Synthesis of novel dihydrotriazine derivatives bearing 1,3-diaryl pyrazole moieties as potential antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2019.02.033

Three novel series of dihydrotriazine derivatives bearing 1,3-diaryl pyrazole moieties were designed, synthesized and evaluated in terms of their antibacterial and antifungal activities. Most of the synthesized compounds showed potent inhibition of several Gram-positive bacterial strains (including multidrug-resistant clinical isolates) and Gram-negative bacterial strains with minimum inhibitory concentration values in the range of 1–64 μg/mL. Compounds 4b and 4c presented the most potent inhibitory activity against Gram-positive bacteria (S. aureus 4220, MRSA 3167, QRSA 3519) and Gram-negative bacteria (E. coli 1924), with minimum inhibitory concentration values of 1 or 2 μg/mL. Compared with previous studies, these compounds exhibited a broad spectrum of inhibitory activity. The cytotoxic activity of the compounds 4a, 4b, 4c and 11n were assessed in L02 cells. In vitro enzyme study implied that compound 4c exerted its antibacterial activity through DHFR inhibition.

Full text of DOI:10.1016/j.bmcl.2019.02.033

Accepted Manuscript
Synthesis of novel dihydrotriazin derivatives bearing 1,3-diaryl pyrazole moi-
eties as potential antibacterial agents
Tian-Yi Zhang, Chang-Ji Zheng, Jie Wu, Liang-Peng Sun, Hu-Ri Piao
PII:
S0960-894X(19)30118-0
https://doi.org/10.1016/j.bmcl.2019.02.033
BMCL 26313
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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4 January 2019
26 February 2019
27 February 2019
Please cite this article as: Zhang, T-Y., Zheng, C-J., Wu, J., Sun, L-P., Piao, H-R., Synthesis of novel dihydrotriazin
derivatives bearing 1,3-diaryl pyrazole moieties as potential antibacterial agents, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.02.033
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Synthesis of novel dihydrotriazin derivatives bearing
1,3-diaryl pyrazole moieties as potential antibacterial agents
Tian-Yi Zhang a,c,*, Chang-Ji Zheng b,c, Jie Wu b, Liang-Peng Sun b, Hu-Ri Piao b,*
aDepartment of Pharmacy, Jilin Medical University, Jilin, Jilin Province 132013, PR
China
bKey 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
*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(432)64561064;
E-mail: tianyizhang@126.com (T. Y. Zhang).
c These authors contributed equally to this work.
1

Abstract
Three novel series of dihydrotriazin derivatives bearing 1,3-diaryl pyrazole
moieties were designed, synthesized and evaluated in terms of their antibacterial and
antifungal activities. Most of the synthesized compounds showed potent inhibition of
several Gram-positive bacterial strains (including multidrug-resistant clinical isolates)
and Gram-negative bacterial strains with minimum inhibitory concentration values in
the range of 1–64 µg/mL. Compounds 4b and 4c presented the most potent inhibitory
activity against Gram-positive bacteria (S. aureus 4220, MRSA 3167, QRSA 3519)
and Gram-negative bacteria (E. coli 1924), with minimum inhibitory concentration
values of 1 or 2 µg/mL. Compared with previous studies, these compounds exhibited
a broad spectrum of inhibitory activity. The cytotoxic activity of the compounds 4a,
4b, 4c and 11n were assessed in L02 cells. In vitro enzyme study implied that
compound 4c exerted its antibacterial activity through DHFR inhibition.
Keywords: Pyrazole; Dihydrotriazine; Antibacterial activity; Antifungal activity;
Cytotoxicity; DHFR inhibition
2

