Synthesis and molecular docking studies of novel pyrimidine derivatives as potential antibacterial agents

Article abstract of DOI:10.1007/s11030-019-10019-8

Abstract: The present work describes the in vitro antibacterial evaluation of some new pyrimidine derivatives. Twenty-two target compounds were designed, synthesized and preliminarily explored for their antimicrobial activities. The antimicrobial assay revealed that some target compounds exhibited significantly inhibitory efficiencies toward bacteria and fungal including drug-resistant pathogens. Compound 7c presented the most potent inhibitory activities against Gram-positive bacteria (e.g., Staphylococcus aureus 4220), Gram-negative bacteria (e.g., Escherichia coli 1924) and the fungus Candida albicans 7535, with an MIC of 2.4?μmol/L. Compound 7c was also the most potent, with MICs of 2.4 or 4.8?μmol/L against four multidrug-resistant, Gram-positive bacterial strains. The toxicity evaluation of the compounds 7c, 10a, 19d and 26b was assessed in human normal liver cells (L02 cells). Molecular docking simulation and analysis suggested that compound 7c has a good interaction with the active cavities of dihydrofolate reductase (DHFR). In vitro enzyme study implied that compound 7c also displayed DHFR inhibition. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-019-10019-8

Molecular Diversity
https://doi.org/10.1007/s11030-019-10019-8
ORIGINAL ARTICLE
Synthesis and molecular docking studies of novel pyrimidine
derivatives as potential antibacterial agents
Xue‑Qian Bai1 · Chun‑Shi Li2 · Ming‑Yue Cui2 · Ze‑Wen Song1,3 · Xing‑Yu Zhou1 · Chao Zhang1 · Yang Zhao1 ·
Tian‑Yi Zhang1 · Tie‑Yan Jiang4
Received: 9 September 2019 / Accepted: 21 November 2019
© Springer Nature Switzerland AG 2019
Abstract
The present work describes the in vitro antibacterial evaluation of some new pyrimidine derivatives. Twenty-two target
compounds were designed, synthesized and preliminarily explored for their antimicrobial activities. The antimicrobial assay
revealed that some target compounds exhibited signifcantly inhibitory efciencies toward bacteria and fungal including
drug-resistant pathogens. Compound 7c presented the most potent inhibitory activities against Gram-positive bacteria (e.g.,
Staphylococcus aureus 4220), Gram-negative bacteria (e.g., Escherichia coli 1924) and the fungus Candida albicans 7535,
with an MIC of 2.4 μmol/L. Compound 7c was also the most potent, with MICs of 2.4 or 4.8 μmol/L against four multidrug-
resistant, Gram-positive bacterial strains. The toxicity evaluation of the compounds 7c, 10a, 19d and 26b was assessed in
human normal liver cells (L02 cells). Molecular docking simulation and analysis suggested that compound 7c has a good
interaction with the active cavities of dihydrofolate reductase (DHFR). In vitro enzyme study implied that compound 7c
also displayed DHFR inhibition.
Xue-Qian Bai and Chun-Shi Li have contributed equally to this
work.
* Tian-Yi Zhang
tianyizhang@126.com
* Tie-Yan Jiang
jty781213@sina.com
1
Jilin Medical University, Jilin 132013,
People’s Republic of China
2
The Third People’s Hospital of Dalian, Dalian 116000,
People’s Republic of China
3
Department of Pharmary, Yanbian University, Yanji 133002,
People’s Republic of China
4
Changning Branch of Shanghai Municipal Public Security
Bureau, Shanghai 200336, People’s Republic of China
Vol.:(0123456789)
1 3

Molecular Diversity
Graphic abstract
Keywords Pyrimidine · Antibacterial activity · Toxicity · Molecular docking · DHFR inhibition
[7]. “The cost in terms of lost global production between
now and 2050 would be an enormous 100 trillion USD
if we do not take action,” as a UK Government report
states [8]. Furthermore, fungal infections pose a serious
threat to human health, especially to immunocompromised
patients [9, 10]. The clinical treatment of life-threatening
invasive fungal infections (IFIs) is also a signifcant global
challenge [11, 12]. Therefore, this highlights the need for
development of new antimicrobial agents that difer from
those of existing agents [13].
Introduction
Infections caused by bacterial resistance to the major
classes of therapeutic drugs have become one of the great-
est threats to public health problems [1]. Drug-resistant
bacteria, such as methicillin-resistant Staphylococcus
aureus (MRSA) and multidrug-resistant Escherichia coli,
cause great difculties in the treatment of nosocomial
infections [2–5], which severely threaten global public
health [6]. Drug-resistant bacteria and the simultaneous
decline in eforts by academic laboratories or pharma-
ceutical companies directed toward the discovery of new
antibacterial agents to combat resistant strains now pose a
serious threat to the treatment of life-threatening infections
Compounds based on the pyrimidine scafold are known
to exhibit many diferent biological actions such as anti-
bacterial, antifungal, anti-infammatory and antitumor
activities [14]. Lots of amino pyrimidine-based deriva-
tives have been reported to exhibit antibacterial activities
1 3

Molecular Diversity
via inhibiting dihydrofolate reductase (DHFR) [15]. Based
on our previous work, several rhodanine or aminoguani-
dine derivatives bearing (benzyloxy)benzylidene [16, 17]
moieties showed moderate activities against several Gram-
positive bacterial strains, including multidrug-resistant
clinical isolates as exemplifed by compounds A and B
(Fig. 1). Furthermore, non-fused pyridines constitute
another important unit of heterocycles, which exhibited
various biological activities [18]. Mohammed A. Seleem
et al. reported that compounds C and D (Fig. 1) exhib-
ited potent activity against MRSA RCMB 2658 strain
(MIC = 0.4 μg/mL or 1.56 μg/mL) in vitro [19]. Herein,
we design of new compounds using A–D as lead com-
pounds, and 22 synthesized compounds were screened for
their antibacterial activities with a minimum inhibitory
concentration method (Fig. 2).
