Design, synthesis and evaluation of dihydrotriazine derivatives-bearing 5-aryloxypyrazole moieties as antibacterial agents

Article abstract of DOI:10.1007/s11030-020-10071-9

Abstract: In the present investigation, a series of dihydrotriazine derivatives-bearing 5-aryloxypyrazole moieties were synthesized and their structures were confirmed by different spectral tools. The biological evaluation in vitro revealed that some of the target compounds exerted good antibacterial and antifungal activity in comparison with the reference drugs. Among these novel hybrids, compound 10d showed the most potent activity with minimum inhibitory concentration values (MIC) of 0.5?μg/mL against S. aureus 4220, MRSA 3506 and E. coli 1924 strain. The cytotoxic activity of the compounds 6d, 6m, 10d and 10g was assessed in MCF-7 and HeLa cells. Growth kinetics study showed significant inhibition of bacterial growth when treated with different conc. of 10d. In vitro enzyme study implied that compound 10d exerted its antibacterial activity through DHFR inhibition. Moreover, significant inhibition of biofilm formation was observed in bacterial cells treated with MIC conc. of 10d as visualized by SEM micrographs. Graphic abstract: Twenty-nine target compounds were designed, synthesized and evaluated in terms of their antibacterial and antifungal activities.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s11030-020-10071-9

Molecular Diversity
https://doi.org/10.1007/s11030-020-10071-9
ORIGINAL ARTICLE
Design, synthesis and evaluation of dihydrotriazine
derivatives‑bearing 5‑aryloxypyrazole moieties as antibacterial agents
Tian‑Yi Zhang1 · Chun‑Shi Li2 · Ming‑Yue Cui2 · Xue‑Qian Bai1 · Jiang‑Hui Chen1,3 · Ze‑Wen Song1,3 · Bo Feng1 ·
Xue‑Kun Liu4
Received: 17 December 2019 / Accepted: 6 March 2020
© Springer Nature Switzerland AG 2020
Abstract
In the present investigation, a series of dihydrotriazine derivatives-bearing 5-aryloxypyrazole moieties were synthesized
and their structures were confrmed by diferent spectral tools. The biological evaluation in vitro revealed that some of the
target compounds exerted good antibacterial and antifungal activity in comparison with the reference drugs. Among these
novel hybrids, compound 10d showed the most potent activity with minimum inhibitory concentration values (MIC) of
0.5 µg/mL against S. aureus 4220, MRSA 3506 and E. coli 1924 strain. The cytotoxic activity of the compounds 6d, 6m,
10d and 10g was assessed in MCF-7 and HeLa cells. Growth kinetics study showed signifcant inhibition of bacterial growth
when treated with diferent conc. of 10d. In vitro enzyme study implied that compound 10d exerted its antibacterial activity
through DHFR inhibition. Moreover, signifcant inhibition of bioflm formation was observed in bacterial cells treated with
MIC conc. of 10d as visualized by SEM micrographs.
Tian-Yi Zhang and Chun-Shi Li have contributed equally to this
work.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10071-9) contains
supplementary material, which is available to authorized users.
* Tian-Yi Zhang
tianyizhang@126.com
* Bo Feng
jyfengb@126.com
* Xue-Kun Liu
liuxuekunth@126.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 Pharmacy, Yanbian University, Yanji 133002,
People’s Republic of China
4
School of Pharmacy Medicine, Tonghua Normal University,
Tonghua 134002, Jilin Province, People’s Republic of China
Vol.:(0123456789)
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Molecular Diversity
Graphic abstract
Twenty-nine target compounds were designed, synthesized and evaluated in terms of their antibacterial and antifungal
activities.
Keywords 5-Aryloxypyrazole · Dihydrotriazine · Antibacterial activity · Growth kinetics · DHFR inhibition
are efective against a broad range of bacteria, including
those which have developed resistance to known drugs, has
become an urgent challenge [4].
Introduction
Antimicrobial resistance (AMR) is a complex global health
issue increasingly accelerated by selective pressure resulting
from the use and misuse of antimicrobial agents in humans
and animals [1]. The CDC currently estimates that antibi-
otic-resistant infections are responsible for at least 2 million
illnesses and 23,000 deaths each year in the USA, with direct
societal costs as high as $20 billion [2]. Compounding the
issue of antibiotic resistance is the fact that very few new
drugs have been approved to treat Gram-negative infections
in recent decades, and the majority of these have come from
well-known classes of drugs discovered half a century ago
[3]. Therefore, discovery of novel classes of antibiotics that
In the quest for new antibacterial agents, several hetero-
cyclic scafolds have been explored. Among them, pyrazoles
represent a key structural motif in heterocyclic chemistry
and occupy a signifcant position in medicinal and pesticide
chemistry because of their capability to exhibit a wide range
of biological activities, including antibacterial [5, 6], anti-
infammatory [7] and fungistatic [8]. The use of pyrazole
derivatives as potential antimicrobial agents has received
considerable attention following the discovery of the natu-
ral pyrazole C-glycoside, pyrazofurin that demonstrated a
broad spectrum of antimicrobial activity [9]. Furthermore,
1 3

Molecular Diversity
dihydrofolate reductase (DHFR), a key enzyme in the folate
pathway, is responsible for reducing dihydrofolic acid to tet-
rahydrofolic acid. Tetrahydrofolate is involved in one carbon
transfer reactions that lead to the biosynthesis of nucleic
acids and amino acids. Thus, the inhibition of DHFR is able
to arrest cell growth by depleting the cellular pool of DNA,
RNA and protein synthesis precursors [10, 11]. DHFR has
been recognized as a validated drug target for antibacterial
agents [12].
bacteria and fungi, including some clinically isolated drug-
resistance microorganisms (Fig. 1) [15, 16]. In the present
study, by use of compounds A–D as a starting template, we
hypothesized that combining the structural features of 5-ary-
loxypyrazole and dihydrotriazine moieties could potentially
have benefcial efect on the antibacterial activity (Fig. 2).
The synthesized compounds were characterized and tested
for their in vitro antibacterial activity. In addition, their
structure–activity relationships, cytotoxicity and preliminary
antibacterial mechanism were studied.
The design of the presented set of compounds was based
on our previous rhodanine derivatives which had more
potent inhibitory activities against Gram-positive bacteria,
as exemplifed by compounds A and B (MIC=2 µg/mL and
16 µg/mL, respectively) (Fig. 1) [13, 14]. Unfortunately,
compounds belonging to this series did not show any bacte-
riostatic activity against Gram-negative bacteria. In contrast,
our previous work indicated that compounds C and D, the
dihydrotriazine derivatives containing pyrazole or quinoline
moieties, had strong antimicrobial activities against many
microorganisms such as Gram-negative, Gram-positive
Results and discussion
Chemistry
As shown in Scheme 1, ethyl acetoacetate was reacted with
phenylhydrazine or 2,4-dichlorophenylhydrazine to aford
3-methyl-1-phenyl-1H-pyrazol-5-ol (2 or 7). The intermedi-
ates (2 or 7) were treated with phosphorus oxychloride to
Fig. 1 Chemical structures of
some reported compounds and
their MICs against S. aureus
A
B
C
D
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Molecular Diversity
Fig. 2 Design of target compounds based on the combination principles
Scheme 1 Synthetic scheme for the synthesis of the target com-
pounds. Reagents and conditions: (a) phenylhydrazine or 2,4-dichlo-
rophenylhydrazine, EtOH, Conc. HCl, 10% NaOH, 1 h, (b) POCl3,
DMF, 0 °C, 0.5 h, 80 °C, 3 h, (c) ROH, K2CO3, DMF, 80 °C, over-
night, (d) metformin hydrochloride or phenformin hydrochloride,
AcOH, 120 °C, 4–8 h
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Molecular Diversity
give 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbalde-
hyde (3 or 8), which was subsequently reacted with diferent
substituted phenols to provide the intermediates (4 or 9).
Then, a series of intermediates (4 or 9) were reacted with
metformin hydrochloride or phenformin hydrochloride in
acetic acid to generate the target compounds. Before biologi-
cal evaluation, all target compounds were characterized via
1H-NMR, 13C-NMR and MS.
physicochemical properties of the diferent substituents on
the phenyl ring. Bioisosteric replacements of a dimethyl
group at the N-position of the dihydro-1,3,5-triazine ring
weakened the antibacterial activity. Based on these results,
we reasonably hypothesized that the phenethyl group of
the dihydro-1,3,5-triazine ring is an important structural
feature that gives rise to promising antibacterial activity.
