Synthesis and evaluation of the antibacterial activities of aryl substituted dihydrotriazine derivatives

Article abstract of DOI:10.1016/j.bmcl.2018.03.037

Five series of dihydrotriazine derivatives containing chalcone (13a–i), phenoxy acetophenone (14a–b), benzyl benzene (15a–c), naphthoxyl acetophenone (16a–b) and benzyl naphthalene (17a–h) moieties were designed and synthesized. The antibacterial and antifungal activities of these compounds were evaluated against several strains of Gram-positive and Gram-negative bacteria, as well as a single fungus. Compound 17h was found to be the most potent of all of the compounds tested, with an MIC value of 0.5 μg/mL against several Gram-positive (Staphylococcus aureus 4220 and QRSA CCARM 3505) and Gram-negative (Escherichia coli 1924) strains of bacteria. However, this compound was inactive against Pseudomonas aeruginosa 2742 and Salmonella typhimurium 2421, indicating that its antibacterial spectrum is similar to those of the positive controls gatifloxacin and moxifloxacin. The cytotoxic activity of the compound 13i, 16b and 17h was assessed in Human normal liver cells.

Full text of DOI:10.1016/j.bmcl.2018.03.037

Accepted Manuscript
Synthesis and evaluation of the antibacterial activities of aryl substituted dihy-
drotriazine derivatives
Tian-Yi Zhang, Zhan-Kui Yu, Xue-Jun Jin, Ming-Yue Li, Liang-Peng Sun,
Chang-Ji Zheng, Hu-Ri Piao
PII:
S0960-894X(18)30230-0
https://doi.org/10.1016/j.bmcl.2018.03.037
BMCL 25691
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 November 2017
25 January 2018
15 March 2018
Please cite this article as: Zhang, T-Y., Yu, Z-K., Jin, X-J., Li, M-Y., Sun, L-P., Zheng, C-J., Piao, H-R., Synthesis
and evaluation of the antibacterial activities of aryl substituted dihydrotriazine derivatives, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.037
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Synthesis and evaluation of the antibacterial activities of aryl
substituted dihydrotriazine derivatives
Tian-Yi Zhang a,b,1, Zhan-Kui Yu a,1, Xue-Jun Jin a, Ming-Yue Li a, Liang-Peng Sun a,
Chang-Ji Zheng a,*, Hu-Ri Piao a,*
aKey Laboratory of Natural Resources and Functional Molecules of the Changbai
Mountain, Affiliated Ministry of Education; Yanbian University College of Pharmacy,
Yanji, Jilin Province 133002, PR China
bDepartment of Pharmacy, Jilin Medical University, Jilin, Jilin Province
132013,China
*Correspondence Author: Tel: + 86(433)243-5003; Fax: + 86(433)243-5026.
E-mail: piaohr@ybu.edu.cn (H. R. Piao).
*Correspondence Author: Tel: + 86(433)243-6015; Fax: + 86(433)243-5026.
E-mail: zhengcj@ybu.edu.cn (C. J. Zheng).
1 These authors contributed equally to this work.

Abstract
Five series of dihydrotriazine derivatives containing chalcone (13a–i), phenoxy
acetophenone (14a–b), benzyl benzene (15a–c), naphthoxyl acetophenone (16a–b)
and benzyl naphthalene (17a–h) moieties were designed and synthesized. The
antibacterial and antifungal activities of these compounds were evaluated against
several strains of Gram-positive and Gram-negative bacteria, as well as a single
fungus. Compound 17h was found to be the most potent of all of the compounds
tested, with an MIC value of 0.5 μg/mL against several Gram-positive
(Staphylococcus aureus 4220 and QRSA CCARM 3505) and Gram-negative
(Escherichia coli 1924) strains of bacteria. However, this compound was inactive
against Pseudomonas aeruginosa 2742 and Salmonella typhimurium 2421, indicating
that its antibacterial spectrum is similar to those of the positive controls gatifloxacin
and moxifloxacin. The cytotoxic activity of the compound 13i, 16b and 17h was
assessed in Human normal liver cells.
Keywords: Antibacterial activitis; Antifungal activities; Cytotoxicity; dihydrotriazine.