Fig. 1 Several molecules of
previously reported antibacterial
agents
A
B
C
D
Fig. 2 Synthesis of target compounds
1 3

Molecular Diversity
Scheme 1 a DMF, K2CO3, 70–80 °C, refux, 4–7 h; b DMF-DMA, DMF, 80 °C, 12 h; c moroxydine hydrochloride or metformin hydrochloride,
AcOH, 120 °C, refux, 8–12 h
In vitro antibacterial activity evaluation
Results and discussion
Totally 22 target compounds were synthesized, and their
structures and activities against susceptible and drug-resist-
ant bacteria are listed in Tables 1 and 2, respectively. Gati-
foxacin, trimethoprim, fuconazole and itraconazole were
used for the antimicrobial activity as positive controls.
The in vitro antimicrobial activity of the target com-
pounds is shown in Table 1. Most of the target compounds
exhibited better bactericidal efect against the diferent bac-
teria. Compound 7c had the strongest activity against the
Gram-positive bacteria (S. aureus 4220), with an MIC value
of 2.4 μmol/L. Against the Gram-negative E. coli 1924,
compound 7c was more potent than the positive controls
trimethoprim, with an MIC of 2.4 μmol/L. Compounds 10a,
17a and 22b were also equipotent potent with the positive
controls gatifoxacin and moxifoxacin. Against the fun-
gus C. albicans 7535, compound 7c displayed the highest
potency of all of the compounds with an MIC value of 2.4
μmol/L, which was more potent than that of fuconazole
(MIC=3.3 μmol/L). Compounds 7a, 10a, 17a, 22b and 26a
Chemistry
The synthesis of target compounds is outlined in
Schemes 1 and 2. Intermediates 2, 5, 8, 11 and 24 were
obtained by reacting hydroxyacetophenone or 2′-hydroxy-
4′-methoxyacetophenone with the appropriate substituted
benzylchlorides or bromine alkane. Intermediates 3, 6,
9, 12 and 25 were prepared using a previously reported
method [17]. Then, the intermediates 3, 6, 9, 12, 16, 18,
21 and 25 were reacted with moroxydine hydrochloride or
metformin hydrochloride to generate the pyrimidine deriv-
atives 4a, 7a–f, 10a and 13a (Scheme 1) [19]. Intermedi-
ate 15 was prepared according to a previously reported
method [20]. Intermediate 20 was prepared using Suzuki
coupling reactions [21]. Compounds 17a–d, 19a–e, 22a–b
and 26a-b were synthesized in the same way as com-
pounds 4a, 7a–f, 10a and 13a (Scheme 2). The structures
of the target compounds were confrmed by 1H NMR, 13
C
NMR, mass spectroscopy and HRMS.
1 3

Molecular Diversity
Scheme 2 a DMF, K2CO3, 70–80 °C, refux, 4–7 h; b DMF-DMA, DMF, 80 °C, 12 h; c metformin hydrochloride, AcOH, 120 °C, refux,
8–12 h; d acetylacetone, AcOH, CH3COONH4, 110 °C, refux, 3 h; e orthoboric acid, Na2CO3, DME, H2O, 85 °C, 4 h
also displayed good potency, with MICs ranging from 4.8
to 5.8 μmol/L.
(SARs) were obtained. The position of the 2,4-di-Cl ben-
zyloxy group with respect to the benzene ring signifcantly
infuenced the antibacterial activity, with an activity order of
p>m>o for compounds 4a, 7c and 10a. Furthermore, the
position of the di-Cl substituent on the benzyloxy moiety
also afected the activity of the compounds with an activity
order of 2,4-di-Cl>2,6-di-Cl, as exemplifed by a compari-
son of the results for the compounds 7a and 7c. A compari-
son of the potency of compounds 4a and 13a revealed that
the introduction of an additional 4-OCH3 moiety to the phe-
nyl ring increased the antimicrobial activity. A comparison
of the potency of compounds 7c and 7f revealed that the
substitution of the dimethylamino group with a morpholino
moiety reduced the antimicrobial activity. The inclusion of
a n-pentyl group at the benzene ring, as exemplifed by the
compounds 17a and 17b, indicated that a suitable length of
the molecule was critical for the activity.
As depicted in Table 2, the target compounds were
tested for their inhibitory activities against the clini-
cal isolates of several different multidrug-resistant
bacterial strains. Compound 7c showed potent activ-
ity against the MRSA (3167 and 3506) strains, with an
MIC value of 2.4 μmol/L. This was equivalent to moxi-
foxacin (MIC = 2.5 μmol/L) and greater than oxacillin
(MIC > 151.2 μmol/L). Compounds 10a and 17a showed
the equipotent or more potent than the positive controls
gatifloxacin (MIC = 5.3 μmol/L) against the MRSA
(3167 and 3506) strains. For the QRSA (CCARM 3505
and 3519) strains, compound 7c also had the strongest
inhibitory efect, with an MIC value of 2.4 μmol/L and
4.8 μmol/L. This was equivalent to oxacillin (MIC = 2.4
μmol/L) and stronger than norfloxacin (MIC > 200.4
μmol/L) and gatifloxacin (MIC = 21.3 μmol/L against
CCARM 3505 and 10.6 μmol/L against CCARM 3519).
Compounds 7a, 7d and 10a were equipotent to the moxi-
foxacin (MIC = 10.0 μmol/L for QRSA 3505).