Antibacterial activity
Cytotoxicity assessment by MTT assay
Minimum inhibitory concentration (MIC) values of these
compounds were calculated using standard methods. Anti-
bacterial activity of the synthesized compounds was com-
pared with trimethoprim, gatifoxacin, moxifoxacin and
fuconazole. The compounds were screened against four
Gram-positive strains (S. aureus 4220, QRSA CCARM
3505, MRSA CCARM 3506 and S. mutans 3289) and
Gram-negative strains (E. coli 1924, P. aeruginosa 2742
and S. typhimurium 2421), as well as one fungus (C. albi-
cans 7535), and the results are shown in Table 1. Against
the Gram-positive S. aureus 4220, compounds (10c, 10d
and 10e) had the highest activity, with an MIC value of
0.5 µg/mL, and this was slightly lower than gatifoxacin
(MIC=0.25 µg/mL) and moxifoxacin (MIC=0.25 µg/mL).
Against the Gram-negative E. coli 1924, compounds (10a–f
and 10h–j) were more potent than the positive controls gati-
foxacin and moxifoxacin (MIC=2 µg/mL), with an MIC
of 0.5 µg/mL. Compounds 10b–f and 10j exhibited twofold
more potent activity relative to trimethoprim (MIC=32 µg/
mL) with MIC values of 16 µg/mL against P. aeruginosa
2742, but weaker than that of gatifoxacin and moxifoxa-
cin (MIC=1 µg/mL). Regretfully, none of the compounds
showed any inhibitory activity against the Gram-negative
strain S. typhimurium 2421 (MICs>64 µg/mL). Compounds
(10b–f and 10h–k) presented the highest activity with MIC
values of 0.5 µg/mL against MRSA CCARM 3506, showed
the twofold more potent activity relative to the standard drug
moxifoxacin (MIC=1 µg/mL), and eightfold more potent
activity relative to trimethoprim (MIC=4 µg/mL). For the
fungus strains, compounds 10d and 10 k also displayed good
potency, with an MIC of 2 µg/mL, which was slightly lower
than that of fuconazole (MIC=1 µg/mL).
The cytotoxicity is one of the most essential criteria to
evaluate the potential of antimicrobial agents. In order to
identify the safety profle, the highly active compounds 6d,
6m, 10d and 10g were further evaluated for their toxicities
against human breast cancer cells (MCF-7) and human
cervical cells (HeLa) line using the colorimetric cell pro-
liferation MTT assay. As shown in Table 2, compound
6d exhibited slightly weaker activity than 10d against the
diferent bacteria, in spite of its greater cytotoxicity than
10d, comparably indicating that the antibacterial activities
exhibited by dihydrotriazine derivatives were not due to
the cytotoxic efects.
Molecular docking
Molecular docking is considered as one of the convenient
and efective theoretical methods in binding studies [18].
The crystal structure data were obtained from the Protein
Data Bank (PDB code: 3fra), which was representative target
to investigate the antibacterial mechanism. The structures
of 5d, 6d and 10d were sketched in 2D and converted into
3D using the DS molecule editor (Fig. 3). Compound 5d is
bound into the active site, in which the benzene ring at the
N1 position of the pyrazole formed alkyl bond with Leu20.
The 2,4-di-Cl-substituted phenyl ring of 5d formed pi-alkyl
bond with Phe92 and Ile14. Compound 6d is bound into
the active site where the thiazine N atom shows interac-
tion with Thr46. The 2,4-di-Cl substituted phenyl ring of
5d shows interaction with Phe92 and Leu5. The pyrazole
formed alkyl bond with Leu20. The phenethyl N atom of 6d
shows interaction with Gln95. Compound 10d is bound into
the active site, in which the position of the di-Cl substituent
on the benzene ring at the N1 position of the pyrazole formed
alkyl bond with Leu20. The most stable contacts observed
in 10d shared four key binding residues including Phe92,
Leu5, Thr46 and Ile14. Furthermore, with respect to DHFR
enzyme, the two compounds were docked with the active
domain and also the results indicated that the binding mode
of compound 10d was better than compound 5d (Fig. 3c, i).
These molecular docking data provide certain theoretical
support for experimental results and the next optimization.
Detailed structure–activity relationships (SAR) analy-
sis on the dihydrotriazine and 5-aryloxypyrazole moie-
ties revealed several structural features that are crucial
to maintain the antibacterial activity. In general, both the
position and the number of 2,4-di-Cl atom at the N1 posi-
tion of the pyrazole ring play a very important role in the
antibacterial potency, as exemplifed by the compounds in
series 10. Furthermore, no clear structure–activity rela-
tionships were observed, indicating that the antibacterial
activity was not signifcantly afected by the position or
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Molecular Diversity
Table 1 Antibacterial data as MICa (μg/mL) for target compounds 5a–d, 6a–n and 10a–k
R1
R2
Gram-positive strains
Fungus
Gram-negative strains
S. aureus QRSA MRSA S. mutans C. albicans E. coli P. aeruginosa S. typhimurium
4220b
3505c 3506d 3289e
7535f
1924g 2742h
2421i
5a
2-Cl
3-Cl
4-Cl
2,4-di-Cl
2-Cl
3-Cl
4-Cl
2,4-di-Cl
3-Br
4-Br
NHMe2
NHMe2
NHMe2
NHMe2
64
64
64
32
2
2
2
1
2
2
4
4
4
4
2
8
2
1
1
>64
>64
>64
64
16
16
16
8
16
16
32
32
64
32
32
64
32
32
16
8
8
8
8
8
16
16
16
16
16
8
4
nd
4
64
32
64
32
2
2
2
1
1
1
4
2
2
2
1
4
2
1
1
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
2
>64
>64
>64
64
8
8
4
4
8
4
16
8
16
8
4
16
4
4
2
2
2
1
2
1
4
2
2
2
2
0.25
0.25
nd
0.5
>64
>64
>64
>64
16
16
16
8
16
16
64
64
32
32
32
64
8
8
4
4
4
2
4
4
16
8
64
32
64
32
4
4
2
1
2
2
4
4
4
2
2
4
2
1
0.5
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
1
64
32
32
32
32
32
64
>64
64
64
64
>64
64
64
64
>64
64
64
32
16
16
16
16
16
32
32
32
16
32
1
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
5b
5c
5d
6a
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
6b
6c
6d
6e
6f
6g
4-F
6h
2-CH3
3-CH3
4-CH3
2,4-di-CH3
3-OCH3
3-CF3
Phenyl(3,4-fused) NH(CH2)2Ph
2-Cl
3-Cl
4-Cl
6i
6j
6k
6l
6m
6n
10a
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph 0.5
NH(CH2)2Ph 0.5
NH(CH2)2Ph 0.5
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
NH(CH2)2Ph
10b
1
10c
10d
2,4-di-Cl
3-Br
4-Br
10e
10f
1
2
1
1
1
1
0.25
0.25
nd
32
10g
4-F
10h
4-CH3
2,4-di-CH3
3-CF3
10i
4
4
2
nd
nd
1
10j
10k
Phenyl(3,4-fused) NH(CH2)2Ph
Gatifoxacin
Moxifoxacin
Fluconazole
Trimethoprim
2
2
nd
4
1
nd
4
1
nd
32
0.5
Nd
2
2
nd not determined
aMICs were determined by microbroth dilution method for microdilution plates
bS. aureus 4220
cQRSA 3505
dMRSA 3506
eS. mutans 3289
fCandida albicans 7535
gE. coli 1924
hPseudomonas aeruginosa 2742
iSalmonella typhimurium 2421
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Molecular Diversity
Table 2 Antibacterial activity and cytotoxicity for 6d, 6m, 10d and
the analogues were active antibacterial compounds inhibit-
ing dihydrofolate reductase enzyme.
10g
Test organisms 6d
MIC (µmol/L) S. aureus 4220 1.87
1.87
6m
3.75
3.75
10d
0.83
0.83
10g
Growth kinetic studies
3.6
1.81
MRSA 3506
MCF-7b
There is always a need to discover rapidly bactericidal agents
to combat antibacterial resistance, and time–kill kinetics is
an important method to judge the efcacy of investigational
antimicrobial agents [17, 18]. Based on the DHFR inhibi-
tion as well as the antibacterial efect on sensitive/resistant
bacterial strains, compound 10d was evaluated for its efect
on the growth of S. aureus bacterial strains. The results
showed that 10d exerted signifcant efect on the growth
pattern of these strains and completely inhibited the growth
of S. aureus even at sub-MIC values with a continuous lag-
phase of 32 h. Furthermore, at 2MIC and 4MIC values, no
growth was observed in case of S. aureus and E. coli even
after 32 h of incubation. Therefore, it can be concluded that
10d is bacteriostatic in nature and efectively arrests the bac-
terial growth (Fig. 5).
