromo-2-thiophenecarboxaldehyde, Pd(PPh3)4, Na2CO3, EtOH, DME, 90 °C; (b)

H2NOH-HCl, pyridine, EtOH, 80 °C; (c) Zn, AcOH; (d) 2,4-dichloropyrimidine,
DIEA, EtOH, 40 °C; (e) appropriate amine, DIEA, n-BuOH, 120 °C.
3. Results and discussion
3.1. Structure-activity relationships against bacteria
In this study, a series of phenylthiazole and phenylthiophene pyrimidindiamine
derivatives were designed and synthesized. The MIC against Gram-negative bacteria
E. coli and Gram-positive bacteria S. aureus was determined to evaluate their
antibacterial efficacies and allowed us to rank their relative potencies. Initially, we
shifted the diaminopyrimidinyl group from the 5’- to the 4’-position of the thiazole
ring for investigating the best linking position of the diaminopyrimidinyl group and
compounds 6 and 7 were synthesized. Regrettably, these compounds completely
abolished their antibacterial activities compared to the hit compound, as shown in
Table 1.
Table 1. MICs of compounds 6, 7 against E. coli ATCC 25922 and S. aureus ATCC
29213.
MIC (µg/mL) α
Compd
R1
E. coli
S. aureus
hit compound
6
-
16
>32
>32
1
16
>32
>32
4
Cl
i-Bu-NH-
7
-
-
-
Vancomycin
Cefazolin
Chloromycetin
2
2
8
8

-
-
32
8
512
8
Polymyxin B
Chloramphenicol
αThe MIC values were measured in two independent experiments with three replicates per
experiment.
In order to improve the antibacterial activities of the hit compound, we replaced the
thiazole ring of it with thiophene ring, and compound 13a was synthesized. The
results indicated the antibacterial activities of compound 13a were increased
compared with the hit compound (Table 2), which may suggest that the thiophene ring
was more beneficial to sustain or enhance the antibacterial activities. To further
explore the structure-activity relationship, compounds 13b-h were synthesized by
replacing isobutylamine of the pyrimidinyl-2-position of compound 13a with other
alkylamines. The results were shown in Table 2, compared with compound 13a, the
introduction of alkylamines group at the pyrimidinyl-2-position did not improve the
antibacterial activities of the compounds. Nonetheless, the mono-substituted
alkylaminopyrimidinyl might be more conducive to the maintenance of the
antibacterial activities than corresponding bis-substituted derivatives (13a vs 13h and
13c vs 13f). Similar results were also observed in the comparison 13e with 13g. Also,
the role of heterocyclic groups was investigated (13i-k), unfortunately, the
introduction of heterocyclic groups didn’t enhance the antibacterial activities of
compounds compared with 13a, and substitutions with piperazinyl (13j) or
morpholinyl (13k) almost completely deprived the antibacterial activities.
Table 2. MICs of compounds 13a-k against E. coli ATCC 25922 and S. aureus ATCC
29213.

MIC (µg/mL) α
E. coli
Compd
R1
S. aureus
hit compound
-
i-Bu-NH-
Et-NH-
n-Pr-NH-
i-Pr-NH-
n-Bu-NH-
(n-Pr)2N-
(n-Bu)2N-
(i-Bu)2N-
pyrrolidinyl
piperazinyl
morpholinyl
-
16
8
16
8
>32
>32
8
>32
>32
8
>32
>32
1
2
8
16
8
>32
16
>32
4
>32
>32
>32
8
>32
>32
4
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
Vancomycin
Cefazolin
Chloromycetin
Polymyxin B
Chloramphenicol
-
-
-
-
2
8
512
8
32
8
αThe MIC values were measured in two independent experiments with three replicates per
experiment.
Next, we focused the modification on the left part phenyl ring. As shown in Table 3,
compared with the unsubstituted compound 13a, the introduction of substituted
groups to the phenyl ring was preferable for sustaining or enhancing the antibacterial
activities. The derivatives with electron-donating substituents (14a-c) shared the
similar antibacterial activities to the ones with electron-withdrawing substituents
(14d-h), which may suggest that the electronic effect of this position has little
influence on the antibacterial activities of compounds. Interestingly, the introduction
of the fluorine substituent (14d) could effectively resist S. aureus with MIC value of 8

