Design, synthesis, and biological evaluation of new 6,: N 2-diaryl-1,3,5-triazine-2,4-diamines as anticancer agents selectively targeting triple negative breast cancer cells

Article abstract of DOI:10.1039/d0ra04970k

New 6,N2-diaryl-1,3,5-triazine-2,4-diamines were designed using the 3D-QSAR model developed earlier. These compounds were prepared and their antiproliferative activity was evaluated against three breast cancer cell lines (MDA-MB231, SKBR-3 and MCF-7) and non-cancerous MCF-10A epithelial breast cells. The synthesized compounds demonstrated selective antiproliferative activity against triple negative MDA-MB231 breast cancer cells. The most active compound in the series inhibited MDA-MB231 breast cancer cell growth with a GI50 value of 1 nM. None of the tested compounds significantly affected the growth of the normal breast cells. The time-dependent cytotoxic effect, observed when cytotoxicity was assessed at different time intervals after the treatment, and morphological features, observed in the fluorescence microscopy and live cell imaging experiments, suggested apoptosis as the main pathway for the antiproliferative activity of these compounds against MDA-MB231 cells.

Full text of DOI:10.1039/d0ra04970k

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PAPER
Design, synthesis, and biological evaluation of new
6,N2-diaryl-1,3,5-triazine-2,4-diamines as
anticancer agents selectively targeting triple
negative breast cancer cells†
Cite this: RSC Adv., 2020, 10, 25517
a
b
Ahmad Junaid,a Felicia Phei Lin Lim,
Edward R. T. Tiekink
ac
*
and Anton V. Dolzhenko
New 6,N2-diaryl-1,3,5-triazine-2,4-diamines were designed using the 3D-QSAR model developed earlier.
These compounds were prepared and their antiproliferative activity was evaluated against three breast
cancer cell lines (MDA-MB231, SKBR-3 and MCF-7) and non-cancerous MCF-10A epithelial breast cells.
The synthesized compounds demonstrated selective antiproliferative activity against triple negative
MDA-MB231 breast cancer cells. The most active compound in the series inhibited MDA-MB231 breast
cancer cell growth with a GI50 value of 1 nM. None of the tested compounds significantly affected the
growth of the normal breast cells. The time-dependent cytotoxic effect, observed when cytotoxicity was
assessed at different time intervals after the treatment, and morphological features, observed in the
fluorescence microscopy and live cell imaging experiments, suggested apoptosis as the main pathway
for the antiproliferative activity of these compounds against MDA-MB231 cells.
Received 5th June 2020
Accepted 28th June 2020
DOI: 10.1039/d0ra04970k
rsc.li/rsc-advances
and the prevalent cause of cancer deaths.1 Breast cancer is
a rather heterogeneous form of cancer with cancer cells signif-
1. Introduction
icantly varying in their properties and thus requiring different
therapeutic approaches.2 On the basis of presence or absence of
molecular markers, breast cancer is classied into 4 main
subtypes: (1) human epidermal growth factor 2 (ERBB2) positive
cancer with cells expressing ERBB2, (2) luminal A breast cancer
with cells expressing estrogen or progesterone receptors but not
ERBB2, (3) luminal B breast cancer with cells expressing
hormone receptors and ERBB2 negative cells, and (4) triple
negative breast cancer with cells lacking molecular markers
used for this classication.
Despite signicant advancements in cancer therapy, cancer
remains one of the diseases having the most negative impact on
society. According to the World Health Organization, cancer
was the second leading cause of patient lethality in 2018
causing almost 10 million deaths.1 Moreover, the cancer prev-
alence and mortality from cancer have been continuously
growing worldwide, in both developing and developed coun-
tries. It was projected that from 14 million people suffering
from cancer in 2012 the number of new cases per year will
double by 2030.