Many different types of disease are caused by bacterial infection. In principle,
pathogenic bacteria and conditional pathogens can produce toxins and secondary
metabolites that can lead to a variety of physical discomforts such as rashes, fever and
chills during blood circulation. The discovery of antibiotics and other antimicrobial
agents has played a critical role in the fight against bacteria. However, recent
increases in drug-resistant bacteria mean that the standard-of-care antibacterial drugs
currently used in clinical practice could soon become ineffective, making it difficult
to manage these diseases. There is therefore an urgent need for the development of
new antibacterial drugs that exert their activity through unique mechanisms of action,
enabling them to inhibit the growth of drug-resistant bacteria. The importance of this
task is highlighted by the large number government agencies, scientific institutions
and clinicians involved in the search for new antimicrobial agents.1
1,3,5-triazine derivatives have been reported to exhibit a wide range of interesting
biological properties including anticancer, anti-HIV and antimicrobial activities.2-6 For
example,
Feng
et
al.
reported
that
N2-methyl-6-(5-methylisoxazol-3-yloxy)-N4-(4-(trifluoromethyl)phenyl)-1,3,5-triazin
e-2,4-diamine displayed an antimicrobial activity of up to 97.7% when it was used at
a concentration of around 200 μg/mL.7 Furthermore, Singga et al. reported that the
growth some Gram-positive and Gram-negative bacteria could be inhibited by triazine
derivatives, which exerted their activity by restricting the growth of the bacterial cell
membranes.8 In our previous work, we reported the identification of a series of
2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives, and demonstrated that all of
the compounds belonging to this series showed outstanding bacteriostatic activity
against Gram-positive bacteria, as exemplified by compounds A, B, C and D (MIC =
2 μg/mL) (Fig. 1).9-12 Unfortunately, however, compounds belonging to this series did
not show any bacteriostatic activity against Gram-negative bacteria. In this study, we
designed and synthesized five novel series of compounds (13a–i, 14a–b, 15a–c, 16a–b,
17a–h) (Fig. 2) using a hybrid strategy with compounds A–D (Fig. 1) as the lead
compounds. Notably, the 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid moiety in
these lead compounds was replaced with a dihydrotriazine ring. These compounds

were subsequently evaluated in terms of their antibacterial activities with an aim of
identifying a new series of potent antimicrobial agents.
Figure 1. Previously reported antibacterial compounds
Figure 2. Designed target compounds
The route used to synthesize the five different series of dihydrotriazine derivatives
is shown in Scheme 1. Metformin hydrochloride was prepared by the reaction of
dicyandiamide with minocycline hydrochloride according to a previously reported
method.13 Acetophenone was reacted with terephthalaldehyde in the presence of
ethylene
glycol
to
afford
a
series
(8a–i).
of
(E)-4-(3-oxo-3-phenylprop-1-en-1-yl)benzaldehydes
Two
4-(2-oxo-2-phenylethoxy)benzaldehydes (9a–b) were prepared by reacting the
corresponding
substituted
in
2-bromo-1-phenylethanones
the presence of
(2)
with
Two
4-hydroxybenzaldehyde
K2CO3.
6-(2-oxo-2-phenylethoxy)-2-naphthaldehydes (11a–b) were prepared using the same
method by reacting with 6-hydroxy-2-naphthaldehyde (6). Three
2