Infections caused by multidrug-resistant (MDR) bacteria, particularly by
Gram-negative pathogens, represent a serious and growing threat to human health.
The“ESKAPE”pathogens, such as Escherichia coli, Klebsiella pneumoniae,
Acinetobacter baumannii and Pseudomonas aeruginosa, are emerging as threatening
pathogens.1,2 Current bacterial infection treatments have become inadequate and are
often associated with higher healthcare costs and increased morbidity and mortality.3,4
Furthermore, fungal infections pose a serious threat to human health, especially to
immunocompromised patients.5,6 More than 90% of the life-threatening invasive
fungal infections (IFIs) are caused by the Candida, Cryptococcus and Aspergillus
species. The Candida species, particularly Candida albicans (C. albicans), are the
most common cause of IFIs and are ranked as the fourth most common cause of
nosocomial bloodstream infections in modern hospitals with approximately 40%
mortality.7,8 Therefore, there is an urgent need for the development of new
antibacterial agents with divergent and unique structures and mechanisms of action
that differ from those of existing antimicrobial agents.9
Heterocyclic chemistry has become one of the most important fields of research
in pharmaceutical industry due to their many fold applications. Pyrazoles and its
derivatives are important nitrogen containing heterocyclic compounds.10 Pyrazoles
constitute the core structures of natural products and pyrazole based well known drugs
are available in the market such as celebrex, viagra and acomplia.11 Medicinal
chemistry studies have shown that a 1,3,5-triazine scaffold is an outstanding
pharmacophore, and many molecules with this scaffold have been reported as
antibacterial agents. 1,2-Dihydro-1,3,5-triazine (Baker triazines) compounds are by
far the best known, with several studies exploring their potential as inhibitors of
eukaryotic dihydrofolate reductase (DHFR), an important enzyme in the de novo
pathway of purine and thymidine synthesis, and small molecules targeting this
enzyme have demonstrated utility as potential antibiotics.12,13 In our previous work,
we reported the identification of a series of 1,3-diphenyl-1H-pyrazoles functionalized
with rhodanine derivatives, and demonstrated that all of the compounds belonging to
this series showed outstanding bacteriostatic activity against Gram-positive bacteria,
3

as exemplified by compounds A and B (MIC = 2 µg/mL and 16 µg/mL, respectively)
(Fig. 1).14,15 Unfortunately, compounds belonging to this series did not show any
bacteriostatic activity against Gram-negative bacteria. In contrast, we synthesized a
series of dihydrotriazine derivatives containing benzyl naphthalene moieties.
Compounds C and D were found to be the most potent of all of the compounds tested,
with an MIC value of 0.5 µg/mL or 1 µg/mL against several Gram-positive (S. aureus
4220 and QRSA CCARM 3505) and Gram-negative (E. coli 1924) strains of bacteria.
(Fig. 1).16,17 In this study, we designed and synthesized three novel series of
compounds (4a-c, 8a-i, 11a-p) (Fig. 2) using a hybrid strategy with compounds A-D
as the lead compounds, in which the rhodamine moiety was replaced by a
dihydrogentriazine scaffold. Simultaneously changing the substituents on the phenyl
ring of the pyrazole, to investigate their effects on activity. Inspired from existing
antibacterial agents bearing nitro groups, such as chloramphenicol that operates via
inhibition of bacterial protein synthesis after the nitro group is metabolized to a
hydroxyamino group, nitro group was introduced into the phenyl ring on the N1
position of the pyrazole. Thus, three series of 28 dihydrotriazin derivatives bearing
1,3-diaryl pyrazoles were synthesized, characterized and screened.
Figure 1. Previously reported antibacterial compounds
4

Figure 2. Designed target compounds
The targets were synthesized according to the route depicted in Scheme 1.
Hydrazone derivatives (compounds 2, 6 and 9) were prepared by reacting substituted
acetophenones with phenylhydrazine, 4-nitrophenylhydrazine or
2,4-dinitrophenylhydrazine in the presence of glacial acetic acid in ethanol.18 A series
of intermediates (2, 6 and 9) reacted under Vilsmeier-Haack (DMF-POCl3) conditions
and afforded corresponding 4-carboxaldehyde functionalized pyrazole intermediates
(3, 7 or 10).19,20 Then, the intermediates were reacted with moroxydine hydrochloride,
metformin hydrochloride or phenformin hydrochloride in acetic acid to generate the
target compounds.17 The structures of the synthesized dihydrotriazine compounds
were characterized by 1H NMR, 13C NMR, MS 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 311 nm under UV.21
5