ICa50 (µmol/L)
7.61 30.13 29.11 35.10
2.32 14.30 22.02 15.68
HeLac
aIC50 is defned as the concentration to inhibit the cell growth by 50%
bHuman breast cancer cells
cHuman cervical cells
Inhibition studies of compound 10d with DHFR
We assayed compounds 10d (MIC of 0.5 µg/mL) and
standard drugs (trimethoprim) against DHFR in order to
confrm that the observed antibacterial activity was clearly
infuenced by this enzyme inhibition (Fig. 4). The results
indicated that compound 10d rendered a good inhibitory
potency against the enzymes as compared to the standard
inhibitors. Furthermore, at concentrations of 10 µmol/L,
compound 10d decreased DHFR activity by 43% com-
pared with the negative control. The results suggested that
Assessment of bioflm formation
Bacterial bioflm, a complex bacterial lifestyle adapta-
tion, provides a protection from environmental stresses
Fig. 3 a Three-dimensional
conformation of compound 5d
docked in DHFR complex. b
2D molecular docking modeling
of compound 5d with 3fra. c
Interaction of 5d with DHFR
inside the binding pocket. d
Three-dimensional conforma-
tion of compound 6d docked in
DHFR complex. e 2D molecular
docking modeling of compound
6d with 3fra. f Interaction of 6d
with DHFR inside the binding
pocket. g Three-dimensional
conformation of compound 10d
docked in DHFR complex. h
2D molecular docking modeling
of compound 10d with 3fra. i
Interaction of 10d with DHFR
inside the binding pocket
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Molecular Diversity
Fig. 3 (continued)
[19]. When bioflms accompany bacterial infections, they
make the infections extremely difcult to treat because
bacterial cells in a bioflm exhibit stronger resistance to
both the host immune system and antibiotic treatments
[20]. Therefore, once bioflms have colonized the wound,
the morbidity and mortality of skin–wound patients, as
well as treatment cost, will greatly increase. To see the
efect of lead inhibitor, SEM analysis was performed to
visualize the efect of 10d on bioflm formation using S.
aureus strain. Signifcant inhibition of bioflm develop-
ment was observed in S. aureus on treatment with MIC
concentration (0.5 µg/mL) of compound 10d. Cluster-
ing of cells which signifes the formation of bioflm was
observed in untreated samples, while scattered cells with
poor cell density were observed in treated cells. So, it can
be concluded that the 10d may have the potential to be an
antibioflm agent as well as an antibacterial agent (Fig. 6).
Fig. 4 Inhibition of DHFR activities of compounds 10d and trimeth-
oprim. Data are represented as the mean standard deviation of three
independent experiments. ***p<0.05, signifcant with respect to the
control
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Molecular Diversity
Fig. 5 Efect of diferent concentrations of compound 10d on the growth kinetic studies
Fig. 6 Scanning electron
microscopy of bioflm: a
untreated, left; b treated with
MIC concentration of com-
pound 10d, right
Conclusions
Materials and methods
To summarize, three novel series of dihydrotriazine deriv-
atives-bearing 5-aryloxypyrazole moieties were prepared
via a convenient synthetic route and their antibacterial
activity was evaluated. The structure–activity relation-
ships were summarized which provided some important
guidance for the development of antibacterial agents.
Compound 10d presented the most potent inhibitory
activity against Gram-positive bacteria (S. aureus 4220,
MRSA 3506) and Gram-negative bacteria (E. coli 1924),
with minimum inhibitory concentration values of 0.5 µg/
mL. Compound 10d also inhibited the growth of various
bacterial strains at diferent concentrations as signifed
by growth kinetics study. In vitro enzyme study implied
that compound 10d possibly displayed their antibacterial
activity through DHFR protein inhibition. Furthermore,
compound 10d also inhibited the formation of bioflm in
S. aureus as seen in SEM micrographs.
General procedures
All commercially available reagents and solvents employed
were used without further purifcation. All reactions were
monitored by thin-layer chromatography (TLC) performed
on silica gel plates. Melting points were determined in open
1
capillaries and were uncorrected. Chemical shifts in H
NMR and 13C NMR spectra are reported in parts per mil-
1
lion (ppm) relative to tetramethylsilane (TMS). H NMR
and 13C NMR spectra were performed on an AV-300 or
AV-500 spectrometer (Bruker, Zurich, Switzerland) oper-
ating at 300 MHz for H and 126 MHz for 13C and using
1
DMSO-d6 as solvent. The following abbreviations were used
to describe peak patterns: s=singlet, d=doublet, t=triplet,
q=quadruplet, m=multiplet. Coupling constants (J) were
expressed in hertz unit (Hz). Mass spectra were measured
on an MALDI-TOF (Shimadzu, Japan).
1 3

Molecular Diversity
48%; m.p. 140–142 °C. 1H NMR (300 MHz, DMSO-d6) δ
8.77 (s, 1H, NH), 8.41 (s, 1H, NH2), 7.49 (t, J=8.5 Hz, 3H,
Ar–H), 7.40 (t, J=7.8 Hz, 2H, Ar–H), 7.29 (d, J=7.2 Hz,
1H, Ar–H), 7.17 (d, J = 7.8 Hz, 1H, Ar–H), 7.06 (d,
J=7.8 Hz, 1H, Ar–H), 6.74 (d, J=8.3 Hz, 1H, Ar–H), 5.78
(s, 1H, CH), 2.81 (s, 6H, CH3), 2.36 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 157.63, 156.11, 151.82, 147.98,
145.09, 137.51, 130.93, 129.78, 128.95, 127.92, 125.07,
122.21, 121.42, 115.45, 108.60, 56.48, 55.84, 55.39, 37.09,
19.03, 13.79. MS (MALDI-TOF) m/z 424 (M+1).
General synthetic procedure for the key
intermediates 2 and 7
To a stirred solution of acetylacetic ether (0.1 mol) and 70%
ethanol (5.0 mL), the solution of phenylhydrazine (0.1 mol)
with absolute alcohol (3.0 mL) was added dropwise at 45 °C,
and the mixture was stirred for 20 min at this temperature.
Cooled to 20 °C, concentrated hydrochloric acid (1.0 mL)
was added dropwise and made to react for 2 h at 45 °C.
Then, the PH value of mixture was adjusted to 7 using 10%
NaOH solution, added 20 mL water and stirred for 1 h at
the room temperature. The precipitated solid was fltered
and washed with cold absolute alcohol to get a white solid
(2 and 7).
6‑(5‑(3‑Chlorophenoxy)‑3‑methyl‑1‑phenyl‑1H‑pyra-
zol‑4‑yl)‑N2,N2‑dimethyl‑3,6‑dihydro‑1,3,5‑tria-
zine‑2,4‑diamine (5b) White solid, Yield 46%; m.p.
1
138–140 °C. H NMR (300 MHz, DMSO-d6) δ 8.79
(s, 1H, NH), 8.41 (s, 1H, NH2), 7.53 (d, J = 7.4 Hz, 2H,
Ar–H), 7.42 (t, J=7.8 Hz, 2H, Ar–H), 7.31 (s, 2H, Ar–H),
7.12 (d, J = 7.5 Hz, 1H, Ar–H), 6.98 (s, 1H, Ar–H), 6.86
(d, J = 8.3 Hz, 1H, Ar–H), 5.76 (s, 1H, CH), 2.82 (s, 6H,
CH3), 2.36 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 157.73, 157.18, 156.00, 148.07, 145.48, 137.54, 134.38,
131.78, 129.88, 127.89, 124.13, 122.38, 115.89, 114.43,
108.05, 56.48, 55.84, 55.39, 37.05, 19.02, 13.81. MS
(MALDI-TOF) m/z 424 (M+1).
General synthetic procedure for the key
intermediates 3 and 8
To a cold, stirred solution of dimethylformamide (1 mmol)
and phosphorous oxychloride (6 mmol) was added com-
pound 2 (1 mmol). The reaction mixture was stirred at
80 °C for 3 h, cooled to room temperature and poured into
ice–cold water whereupon a solid separated out that was
fltered, washed with excess of cold water and dried to aford
a yellow solid (3 and 8).
6‑(5‑(4‑Chlorophenoxy)‑3‑methyl‑1‑phenyl‑1H‑pyra-
zol‑4‑yl)‑N2,N2‑dimethyl‑3,6‑dihydro‑1,3,5‑tria-
zine‑2,4‑diamine (5c) White solid, Yield 45%; m.p. 141–
143 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.82 (s, 1H, NH),
8.41 (s, 1H, NH2), 7.52 (d, J=7.4 Hz, 2H, Ar–H), 7.41 (t,
J=7.8 Hz, 2H, Ar–H), 7.32 (t, J=9.2 Hz, 3H, Ar–H), 6.93
(d, J = 9.0 Hz, 2H, Ar–H), 5.73 (s, 1H, CH), 2.83 (s, 6H,
CH3), 2.35 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 157.76, 156.11, 155.38, 148.03, 145.80, 137.56, 130.14,
129.85, 127.8, 122.41, 117.44, 107.85, 56.48, 55.86, 55.39,
37.08, 19.03, 13.85. MS (MALDI-TOF) m/z 424 (M+1).