4-(benzyloxy)benzaldehydes (10a–c) and eight 6-(benzyloxy)-2-naphthaldehydes
(12a–h) were obtained by reacting substituted chloromethylbenzene (3) with
4-hydroxybenzaldehydes (5) and 6, respectively. The reductive cyclization reactions
of the five intermediate series (8a–i, 9a–b, 10a–c, 11a–b, 12a–h) with metformin
hydrochloride (7) afforded the corresponding novel dihydrotriazine derivatives (13a–i,
14a–b, 15a–c, 16a–b, 17a–h). The structures of the synthesized dihydrotriazine
1
compounds were characterized by H NMR, 13C NMR and HRMS analyses. The
purity (≥95%) of the compound is verified by the HPLC study performed on
Develosil C18 (4.6 mm×250 mm, 5 μm) column using a mixture of solvent 0.1FA
acetonitrile/0.1FA water at the flow rate of 1 mL/min and peak detection at 334 nm
under UV. 14
Scheme 1. Synthetic scheme for the synthesis of compounds 13a-i, 14a-c, 15a-c,
16a-b and 17a-h. Reagents and conditions : (a) NaOH, EtOH, 23 oC, 3-4 h (b) K2CO3 , DMF (c)
K2CO3 , DMF, 60 oC (d) AcOH, 120 oC, 4-8h
The in vitro antimicrobial and antifungal activities of the synthesized compounds
were evaluated against various bacteria, including multidrug-resistant clinical isolates,

as well as one fungus. These screening assays were conducted in 96-well microtiter
plates to obtain the minimum inhibitory concentration (MIC) values of the different
compounds.15 Gatifloxacin, moxifloxacin and fluconazole were used as positive
controls. The compounds were screened against several Gram-positive strains (S.
aureus 4220, QRSA CCARM 3505, MRSA CCARM 3167, S. mutans 3289) and
Gram-negative strains (Escherichia coli 1924, Pseudomonas aeruginosa 2742,
Salmonella typhimurium 2421), as well as one fungus (Candida albicans 7535), and
the results are shown in Table 1. Most of the newly synthesized compounds exhibited
potent inhibitory activities against the different bacteria and single fungus tested in the
current study with MICs in the range of 0.5 to 64 µg/mL.
Compounds belonging to series 13, 14, 15 and 16 showed moderate levels of
antimicrobial activity against the different strains of Gram-positive bacteria and
fungus, whereas compounds belonging to series 17 exhibited potent activity against
the Gram-positive bacteria and fungus with MIC values of 0.5–2 μg/mL (except for
17a). It is noteworthy that compound 17h exhibited the greatest activities of all of the
compounds prepared in the current study with MIC values in the range of 0.5–1
µg/mL, making its potency comparable to those gatifloxacin and moxifloxacin.
Furthermore, compound 17h was determined to be 8-fold more potent than
moxifloxacin (MIC = 4 μg/mL) and 16-fold more potent than Gatifloxacin (MIC = 8
μg/mL) towards QRSA CCARM 3505. In terms of its activity towards the fungus C.
albicans 7535, compound 17h displayed the strongest activity of all of the compounds
synthesized in the current study with an MIC value of 1 µg/mL, making it equipotent
to fluconazole. All of the compounds prepared in this study were found to be inactive
against the Gram-negative bacteria P. aeruginosa 2742 and S. typhimurium 2421, but
did display good activity against E. coli 1924 with MIC values in the range of 0.5–64
μg/mL. Once again, compounds belonging to series 17 showed the greatest
antibacterial activities of all the compounds tested, with MIC values in the range of
0.5–8 μg/mL. Compound 17h, in particular, exhibited the highest potency with an
MIC value of 0.5 μg/mL, making it 4-fold more potent than both of the controls (MIC
= 2 μg/mL).