Toxicity evaluation
We evaluated the toxicity of a normal human liver cell line
(L02) using a standard technique. As can be seen from
Table 3, compound 19d exhibited weaker activity than 10a
Based on the analysis of the activities of the synthesized
compounds, the following structure–activity relationships
1 3

Molecular Diversity
Table 1 Inhibitory activity (MICa, μmol/L) of compounds 4a, 7a–f, 10a, 13a, 17a–d, 19a–e, 22a–b and 26a–b against various bacteria
Compd
R1/R3/R4
R2
Gram-positive strains
Gram-negative strains
Fungus
S. aureus
4220b
S. mutans
3289c
E. coli
1924d
C. albicans
7535e
4a
2,4-di-Cl
2,6-di-Cl
4-CH3
2,4-di-Cl
4-Br
-
>153.8
4.8
11.0
2.4
9.4
85.8
>139.7
4.8
17.9
5.6
>184.4
>159.6
77.9
>206.5
109.6
>199.4
25.6
>224.6
50.3
5.2
5.8
24.4
0.7
0.6
>153.8
9.6
22.1
4.8
18.7
171.6
>139.7
9.6
>143.5
11.2
>184.4
>159.6
155.7
>206.5
219.2
>199.4
51.3
>224.6
50.3
5.2
11.7
48.8
0.7
0.6
>153.8
9.6
22.1
2.4
18.7
85.8
>139.7
4.8
35.9
5.6
>184.4
>159.6
155.7
>206.5
109.6
>199.4
25.6
>224.6
50.3
5.2
11.7
48.8
5.3
5.0
>153.8
4.8
22.1
2.4
9.4
85.8
>139.7
4.8
>143.5
5.6
>184.4
159.6
155.7
>206.5
109.6
>199.4
25.6
>224.6
50.3
5.2
5.8
48.8
1.3
1.2
7a
NMe2
NMe2
NMe2
NMe2
NMe2
Morpholino
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7b
7c
7d
7e
4-CN
7f
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
n-pentyl
4-CH3
2,4-di-Cl
4-Br
2,4-di-Cl
Phenyl(3,4-fused)
4-Br
n-pentyl
NHMe2
H
10a
13a
17a
17b
17c
17d
19a
19b
19c
19d
19e
22a
22b
2,4-di-Cl
n-hexyl
n-pentyl
26a
26b
Gatifoxacin
Moxifoxacin
Trimethoprim
Itraconazole
Fluconazole
110.2
1.7
13.8
6.9
0.85
3.3
n.df
n.df
n.df
n.df
n.df
n.df
aMICs were determined by microbroth dilution method for microdilution plates
bStaphylococcus aureus RN 4220
cStreptococcus mutans 3289
dEscherichia coli KCTC 1924
eCandida albicans 7535
fn.d. not determined
against the diferent bacteria, in spite of its greater cyto-
toxicity than 10a, comparably indicating that the promising
antibacterial activity of these compounds may not be due to
their cytotoxicity, but some unknown mechanism of action.
22b being the most potent were selected as a template
molecule. It was also interesting to start a comparative
modeling study of the most active compounds 7c and 22b
against Iclaprim (Fig. 3). The structure data were obtained
from the protein data bank (PDB ID: 3fra) [22, 23]. The
water molecules and heavy atom in protein were removed,
the protein was prepared by adding hydrogen and correct-
ing incomplete residues using Clean Protein tool of DS,
and then the protein was refined with CHARMm. The
structures of 7c and 22b were sketched in 2D and con-
verted into 3D using the DS molecule editor (Fig. 3b, e).
Compound 7c is bound into the active site, in which the
benzene ring formed alkyl bond (4.34 Å) with Arg44. The
Molecular docking
Molecular docking is a significant computational method
used to forecast the binding of the ligand to the receptor
binding site by varying position and conformation of the
ligand keeping the receptor rigid. To evaluate the anti-
bacterial mechanism of compounds action, a molecular
docking investigation was undertaken. Compounds 7c and
1 3

Molecular Diversity
Table 2 Inhibitory activity
Compd
R1/R3/R4
R2
Multidrug-resistant Gram-positive strains
(MICa, μmol/L) of compounds
4a, 7a–f, 10a, 13a, 17a–d, 19a–
e, 22a–b and 26a–b against
clinical isolates of multidrug-
resistant Gram-positive strains
MRSA
3167b
QRSA
3505d
3506c
3519e
4a
2,4-di-Cl
2,6-di-Cl
4-CH3
2,4-di-Cl
4-Br
–
>153.8
9.6
22.1
>153.8
9.6
11
>153.8
9.6
22.1
>153.8
9.6
22.1
7a
NMe2
NMe2
NMe2
NMe2
NMe2
Morpholino
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7b
7c
2.4
9.4
2.4
18.7
2.4
9.4
4.8
9.4
7d
7e
4-CN
85.8
>139.7
4.8
85.8
>139.7
4.8
85.8
>139.7
9.6
85.8
>139.7
4.8
7f
2,4-di-Cl
2,4-di-Cl
2,4-di-Cl
n-pentyl
4-CH3
2,4-di-Cl
4-Br
2,4-di-Cl
Phenyl(3,4-fused)
4-Br
n-pentyl
NHMe2
H
10a
13a
35.9
5.6
71.7
5.6
71.7
11.2
143.5
11.2
17a
17b
>184.4
>159.6
>155.7
>206.5
109.6
>199.4
25.6
>224.6
50.3
5.2
>184.4
>159.6
77.9
>206.5
109.6
>199.4
25.6
>224.6
50.3
5.2
>184.4
>159.6
>155.7
>206.5
219.2
>199.4
25.6
>224.6
100.6
5.2
>184.4
>159.6
77.9
>206.5
109.6
>199.4
26.5
>224.6
100.6
5.2
17c
17d
19a
19b
19c
19d
19e
22a
22b
2,4-di-Cl
n-hexyl
n-pentyl
26a
11.7
48.8
5.8
24.4
11.7
48.8
11.7
48.8
26b
Gatifoxacin
Moxifoxacin
Norfoxacin
Oxacillin
Trimethoprim
5.3
2.5
5.3
2.5
21.3
10.0
10.6
10.0
25.1
12.5
>151.2
13.8
>200.4
2.4
13.8
>200.4
2.4
>151.2
n.df
n.df
aThe antibacterial testing was carried out three times, and the MICs are average of them
bMethicillin-resistant S. aureus 3167
cMethicillin-resistant S. aureus 3506
dQuinolone-resistant S. aureus 3505
eQuinolone-resistant S. aureus 3519
fn.d. not determined
Table 3 Antibacterial activity and cytotoxicity (IC5a0 µM) for 7c, 10a,
19d and 26b against L02 cell
alkyl bond (3.97 Å) with Leu62. The NMe2 atom of 22b
formed carbon hydrogen bond (2.46 Å) with Thr46. The
pyrimidine ring of 22b formed alkyl bond (4.19 Å) with
Arg44. Compound 22b is bound into the active site where
benzene ring shows interaction (4.19 and 5.33 Å) with
Leu62. Otherwise, the enzyme surface model is shown
in Fig. 3c, f, I, which revealed that compounds 7c and
22b were well inserted into the active pocket of S. aureus
DHFR, which were the same to that of Iclaprim. These
data provide certain theoretical support for experimental
results and the next optimization.