General synthetic procedure for the key
intermediates 4 and 9
To a solution of compound 3 (10 mmol) and K2CO3
(15 mmol) in dry dimethylformamide (10 mL), correspond-
ing substituted phenols (11 mmol) were added and mixture
was stirred overnight at 80 °C. After the completion of reac-
tion, the mixture was poured into 100 mL ice–water and
fltered to obtain a white solid. The resulting crude solid (4
and 9) was directly used in the next step without purifcation.
6‑(5‑(2,4‑Dichlorophenoxy)‑3‑methyl‑1‑phenyl‑1H‑pyra-
zol‑4‑yl)‑N2,N2‑dimethyl‑3,6‑dihydro‑1,3,5‑tria-
zine‑2,4‑diamine (5d) White solid, Yield 45%; m.p. 152–
154 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.79 (s, 1H, NH),
8.47 (s, 1H, NH2), 7.66 (d, J=2.5 Hz, 1H, Ar–H), 7.51–7.39
(m, 4H, Ar–H), 7.29 (dd, J=14.6, 7.9 Hz, 2H, Ar–H), 6.76
(d, J = 8.9 Hz, 1H, Ar–H), 5.80 (s, 1H, CH), 2.85 (s, 6H,
CH3), 2.36 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 157.57, 156.06, 150.94, 148.05, 144.74, 137.35, 130.30,
129.86, 128.83, 128.13, 122.61, 122.33, 116.78, 108.74,
56.48, 55.78, 55.39, 37.11, 19.03, 13.73. MS (MALDI-TOF)
m/z 458 (M+1).
General synthetic procedure for the target
compounds 5a–d, 6a–n and 10a–k
Synthesized intermediate compounds (4 and 9)
(1 mmol) and metformin hydrochloride or phen-
formin hydrochloride (1 mmol) were refluxed in gla-
cial acetic acid (10 mL) at 120 °C for 4–6 h. The whole
processes of the reactions were traced by TLC
and then removed solvent under reduced pres-
sure. The crude products were purified by col-
u m n c h r o m a t o g r a p h y ( d i c h l o r o m e t h a n e /
methanol=20:1).
6 ‑ ( 5 ‑ ( 2 ‑ C h l o r o p h e n o x y ) ‑ 3 ‑ m e t h y l ‑ 1 ‑ p h e -
nyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (6a) White solid, Yield 52%; m.p.
6 ‑ ( 5 ‑ ( 2 ‑ C h l o r o p h e n o x y ) ‑ 3 ‑ m e t h y l ‑ 1 ‑ p h e -
nyl‑ 1H‑ pyrazol‑ 4‑ yl)‑ N2,N2‑ dimethyl‑ 3,6‑ d-
ihydro‑1,3,5‑triazine‑2,4‑diamine (5a) White solid, Yield
1 3

Molecular Diversity
101–103 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.78 (s, 1H,
NH), 8.69 (s, 1H, NH), 8.48 (s, 1H, NH2), 7.89 (s, 1H, NH2),
7.50 (d, J=7.5 Hz, 2H, Ar–H), 7.42 (dd, J=15.3, 7.9 Hz,
3H, Ar–H), 7.32–7.26 (m, 3H, Ar–H), 7.19 (dd, J= 14.7,
7.2 Hz, 4H, Ar–H), 7.05 (dd, J=10.8, 4.5 Hz, 1H, Ar–H),
6.81 (t, J=13.4 Hz, 1H, Ar–H), 5.73 (s, 1H, CH), 2.71 (t,
J=7.2 Hz, 2H, CH2), 2.51–2.48 (m, 2H, CH2), 2.32 (s, 3H,
CH3). 13C NMR (126 MHz, DMSO-d6) δ 158.22, 157.13,
151.82, 147.90, 145.53, 139.34, 137.45, 131.03, 129.80,
129.15, 128.81, 128.01, 126.73, 125.23, 122.33, 121.51,
115.69, 107.94, 56.49, 55.39, 41.90, 35.58, 19.03, 13.80.
MS (MALDI-TOF) m/z 500 (M+1).
NMR (126 MHz, DMSO-d6) δ 158.12, 157.00, 150.96,
147.95, 145.04, 139.31, 137.30, 130.38, 129.88, 129.12,
128.82, 128.21, 126.73, 122.68, 122.42, 117.01, 108.27,
56.49, 55.39, 41.92, 35.57, 19.03, 13.69. MS (MALDI-TOF)
m/z 534 (M+1).
6 ‑ ( 5 ‑ ( 3 ‑ B r o m o p h e n o x y ) ‑ 3 ‑ m e t h y l ‑ 1 ‑ p h e -
nyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (6e) White solid, Yield 48%; m.p.
1
95–96 °C. H NMR (300 MHz, DMSO-d6) δ 8.71 (s, 1H,
NH), 8.62 (s, 1H, NH), 8.41 (s, 1H, NH2), 7.89 (s, 1H, NH2),
7.51 (d, J = 7.7 Hz, 2H, Ar–H), 7.42 (t, J = 7.8 Hz, 2H,
Ar–H), 7.35–7.26 (m, 4H, Ar–H), 7.22 (d, J=6.9 Hz, 4H,
Ar–H), 7.14 (s, 1H, Ar–H), 6.92 (d, J=26.0 Hz, 1H, Ar–H),
5.71 (d, J=17.8 Hz, 1H, CH), 2.72 (t, J=7.3 Hz, 2H, CH2),
2.51 (d, J=1.7 Hz, 2H, CH2), 2.31 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 158.27, 157.15, 147.92, 145.66,
139.34, 137.51, 132.20, 129.88, 129.16, 128.81, 127.94,
127.13, 126.72, 122.68, 122.57, 119.13, 114.64, 107.51,
56.49, 55.41, 41.92, 35.61, 19.03, 13.80. MS (MALDI-TOF)
m/z 544 (M+1).
6 ‑ ( 5 ‑ ( 3 ‑ C h l o r o p h e n o x y ) ‑ 3 ‑ m e t h y l ‑ 1 ‑ p h e -
nyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (6b) White solid, Yield 50%; m.p.
1
79–81 °C. H NMR (300 MHz, DMSO-d6) δ 8.77 (s, 1H,
NH), 8.68 (s, 1H, NH), 8.46 (s, 1H, NH2), 7.89 (s, 1H, NH2),
7.51 (d, J = 7.6 Hz, 2H, Ar–H), 7.42 (t, J = 7.8 Hz, 3H,
Ar–H), 7.33–7.25 (m, 4H, Ar–H), 7.22 (d, J=6.6 Hz, 2H,
Ar–H), 7.11 (d, J=7.9 Hz, 1H, Ar–H), 7.02 (s, 1H, Ar–H),
6.85 (s, 1H, Ar–H), 5.74 (s, 1H, CH), 2.72 (dd, J= 8.6,
5.3 Hz, 2H, CH2), 2.50 (d, J=1.7 Hz, 2H, CH2), 2.32 (s, 3H,
CH3). 13C NMR (126 MHz, DMSO-d6) δ 158.31, 157.15,
147.94, 145.67, 139.34, 137.50, 134.46, 131.92, 129.88,
129.16, 128.80, 127.93, 126.72, 124.24, 122.46, 116.35,
114.30, 107.52, 56.48, 55.40, 41.91, 35.61, 19.03, 13.81.
MS (MALDI-TOF) m/z 500 (M+1).
6 ‑ ( 5 ‑ ( 4 ‑ B r o m o p h e n o x y ) ‑ 3 ‑ m e t h y l ‑ 1 ‑ p h e -
nyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (6f) White solid, Yield 50%; m.p. 101–
102 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.73 (s, 1H, NH),
8.65 (s, 1H, NH), 8.44 (s, 1H, NH2), 7.88 (s, 1H, NH2),
7.51 (d, J=7.5 Hz, 2H, Ar–H), 7.47–7.39 (m, 4H, Ar–H),
7.34–7.27 (m, 3H, Ar–H), 7.22 (d, J=7.1 Hz, 3H, Ar–H),
6.89 (d, J = 8.1 Hz, 2H, Ar–H), 5.71 (s, 1H, CH), 2.72 (t,
J = 7.1 Hz, 2H, CH2), 2.50 (d, J= 1.7 Hz, 2H, CH2), 2.30
(s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6) δ 158.29,
157.18, 155.88, 147.92, 145.87, 139.33, 137.50, 133.23,
129.87, 129.16, 128.80, 127.91, 126.72, 122.45, 117.98,
115.81, 107.41, 56.49, 55.42, 41.91, 35.63, 19.03, 13.81.