Table 1. Inhibitory activities (MICa, μg/mL) of the five different series of compounds
against clinical isolates of Gram-positive and Gram-negative bacteria, as well as one
fungus.
Gram-positive strains
Fungus
C. albicans
7535f
Gram-negative strains
QRSA
MRSA
P.
S.
S. aureusb
S.mutans
E.coli
Compd
R
CCARM
CCARM
aeruginosa
typhinurium
4220b
3505c
3167d
3289e
1924g
2742h
2421i
13a
13b
13c
H
4-F
2-Br
32
32
64
32
32
32
32
32
32
32
16
16
32
32
32
64
32
64
>64
>64
>64
>64
>64
>64
13d
13e
13f
4-Br
16
64
32
32
32
8
8
64
16
16
32
8
8
64
16
16
32
8
16
64
16
16
32
4
16
32
16
16
32
8
16
64
16
32
32
8
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
2-OCH3
3-OCH3
4-CH3
2,4-(CH3)2
2,4-(Cl)2
H
13g
13h
13i
14a
14b
15a
15b
15c
16a
16b
>64
32
64
32
64
32
8
>64
64
32
32
64
32
4
>64
32
32
32
32
32
4
>64
32
32
16
64
64
8
>64
32
32
16
64
32
16
>64
64
32
16
64
64
8
>64
>64
>64
>64
>64
>64
64
4-Br
H
4-CH3
4-F
H
4-Br
17a
17b
17c
H
16
2
8
2
1
8
2
1
8
2
2
8
4
1
8
2
1
>64
64
>64
64
4-CH3
2-Cl
2
64
64
17d
17e
17f
3-Cl
2
1
1
1
1
2
1
1
2
1
4
1
1
1
1
4
1
1
1
64
64
64
64
64
64
64
64
64
64
4-Cl
1
1
2-F
2
4
4
17g
17h
4-Br
1
1
1
2,4-(Cl)2
0.5
0.5
0.5
Gatifloxacin
Moxifloxacin
Fluconazole
0.25
0.25
nd j
8
4
nd
2
1
nd
0.25
0.25
nd
0.5
0.5
1
2
2
nd
1
1
nd
0.5
0.5
nd
a The antibacterial tests was carried out three times, and the average values were taken as the
MICs.
b Staphylococcus aureus 4220. c Quinolone-resistant Staphylococcus aureus 3505.
d
e
f
Methicillin-resistant Staphylococcus aureus 3167. Streptococcus mutans 3289. Candida
albicans 7535. g Escherichia coli 1924.
h Pseudomonas aeruginosa 2742. i Salmonella typhimurium 2421. j nd: Not determined.
Analysis of the structure activity relationships revealed that the inclusion of a
dihydrotriazine ring was critical for the antibacterial activity because all of the
corresponding aldehyde intermediates were inactive, as reported in our previous

work.9-12 Compounds belonging to series 13, 14 and 15 exhibited similar levels of
antibacterial activity, indicating that the length of the linker between the two benzene
rings had very little effect on the activity of these compounds. In contrast, the
inclusion of a naphthalene nucleus, as exemplified by the compounds in series 16 and
17, resulted in a significant difference in the activity, which indicated that the length
of the bridge was critical to the activities of these compounds. Furthermore, the
introduction of substituents to the phenyl ring had very little impact on the
antimicrobial activity except for the compounds in series 17, where the introduction
of substituents to the benzene ring resulted in an increase in the activity. It is also
noteworthy that compound 17h, bearing a 2,4-dichloro-substituted phenyl ring,
showed excellent antimicrobial activities. These results therefore provide further
evidence that the inclusion of a 2,4-dichloro-substituted benzene ring is critical to the
activity of these compounds, which is consistent with the results obtained for a
previously reported series of rhodamine and aminoguanidine derivatives.16,17
To determine whether the compounds synthesized in the current study were
selectively toxic towards bacteria, we evaluated the cytotoxicities of several
representative compounds (13i, 16b, 17h) using a standard technique.18 As shown in
the Table 2, compound 13i exhibited weaker activity than 17h against the different
bacteria, in spite of its slight greater cytotoxicity than 17h, These results therefore
suggested that the promising antibacterial activities of these compounds could be
attributed to some unknown mechanism of action rather than their general toxicity.19
Table 2. Antibacterial activity and cytotoxicity for 13i, 16b and 17h
Test organisms
S. aureus 4220
MRSA 3167
L02b
13i
16b
17h
19.27
19.27
45.06
16.70
8.35
1.13
2.27
58.86
MIC (µmol/L)
IC50a (µmol/L)
58.70
a IC50 is the concentration of compound required to inhibit the growth of the cells by 50%. Values
represent the average of three independent experiments running in triplicate. Variation was
generally between 5–10%.
b Human normal liver cells.
To rationalize the observed antibacterial activity and understand the possible
mechanism of action of these compounds, a libdocking investigation was undertaken.