µg/mL, however, it didn’t has any inhibitory activity against E.coli at the same
concentration. In addition, the bromine group was introduced to different positions (o-,
m- and p-position) of the phenyl ring (14f-h) to investigate the effect of the
substitution position on antibacterial activity. As shown in Table 3, m-position
substitution (14g) may be more favorable against both E. coli and S. aureus than o- or
p-position (14h and 14f), but it still needs more compounds to validate. Among the
investigated compounds, compounds 14f and 14g exhibited the most excellent
antibacterial activities with the MIC values of 2 µg/mL for E. coli and 3 or 4 µg/mL
for S. aureus, which were comparable to or even better than the widely used positive
controls vancomycin, cefazolin, chloromycetin, polymyxin B and chloramphenicol.
Given compounds 14f and 14g shared very similar structures except of difference in
the position of the bromine group, we speculated that their antibacterial activities and
mechanism were also similar. Therefore, 14g was selected as the representative
compound for further research.
Table 3. MICs of compounds 14a-h against E. coli ATCC 25922 and S. aureus ATCC
29213.
MIC (µg/mL) α
Compd
R2
E. coli
S. aureus
hit compound
-
H
4-OBn
4-OMe
4-t-Bu
16
8
8
4
2
16
8
8
4
8
13a
14a
14b
14c

4-F
4-Cl
>32
8
8
8
14d
14e
4-Br
2
4
14f
3-Br
2
3
14g
2-Br
4
8
14h
-
-
-
-
-
1
2
8
32
8
4
2
8
512
8
Vancomycin
Cefazolin
Chloromycetin
Polymyxin B
Chloramphenicol
αThe MIC values were measured in two independent experiments with three replicates per
experiment.
3.2. Compound 14g causes no significant hemolysis
In order to determine whether compound 14g is cytotoxic to mammalian cells, the
hemolysis assays were performed using the rabbit’s red blood cells. After exposure to
different concentrations of compound 14g for 1 h, hemolysis levels were below 20%,
which indicated compound 14g was not significantly hemolytic within its antibacterial
activity range (Fig. 2) and suggested that compound 14g could differentiate between
microbial and erythrocyte cellular membrane. The difference between eukaryotic and
prokaryotic cell membranes lies in the content and distribution of phospholipids [23,
29], which may contribute to the antibacterial activity of compound 14g.

Fig 2. The hemolysis rate of rabbit red blood cell induced by positive control (Triton
X-100), negative control (PBS) and 14g at 1 ×, 2 ×, 3 ×, 4 ×, 5 ×, and 7.5 × MIC,
respectively. Bars represent the standard deviation (n = 3).
3.3. Compound 14g inhibits bacterial growth
To further determine the antibacterial efficacy of compound 14g and how long it
will start to exhibit the antibacterial effect, the effects of 14g on the bacterial growth
process were monitored at different concentrations. The results were recorded every
two hours within 24 h, giving the complete growth cycle for bacteria. As shown in Fig.
3A-B, when the bacteria in the control group began to enter the logarithmic growth
phase at the 6th hour of incubation, the growth of the bacteria was still wholly
inhibited in the group treated with 1 × and 2 × MIC of compound 14g. It suggested
that the compound could exert an antibacterial effect within 6 h. Although 0.5 × MIC
of compound 14g showed little effects on the growths of E. coli and S. aureus, it still
could delay the growth cycle of bacteria.
Fig 3. (A) Effect of 14g on E. coli growth for 24 h. (B) Effect of 14g on S. aureus
growth for 24 h.
3.4. Compound 14g changes bacterial cell morphology

The above results showed that compound 14g could exert effect quickly within 6 h,
possibly because it acted on the cell’s first line of defense, the cell membrane, causing
changes in cell morphology. To test whether the treatment of compound 14g could
change the cell morphology, Scanning Electron Microscopy (SEM) and Transmission
Electron Microscopy (TEM) were performed.
As shown by SEM, a visible morphological change occurred on the surface of the
treated strain compared to the untreated control. Untreated cells were regular, intact
and had a smooth surface (Fig. 4A and 4C), while some bacterial cells treated with
compound 14g became deformed, pitted and shriveled. (Fig. 4B and 4D). TEM was
used to intensify the investigation of the morphological changes of E. coli and S.
aureus. Same as observed by SEM, the cell morphology of untreated bacteria was
regular, the double membrane structure was smooth and continuous, and the
cytoplasm was homogenous (Fig. 4E and 4G). In contrast, after treatment with
compound 14g, the entire cell shape was significantly modified or damaged, and the
bacterial cells became ghost-like structures, resulting in electron density heterogeneity
(Fig. 4F and 4H). The cell debris and electron-dense particles were found around the
disrupted cells. The above results indicated that compound 14g could destroy the cell
membrane of E. coli and S. aureus.