The current therapeutic options and agents under develop-
ment for the treatment of different types of breast cancer vary
signicantly. The cancer cells overexpressing hormone recep-
tors can be targeted by anti-estrogenic medicines, like tamox-
ifen, by aromatase inhibitors, like letrozole, or other medicines
for endocrine therapy. To improve therapeutic outcome of the
endocrine therapy, other agents with different mechanisms
have been investigated: pan-class I phosphatidylinositol 3-
kinase (PI3K) inhibitors (e.g. alpelisib and buparlisib),3,4
mammalian target of rapamycin (mTOR) inhibitors (e.g. ever-
olimus),5,6 and cyclin-dependent kinase CDK4 and CDK6
inhibitors (e.g. palbociclib and ribociclib).7–9 For the treatment
of ERBB2-positive breast cancer, PI3K and mTOR inhibitors are
used together with ERBB2-targeted antibodies. Due to the
absence of any targeted therapy for triple negative breast cancer,
the general chemotherapy remains the main option available
Breast cancer had the highest incidence rates among all
types of cancer in 2018 (46.3 per 100 000 females). In females,
breast cancer is the most frequently diagnosed type of cancer
aSchool of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, Bandar
Sunway, Selangor Darul Ehsan 47500, Malaysia. E-mail: anton.dolzhenko@monash.
edu
bResearch Centre for Crystalline Materials, School of Science and Technology, Sunway
University, 5 Jalan Universiti, Bandar Sunway, Selangor Darul Ehsan 47500, Malaysia
cSchool of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research
Institute, Faculty of Health Sciences, Curtin University, GPO Box U1987, Perth,
Western Australia 6845, Australia
† Electronic supplementary information (ESI) available: Copies of 1H and 13C
NMR spectra of prepared compounds 1–21; details of crystallographic study for
compound 20, plots of concentration–response curves for cytotoxicity
experiments, video of live cell imaging of MDA-MB231 cells treated with
compound 18. CCDC 1934496. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/d0ra04970k
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Table
1
6,N2-Diaryl-1,3,5-triazine-2,4-diamines (1–21) and their
for the treatment of this most aggressive and mortal subtype of
breast cancer. Typical medicines used against triple negative
breast cancer include platinum drugs, taxanes, and anthracy-
cline.10 A group of promising emerging medicines, poly(ADP-
ribose) polymerase inhibitors (e.g. olaparib and talazoparib),
have been identied as a more specic therapy for a subgroup of
triple negative breast cancer with cells having a mutation of
BRCA1/BRCA2 genes.11 New effective and selective anticancer
agents are urgently needed for the safer and more effective
treatment of triple negative breast cancer. The search for new
potent compounds targeting breast cancer broadly covers
various types of chemical structures.12–14
antiproliferative activity against MDA-MB231 cells predicted using the
QSAR model developed earlier16
1,3,5-Triazine ring has been effectively used as a skeleton for
the construction of new anticancer agents.15 Recently, we
identied 6,N2-diaryl-1,3,5-triazine-2,4-diamines selectively tar-
geting triple negative MDA-MB231 breast cancer cells.16 We also
developed a 3D-QSAR model for the prediction of anti-
proliferative activity of this type of compounds against MDA-
MB231 breast cancer cells. Herein, we are testing predictive
power of this model for the design of new anticancer agents
with the 6,N2-diaryl-1,3,5-triazine-2,4-diamine scaffold and
continue our efforts on the development of highly potent and
selective anticancer agents.
Predicted
pGI50
Compound
R1
R2
1
2
3
4
5
6
7
8
3-F
4-Cl
4-CF3
4-CF3O
4-Me2N
4-Me
4-Me
4-MeO
2-MeO
4-Me
2-Cl
4-Cl
2-Cl
2-Cl
4-Cl
H
4-Me
H
2-F
2-Cl
2-MeO
3-Cl
3-Me
4-Cl
4-Br
4-Me
4-MeO
4-CF3O
4-iPr
5.54
5.51
5.22
4.95
5.79
5.65
5.51
6.58
5.41
5.58
4.85
5.10
4.78
4.62
4.49
4.95
4.45
4.40
4.79
4.49
5.01
9
4-MeO
10
11
12
13
14
15
16
17
18
19
20
21
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
2. Results and discussion
2.1. QSAR-guided design of compounds
We previously reported synthesis of 6,N2-substituted 1,3,5-
triazine-2,4-diamines (126 compounds) and their cytotoxic
activity against breast cancer cell lines (MDA-MB231, SKBR-3 and
MCF-7) and non-cancerous epithelial breast cells (MCF-10A).16
Some of the prepared compounds demonstrated selective activity
against triple negative breast cancer cells (MDA-MB231). Twenty-
ve most active compounds were further evaluated and their GI50
values were estimated and used for the development of a 3D-
QSAR model suitable for the design of new potent anticancer
agents.16 The model is based on the activity of compounds with
different substituents in phenyl rings A and B (Fig. 1).