Scheme 1. Synthetic scheme for the synthesis of the target compounds. Reagents and
conditions: (a) EtOH, AcOH or AcONa, Reflux 5-7 h, (b) POCl3 , DMF, 0℃, 0.5 h,
70℃, 6h, (c) Moroxydine hydrochloride or metformin hydrochloride or phenformin
hydrochloride, AcOH, 120℃, 4-8 h.
The MIC values of the synthesized compounds were determined against S.
aureus, E. coli, Candida albicans, Streptococcus mutans and the clinical isolates of
multidrug-resistant Gram-positive bacterial strains (Tables 1 and 2).22 Gatifloxacin,
moxifloxacin, norfloxacin, and oxacillin were used for the antibacterial activity,
fluconazole and itraconazole for the antifungal activity as positive controls.
As shown in Table 1, most of the synthesized compounds showed good
inhibitory activities against the different bacteria and the one fungus tested in the
current study with MICs ranging from 1-64 µg/mL. Compound 4c had the highest
activity against the two different types of Gram-positive bacteria, with an MIC value
of 1 µg/mL, this was slightly lower than gatifloxacin (MIC = 0.25 µg/mL) and
moxifloxacin (MIC = 0.25 µg/mL). Against the Gram-negative E. coli 1924,
compounds 4b and 4c were equipotent or more potent than the positive controls
gatifloxacin and moxifloxacin (MIC = 2 µg/mL), with an MIC of 1 µg/mL. In terms
of its activity towards the fungus C. albicans 7535, compound 4c displayed the
strongest potency of all of the compounds synthesized in series 4 with an MIC value
6

of 1 µg/mL, which was equal to that of fluconazole. Compounds 4a and 4b also
displayed good potency, with an MIC of 4 µg/mL, which was slightly lower than that
of fluconazole (MIC = 1 µg/mL).
As indicated in Table 2, all of the synthesized compounds were also tested for
their inhibitory activities against the clinical isolates of several different
multidrug-resistant Gram-positive bacterial strains (MRSA CCARM 3167 and 3506,
QRSA CCARM 3505 and 3519). Compound 4c showed potent activity against the
MRSA (3167 and 3506) strains, with an MIC value of 1 µg/mL. This was equivalent
to moxifloxacin (MIC = 1 µg/mL) and greater than gatifloxacin (MIC = 2 µg/mL),
oxacillin (MIC > 64 µg/mL) and norfloxacin (MIC = 8 µg/mL for MRSA 3167 and 4
µg/mL for MRSA 3056). Compounds 4a and 4b showed the same potenty as
gatifloxacin (MIC = 2 µg/mL) against the MRSA (3167 and 3506) strains. For the
QRSA (CCARM 3505 and 3519) strains, compound 4c also had the greatest
inhibitory effect, with an MIC value of 1 µg/mL. This was equivalent to oxacillin
(MIC = 1 µg/mL) and stronger than moxifloxacin (MIC = 4 µg/mL), norfloxacin
(MIC > 64 µg/mL) and gatifloxacin (MIC = 8 µg/mL against CCARM 3505 and 4
µg/mL against CCARM 3519). Compounds 4a, 4b, 11g and 11m were equipotent to
the positive controls moxifloxacin (MIC = 4 µg/mL for QRSA 3505), with an MIC of
4 µg/mL.
Based on the present data of the synthesized compounds, simple
structure-activity relationships (SAR) could be proposed. The introduction of a
dihydrotriazin moiety resulted in moderate to good levels of inhibition against the
Gram-negative strains. Comparing the antibacterial activity of the compounds in
series 4, 8 and 11, compounds in series 4 showed better activity than those in series 8
and 11 against most of the test strains, which suggested that the 4-nitrophenyl moiety
at the N1 position of the pyrazole is critical. Nevertheless, the introduction of a
2-nitrophenyl moiety on the phenyl ring, as exemplified by the compounds 11d and
11p, resulted in a significant difference in the activity, which indicated that the
increase of 2-nitrophenyl moiety on the phenyl ring did not exhibit a positive impact
on the activity. Furthermore, no clear pattern was found for the SAR between the
7