Fig 4. (A-D) Scanning electron microscopy images of E. coli and S. aureus. (A)
Untreated E. coli. (B) E. coli treated with 14g at 1 × MIC for 12 h. (C) Untreated S.
aureus. (D) S. aureus treated with 14g at 1 × MIC for 12 h. The black arrow indicates
a rough cell membrane with some cracks and holes after treatment with 14g. Bar = 1
µm. (E-H) Transmission electron microscopy images of E. coli and S. aureus. (E)
Untreated E. coli. (F) E. coli treated with 14g at 1 × MIC for 12 h. (G) Untreated S.
aureus. (H) S. aureus treated with 14g at 1 × MIC for 12 h. The black arrow indicates
the leakage of intracellular contents, cell lysis and the appearance of ghost cells. Bar =
200 nm.
3.5. Compound 14g destroys bacteria membrane
3.5.1 Compound 14g causes the leakage of cell contents
To examine how the compound achieves antibacterial efficacy by altering the
morphology of bacterial cells, changes in the physicochemical properties of the cell
membrane after compound 14g treatment were detected. The cell membrane is a vital
component of bacteria, and it is responsible for providing a stable internal
environment for the cells and for transporting materials or transmitting information
function [30]. If the cell membranes are destroyed, the intracellular components, such
as small ions, proteins and nucleotide will leak out. Therefore, the integrity of the cell
membrane can be detected by some fluorescent stains of these components [31].
Ethidium bromide (EB) is an indicator of DNA structure. It binds nucleic acids via
intercalative binding mode with changes in EB fluorescence [32]. When bacteria were
exposed to compound 14g, EB will be able to bind to the DNA released in culture or

damaged cells, causing an increase in fluorescence, in case compound 14g could
disrupt the bacterial membrane. As shown in Fig. 5A-B, compared to the solvent
control group, the fluorescence of compound 14g treatment groups significantly
increased within 20 min, which indicated that compound 14g could destroy the
bacterial cell membranes. The red-fluorescent propidium iodide (PI), which can
penetrate only membrane-broken cells and embed DNA to emit fluorescence, was
used to further test the effects of compound 14g on bacterial cell membranes. As
shown in Fig. 5C-F, 77.16% of the compound 14g-treated E. coli cells were stained by
PI (Fig. 5D), while the stained cells only accounted for 4.34% in the solvent control
group (Fig. 5C). The same result was also found in S. aureus group, the percentage of
the damaged cells increased from 1.53% (Fig. 5E) to 58.47% after incubation with 1 ×
MIC compound 14g for 1 h (Fig. 5F). The results showed that when exposed to
compound 14g, the integrity of the bacterial cell membranes was compromised and
the contents leaked.
3.5.2 Compound 14g depolarizes bacterial cell membranes
The structural damage of cell membranes often leads to potential variations in cell
membranes [33]. The fluorescent probe DiSC3(5) can quench the fluorescence when
inserted into a hyperpolarized membrane, and if the cell membranes are depolarized,
it can be released to enhance the fluorescent signal. Therefore, the molecule DiSC3(5)
was selected to detect changes in membrane potential caused by compound 14g. As
shown in Fig. 5G-H compound 14g increased the fluorescence intensity in the
co-incubation group compared to the solvent control or negative control vancomycin

in both E. coli and S. aureus group. The behavior was similar to that of the positive
control of polymyxin B, which also produced an increase in fluorescence intensity. In
the S. aureus group (Fig. 5H), the change in fluorescence intensity showed a more
obvious dose-dependent manner and concentration-dependent characteristic. The
results suggested that compound 14g could dissipate the bacteria membrane potential.
Fig 5. (A-B) Membrane disruption as measured by ethidium bromide uptake against
(A) E. coli and (B) S. aureus. (C-F) FACScan analysis of PI staining in bacteria cells