Test organisms
7c
10a
19d
26b
MIC (µmol/L) S. aureus 4220
2.4
2.4
4.8
4.8
25.6
25.6
24.4
24.4
MRSA 3506
ICa50 (µM)
L02b
18.53 45.06 12.19 18.45
aIC50 is the concentration of compound required to inhibit the growth
of the cells by 50%
bHuman normal liver cells
NMe2 atom of 7c formed carbon hydrogen bond (1.94 Å)
with Ser49. The Cl-substituted phenyl ring of 7c formed
1 3

Molecular Diversity
Fig. 3 a 3D conformation of 7c
docked in DHFR complex. b
Predicted interactions between
7c and the amino acids of 3fra.
c Proposed pose of 7c in the
binding pocket of DHFR. d 3D
conformation of 22b docked
in DHFR complex. e Predicted
interactions between 22b and
the amino acids of 3fra. f
Proposed pose of 22b in the
binding pocket of DHFR. g
3D conformation of Iclaprim
docked in DHFR complex. h
Predicted interactions between
Iclaprim and the amino acids
of 3fra. i Proposed pose of
Iclaprim in the binding pocket
of DHFR
1 3

Molecular Diversity
Materials and methods
Chemistry
Melting points were determined in open capillary tubes
and are uncorrected. The reactions were monitored by thin
1
layer chromatography (TLC). The H NMR spectra were
recorded on 300 MHz spectrometers using DMSO as a
solvent. The 13C NMR spectra were recorded on 126 MHz
instruments using DMSO as a solvent. Mass spectra were
measured on an MALDI-TOF (Shimadzu, Japan). High-
resolution mass spectrometry was measured on a Thermo
Scientifc LTQ Orbitrap XL spectrometer. The other raw
materials and solvents were purchased from their respec-
tive suppliers and underwent no further purifcation. Puri-
fcations by column chromatography were conducted over
silica gel (200–300 mesh).
Fig. 4 DHFR inhibition assay. Data are represented as the
mean standard deviation of three independent experiments.
*p<0.05, signifcant with respect to the control
DHFR inhibition assay
We performed in vitro enzyme assays to test the inhibitory
efect of compound 7c and standard drugs (trimethoprim) on
DHFR activity (Fig. 4). The results indicated that compound
7c was potent DHFR inhibitors when compared to trimetho-
prim. Furthermore, at concentration of 10 μmol/L, compound
7c decreased DHFR activity to 70% compared with the nega-
tive control. These results imply that compound 7c possibly
displays their antibacterial activity through DHFR protein
inhibition.
General procedures for the synthesis of 4a, 7a–f,
10a and 13a
A mixture of hydroxyacetophenone (5 mmol) or paeonol
(5 mmol) and K2CO3 (5 mmol) in dry DMF (10 mL), the
corresponding substituted benzyl chloride (5 mmol) was
added to the stirred solution 4–7 h at 80 °C. After the reac-
tion is completed, the mixture was poured into ice water.
The resulting precipitate was fltered and recrystallized
with ethanol to obtain crude products (intermediates 2, 5,
8 and 11) which were purifed by column chromatogra-
phy (dichloromethane/petroleum ether = 10:1). To com-
pounds 2, 5, 8 and 11 (10 mmol), DMF-DMA (20 mmol)
was added, and the reaction mixture was heated at 80 °C
for 12 h to yield the desired products (intermediates 3,
6, 9 and 12). To a solution of compounds 3, 6, 9 and 12
(10 mmol) in acetic acid (10 mL), proper moroxydine
hydrochloride or metformin hydrochloride (10 mmol)
was added. The reaction mixture was heated at refux for
12 h, and acetic acid was evaporated under reduced pres-
sure to obtain crude products (compounds 4a, 7a–f, 10a
and 13a) which were purifed by column chromatography
(dichloromethane/methanol = 15:1).
Conclusions
In summary, totally 22 new analogues and hybrids bearing
the pyrimidine scafold were synthesized and their in vitro
antibacterial activities were investigated comprehen-
sively. Compounds 7c and 22b presented the most potent
inhibitory activity against Gram-positive bacteria and
Gram-negative bacteria, with MICs ranging from 2.4 to
5.2 μmol/L. Furthermore, compound 7c showed the most
potent antimicrobial activity (MIC = 2.4 μmol/L) against
selected MRSA strains. In vitro enzyme study implied that
compound 7c possibly displayed their antibacterial activity
through DHFR protein inhibition. These results suggested
that the pyrimidine derivatives bearing a benzyloxyben-
zaldehyde moiety, which play a critical role in increasing
the antibacterial properties of the compounds, represented
promising lead compounds for the development of novel
antibacterial agents.