The crystal structure data (S. aureus DHFR) were obtained from the protein data bank
(PDB ID: 3fra) 20-22. Enzyme structures were checked for missing atoms, bonds and
contacts. Hydrogen atoms were added to the enzyme structure. Water molecules and
bound ligands were manually deleted. Preferred coordination modes of 17a and 17h
with dihydrofolate reductase (DHFR) protein are presented in figure 3. Fragment B of
17a is bound into the active site, in which the benzyl group shows Amide-Pi stacked
interaction with Leu 5. Fragment A of 17h is bound into the active site where the
dihydrotriazine ring shows H-bond interaction with Phe 92, Leu 5, and Asp 27,
comparably indicated its more potent activity than that of 17a. The preliminary
docking results imply that compounds 17a and 17h possibly display their antibacterial
activity through the interaction with DHFR protein by targeting residues of the active
cavities of DHFR.
Figure 3. Docking result of compound 17a and 17h. (A) Key residues in binding site
surrounding 17h. (B) 2D molecular docking modeling of compound 17h with 3fra. (C)
Key residues in binding site surrounding 17a. (D) 2D molecular docking modeling of
compound 17a with 3fra.

In order to confirm this phenomenon, compound 17h (MIC= 0.5 μg/mL) was tested
for its ability to inhibit DHFR activity in vitro enzyme assays (Fig. 4). 23 The test
molecule rendered a good inhibitory against the dihydrofolate reductase at 10 μmol/L,
comparably indicated its cause 58% decrease than that of the control group. The
results imply that compound 17h exert its antibacterial activity through DHFR
inhibition.
Figure 4. Inhibition of DHFR activities in vitro.
In conclusion, five new series of dihydrotriazine derivatives were synthesized using
a hybrid strategy and their antibacterial activities were evaluated in vitro using
standard techniques. The results showed that some of the compounds exhibited
pronounced inhibitory activities towards the growth of several strains of
Gram-positive bacteria with MIC values in the range of 0.5–64 μg/mL. All five
compound series exhibited good antibacterial activities with MIC values in the range
of 0.5–64 μg/mL. Notably, all of the compounds in series 17 (except for 17a) showed
strong activity against Gram-positive bacteria and fungus with MIC values of 0.5–2
μg/mL. Compound 17h exhibited the most potent antibacterial activity of all the
compounds in this series, making it comparable to gatifloxacin and moxifloxacin.
Furthermore, compound 17h (MIC = 0.5 μg/mL) was found to be 8- and 16-fold more
potent towards QRSA CCARM 3505 than moxifloxacin and gatifloxacin, respectively.
This compound also showed excellent activity against E. coli 1924 and C. albicans
7535 with MIC values of 0.5 and 1 μg/mL, respectively. However, compound 17h
was ineffective against P. aeruginosa 2742 and S. typhimurium 2421. These results
therefore suggest that dihydrotriazine derivatives could potentially be used to develop

potent antibacterial and antifungal agents for the clinical treatment of infectious
diseases. Preliminary docking study showed that these compounds have a good
interaction with the active cavities of DHFR, possibly exhibit their potency via
inhibiting DHFR. In vitro enzyme study implies that compound 17h exert its
antibacterial activity through DHFR inhibition.
Acknowledgment
This work was supported by the National Natural Science Foundation of China
(Grant numbers 81260468, 81460524 and 81060257) and the Natural Science
Foundation of Jilin Province (No. 20160101218JC).
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14. Preparation of 17h: Synthesized intermediate compounds (1 mmol) and
Metformin hydrochloride (1 mmol) were refluxed in glacial acetic acid (7 mL) at
o
120 C for 4–6 h. The whole processes of the reactions were traced by TLC, then