treated with 14g for 1 h. (C) Staining of E. coli cells without 14g treatment. (D)
Staining of E. coli cells treated with 1 × MIC 14g. (E) Staining of S. aureus cells
without 14g treatment. (F) Staining of S. aureus cells treated with 1 × MIC 14g. The
percentage of PI-stained cells is indicated. M1 is the percentage of membrane
disrupted cells. The x-axis shows the relative fluorescence intensity. The y-axis shows
the relative number of cells. (G-H) Effect of 14g on the membrane potential of
bacterias. (G) E. coli treated with 1% DMSO, polymyxin B, vancomycin and 14g at 1
× MIC, 2 × MIC for 80 min. (H) S. aureus treated with 1% DMSO, polymyxin B,
vancomycin and 14g at 1 × MIC, 2 × MIC for 80 min.
3.6. Antibacterial activity of compound 14g in vivo
Encouraged by the excellent antibacterial activity of compound 14g in vitro, the
mouse bacteremia models infected by S. aureus were used to further assess the
antibacterial activity of compound 14g in vivo. It is known that murine models of
bacteremia are commonly used to detect the systemic effects of antibiotics in the
pharmaceutical industry [34]. The treatment was performed after the 1-hour injection,
which is sufficient for the bacteria to spread throughout the mouse [35]. As shown in
Fig. 6, compound 14g could reduce the number of viable pathogens in the peritoneal
cavity of infected mice at low dosage (12 mg/kg), but the efficacy was not as good as
the same concentration of polymyxin B. The antibacterial efficacy of high dose
compound 14g (24 mg/kg) was better than polymyxin B. The results indicated that
compound 14g exhibited good antimicrobial activity in vivo.

***
*
**
Fig 6. Viable bacterial counts of the abdominal cavity in the bacteremia model
infected with S. aureus strain. The infected mice were treated with 14g (12 and 24
mg/kg) or polymyxin B (12 mg/kg) via intraperitoneally injection (n = 7). Each point
represents the determination of a single animal, and the line shows the mean value. *P
< 0.05, **P < 0.01, ***P < 0.001, compared with untreated control group.
4. Conclusion
With the overuse and abuse of antibiotics, the problem of bacterial resistance has
become more and more prominent, and new antibacterial agents are urgently needed
to alleviate this situation [36, 37]. In this study, an in-house library was screened to
discovery novel antibacterial candidates. The compound N2-isobutyl-N4-
((4-methyl-2-phenylthiazol-5-yl)methyl)pyrimidine-2,4-diamine
displayed
mild
antibacterial activity and presented as a hit compound. A series of phenylthiazole and
phenylthiophene pyrimidindiamine derivatives with antibacterial potential were
designed and synthesized by modifying the hit compound. Among the investigated
compounds, compound 14g showed the best antibacterial activities, which was not

only capable of inhibiting E. coli and S. aureus in vitro, but also was efficacious in a
mice model of bacteremia in vivo. Moreover, further mechanism study revealed that
compound 14g plays an antibacterial role by inducing cytoplasmic membrane rupture,
leading to the release of cell contents, and disordering membrane properties and
functions, which can decrease the possibility of inducing bacterial resistance. Hence,
compound 14g is likely to be developed as a promising antibacterial candidate or to
provide a kind of new chemical skeleton for the development of therapeutic agents.
5. Materials and methods
5.1. General methods for chemistry
All reagents were purchased from Bide Pharmatech Ltd, Leyan Ltd. or
Aladdin-reagents Inc.. Chemical reactions were completed through standard
techniques under inert gas (N2). Using Bruker 500 MHz instruments to generate NMR
spectra. High-resolution mass spectra were collected on a Bruker MicroTOF-Q II
LCMS instrument operating in electrospray ionization (ESI). The purity of the
compounds was determined by high-pressure liquid chromatography (HPLC, Agilent
Technologies 1200 Series) and the final purity of all biologically targeted compounds
was ≥ 95%.
5.2. General procedure for the synthesis of compounds 6, 7, 13a-k and 14a-h
5.2.1. 2-Chloro-N-((2-phenyl-4-thiazolyl)methyl)-4-pyrimidinamine (6).
Compound 6, white solid (76% yield), was prepared according to our previous
report [38], except using ethyl bromopyruvate instead of ethyl 2-chloroacetoacetate.
1H NMR (500 MHz, CDCl3) δ 8.04 (d, J = 1.1 Hz, 1H), 7.92 (dd, J = 6.5, 3.2 Hz, 2H),