antibacterial activity and the position and physicochemical properties of different
substituents on the phenyl ring, indicating that the electronic effect of the substituent
on the benzene ring is not critical. Isosteric replacement with a phenethyl group at the
N-position of the dihydro-1,3,5-triazine ring, as exemplified by the compounds in
series 4, resulted in a significant difference in the activity, indicating that the
phenethyl group was critical to the activities of these compounds.
Table 1. Inhibitory activity (MIC, µg/mL) of compounds 4a–c, 8a–i and 11a–p against various
bacteria.
Gram-positive strains
Gram-negative strains
Fungus
Compound R1
R2
4220a
2
3289b
8
1924c
4
7535d
4a
2,4-di-Cl morpholine
2,4-di-Cl NHMe2
4
4b
2
4
2
4
4c
2,4-di-Cl NH(CH2)2Ph
1
1
1
1
8a
3-Cl
4-Cl
4-Br
4-CH3
4-CH3
3-Cl
4-Br
4-Cl
H
morpholine
morpholine
morpholine
morpholine
NHMe2
16
16
8
32
32
32
64
64
16
16
32
>64
64
>64
16
>64
16
64
8
16
16
16
64
32
8
16
16
16
64
32
8
8b
8c
8d
64
32
4
8e
8f
NHMe2
8g
NHMe2
8
8
8
8h
NHMe2
8
8
8
8i
NHMe2
32
32
64
8
64
64
64
8
>64
64
64
8
11a
11b
11c
11d
11e
11f
11g
11h
11i
2-Cl
2-F
morpholine
morpholine
morpholine
morpholine
4-Br
2-NO2
>64
16
32
4
>64
8
>64
8
2,4-di-Cl morpholine
2-Cl
3-Cl
4-CH3
3-OCH3
2-F
NHMe2
NHMe2
NHMe2
NHMe2
NHMe2
NHMe2
NHMe2
NHMe2
32
8
64
8
16
32
64
32
32
8
16
64
64
32
64
16
8
16
32
32
32
32
8
64
32
32
64
32
8
11j
11k
11l
2-Br
H
11m
11n
11o
11p
Gatifloxacin
4-Br
2,4-di-Cl NHMe2
8
8
8
4-F
NHMe2
NHMe2
32
>64
0.25
32
>64
0.25
32
>64
2
32
>64
n.d
2-NO2
8

Moxifloxacin
Fluconazole
Itraconazole
0.25
n.d
0.25
n.d
2
n.d
1
n.d
n.d
n.d
n.d
0.6
a Staphylococcus aureus RN 4220. b Streptococcus mutans 3289.
c Escherichia coliKCTC 1924. d Candida albicans 7535.
S. aureus RN 4220 is a genotype of S. aureus. KCTC, Korean Collection for Type Cultures;
CCARM, Culture Collection of Antimicrobial Resistant Microbes; n.d.: Not determined
Table 2. Inhibitory activity (MIC, µg/mL) of compounds 4a–c, 8a–i and 11a–p against clinical
isolates of multidrug-resistant Gram-positive strains.
Multidrug-resistant Gram-positive strains
Compound R1
R2
MRSA
3167a
2
QRSA
3505c
4
3506b
2
3519d
4
4a
2,4-di-Cl
morpholine
NHMe2
4b
2,4-di-Cl
2,4-di-Cl
3-Cl
2
2
4
2
4c
NH(CH2)2Ph
morpholine
morpholine
morpholine
morpholine
NHMe2
1
1
1
1
8a
16
16
8
16
16
4
16
16
16
64
64
32
16
16
32
32
64
8
16
16
8
8b
4-Cl
8c
4-Br
8d
4-CH3
4-CH3
3-Cl
32
16
16
4
32
32
16
4
64
64
32
16
16
64
64
64
8
8e
8f
NHMe2
8g
4-Br
NHMe2
8h
4-Cl
NHMe2
16
32
32
64
8
8
8i
H
NHMe2
32
32
32
4
11a
11b
11c
11d
11e
11f
11g
11h
11i
2-Cl
morpholine
morpholine
morpholine
morpholine
morpholine
NHMe2
2-F
4-Br
2-NO2
2,4-di-Cl
2-Cl
>64
8
>64
8
>64
8
>64
16
64
16
16
64
64
64
64
8
32
4
64
4
32
4
3-Cl
NHMe2
4-CH3
3-OCH3
2-F
NHMe2
8
8
8
NHMe2
32
64
32
32
8
32
32
32
64
4
64
64
32
32
4
11j
11k
11l
NHMe2
2-Br
NHMe2
H
NHMe2
11m
11n
11o
11p
Gatifloxacin
4-Br
NHMe2
2,4-di-Cl
4-F
NHMe2
4
4
8
16
64
>64
4
NHMe2
32
>64
2
32
>64
2
64
>64
8
2-NO2
NHMe2
9