3‑(4‑(2‑(2,4‑Dichlorobenzyloxy)phenyl)pyrimidin‑2‑yl)dimeth‑
1
ylguanidine (4a) Yield 47%, m.p. 153–155 °C. H NMR
(300 MHz, DMSO-d6, ppm): δ 9.04 (br s, 2H, NH), 8.64 (d,
J=5.3 Hz, 1H, Ar–H), 7.86 (dd, J=7.7, 1.7 Hz, 1H, Ar–H),
7.69 (d, J = 2.1 Hz, 1H, Ar–H), 7.60 (dd, J = 6.8, 4.4 Hz,
2H, Ar–H), 7.53–7.57 (m, 1H, Ar–H), 7.47 (dd, J = 8.3,
2.1 Hz, 1H, Ar–H), 7.34 (d, J=8.4 Hz, 1H, Ar–H), 7.17 (t,
J=7.5 Hz, 1H, Ar–H), 5.29 (s, 2H, CH2), 3.14 (s, 6H, CH3).
MS (MALDI-TOF) m/z 416 (M+ +H).
1 3

Molecular Diversity
3‑(4‑(4‑(2,6‑Dichlorobenzyloxy)phenyl)pyrimidin‑2‑yl)dimeth‑
ylguanidine (7a) Yield 47%, m.p. 169–170 °C. H NMR
1H NMR (300 MHz, DMSO-d6, ppm): δ 8.70 (br s, 2H,
NH), 8.53 (s, 1H, Ar–H), 8.09 (d, J = 7.9 Hz, 2H, Ar–H),
7.69 (d, J = 24.9 Hz, 2H, Ar–H), 7.52 (s, 2H, Ar–H),
7.18 (d, J = 7.9 Hz, 2H, Ar–H), 5.25 (s, 2H, CH2), 3.04
(d, J = 21.3 Hz, 8H, CH2). MS (MALDI-TOF) m/z 458
(M+ +H).
1
(300 MHz, DMSO-d6, ppm): δ 8.89 (br s, 2H, NH), 8.63 (d,
J=5.3 Hz, 1H, Ar–H), 8.16 (d, J=8.8 Hz, 2H, Ar–H), 7.63
(d, J=5.4 Hz, 1H, Ar–H), 7.59 (d, J=8.0 Hz, 2H, Ar–H),
7.50 (dd, J = 8.7, 7.5 Hz, 1H, Ar–H), 7.25 (d, J = 8.8 Hz,
2H, Ar–H), 5.35 (s, 2H, CH2), 3.14 (s, 6H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 163.74, 161.35, 158.54, 156.69,
136.55, 132.21, 131.81, 129.70, 129.25, 115.40, 109.35,
65.62, 38.32. MS (MALDI-TOF) m/z 416 (M+ +H).
3‑(4‑(3‑(2,4‑Dichlorobenzyloxy)phenyl)pyrimidin‑2‑yl)dimeth‑
ylguanidine (10a) Yield 42%, m.p. 116–117 °C. 1H NMR
(300 MHz, DMSO-d6, ppm): δ 8.60 (br s, 2H, NH), 7.65–
7.70 (m, 3H, Ar–H), 7.50–7.54 (m, 3H, Ar–H), 7.24 (s, 1H,
Ar–H), 6.62 (s, 2H, Ar–H), 5.24 (s, 2H, CH2), 3.08 (s, 6H,
CH3). 13C NMR (126 MHz, DMSO-d6) δ 172.18, 163.77,
136.94, 136.83, 135.41, 134.19, 133.89, 132.91, 125.21,
122.29, 118.41, 71.76, 70.24, 35.23, 23.87. HRMS (ESI)
m/z calcd for C20H20Cl2N5O (M+ + H): 416.10394, found
416.10394.
3‑(4‑(4‑(4‑Methylbenzyloxy)phenyl)pyrimidin‑2‑yl)dimeth‑
1
ylguanidine (7b) Yield 42%, m.p. 141–143 °C. H NMR
(300 MHz, DMSO-d6, ppm): δ 8.90 (br s, 2H, NH), 8.62 (d,
J=5.4 Hz, 1H, Ar–H), 8.12 (d, J=8.8 Hz, 2H, Ar–H), 7.63
(d, J=5.4 Hz, 1H, Ar–H), 7.36 (d, J=7.9 Hz, 2H, Ar–H),
7.21 (d, J = 7.9 Hz, 2H, Ar–H), 7.17 (d, J = 8.8 Hz, 2H,
Ar–H), 5.16 (s, 2H, CH2), 3.13 (s, 6H, CH3), 2.31 (s, 3H,
CH3). 13C NMR (126 MHz, DMSO-d6) δ 162.62, 161.25,
158.42, 156.83, 137.70, 134.12, 129.49, 129.26, 129.16,
129.06, 128.35, 115.62, 69.82, 38.21, 31.15. MS (MALDI-
TOF) m/z 362 (M+ +H).
3‑(4‑(2‑(2,4‑Dichlorobenzyloxy)‑4‑methoxyphenyl)pyrimi‑
din‑2‑yl)dimethylguanidine (13a) Yield 40%, m.p. 181–
183 °C. 1H NMR (300 MHz, DMSO-d6, ppm): δ 9.02 (br s,
2H, NH), 8.58 (s, 1H, Ar–H), 7.92 (s, 1H, Ar–H), 7.74 (s,
1H, Ar–H), 7.62 (s, 2H, Ar–H), 7.52 (s, 1H, Ar–H), 6.88 (s,
1H, Ar–H), 6.76 (dd, J=8.8, 2.2 Hz, 1H), 5.33 (s, 2H, CH2),
3.87 (s, 3H, CH3), 3.13 (s, 6H, CH3). MS (MALDI-TOF)
m/z 446 (M+ +H).
3‑(4‑(4‑(2,4‑Dichlorobenzyloxy)phenyl)pyrimidin‑2‑yl)dimeth‑
1
ylguanidine (7c) Yield 45%, m.p. 133–135 °C. H NMR
(300 MHz, DMSO-d6, ppm): δ 11.25 (s, 1H), 7.79 (s, 3H),
7.58–7.40 (m, 3H), 7.23 (dd, J = 9.8, 6.0 Hz, 2H), 6.90 –
6.53 (m, 2H), 5.16 (s, 2H), 3.80 (s, 3H), 2.24 (s, 3H). 13C
NMR (126 MHz, DMSO-d6) δ 166.36, 162.91, 160.37,
158.53, 158.12, 134.12, 133.78, 131.92, 130.77, 129.48,
128.81, 128.07, 115.43, 106.76, 66.92, 37.28. HRMS (ESI)
m/z calcd for C20H20Cl2N5O (M+ + H): 416.10394, found
416.10397.