13
7.47 – 7.43 (m, 3H), 7.18 (s, 1H), 6.34 (d, J = 5.8 Hz, 1H), 4.70 (s, 2H). C NMR
(125 MHz, CDCl3) δ 169.08, 160.83, 152.75, 145.78, 133.20, 130.33, 129.01, 126.48,
115.39, 95.31, 29.67. HR MS (ESI): calcd for C14H11ClN4NaS [M + Na]+; 325.0285;
found 325.0295.
5.2.2. N2-isobutyl-N4-((2-phenyl-4-thiazolyl)methyl)pyrimidine-2,4-diamine (7).
A mix of compound 6 (70 mg, 0.23 mM) and isobutylamine (70 mg, 1.0 mM) in
n-butyl alcohol (5 mL), then N,N-diisopropylethylamine (0.1 mL, 0.6 mM) was added.
Then the mix was heated to 120 °C overnight. After reaction was completed, the
solvent (n-butyl alcohol) was removed by evaporation and diluted with water,
extracted with CH2Cl2. The CH2Cl2 layer was dried over anhydrous sodium sulfate,
concentrated. The resulting crude product was purified by column chromatography
1
(CH2Cl2/MeOH 100:1-20:1) to afford 7, brown oily liquid (63 mg, 81%). H NMR
(500 MHz, CDCl3) δ 7.92 (dd, J = 6.6, 2.9 Hz, 2H), 7.53 (s, 1H), 7.47 – 7.42 (m, 3H),
7.16 (s, 1H), 5.92 (d, J = 5.7 Hz, 1H), 4.74 (s, 2H), 3.23 (t, J = 6.2 Hz, 2H), 1.92 –
1.84 (m, 1H), 0.95 (d, J = 6.7 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 168.96, 162.57,
152.86, 146.06, 133.23, 130.31, 129.01, 126.48, 115.18, 95.30, 48.69, 41.72, 28.30,
20.24. HR MS (ESI): calcd for C18H22N5S [M + H]+; 340.1590; found 340.1603.
5.2.3. N2-isobutyl-N4-((5-phenylthiophen-2-yl)methyl)pyrimidine-2,4-diamine (13a).
A mix of compound 5-bromothiophene-2-carbaldehyde (1.55 mL, 13 mM) and
catalytic tetrakis (triphenylphosphine) palladium in DME (20 mL), an aqueous
solution of Na2CO3 (2.12 g in 10 mL of H2O) was added at r.t, the vial was blown
three times with N2. After stirring for 5 min, compound phenylboronic acid (1.22 g,

10.0 mM) and EtOH (20 mL) were added, the resulting mix was degassed under
vacuum and was heated to 90 °C overnight under N2, then evaporated and extracted
with CH2Cl2 and H2O. The CH2Cl2 layer was dried over anhydrous sodium sulfate,
concentrated. The residue was subjected to column chromatography (PE/EA = 20:1)
to afford compound 5-phenylthiophene-2-carbaldehyde (1224 mg, 65%). A mix of
compound 5-phenylthiophene-2-carbaldehyde (1224 mg, 6.5 mM) in anhydrous EtOH
(40 mL), hydroxylamine hydrochloride (903 mg, 13 mM) and pyridine (1.05 mL, 13
mM) were added, then heated to 80 °C for 1 h, evaporated to afford crude
5-phenylthiophene-2-carbaldehyde oxime. To
a
solution of compound
5-phenylthiophene-2-carbaldehyde oxime in AcOH (10 mL), Zn (1.643 g, 25.13 mM)
was added. The mix was stirred at r.t overnight. The resulting mix was filtered via a
pad of Celite and the pad was washed with CH2Cl2 (15 mL × 3). After removal of the
solvent by concentration, crude compound (5-phenyl-2-thiophenyl)methanamine was
obtained. A mix of compound (5-phenyl-2-thiophenyl)methanamine (1041 mg, 5.5
mM) and 2,4-dichloropyrimidine (1639 mg, 11 mM) in anhydrous EtOH (50 mL),
N,N-diisopropylethylamine (2.8 mL, 16.5 mM) was added, the mix was stirred at
40 °C overnight. Then the solvent was removed by evaporation and diluted with water,
extracted with CH2Cl2. The CH2Cl2 layer was dried over anhydrous sodium sulfate,
concentrated. The residue was subjected to column chromatography (PE/EA, 8:1-2:1)
to obtain compound N-((5-phenyl-2-thiophenyl)-methyl)-2-chloropyrimidin-4-amine
(1021 mg, 62%). A mix of compound N-((5-phenyl-2-thiophenyl)methyl)-2-chloro-
pyrimidin-4-amine (60 mg, 0.2 mM) and isobutylamine (1.0 mL) in n-butyl alcohol (5