Moxifloxacin
Norfloxacin
Oxacillin
1
1
4
4
8
4
>64
1
>64
1
>64
>64
a Methicillin-resistant S. aureus CCARM 3167. b Methicillin-resistant S. aureus CCARM 3506.
c Quinolone-resistant S. aureus CCARM 3505. d Quinolone-resistant S. aureus CCARM 3519.
To determine whether the antibacterial and antifungal activities of the
synthesized compounds were selectively toxic towards bacteria and the fungus, we
evaluated the cytotoxicity of the compounds 4a, 4b, 4c and 11n using a standard
technique.23 As shown in Table 3, the result indicated that the compounds 4a, 4b and
11n showed any appreciable cytotoxic activity (IC50 > 100 µmol/L against L02 cells),
suggesting that the promising antibacterial activity of these compounds is not due to
their toxicity, but some unknown mechanism of action.
Table 3. Antibacterial activity and cytotoxicity for 4a, 4b, 4c and 11n
Test organisms 4a
4b
4c
11n
S. aureus 4220 3.9
4.2
1.8
15.5
7.7
MIC (µmol/L)
IC50a (µmol/L)
MRSA 3167
L02b
3.9
4.2
1.8
>100
>100
50.46
>100
a IC50 is the concentration required to inhibit the cell growth by 50%. Data represent the average
of three independent experiments running in triplicate. Variation was generally between 5–10%.
b Human normal hepatic cells.
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 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).24,25 The
water molecules and heavy atom in protein were removed and the protein was
prepared by adding hydrogen and correcting incomplete residues using Clean Protein
tool of DS, then the protein was refined with CHARMm. The structures of 4b, 4c and
11l were sketched in 2D and converted into 3D using the DS molecule editor (Fig. 3).
Fragment A of 4b is bound into the active site, in which the benzene ring formed
pi-alkyl bond with Phe 92. The pyrazole N atom of 4b formed alkyl bond with Leu 20
10

and nitro group of 4b formed hydrogen bond with the backbone of Gly 94 and Gln 95.
The dihydrotriazin ring N atom of 4b interacted with Gln 19 via Conventional
hydrogen bond. Fragment B of 4c is bound into the active site, in which the benzene
ring formed alkyl bond with Leu 5, Val 31 and Ala 7. The Cl atom of 4c interacted
with the backbone of Ile 14 and Leu 20. Fragment C of 11l is bound into the active
site where the pyrazole N atom shows interaction with Gln 95 via Conventional
hydrogen bond. Nitro group of 11l also formed Conventional hydrogen bond with the
backbone of Gly 94 and Thr 96. The benzene ring of 11l formed pi-alkyl bond with Ile
14. Our docking results indicated that more active compounds 4b and 4c, showed the
more favorable intermolecular interactions with the DHFR active site than compound
11l. The preliminary docking results imply that compounds 4b, 4c and 11l possibly
display their antibacterial activity through the interaction with DHFR protein by
targeting residues of the active cavities of DHFR.
11