removed solvent under reduced pressure. The crude products were purified by
column chromatography (dichloromethane : methanol = 20 : 1). Yield 82%; m.p.
1
266-268 oC. H NMR (300 MHz, DMSO-d6, ppm) δ 9.04 (s, 2H, NH2), 7.92 (d,
2H, J = 9.0 Hz, Ar-H), 7.83 (s, 1H, Ar-H), 7.73-7.68 (m, 2H, Ar-H), 7.58-7.48 (m,
4H, Ar-H and NH), 7.30 (dd, 1H, J = 9.0, 3.0 Hz, Ar-H) 5.96 (s, 1H, CH), 5.28 (s,
13
2H, CH2), 3.06 (s, 6H, CH3). C NMR (75 MHz, DMSO-d6, ppm) δ 157.85,
156.91, 156.24, 136.48, 134.72, 134.14, 134.07, 133.80, 131.92, 130.28, 129.42,
128.44, 128.14, 128.05, 125.20, 124.93, 119.74, 107.74, 66.84, 62.70, 37.36 (2C).
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HPLC purity of 96.94% (retention time = 15.33 min).
15. Evaluation of anti-bacterial activity in vitro: The micro-organisms used in the
present study were S. aureus 4220, E. Coli 1942, S.mutans 3289, P. Aeruginosa
2742, S. Typhinurium 2421 and C. Albicans 7535. The strains of
multidrug-resistant clinical isolates were methicillin-resistant S. aureus (MRSA
CCARM 3167) 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 MuellereHinton 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 norfloxacin were used. Test compounds
were prepared in DMSO, the final concentration of which did not exceed 0.05%.
A two-fold serial dilution technique was used to obtain final concentrations of
64-0.25 mg/mL. The MIC was defined as the concentration of a test compound
o
that completely inhibited bacteria growth during 24 h incubation at 37 C.
Bacteria growth was determined by measuring the absorption at 650 nm using a
microtiter enzyme-linked immunosorbent assay (ELISA) reader. All experiments
were carried out three times.
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measured through the colorimetric MTT assay. Human normal liver cells (L02)
suspension in DMEM medium supplemented with 10% FBS and antimycotic was
added in 96-well microplates at 1.8×104 cells/well. A variety of concentrations of
the test compounds (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.625 µM/L) dissolved by
distilled 10% DMSO was added to each well. Incubation for 24h at 37℃ under
5% CO2, 2.5mg/mL of MTT solution was added to each well. Further the plate
was incubated for 4h. 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 positive control, whereas untreated cells were used as
negative controls. The IC50 values were defined as the concentrations inhibiting
50% of cell growth. All experiments were performed in triplicate.
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23. Inhibition of DHFR activities in vitro: The test of compound 17h was measured
through the ELISA assay. Solid-phase antibody was prepared by coating the
microtiter plate wells with purified human dihydrofolate reductase (DHFR)
antibody. A variety of concentrations of the test compound (0, 0.1, 0.3, 1, 3, 10
µmol/L) was combined with DHFR. Incubation for 1.5 h at 37℃, HRP labeled
was added to become antibody-antigen-enzyme-antibody complex. After washing
completely, TMB substrate was added, TMB substrate was becoming blue color at
HRP enzyme-catalyzed. Reaction was terminated by the addition of a sulphuric
acid solution and the color change was measured spectrophotometrically at a
wavelength of 450nm. The concentration of DHFR activity was calculated by a
standard curve.

Synthesis and evaluation of the antibacterial activities of aryl
substituted dihydrotriazine derivatives
Tian-Yi Zhang a,b,1, Zhan-Kui Yu a,1, Xue-Jun Jin a, Ming-Yue Li a, Liang-Peng Sun a,
Chang-Ji Zhenga,*, Hu-Ri Piao a,*
aKey Laboratory of Natural Resources and Functional Molecules of the Changbai
Mountain, Affiliated Ministry of Education; Yanbian University College of Pharmacy,
Yanji, Jilin Province 133002, PR China
bDepartment of Pharmacy, Jilin Medical University, Jilin, Jilin Province 132013,China
Five novel series of dihydrotriazine derivatives were designed, synthesized,
and evaluated for their antibacterial and antifungal activity.

Research Highlights
1) Five series of dihydrotriazine derivatives were designed and synthesized.
2) 17h showed the most potent with MIC values of 0.5 g/mL against selected strains
of bacteria.
3) Cytotoxicity results indicate that these compounds are selectively toxic towards
bacteria.