mL), N,N-diisopropylethylamine (0.1 mL, 0.6 mM) was added, and the resulting was
stirred at 120 °C overnight. Then the solvent was removed by concentration and
extracted with DCM and H2O. The CH2Cl2 layer was dried over anhydrous sodium
sulfate, concentrated. The residue was subjected to column chromatography
(CH2Cl2/MeOH 100:1-20:1) to afford compound N2-isobutyl-N4-((5-phenylthiophen-2
1
-yl)methyl)pyrimidine-2,4-diamine 13a, light yellow solid (56 mg, 90%). H NMR
(500 MHz, CDCl3) δ 7.68 (d, J = 7.4 Hz, 1H), 7.57 – 7.48 (m, 2H), 7.36 (dd, J = 10.5,
4.8 Hz, 2H), 7.28 (d, J = 7.4 Hz, 1H), 7.14 (d, J = 3.6 Hz, 1H), 6.96 (d, J = 3.6 Hz,
1H), 5.79 (d, J = 6.2 Hz, 1H), 4.72 (d, J = 4.1 Hz, 2H), 3.25 (t, J = 6.3 Hz, 2H), 1.93 –
1.85 (m, 1H), 0.97 (d, J = 6.7 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 162.39, 144.06,
140.65, 134.16, 128.85, 127.50, 126.77, 125.61, 122.56, 95.29, 48.80, 40.00, 28.43,
20.24. HR MS (ESI): calcd for C19H23N4S [M + H]+; 339.1638; found 339.1651.
5.2.4. N2-ethyl-N4-((5-phenylthiophen-2-yl)methyl)pyrimidine-2,4-diamine (13b).
Similar procedures to the preparation of compound 13a were used to synthesize
compound 13b, light yellow solid (90% yield), except using ethylamine replace with
1
2-methylpropanamine. H NMR (500 MHz, CDCl3) δ 7.55 – 7.51 (m, 2H), 7.50 –
7.39 (m, 1H), 7.35 (t, J = 7.6 Hz, 2H), 7.27 (d, J = 7.4 Hz, 1H), 7.12 (d, J = 3.6 Hz,
1H), 6.96 (d, J = 3.6 Hz, 1H), 5.95 (d, J = 5.6 Hz, 1H), 4.75 (d, J = 4.4 Hz, 2H), 3.51
– 3.42 (m, 2H), 1.24 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 162.36,
144.16, 139.80, 134.04, 128.86, 127.55, 127.17, 125.57, 122.53, 96.40, 39.79, 36.14,
14.60. HR MS (ESI): calcd for C17H19N4S [M + H]+; 311.1325; found 311.1339.
5.2.5. N4-((5-phenylthiophen-2-yl)methyl)-N2-propylpyrimidine-2,4-diamine (13c).

Similar procedures to the preparation of compound 13a were used to synthesize
compound 13c, light yellow solid (99% yield), except using propylamine replace with
1
2-methylpropanamine. H NMR (500 MHz, CDCl3) δ 7.81 (d, J = 4.4 Hz, 1H), 7.55
(d, J = 7.8 Hz, 2H), 7.36 (t, J = 7.6 Hz, 2H), 7.27 (d, J = 7.7 Hz, 1H), 7.14 (d, J = 3.6
Hz, 1H), 6.95 (d, J = 3.5 Hz, 1H), 5.73 (d, J = 5.8 Hz, 1H), 5.12 (s, 1H), 4.70 (d, J =
5.0 Hz, 2H), 3.36 (dd, J = 13.3, 6.6 Hz, 2H), 1.67 – 1.56 (m, 2H), 0.97 (t, J = 7.4 Hz,
3H). 13C NMR (125 MHz, CDCl3) δ 162.47, 144.02, 141.21, 134.25, 128.85, 127.48,
126.54, 125.67, 122.61, 94.59, 43.18, 40.14, 23.00, 11.51. HR MS (ESI): calcd for
C18H21N4S [M + H]+; 325.1481; found 325.1491.
5.2.6.
N2-isopropyl-N4-((5-phenylthiophen-2-yl)methyl)pyrimidine-2,4-diamine
(13d).
Similar procedures to the preparation of compound 13a were used to synthesize
compound 13d, light yellow solid (44% yield), except using isopropylamine replace
with 2-methylpropanamine. 1H NMR (500 MHz, CDCl3) δ 7.60 – 7.48 (m, 3H), 7.36
(t, J = 7.6 Hz, 2H), 7.29 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 3.6 Hz, 1H), 6.97 (d, J = 3.5
Hz, 1H), 5.83 (d, J = 6.5 Hz, 1H), 4.76 (d, J = 3.9 Hz, 2H), 4.21 – 4.17 (m, 1H), 1.26
13
(d, J = 6.5 Hz, 6H). C NMR (125 MHz, CDCl3) δ 162.39, 144.28, 139.91, 134.08,
128.90, 127.60, 127.08, 125.63, 122.62, 94.95, 43.18, 39.98, 22.65. HR MS (ESI):
calcd for C18H21N4S [M + H]+; 325.1481; found 325.1495.
5.2.7. N2-butyl-N4-((5-phenylthiophen-2-yl)methyl)pyrimidine-2,4-diamine (13e).
Similar procedures to the preparation of compound 13a were used to synthesize
compound 13e, light yellow solid (44% yield), except using 1-butylamine replace