Figure 3. Docking result of compound 4b, 4c and 11l. (A) Key residues in binding
site surrounding 4b. (B) 2D molecular docking modeling of compound 4b with 3fra.
(C) Key residues in binding site surrounding 4c. (D) 2D molecular docking modeling
of compound 4c with 3fra. (E) Key residues in binding site surrounding 11l. (F) 2D
molecular docking modeling of compound 11l with 3fra.
In order to confirm this phenomenon, compound 4c (MIC= 1 μg/mL) was tested for
its ability to inhibit DHFR activity in vitro enzyme assays (Fig. 4).26 The test molecule
rendered a inhibitory against the dihydrofolate reductase at 10 μmol/L, comparably
indicated its cause 92% decrease than that of the control group. The results implied
that compound 4c exerted its antibacterial activity through DHFR inhibition.
Figure 4. Inhibition of DHFR activities in vitro.
In summary, we have synthesized three novel series of dihydrotriazin derivatives
bearing 1,3-diaryl pyrazole moieties and evaluated their antibacterial and antifungal
activities. The results showed that most compounds exhibited moderate to good levels
of antibacterial activities. In particular, all of the compounds in series 4 exhibited
good levels of antibacterial activities against Gram-negative strains (E. coli 1924) and
12

antifungal activity (C. albicans 7535). These results suggested that the dihydrotriazine
derivatives bearing 1,3-diaryl pyrazole moieties, which play a critical role in
increasing the antibacterial properties of the compounds, represented promising lead
compounds for the development of novel antibacterial agents. In vitro enzyme study
implied that compound 4c exerted 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 Department of
Education of Jilin Province (No. JJKH20191070KJ).
References and notes
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21. Preparation of 4b: Synthesized intermediate compounds (1 mmol) and Metformin
hydrochloride (1 mmol) were refluxed in glacial acetic acid (7 mL) at 120oC for 4–8 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 50%; m.p. 234-236 oC. 1H NMR
(DMSO-d6, 300 MHz, ppm): δ 9.06 (s, 1H, NH), 8.86 (s, 1H, NH), 8.59 (s, 1H, NH),
8.41 (d, J = 9.1 Hz, 2H, Ar-H), 8.18 (d, J = 9.1 Hz, 2H, Ar-H), 7.78 (s, 1H, Ar-H),
7.55 (s, 2H, Ar-H), 7.37 (s, 1H, CH), 5.82 (s, 1H, CH),2.90 (s, 6H, CH3). 13C NMR
(126 MHz, DMSO) δ 157.64, 155.90, 149.62, 145.77, 143.83, 135.06, 134.65, 133.86,
130.35, 129.64, 129.46, 127.80, 126.07, 125.15, 119.19, 56.50. HRMS (ESI) m/z
calcd for C20H19Cl2N8O2 (M+H) 473.10025, found 473.10022. HPLC purity of
95.34% (retention time = 13.04 min).
22. Evaluation of anti-bacterial activity in vitro: The micro-organisms used in the
present study were S. aureus 4220, S.mutans 3289, E. Coli 1942 and C. Albicans 7535.
The strains of multidrug-resistant clinical isolates were methicillin-resistant S. aureus
(MRSA CCARM 3167 and 3506) and quinolone-resistant S. aureus (QRSA CCARM
3505 and 3519). 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 µg/mL. The
MIC was defined as the concentration of a test compound that completely inhibited
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bacteria growth during 24 h incubation at 37 oC. 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.
23. Cytotoxicity on human cells: The cytotoxicity test of selected compounds was
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.
24. Lam T, Hilgers MT, Cunningham ML, et al. J Med Chem. 2014;57:651.
25. Vijesh AM, Isloor AM, Peethambar SK, et al. Eur J Med Chem. 2011;46:5591.
26. Inhibition of DHFR activities in vitro: The test of compound 4c 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, 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 450 nm. The concentration of DHFR activity was calculated by a standard curve.
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Three novel series of dihydrotriazine derivatives were designed, synthesized, and
evaluated for their antibacterial and antifungal activity.
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