MS (MALDI-TOF) m/z 544 (M+1).
6 ‑ ( 5 ‑ ( 4 ‑ C h l o r o p h e n o x y ) ‑ 3 ‑ m e t h y l ‑ 1 ‑ p h e -
nyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (6c) White solid, Yield 50%; m.p.
1
87–88 °C. H NMR (300 MHz, DMSO-d6) δ 8.78 (s, 1H,
NH), 8.71 (s, 1H, NH), 8.50 (s, 1H, NH2), 7.88 (s, 1H,
NH2), 7.51 (d, J= 7.5 Hz, 2H, Ar–H), 7.42 (t, J= 7.8 Hz,
2H, Ar–H), 7.35–7.27 (m, 5H, Ar–H), 7.22 (d, J=7.2 Hz,
3H, Ar–H), 6.95 (d, J = 7.8 Hz, 2H, Ar–H), 5.70 (s, 1H,
CH), 2.72 (dd, J=8.9, 5.3 Hz, 2H, CH2), 2.52–2.49 (m, 2H,
CH2), 2.31 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 158.26, 157.10, 155.40, 147.92, 145.97, 139.34, 137.51,
130.31, 129.87, 129.15, 128.81, 127.91, 126.73, 122.46,
117.56, 107.38, 56.49, 55.41, 41.90, 35.62, 19.03, 13.80.
MS (MALDI-TOF) m/z 500 (M+1).
6 ‑ ( 5 ‑ ( 4 ‑ F l u o r o p h e n o x y ) ‑ 3 ‑ m e t h y l ‑ 1 ‑ p h e -
nyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (6g) White solid, Yield 50%; m.p.
1
87–89 °C. H NMR (300 MHz, DMSO-d6) δ 8.72 (s, 1H,
NH2), 8.64 (s, 1H, NH), 8.43 (s, 1H, NH2), 7.88 (s, 1H,
NH2), 7.52 (d, J = 7.5 Hz, 2H, Ar–H), 7.42 (dd, J = 10.4,
5.2 Hz, 2H, Ar–H), 7.34–7.26 (m, 3H, Ar–H), 7.22 (d,
J=7.0 Hz, 3H, Ar–H), 7.11 (dd, J=11.9, 5.5 Hz, 2H, Ar–H),
6.96 (s, 2H, Ar–H), 5.69 (s, 1H, CH), 2.74 (d, J =6.9 Hz,
2H, CH2), 2.51 (d, J=1.8 Hz, 2H, CH2), 2.30 (s, 3H, CH3).
13C NMR (126 MHz, DMSO-d6) δ 13C NMR (126 MHz,
DMSO) δ 159.42, 158.32, 157.52, 157.26, 152.78, 147.90,
146.55, 139.34, 137.59, 129.83, 129.16, 128.81, 127.86,
126.73, 122.50, 117.43, 116.97, 116.87, 116.72, 107.00,
56.49, 55.46, 41.90, 35.63, 19.03, 13.85. MS (MALDI-TOF)
m/z 484 (M+1).
6‑ (5‑ (2,4‑ Dichlorophenoxy)‑ 3‑ methyl‑ 1‑ phe-
nyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (6d) White solid, Yield 48%; m.p.
108–110 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.79 (s, 1H,
NH), 8.73 (s, 1H, NH), 8.52 (s, 1H, NH2), 7.90 (s, 1H, NH2),
7.66 (d, J=2.5 Hz, 1H, Ar–H), 7.52–7.39 (m, 5H, Ar–H),
7.34–7.27 (m, 3H, Ar–H), 7.26–7.21 (m, 3H, Ar–H), 6.79 (d,
J=9.0 Hz, 1H, Ar–H), 5.77 (s, 1H, CH), 2.71 (t, J=7.3 Hz,
2H, CH2), 2.52–2.49 (m, 2H, CH2), 2.32 (s, 3H, CH3). 13
C
1 3

Molecular Diversity
6‑(3‑Methyl‑1‑phenyl‑5‑(o‑tolyloxy)‑1H‑pyra-
zol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑triazine‑2,4‑di-
amine (6h) White solid, Yield 46%; m.p. 87–89 °C. H
3H, Ar–H), 6.99 (s, 1H, Ar–H), 6.82 (d, J = 7.6 Hz, 1H,
Ar–H), 6.44 (d, J = 8.0 Hz, 1H, Ar–H), 5.63 (s, 1H, CH),
2.68 (s, 2H, CH2), 2.51 (d, J = 1.8 Hz, 2H, CH2), 2.30 (s,
3H, CH3), 2.26 (s, 3H, CH3), 2.15 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 160.69, 157.81, 156.43, 154.33,
135.04, 133.30, 131.23, 121.25, 116.98, 110.89, 68.41,
59.20, 56.47, 37.48 (2C), 28.83, 28.19, 22.35, 14.38. MS
(MALDI-TOF) m/z 494 (M+1).
1
NMR (300 MHz, DMSO-d6) δ 8.72 (s, 1H, NH), 8.63 (s,
1H, NH), 8.43 (s, 1H, NH2), 7.82 (s, 1H, NH2), 7.48 (s,
2H, Ar–H), 7.45–7.37 (m, 2H, Ar–H), 7.28 (s, 3H, Ar–H),
7.21 (s, 4H, Ar–H), 7.03 (s, 1H, Ar–H), 6.93 (s, 1H, Ar–H),
6.57 (s, 1H, Ar–H), 5.65 (s, 1H, CH), 2.68 (s, 2H, CH2),
2.51 (s, 2H, CH2), 2.30 (d, J=3.2 Hz, 6H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 158.29, 157.18, 154.91, 147.83,
146.49, 139.34, 137.75, 131.70, 129.75, 129.14, 128.80,
127.84, 126.71, 126.14, 123.72, 122.33, 113.28, 107.20,
56.49, 55.47, 41.88, 35.57, 19.03, 16.19, 13.84. MS
(MALDI-TOF) m/z 480 (M+1).
6‑ (5‑ (3‑ Methox yphenox y)‑ 3‑ methyl‑ 1‑ phe-
nyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (6l) Yellow solid, Yield 52%; m.p.
1
78–80 °C. H NMR (300 MHz, DMSO-d6) δ 8.66 (s, 1H,
NH), 8.55 (s, 1H, NH), 8.34 (s, 1H, NH2), 7.88 (s, 1H,
NH2), 7.53 (d, J= 7.6 Hz, 2H, Ar–H), 7.42 (t, J= 7.8 Hz,
2H, Ar–H), 7.33–7.27 (m, 3H, Ar–H), 7.19 (dd, J= 19.2,
7.4 Hz, 4H, Ar–H), 6.63 (d, J = 8.3 Hz, 1H, Ar–H), 6.48
(d, J=12.9 Hz, 2H, Ar–H), 5.66 (s, 1H, CH), 3.69 (s, 3H,
OCH3), 2.73 (s, 2H, CH2), 2.51 (d, J = 1.7 Hz, 2H, CH2),
2.30 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6) δ
161.00, 158.37, 157.71, 157.30, 146.34, 139.34, 137.70,
131.06, 129.82, 129.16, 128.80, 127.77, 126.72, 122.35,
109.94, 107.45, 106.94, 102.17, 56.49, 55.65, 41.90, 35.62,
19.03, 13.93. MS (MALDI-TOF) m/z 496 (M+1).
6‑(3‑Methyl‑1‑phenyl‑5‑(m‑tolyloxy)‑1H‑pyra-
zol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑triazine‑2,4‑di-
1
amine (6i) White solid, Yield 46%; m.p. 81–83 °C. H
NMR (300 MHz, DMSO-d6) δ 8.71 (s, 1H, NH), 8.62 (s,
1H, NH), 8.39 (s, 1H, NH2), 7.88 (s, 1H, NH2), 7.52 (d,
J = 7.5 Hz, 2H, Ar–H), 7.42 (t, J = 7.8 Hz, 2H, Ar–H),
7.34–7.27 (m, 3H, Ar–H), 7.26–7.18 (m, 3H, Ar–H), 7.14 (d,
J=7.8 Hz, 1H, Ar–H), 6.87 (d, J=7.3 Hz, 1H, Ar–H), 6.76
(s, 2H, Ar–H), 5.64 (s, 1H, CH), 2.72 (s, 2H, CH2), 2.50 (d,
J=1.7 Hz, 2H, CH2), 2.30 (s, 3H, CH3), 2.24 (s, 3H, CH3).