General procedures for the synthesis of 17a–d
To a stirred solution of the corresponding substituted ace-
tophenone (2 mmol), acetylacetone (2.2 mmol) and ammo-
nium acetate (16 mmol) in glacial acetic acid (10 mL) were
added, and the reaction mixture was stirred under refux for
3 h. After the completion of reaction, the reaction mixture
was poured into water and the obtained precipitate was
fltered.
3‑(4‑(4‑(4‑Bromobenzyloxy)phenyl)pyrimidin‑2‑yl)dimeth‑
1
ylguanidine (7d) Yield 45%, m.p. 242–243 °C. H NMR
(300 MHz, DMSO-d6, ppm): δ 9.10 (br s, 2H, NH), 8.68 (d,
J=5.4 Hz, 1H, Ar–H), 8.17 (d, J=8.6 Hz, 2H, Ar–H), 7.76
(d, J=5.4 Hz, 1H, Ar–H), 7.62 (d, J=8.2 Hz, 2H, Ar–H),
7.45 (d, J = 8.0 Hz, 2H, Ar–H), 7.19 (d, J = 8.6 Hz, 2H,
Ar–H), 5.22 (s, 2H, CH2), 3.16 (s, 6H, CH3). MS (MALDI-
TOF) m/z 427 (M+ +H).
Dimethyl‑3‑(4‑(2‑methyl‑6‑(4‑pentylphenyl)pyridin‑3‑yl)
pyrimidin‑2‑yl)guanidine (17a) Yield 47%, m.p. 135–137 °C.
1H NMR (300 MHz, DMSO-d6, ppm): δ 8.50 (d, J=5.0 Hz,
1H, Ar–H), 8.24 (br s, 2H, NH), 8.04 (d, J = 8.2 Hz, 2H,
Ar–H), 7.87 (q, J=8.1 Hz, 2H, Ar–H), 7.32 (d, J=8.2 Hz,
2H, Ar–H), 6.94 (d, J = 5.0 Hz, 1H, Ar–H), 3.02 (s, 6H,
CH3), 2.60–2.65 (m, 5H, CH2CH3), 1.57–1.65(m, 2H, CH2),
3‑(4‑(4‑((4‑Cyanobenzyl)oxy)phenyl)pyrimidin‑2‑yl)‑1,1‑di‑
methylguanidine (7e) Yield 38%, m.p. 159–161 °C. H
1.27–1.34 (m, 4H, CH2), 0.86 (t, J=7.0 Hz, 3H, CH3). 13
C
1
NMR (300 MHz, DMSO-d6, ppm): δ 8.46 (br s, 2H, NH),
8.06 (s, 2H, Ar–H), 7.89 (d, J=8.1 Hz, 2H, Ar–H), 7.69 (s,
3H, Ar–H), 7.28 (s, 1H, Ar–H), 7.17 (s, 2H, Ar–H), 5.32 (s,
2H, CH2), 3.04 (s, 6H, CH3). MS (MALDI-TOF) m/z 373
(M+ +H).
NMR (126 MHz, DMSO-d6) δ 167.44, 165.19, 159.63,
158.24, 155.73, 144.25, 138.42, 136.09, 129.18, 127.09,
117.75, 111.66, 37.40, 36.06, 35.32, 31.34, 30.95, 24.10,
22.41, 14.37. MS m/z: 358 (M+ +H). HRMS (MALDI-TOF)
calcd for C17H20N5O2 (M+ +H): 358.1510, found: 358.1515.
N‑(4‑(4‑(2,4‑Dichlorobenzyloxy)phenyl)pyrimidin‑2‑yl)mor‑
pholine‑4‑carboxamidine (7f) Yield 45%, m.p. 128–130 °C.
Dimethyl‑3‑(4‑(2‑methyl‑6‑p‑tolylpyridin‑3‑yl)pyrimidin‑2‑yl)
guanidine (17b) Yield 42%, m.p. 174–176 °C. H NMR
1
1 3

Molecular Diversity
(300 MHz, DMSO-d6, ppm): δ 8.61 (d, J = 5.0 Hz, 1H,
Ar–H), 8.50 (br s, 2H, NH), 8.06 (d, J=8.1 Hz, 2H, Ar–H),
7.92 (dd, J=18.8, 8.1 Hz, 2H, Ar–H), 7.33 (d, J=8.1 Hz,
2H, Ar–H), 7.16 (d, J = 4.9 Hz, 1H, Ar–H), 3.07 (s, 6H,
CH3), 2.65 (s, 3H, CH3), 2.38 (s, 3H, CH3). MS (MALDI-
TOF) m/z 347 (M+ +H).
127.25, 108.47, 37.79, 35.39, 31.34, 30.88, 22.40, 14.36.
MS (MALDI-TOF) m/z 312 (M+ +H).
3‑(4‑(4‑(Dimethylamino)phenyl)pyrimidin‑2‑yl)dimethyl‑
1
guanidine (19e) Yield 42%, m.p. 221–222 °C. H NMR
(300 MHz, DMSO-d6, ppm): δ 8.29 (s, 2H), 7.28 (t,
J=6.0 Hz, 6H), 6.48 (ddd, J=16.3, 10.5, 4.1 Hz, 2H), 5.00
(s, 2H), 3.73 (s, 3H), 2.14 (s, 3H). 13C NMR (126 MHz,
DMSO-d6) δ 167.21, 164.84, 159.42, 157.43, 155.70,
153.04, 129.01, 112.08, 38.91, 31.15. MS (MALDI-TOF)
m/z 285 (M+ +H).