1
with 2-methylpropanamine. H NMR (500 MHz, CDCl3) δ 7.53 (d, J = 8.1 Hz, 2H),
7.36 (t, J = 7.6 Hz, 2H), 7.27 (d, J = 7.2 Hz, 1H), 7.13 (d, J = 3.5 Hz, 1H), 6.96 (d, J =
3.5 Hz, 1H), 5.87 (s, 1H), 4.74 (s, 2H), 3.43 (dd, J = 12.7, 6.4 Hz, 2H), 1.64 – 1.55 (m,
13
2H), 1.46 – 1.36 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). C NMR (125 MHz, CDCl3) δ
162.38, 144.28, 139.89, 134.08, 128.88, 127.59, 127.08, 125.63, 122.57, 95.82, 41.03,
39.98, 31.50, 20.10, 13.80. HR MS (ESI): calcd for C19H23N4S [M + H]+; 339.1638;
found 339.1644.
5.2.8.
N4-((5-phenylthiophen-2-yl)methyl)-N2,N2-dipropylpyrimidine-2,4-diamine
(13f).
Similar procedures to the preparation of compound 13a were used to synthesize
compound 13f, brown oily liquid (82% yield), except using dipropylamine replace
1
with 2-methylpropanamine. H NMR (500 MHz, CDCl3) δ 7.85 (d, J = 6.0 Hz, 1H),
7.56 (d, J = 7.5 Hz, 2H), 7.38 (t, J = 7.7 Hz, 2H), 7.30–7.29 (m, 1H), 7.16 (d, J = 3.6
Hz, 1H), 6.97 (d, J = 3.6 Hz, 1H), 5.85 (d, J = 5.3 Hz, 1H), 4.75 (d, J = 5.7 Hz, 2H),
13
3.60 – 3.49 (m, 4H), 1.70 – 1.62 (m, 4H), 0.94 (t, J = 7.4 Hz, 6H). C NMR (125
MHz, CDCl3) δ 161.99, 143.78, 141.33, 140.05, 134.29, 128.84, 127.41, 126.44,
125.62, 122.59, 94.71, 49.73, 39.89, 21.06, 11.41. HR MS (ESI): calcd for C21H27N4S
[M + H]+; 367.1951; found 367.1967.
5.2.9.
N2,N2-dibutyl-N4-((5-phenylthiophen-2-yl)methyl)pyrimidine-2,4-diamine
(13g).
Similar procedures to the preparation of compound 13a were used to synthesize
compound 13g, brown oily liquid (86% yield), except using dibutylamine replace