13C NMR (126 MHz, DMSO-d6) δ 158.41, 157.34, 156.67,
147.90, 146.51, 140.44, 139.34, 137.72, 130.24, 129.80,
129.16, 128.80, 127.74, 126.72, 124.87, 122.33, 116.05,
112.75, 106.76, 56.49, 55.49, 41.90, 35.64, 21.40, 19.03,
13.94. MS (MALDI-TOF) m/z 480 (M+1).
6‑ (3‑ Methyl‑ 1‑ phenyl‑ 5‑ (3‑ (trifluoromethyl)
phenoxy)‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑di-
hydro‑1,3,5‑triazine‑2,4‑diamine (6m) White solid, Yield
50%; m.p. 87–89 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.91
(s, 1H, NH), 8.66 (s, 1H, NH2), 7.86 (s, 1H, Ar–H), 7.78
(d, J = 2.2 Hz, 1H, Ar–H), 7.63 (s, 1H, Ar–H), 7.47 (dd,
J=13.0, 4.5 Hz, 2H, Ar–H), 7.41 (d, J=7.3 Hz, 2H, Ar–H),
7.33–7.25 (m, 2H, Ar–H), 7.21 (dd, J = 9.7, 4.5 Hz, 3H,
Ar–H), 7.12 (s, 1H, Ar–H), 5.81 (s, 1H, CH), 2.68 (s, 2H,
CH2), 2.51–2.50 (m, 2H, CH2), 2.31 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 158.28, 157.18, 156.60, 147.97,
145.63, 139.30, 137.45, 131.92, 131.10, 130.84, 129.85,
129.12, 128.79, 128.00, 126.71, 125.04, 122.87, 122.63,
120.82, 119.47, 113.28, 107.53, 56.48, 55.42, 41.86, 35.60,
19.02, 13.78. MS (MALDI-TOF) m/z 534 (M+1).
6‑(3‑Methyl‑1‑phenyl‑5‑(p‑tolyloxy)‑1H‑pyra-
zol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑triazine‑2,4‑di-
1
amine (6j) White solid, Yield 48%; m.p. 88–89 °C. H
NMR (300 MHz, DMSO-d6) δ 8.72 (s, 1H, NH), 8.63 (s,
1H, NH), 8.41 (s, 1H, NH2), 7.88 (s, 1H, NH2), 7.52 (d,
J=7.7 Hz, 2H, Ar–H), 7.41 (t, J=7.9 Hz, 2H, Ar–H), 7.28
(s, 3H, Ar–H), 7.23 (s, 3H, Ar–H), 7.08 (d, J=8.4 Hz, 2H,
Ar–H), 6.84 (s, 2H, Ar–H), 5.63 (s, 1H, CH), 2.73 (s, 2H,
CH2), 2.51 (d, J=1.7 Hz, 2H, CH2), 2.30 (s, 3H, CH3), 2.19
(s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6) δ 158.44,
157.37, 154.65, 147.87, 146.63, 139.35, 137.71, 133.04,
130.86, 129.79, 129.15, 128.80, 127.71, 126.72, 122.29,
115.43, 106.85, 56.49, 55.48, 41.91, 35.64, 20.54, 19.03,
13.96. MS (MALDI-TOF) m/z 480 (M+1).
6‑(3‑Methyl‑5‑(naphthalen‑2‑yloxy)‑1‑phenyl‑1H‑pyra-
zol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑triazine‑2,4‑di-
amine (6n) Yellow solid, Yield 42%; m.p. 128–130 °C. 1H
NMR (300 MHz, DMSO-d6) δ 8.77 (s, 1H, NH), 8.70 (s, 1H,
NH), 8.43 (s, 1H, NH2), 8.31 (d, J=8.3 Hz, 1H, NH2), 7.97–
7.88 (m, 1H, Ar–H), 7.75 (s, 1H, Ar–H), 7.67–7.52 (m, 5H,
Ar–H), 7.34–7.24 (m, 5H, Ar–H), 7.23–7.10 (m, 4H, Ar–H),
6.75 (t, J= 18.3 Hz, 1H, Ar–H), 5.67 (d, J= 19.2 Hz, 1H,
CH), 3.17 (d, J=22.0 Hz, 2H, CH2), 2.58 (s, 2H, CH2), 2.35
(s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6) δ 158.23,
157.16, 152.43, 147.98, 146.28, 139.26, 137.71, 134.64,
129.73, 129.09, 128.76, 128.08, 127.79, 127.52, 126.87,
6‑(5‑(2,4‑Dimethylphenoxy)‑3‑methyl‑1‑phe-
nyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (6 k) White solid, Yield 52%; m.p.
98–100 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.67 (s, 1H,
NH), 8.58 (s, 1H, NH), 8.38 (s, 1H, NH2), 7.83 (s, 1H,
NH2), 7.49 (d, J= 7.7 Hz, 2H, Ar–H), 7.41 (t, J= 7.8 Hz,
2H, Ar–H), 7.32–7.26 (m, 3H, Ar–H), 7.21 (t, J=5.6 Hz,
1 3

Molecular Diversity
126.55, 124.13, 123.58, 122.24, 121.50, 108.38, 107.49,
56.49, 55.46, 41.75, 35.41, 19.03, 13.88. MS (MALDI-TOF)
m/z 516 (M+1).
6‑(5‑(2,4‑Dichlorophenoxy)‑1‑(2,4‑dichloropheny
l)‑3‑methyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑di-
hydro‑1,3,5‑triazine‑2,4‑diamine (10d) White solid,
Yield 42%; m.p. 119–120 °C. 1H NMR (300 MHz, DMSO-
d6) δ 8.77 (s, 1H, NH), 8.66 (s, 1H, NH), 8.47 (s, 1H, NH2),
7.91 (s, 1H, NH2), 7.84 (d, J = 2.2 Hz, 1H, Ar–H), 7.58
(d, J = 2.5 Hz, 1H, Ar–H), 7.49 (dd, J = 8.5, 2.3 Hz, 1H,
Ar–H), 7.39 (d, J=8.7 Hz, 2H, Ar–H), 7.29 (d, J=7.1 Hz,
2H, Ar–H), 7.25–7.19 (m, 3H, Ar–H), 7.00 (d, J=9.0 Hz,
1H, Ar–H), 5.82 (s, 1H, CH), 2.72 (d, J=6.2 Hz, 2H, CH2),
2.51 (d, J=1.8 Hz, 2H, CH2), 2.30 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 158.21, 157.13, 151.01, 148.52,
146.89, 139.30, 135.68, 133.26, 132.71, 131.33, 130.24,
129.24, 128.67, 128.54, 126.73, 122.96, 118.26, 107.12,
56.48, 55.39, 41.89, 35.59, 19.03, 13.77. MS (MALDI-TOF)
m/z 602 (M+1).
6‑ (5‑ (2‑ Chlorophenoxy)‑ 1‑ (2,4‑ dichlorophen
yl)‑ 3‑ methyl‑ 1H‑ pyrazol‑ 4‑ yl)‑ N2‑ phenethyl-
‑3,6‑dihydro‑1,3,5‑triazine‑2,4‑diamine (10a) White
1
solid, Yield 42%; m.p. 112–113 °C. H NMR (300 MHz,
DMSO-d6) δ 8.76 (s, 1H, NH), 8.63 (s, 1H, NH), 8.44 (s,
1H, NH2), 7.91 (s, 1H, NH2), 7.80 (d, J=2.2 Hz, 1H, Ar–H),
7.45 (dd, J = 8.5, 2.3 Hz, 1H, Ar–H), 7.40–7.34 (m, 2H,
Ar–H), 7.33–7.26 (m, 2H, Ar–H), 7.21 (d, J=7.6 Hz, 4H,
Ar–H), 7.10–7.02 (m, 1H, Ar–H), 6.97 (d, J=7.0 Hz, 1H,
Ar–H), 5.79 (s, 1H, CH), 2.73 (d, J = 6.6 Hz, 2H, CH2),
2.51 (d, J=1.7 Hz, 2H, CH2), 2.30 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 158.27, 157.24, 151.88, 148.42,
147.43, 139.31, 135.50, 133.38, 132.73, 131.26, 130.78,
130.24, 129.14, 128.72, 128.64–128.41, 126.72, 125.53,
121.88, 117.11, 106.82, 56.49, 55.38, 41.89, 35.58, 19.03,
13.86. MS (MALDI-TOF) m/z 568 (M+1).
6‑(5‑(3‑Bromophenoxy)‑1‑(2,4‑dichlorophenyl)‑3‑meth
yl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (10e) White solid, Yield 44%; m.p.