3‑(4‑(6‑(2,4‑Dichlorophenyl)‑2‑methylpyridin‑3‑yl)pyrimi‑
din‑2‑yl)dimethylguanidine (17c) Yield 45%, m.p. 135–
1
137 °C. H NMR (300 MHz, DMSO-d6, ppm): δ 8.58
(d, J = 5.0 Hz, 1H, Ar–H), 8.37 (br s, 2H, NH), 7.95 (d,
J = 8.0 Hz, 1H, Ar–H), 7.77 (d, J = 2.0 Hz, 1H, Ar–H),
7.66 (t, J = 8.3 Hz, 2H, Ar–H), 7.58 (dd, J = 8.3, 2.0 Hz,
1H, Ar–H), 7.08 (d, J = 5.0 Hz, 1H, Ar–H), 3.05 (s, 6H,
CH3), 2.61(s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 165.05, 158.47, 157.66, 155.84, 155.05, 137.82, 134.43,
133.43, 133.20, 132.69, 129.86, 128.11, 122.52, 112.55,
37.76, 31.14, 23.83. MS (MALDI-TOF) m/z 401 (M+ +H).
3‑(4‑([1,1′‑Biphenyl]‑4‑yl)pyrimidin‑2‑yl)‑1,1‑dimethyl‑
1
guanidine (22a) Yield 45%, m.p. 175–177 °C. H NMR
(300 MHz, DMSO-d6, ppm): δ 8.51 (s, 1H, NH), 8.36 (s,
1H, NH), 8.15 (d, J=7.7 Hz, 2H, Ar–H), 7.79 (dd, J=19.9,
7.7 Hz, 4H, Ar–H), 7.29–7.59 (m, 4H, Ar–H), 6.60 (s, 1H,
Ar–H), 3.05 (s, 6H, CH3). MS (MALDI-TOF) m/z 318
(M+ +H).
3‑(4‑(6‑(4‑Bromophenyl)‑2‑methylpyridin‑3‑yl)pyrimidin‑2‑yl)
dimethylguanidine (17d) Yield 45%, m.p. 182–184 °C. 1H
NMR (300 MHz, DMSO-d6, ppm): δ 8.56 (s, 1H, Ar–H),
8.34 (br s, 2H, NH), 8.12 (d, J = 8.5 Hz, 2H, Ar–H), 7.95
(s, 1H, Ar–H), 7.22 (d, J=8.4 Hz, 2H, Ar–H), 7.05 (s, 1H,
Ar–H), 3.04 (s, 6H, CH3), 2.64 (s, 3H, CH3). MS (MALDI-
TOF) m/z 411 (M+ +H).
3‑(4‑(2′ , 4′ ‑Dichloro ‑[1, 1′ ‑biphenyl]‑4‑yl)pyrimi‑
din‑2‑yl)‑1,1‑dimethylguanidine (22b) Yield 38%, m.p.
107–109 °C. 1H NMR (300 MHz, DMSO-d6, ppm): δ 8.29
(s, 2H), 7.28 (t, J=6.0 Hz, 6H), 6.48 (ddd, J =16.3, 10.5,
4.1 Hz, 2H), 5.00 (s, 2H), 3.73 (s, 3H), 2.14 (s, 3H). HRMS
(ESI) m/z calcd for C20H20Cl2N5O (M+ + H): 386.09338,
found 386.09348.
3‑(4‑(2,4‑Dichlorophenyl)pyrimidin‑2‑yl)dimethylguanidine
(19a) Yield 42%, m.p. 153–155 °C. 1H NMR (300 MHz,
DMSO-d6, ppm): δ 8.51 (d, J = 4.8 Hz, 1H, Ar–H), 8.22
(br s, 2H, NH), 7.78 (s, 1H, Ar–H), 7.59 (q, J=8.3 Hz, 2H,
Ar–H), 6.94 (d, J=4.9 Hz, 1H, Ar–H), 3.02 (s, 6H, CH3).
MS (MALDI-TOF) m/z 310 (M+ +H).
3‑(4‑(4‑(Hexyloxy)phenyl)pyrimidin‑2‑yl)dimethylguanidine
(26a) Yield 42%, m.p. 140–142 °C. 1H NMR (300 MHz,
DMSO-d6, ppm): δ 8.29 (s, 2H), 7.28 (t, J= 6.0 Hz, 6H),
6.48 (ddd, J = 16.3, 10.5, 4.1 Hz, 2H), 5.00 (s, 2H), 3.73
(s, 3H), 2.14 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
163.30, 161.29, 158.15, 158.00, 129.64, 128.78, 115.09,
107.36, 68.14, 37.55, 31.48, 29.07, 25.64, 22.54, 14.37. MS
(MALDI-TOF) m/z 342 (M+ +H).
Dimethyl‑3‑(4‑(naphthalen‑3‑yl)pyrimidin‑2‑yl)guanidine
(19b) Yield 38%, m.p. 131–132 °C. 1H NMR (300 MHz,
DMSO-d6, ppm): δ 8.29 (s, 2H), 7.28 (t, J= 6.0 Hz, 6H),
6.48 (ddd, J=16.3, 10.5, 4.1 Hz, 2H), 5.00 (s, 2H), 3.73 (s,
3H), 2.14 (s, 3H). MS (MALDI-TOF) m/z 292 (M+ +H).
Dimethyl‑3‑(4‑(4‑(pentyloxy)phenyl)pyrimidin‑2‑yl)guanidine
(26b) Yield 48%, m.p. 159–160 °C. 1H NMR (300 MHz,
DMSO-d6, ppm): δ 8.29 (s, 2H), 7.28 (t, J= 6.0 Hz, 6H),
6.48 (ddd, J = 16.3, 10.5, 4.1 Hz, 2H), 5.00 (s, 2H), 3.73
(s, 3H), 2.14 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
170.76, 168.08, 165.94, 163.13, 162.96, 134.59, 133.46,
123.63, 119.82, 72.86, 42.11, 33.55, 32.91, 27.12, 19.13.