1
with 2-methylpropanamine. H NMR (500 MHz, CDCl3)
δ 7.91 (d, J = 5.7 Hz, 1H),
7.57 (dd, J = 8.1, 0.9 Hz, 2H), 7.37 (t, J = 7.7 Hz, 2H), 7.29 – 7.27 (m, 1H), 7.16 (d, J
= 3.6 Hz, 1H), 6.95 (d, J = 3.6 Hz, 1H), 5.67 (d, J = 5.7 Hz, 1H), 4.90 (s, 1H), 4.74 (d,
J = 5.7 Hz, 2H), 3.57 – 3.49 (m, 4H), 1.63–1.57 (m, 4H), 1.36 – 1.32 (m, 4H), 0.94 (t,
J = 5.8 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 161.28, 156.12, 143.68, 142.13,
134.36, 128.81, 127.36, 126.15, 125.63, 122.55, 93.42, 60.38, 47.32, 30.20, 20.35,
14.08. HR MS (ESI): calcd for C23H31N4S [M + H]+; 395.2264; found 395.2277.
5.2.10. N2,N2-diisobutyl-N4-((5-phenylthiophen-2-yl)methyl)pyrimidine-2,4-diamine
(13h).
Similar procedures to the preparation of compound 13a were used to synthesize
compound 13h, brown oily liquid (30% yield), except using diisobutylamine replace
1
with 2-methylpropanamine. H NMR (500 MHz, CDCl3) δ 7.88 (d, J = 5.7 Hz, 1H),
7.55 (d, J = 7.4 Hz, 2H), 7.36 (t, J = 7.7 Hz, 2H), 7.27 – 7.26 (m, 1H), 7.14 (d, J = 3.6
Hz, 1H), 6.94 (d, J = 3.6 Hz, 1H), 5.65 (d, J = 5.7 Hz, 1H), 4.81 (s, 1H), 4.71 (d, J =
5.6 Hz, 2H), 3.39 (d, J = 7.4 Hz, 4H), 2.19 – 2.10 (m, 2H), 0.88 (d, J = 6.7 Hz, 12H).
13C NMR (125 MHz, CDCl3) δ 161.83, 156.05, 143.73, 142.04, 134.38, 128.84,
127.39, 126.22, 125.65, 122.58, 93.01, 55.78, 40.02, 27.01, 20.36. HR MS (ESI):
calcd for C23H31N4S [M + H]+; 395.2264; found 395.2273.
5.2.11. N-((5-phenylthiophen-2-yl)methyl)-2-(1-pyrrolidinyl)pyrimidin-4-amine (13i).
Similar procedures to the preparation of compound 13a were used to synthesize
compound 13i, light yellow crystals (99% yield), except using pyrrolidine replace
1
with 2-methylpropanamine. H NMR (500 MHz, CDCl3) δ 7.90 (d, J = 5.7 Hz, 1H),

7.55 (d, J = 7.8 Hz, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.27 – 7.26 (m, 1H), 7.14 (d, J = 3.5
Hz, 1H), 6.95 (d, J = 3.4 Hz, 1H), 5.70 (d, J = 5.8 Hz, 1H), 5.01 (s, 1H), 4.72 (d, J =
13
5.5 Hz, 2H), 3.57 (t, J = 6.4 Hz, 4H), 1.95 (t, J = 6.4 Hz, 4H). C NMR (125 MHz,
CDCl3) δ 162.09, 143.78, 141.78, 134.31, 128.80, 127.36, 126.42, 125.62, 122.51,
93.72, 46.47, 39.93, 25.48. HR MS (ESI): calcd for C19H21N4S [M + H]+; 337.1481;
found 337.1496.
5.2.12. N-((5-phenylthiophen-2-yl)methyl)-2-(1-piperazinyl)pyrimidin-4-amine (13j).
Similar procedures to the preparation of compound 13a were used to synthesize
compound 13j, brown crystals (64% yield), except using piperazine replace with
1
2-methylpropanamine. H NMR (500 MHz, CDCl3)
δ 7.90 (d, J = 5.7 Hz, 1H), 7.54
(d, J = 7.4 Hz, 2H), 7.36 (t, J = 7.7 Hz, 2H), 7.28 (s, 1H), 7.14 (d, J = 3.6 Hz, 1H),
6.94 (d, J = 3.5 Hz, 1H), 5.74 (d, J = 5.7 Hz, 1H), 4.99 (s, 1H), 4.70 (d, J = 5.6 Hz,
13
2H), 3.90 – 3.80 (m, 4H), 3.02 – 2.94 (m, 4H), 1.24 (d, J = 6.4 Hz, 1H). C NMR
(125 MHz, CDCl3) δ 161.31, 143.98, 141.35, 134.22, 128.85, 127.47, 126.48, 125.64,
122.54, 94.93, 51.61, 45.05, 43.58. HR MS (ESI): calcd for C19H22N5S [M + H]+;
352.1590; found 352.1604.
5.2.13. 2-morpholino-N-((5-phenylthiophen-2-yl)methyl)pyrimidin-4-amine (13k).
Similar procedures to the preparation of compound 13a were used to synthesize
compound 13k, yellow solid (99% yield), except using morpholine replace with
2-methylpropanamine. 1H NMR (500 MHz, CDCl3) δ 7.93 (d, J = 5.7 Hz, 1H), 7.59 –
7.54 (m, 2H), 7.38 (t, J = 7.7 Hz, 2H), 7.30 (d, J = 7.4 Hz, 1H), 7.16 (d, J = 3.6 Hz,
1H), 6.97 (d, J = 3.6 Hz, 1H), 5.78 (d, J = 5.7 Hz, 1H), 5.03 (s, 1H), 4.73 (d, J = 5.7