1
90–92 °C. H NMR (300 MHz, DMSO-d6) δ 8.85 (s, 1H,
6‑ (5‑ (3‑ Chlorophenoxy)‑ 1‑ (2,4‑ dichlorophen
yl)‑ 3‑ methyl‑ 1H‑ pyrazol‑ 4‑ yl)‑ N2‑ phenethyl-
‑3,6‑dihydro‑1,3,5‑triazine‑2,4‑diamine (10b) White
NH), 8.74 (s, 1H, NH), 8.59 (s, 1H, NH2), 7.88 (s, 1H, NH2),
7.81 (d, J=2.2 Hz, 1H, Ar–H), 7.55 (s, 1H, Ar–H), 7.49 (dd,
J=8.5, 2.2 Hz, 1H, Ar–H), 7.40 (d, J=8.3 Hz, 1H, Ar–H),
7.32–7.25 (m, 2H, Ar–H), 7.24–7.15 (m, 4H, Ar–H), 7.04 (s,
1H, Ar–H), 6.91 (s, 1H, Ar–H), 5.77 (s, 1H, CH), 2.77–2.66
(m, 2H, CH2), 2.51 (d, J = 1.7 Hz, 2H, CH2), 2.30 (s, 3H,
CH3). 13C NMR (126 MHz, DMSO-d6) δ 158.27, 157.09,
147.29, 135.43, 133.59, 132.15, 131.80, 131.52, 131.25,
130.35, 129.14, 128.80, 127.37, 126.72, 122.45, 119.39,
115.46, 56.49, 55.39, 41.89, 35.59, 22.38, 19.03, 13.82. MS
(MALDI-TOF) m/z 612 (M+1).
1
solid, Yield 40%; m.p. 91–93 °C. H NMR (300 MHz,
DMSO-d6) δ 8.70 (s, 1H, NH), 8.56 (s, 1H, NH), 8.37 (s,
1H, NH2), 7.90 (s, 1H, NH2), 7.81 (d, J=2.2 Hz, 1H, Ar–H),
7.49 (dd, J = 8.5, 2.3 Hz, 1H, Ar–H), 7.41 (d, J = 8.3 Hz,
1H, Ar–H), 7.33–7.25 (m, 3H, Ar–H), 7.25–7.18 (m, 3H,
Ar–H), 7.12 (d, J=7.3 Hz, 1H, Ar–H), 6.89 (d, J=20.3 Hz,
2H, Ar–H), 5.79 (s, 1H, CH), 2.72 (d, J=7.0 Hz, 2H, CH2),
2.51 (d, J=1.8 Hz, 2H, CH2), 2.30 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 158.24, 157.08, 156.86, 156.71,
148.93, 148.48, 147.88, 139.31, 135.44, 134.28, 133.58,
131.76, 131.26, 131.26, 130.35, 129.14, 128.82, 126.73,
124.47, 116.57, 115.05, 106.52, 56.48, 55.39, 41.88, 35.59,
19.03, 13.82. MS (MALDI-TOF) m/z 568 (M+1).
6‑(5‑(4‑Bromophenoxy)‑1‑(2,4‑dichlorophenyl)‑3‑meth
yl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (10f) White solid, Yield 40%; m.p.
105–107 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.72 (s, 1H,
NH), 8.58 (s, 1H, NH), 8.43 (s, 1H, NH2), 7.89 (s, 1H, NH2),
7.82 (d, J = 2.2 Hz, 1H, Ar–H), 7.50 (dd, J = 8.5, 2.3 Hz,
1H, Ar–H), 7.46–7.38 (m, 3H, Ar–H), 7.36–7.27 (m, 2H,
Ar–H), 7.26–7.18 (m, 3H, Ar–H), 6.86 (d, J=8.5 Hz, 2H,
Ar–H), 5.74 (s, 1H, CH), 2.71 (t, J = 7.2 Hz, 2H, CH2),
2.51 (d, J=1.8 Hz, 2H, CH2), 2.29 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 158.30, 157.20, 155.78, 148.55,
147.42, 139.32, 135.46, 133.64, 133.03, 132.38, 131.32,
130.33, 129.14, 128.81, 126.72, 118.34, 115.99, 106.26,
56.49, 55.39, 41.88, 35.61, 21.67, 19.03, 13.84. MS
(MALDI-TOF) m/z 612 (M+1).
6‑ (5‑ (4‑ Chlorophenoxy)‑ 1‑ (2,4‑ dichlorophen
yl)‑ 3‑ methyl‑ 1H‑ pyrazol‑ 4‑ yl)‑ N2‑ phenethyl-
‑3,6‑dihydro‑1,3,5‑triazine‑2,4‑diamine (10c) White
1
solid, Yield 42%; m.p. 108–110 °C. H NMR (300 MHz,
DMSO-d6) δ 8.72 (s, 1H, NH), 8.58 (d, J = 21.4 Hz, 1H,
NH), 8.42 (s, 1H, NH2), 7.82 (d, J = 2.1 Hz, 1H, Ar–H),
7.50 (dd, J = 8.5, 2.2 Hz, 1H, Ar–H), 7.42 (d, J = 7.8 Hz,
2H, Ar–H), 7.29 (d, J=8.5 Hz, 3H, Ar–H), 7.25–7.19 (m,
3H, Ar–H), 6.91 (d, J = 8.6 Hz, 2H, Ar–H), 5.74 (s, 1H,
CH), 2.71 (t, J=7.0 Hz, 2H, CH2), 2.51 (d, J=1.7 Hz, 2H,
CH2), 2.29 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6)
δ 158.28, 157.21, 155.29, 148.53, 147.55, 139.32, 135.45,
133.66, 132.37, 131.34, 130.32, 130.12, 129.14, 128.81,
128.08, 126.73, 117.96, 106.22, 56.49, 55.39, 41.87, 35.60,
19.03, 13.84. MS (MALDI-TOF) m/z 568 (M+1).
6‑(1‑(2,4‑Dichlorophenyl)‑5‑(4‑fuorophenoxy)‑3‑meth
yl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑dihydro‑1,3,5‑tri-
azine‑2,4‑diamine (10g) White solid, Yield 40%; m.p.
103–104 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.85 (s, 1H,
1 3

Molecular Diversity
NH), 8.61 (s, 1H, NH), 7.81 (d, J=2.2 Hz, 1H, NH2), 7.60
(s, 1H, Ar–H), 7.49 (dd, J = 8.5, 2.2 Hz, 1H, Ar–H), 7.40
(d, J=8.3 Hz, 1H, Ar–H), 7.34–7.26 (m, 2H, Ar–H), 7.24–
7.20 (m, 2H, Ar–H), 7.06 (t, J = 8.7 Hz, 2H, Ar–H), 6.92
(dd, J=9.0, 4.1 Hz, 2H, Ar–H), 5.72 (s, 1H, CH), 2.72 (t,
J=7.1 Hz, 2H, CH2), 2.52–2.50 (m, 2H, CH2), 2.28 (s, 3H,
CH3). 13C NMR (126 MHz, DMSO-d6) δ 159.52, 158.35,
157.61, 157.30, 152.66, 148.49, 148.21, 139.35, 135.37,
133.78, 132.42, 131.34, 130.27, 129.14, 128.80, 126.72,
118.03, 116.84, 116.65, 105.90, 56.48, 55.44, 41.88, 35.63,
19.03, 13.86. MS (MALDI-TOF) m/z 552 (M+1).
J=13.0, 4.5 Hz, 2H, Ar–H), 7.41 (d, J=7.3 Hz, 2H, Ar–H),
7.33–7.25 (m, 2H, Ar–H), 7.21 (dd, J = 9.7, 4.5 Hz, 3H,
Ar–H), 7.12 (s, 1H, Ar–H), 5.81 (s, 1H, CH), 2.68 (s, 2H,
CH2), 2.51–2.50 (m, 2H, CH2), 2.31 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 158.28, 157.22, 156.43, 148.54,
147.29, 139.31, 135.47, 133.56, 132.31, 131.72, 131.26,
130.93, 130.68, 130.30, 129.10, 128.79, 126.71, 124.95,
122.78, 121.15, 120.55, 113.42, 106.66, 56.48, 55.38,
41.84, 35.58, 22.47, 19.02, 13.79. MS (MALDI-TOF) m/z
602 (M+1).