MS (MALDI-TOF) m/z 328 (M+ +H).
3‑(4‑(4‑Bromophenyl)pyrimidin‑2‑yl)dimethylguanidine
1
(19c) Yield 40%, m.p. 144–146 °C. H NMR (300 MHz,
DMSO-d6, ppm): δ 8.29 (s, 2H), 7.28 (t, J= 6.0 Hz, 6H),
6.48 (ddd, J=16.3, 10.5, 4.1 Hz, 2H), 5.00 (s, 2H), 3.73 (s,
3H), 2.14 (s, 3H). MS (MALDI-TOF) m/z 321 (M+ +H).
Dimethyl‑3‑(4‑(4‑pentylphenyl)pyrimidin‑2‑yl)guanidine
(19d) Yield 42%, m.p. 129–131 °C. 1H NMR (300 MHz,
DMSO-d6, ppm): δ 8.29 (s, 2H), 7.28 (t, J= 6.0 Hz, 6H),
6.48 (ddd, J = 16.3, 10.5, 4.1 Hz, 2H), 5.00 (s, 2H), 3.73
(s, 3H), 2.14 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ
163.75, 163.37, 158.57, 157.62, 145.97, 134.78, 129.27,
Biological evaluation
In vitro antibacterial and antifungal activity
MIC assay for each test compound was performed as
described previously [24]. Testing was performed by the
1 3

Molecular Diversity
9. Wirnsberger G, Zwolanek F, Asaoka T, Kozieradzki I, Tortola L,
Wimmer RA, Kavirayani A, Fresser F, Baier G, Langdon WY, Ikeda
F, Kuchler K, Penninger JM (2016) Inhibition of CBLB protects
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didiasis: a persistent public health problem. Clin Microbiol Rev
20:133–163
standard broth microdilution method with trimethoprim and
gatifoxacin. All stock solutions of the compounds were dis-
solved in DMSO. Bacteria growth was determined by meas-
uring the absorption at 630 nm using a microtiter enzyme-
linked immunosorbent assay (ELISA) reader.
11. Denning DW, Bromley MJ (2015) How to bolster the antifungal
pipeline. Science 347:1414–1416
Toxicity evaluation
12. Brown GD, Denning DW, Levitz SM (2012) Tackling human fungal
infections. Science 336:647
Toxicity test was performed using MTT assay, where all
instructions were performed typically to our previous work
[24]. For this study, all compounds were tested against a
normal human liver cell line (L02) for 48 h at 37 °C.
13. Khalaf-Nezhad A, Soltani Rad MN, Mohabatkar H, Asrari Z, Hem-
mateenejad B (2005) Design, synthesis, antibacterial and QSAR
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Krishna NS (2014) Synthesis, antimicrobial and cytotoxic activi-
ties of pyrimidinyl benzoxazole, benzothiazole and benzimidazole.
Eur J Med Chem 77:1–7
Molecular modeling
15. Desai NC, Kotadiya GM, Trivedi AR (2014) Studies on molecular
properties prediction, antitubercular and antimicrobial activities of
novel quinoline based pyrimidine motifs. Bioorg Med Chem Lett
24:3126–3130
Molecular docking protocol was followed according to the
reported method [24]. All docking runs were carried out
using Discovery Studio v17.1.0.16143. The 3D structure of
3FRA in docking study was downloaded from Protein Data
Bank. For protein preparation, the hydrogen atoms were
added, and water and impurities were removed.
16. Jin X, Zheng CJ, Song MX, Wu Y, Sun LP, Li YJ, Yu LJ, Piao HR
(2012) Synthesis and antimicrobial evaluation of l-phenylalanine-
derived C5-substituted rhodanine and chalcone derivatives con-
taining thiobarbituric acid or 2-thioxo-4-thiazolidinone. Eur J Med
Chem 56:203–209
17. Zhang TY, Li C, Li YR, Li XZ, Sun LP, Zheng CJ, Piao HR (2016)
Synthesis and antimicrobial evaluation of aminoguanidine and
3-amino-1,2,4-triazole derivatives as potential antibacterial agents.
Lett Drug Des Discov 13:1063–1075
DHFR inhibition assay
Solid-phase antibody was prepared by coating the microti-
ter plate wells with purifed human dihydrofolate reductase
(DHFR) antibody. To see the efect of lead inhibitor (7c) on
DHFR activities, ELISA assay was performed as described
previously [24]. All assays were performed in triplicate.
18. Davari AS, Abnous K, Mehri S, Ghandadi M, Hadizadeh F (2014)
Synthesis and biological evaluation of novel pyridine derivatives
as potential anticancer agents and phosphodiesterase-3 inhibitors.
Bioorg Chem 57:83–89
19. Seleem MA, Disouky AM, Mohammad H, Abdelghany TM, Mancy
AS, Bayoumi SA, Elshafeey A, El-Morsy A, Seleem MN, May-
houb AS (2016) Second-generation phenylthiazole antibiotics with
enhanced pharmacokinetic properties. J Med Chem 59:4900–4912
20. Eldehna WM, Altoukhy A, Mahrous H, Abdel-Aziz HA (2015)
Design, synthesis and QSAR study of certain isatin-pyridine hybrids
as potential anti-proliferative agents. Eur J Med Chem 90:684–694
21. Li B, Pai R, Di M, Aiello D, Barnes MH, Butler MM, Tashjian TF,
Peet NP, Bowlin TL, Moir DT (2012) Coumarin-based inhibitors of
Bacillus anthracis and Staphylococcus aureus replicative DNA heli-
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activities. J Med Chem 55:10896–10908
Acknowledgements This work was supported by the Doctoral Foun-
dation of Jilin Medical University (No. JYBS2018007), the Health
Department of Jilin Province (2018ZC034) and the Department of
Education of Jilin Province (No. JJKH20191070KJ).
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