6 ‑ ( 1 ‑ ( 2 , 4 ‑ D i c h l o r o p h e n y l ) ‑ 3 ‑ m e -
thyl‑5‑(naphthalen‑2‑yloxy)‑1H‑pyrazol‑4‑yl)‑N2‑phe-
nethyl‑3,6‑dihydro‑1,3,5‑triazine‑2,4‑diamine
(10 k) Yellow solid, Yield 38%; m.p. 116–118 °C. 1H NMR
(300 MHz, DMSO-d6) δ 8.83 (s, 1H, NH), 8.73 (s, 1H, NH),
8.49 (s, 1H, NH2), 8.09 (s, 1H, NH2), 7.87 (d, J=8.5 Hz, 1H,
Ar–H), 7.76 (s, 1H, Ar–H), 7.67 (d, J=2.0 Hz, 1H, Ar–H),
7.61–7.50 (m, 3H, Ar–H), 7.42 (d, J=8.2 Hz, 1H, Ar–H),
7.33–7.24 (m, 4H, Ar–H), 7.20 (d, J=6.9 Hz, 1H, Ar–H),
7.14 (s, 2H, Ar–H), 6.86 (d, J=7.6 Hz, 1H, Ar–H), 5.79 (s,
1H, CH), 3.21 (s, 2H, CH2), 2.57 (s, 2H, CH2), 2.34 (s, 3H,
CH3). 13C NMR (126 MHz, DMSO-d6) δ 158.23, 157.20,
152.40, 147.97, 139.25, 135.12, 134.40, 133.67, 132.29,
131.19, 130.13, 129.05, 128.75, 128.57, 127.97, 127.36,
126.66, 126.18, 124.17, 123.76, 121.15, 109.39, 56.49,
55.44, 41.73, 35.39, 21.97, 19.02, 13.88. MS (MALDI-TOF)
m/z 584 (M+1).
6 ‑ ( 1 ‑ ( 2 , 4 ‑ D i c h l o r o p h e n y l ) ‑ 3 ‑ m e -
thyl‑5‑(p‑tolyloxy)‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3-
,6‑dihydro‑1,3,5‑triazine‑2,4‑diamine (10h) White solid,
Yield 42%; m.p. 88–90 °C. 1H NMR (300 MHz, DMSO-d6)
δ 8.79 (s, 1H, NH), 8.50 (s, 1H, NH), 7.81 (d, J = 2.2 Hz,
1H, NH2), 7.48 (dd, J = 8.5, 2.3 Hz, 1H, Ar–H), 7.38 (d,
J=8.6 Hz, 1H, Ar–H), 7.34–7.27 (m, 2H, Ar–H), 7.22 (t,
J = 5.3 Hz, 3H, Ar–H), 7.03 (d, J = 8.4 Hz, 2H, Ar–H),
6.77 (d, J=7.8 Hz, 2H, Ar–H), 5.66 (s, 1H, CH), 2.72 (d,
J = 6.5 Hz, 2H, CH2), 2.51 (d, J= 1.8 Hz, 2H, CH2), 2.28
(s, 3H, CH3), 2.20 (d, J = 9.2 Hz, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 158.45, 157.41, 154.53, 149.06,
148.49, 148.36, 139.37, 135.23, 133.89, 133.23, 132.36,
131.25, 130.59, 130.27, 129.14, 128.76, 126.71, 115.90,
105.63, 55.46, 41.88, 35.62, 22.33, 20.57, 19.03, 13.96. MS
(MALDI-TOF) m/z 548 (M+1).
6‑(1‑(2,4‑Dichlorophenyl)‑5‑(2,4‑dimethylphenox
y)‑3‑methyl‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑di-
hydro‑1,3,5‑triazine‑2,4‑diamine (10i) White solid, Yield
42%; m.p. 108–110 °C. 1H NMR (300 MHz, DMSO-d6) δ
8.77 (s, 1H, NH), 8.66 (s, 1H, NH), 8.51 (s, 1H, NH2), 7.81
(d, J=2.2 Hz, 2H, NH2 and Ar–H), 7.46 (dd, J=8.5, 2.3 Hz,
2H, Ar–H), 7.36 (s, 1H, Ar–H), 7.31 (dd, J=13.1, 5.9 Hz,
3H, Ar–H), 7.21 (t, J = 6.5 Hz, 3H, Ar–H), 6.89 (s, 1H,
Ar–H), 6.84 (d, J=8.4 Hz, 1H, Ar–H), 6.61 (d, J=8.4 Hz,
1H, Ar–H), 5.68 (s, 1H, CH), 2.69 (s, 2H, CH2), 2.51–2.50
(m, 2H, CH2), 2.29 (s, 3H, CH3), 2.14 (s, 3H, CH3), 2.05
(s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6) δ 158.38,
157.29, 152.91, 148.36, 139.34, 135.20, 133.83, 132.71,
132.23, 131.92, 131.75, 131.18, 130.20, 129.10, 128.72,
127.84, 126.71, 125.93, 114.12, 106.13, 56.48, 55.44, 41.86,
35.57, 22.41, 20.52, 19.02, 15.76, 13.88. MS (MALDI-TOF)
m/z 562 (M+1).
In vitro inhibition of bacterial and fungal growth
Determination of minimal inhibitory concentrations (MICs)
was performed as described previously [16]. The microor-
ganisms used in the present study were S. aureus 4220, E.
Coli 1924, S. mutans 3289, P. aeruginosa 2742, S. typhimu-
rium 2421 and C. albicans 7535. The strains of multidrug-
resistant clinical isolates were methicillin-resistant S. aureus
(MRSA CCARM 3506) and quinolone-resistant S. aureus
(QRSA CCARM 3505). Clinical isolates were collected
from various patients hospitalized in several clinics. Test
bacteria were grown to mid-log phase in Mueller–Hinton
broth (MHB) and diluted 1000-fold in the same medium.
The bacteria of 105 CFU/mL were inoculated into MHB
and dispensed at 0.2 mL/well in a 96-well microtiter plate.
As positive controls, oxacillin and norfoxacin were used.
Test compounds were prepared in DMSO, the fnal con-
centration of which did not exceed 0.05%. A twofold serial
dilution technique was used to obtain fnal concentrations
of 64–0.25 µg/mL. The MIC was defned as the concentra-
tion of a test compound that completely inhibited bacteria
growth during 24 h incubation at 37 °C. Bacteria growth was
determined by measuring the absorption at 650 nm using
6‑(1‑(2,4‑Dichlorophenyl)‑3‑methyl‑5‑(3‑(trifluorome
thyl)phenoxy)‑1H‑pyrazol‑4‑yl)‑N2‑phenethyl‑3,6‑di-
hydro‑1,3,5‑triazine‑2,4‑diamine (10j) White solid,
Yield 40%; m.p. 86–88 °C. 1H NMR (300 MHz, DMSO-d6)
δ 8.91 (s, 1H, NH), 8.66 (s, 1H, NH), 7.86 (s, 1H, NH2),
7.78 (d, J=2.2 Hz, 1H, Ar–H), 7.63 (s, 1H, Ar–H), 7.47 (dd,
1 3

Molecular Diversity
a microtiter enzyme-linked immunosorbent assay (ELISA)
reader. All experiments were carried out three times.
spectrophotometrically at a wavelength of 450 nm. The con-
centration of DHFR activity was calculated by a standard
curve.
Cytotoxicity assessment by MTT assay
Acknowledgements This work was supported by the National Natu-
ral Science Foundation of China (No. 81773692); the Science and
Technology Department of Jilin Province (No. 20180311016YY), the
Department of Education of Jilin Province (No. JJKH20191070KJ);
the Health Department of Jilin Province (No. 2018ZC034); and the
Doctoral Foundation of Jilin Medical University (No. JYBS2018007).
The cytotoxicity test of selected compounds was measured
through the colorimetric MTT assay. Human cancer cell
lines MCF-7 and HeLa were bought from the Cell Bank of
Chinese Academy of Sciences (Shanghai, China). MCF-7
and HeLa were grown in the Dulbecco’s modifcation of
eagle’s medium (DMEM), (Corning) supplemented with
10% fetal bovine serum (FBS). A variety of concentrations
of the test compounds (200, 100, 50, 25, 12.5, 6.25, 3.125,
1.625 μmol/L) dissolved by 10% distilled DMSO was added
to each well. After incubation for 24 h at 37 °C under 5%
CO2, 2.5 μg/mL of MTT solution was added to each well.
Further the plate was incubated for 4 h. Then, the medium
was removed and the resulting formazan crystals were
dissolved with 100 µL DMSO. After shaking 10 min, the
optical density was measured at 570 nm using a microtiter
ELISA reader. The selected compounds were used as posi-
tive control, whereas untreated cells were used as negative
controls. The IC50 values were defned as the concentrations
inhibiting 50% of cell growth. All experiments were per-
formed in triplicate.
Compliance with ethical standards
Conflict of interest The authors state no confict of interest.
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1 3

Molecular Diversity
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