To test the earlier developed model, two main groups of
compounds were selected for the synthesis. Number of
substituents in each of the phenyl rings A and B for the rst
group of compounds (1–9) was limited to one functional group.
The second group included 3,4,5-trimethoxyphenyl substituted
compounds 10–21 to test effect of multiple substituents in ring
A on the activity. Previously, we noticed that compounds with
the R1 group in meta-position of ring A retained activity with
a greater variety of substituents at another phenyl ring.
Contrary, activity of compounds with para-position of R1 was
very sensitive to the type and position of R2, disappearing when
R2 was located in the para-position of ring B. Selecting
compounds 10–21 with the preferred methoxy groups located in
positions equivalent to the para- and both meta-positions of ring
A, we intended to test which activity pattern they will follow. The
predicted pGI50 values obtained from the 3D-QSAR model
justied synthesis of the compounds.
The developed 3D-QSAR model indicated that bulky electron
donating groups at the phenyl in the position 6 of the triazine,
i.e. ring
A would improve antiproliferative activity of
compounds against MDA-MB231 cells. Based on this model, we
designed a group of compounds bearing suitable functional
groups at the phenyl rings A and B, with an expectation of
higher activity against triple negative breast cancer, and applied
the model to predict pGI50 values for these compounds (Table
1).
2.2. Synthesis
Microwave irradiation has been widely used to facilitate
synthesis of 1,3,5-triazines.17 Sometimes, microwave irradiation
Fig. 1 General structure of the designed compounds.
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also changes outcome of reactions. The one-pot reaction of
cyanoguanidine, benzaldehydes, and anilines in ethanol in the
presence hydrochloric acid under conventional heating, fol-
lowed by the treatment with aqueous sodium hydroxide (excess)
was reported to produce 6,N2-diaryl-5,6-dihydro-1,3,5-triazine-
2,4-diamines.18 However, a similar reaction under focused
microwave irradiation resulted in the formation their fully
aromatic analogues.19 This microwave-assisted methodology we
applied for synthesis of new 6,N2-diaryl-1,3,5-triazine-2,4-
diamines (1–21), which were designed using the 3D-QSAR
model as describe above.
The reactions were performed in a one-pot manner with the
three-component condensation of cyanoguanidine, benzalde-
hydes, and anilines at the rst stage and the rearrangement
accompanied with dehydrogenative aromatization at the
second one (Scheme 1). The structure of the resulting 6,N2-
diaryl-1,3,5-triazine-2,4-diamines (1–21) was conrmed by the
NMR spectroscopic data and X-ray crystallographic study on one
representative product, compound 20. The three diagnostic
signals of the aromatic triazine ring quaternary carbon atoms
appear in 13C NMR spectra of 1–21 in the region 164.4–
170.2 ppm. In the 1H NMR spectra, the downeld shi of
signals for protons in the ortho-positions of the phenyl ring
directly attached to the 1,3,5-triazine ring should be attributed
to the anisotropic effect of the coplanar triazine p-electron
system.
Fig. 2 Molecular structure of 20, showing atom labelling scheme and
anisotropic displacement parameters at the 70% probability level.
equivalence of the C–N bond lengths is indicative of substantial
delocalisation of p-electron density over the ring. Details of
crystallographic analysis are available in ESI.†
2.3. Biological evaluation
2.3.1. Cytotoxicity evaluation. The prepared compounds 1–
21 were tested against three breast tumor cell lines: hormone
(estrogen and progesterone) negative MDA-MB231 and
hormone positive SKBR-3 and MCF-7. The initial screening of
6,N2-diaryl-1,3,5-triazine-2,4-diamines (1–21) was performed at
X-ray crystallography of 20 (Fig. 2) showed that to a rst
approximation the molecule is planar and has the shape of the
letter U as both appended aromatic rings are orientated to the
same side of the molecule. Within the triazine ring, the near
Scheme 1 Synthesis of 6,N2-diaryl-1,3,5-triazine-2,4-diamines (1–21).
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Table 2 Preliminary cytotoxic screening of 6,N2-1,3,5-triazine-2,4-
diamines (1–21) on breast cancer cell lines at 10 mM
the ring A. This substitution was exceptionally benecial for the
anticancer activity, particularly in a combination with the para-
substitution at the phenyl ring B. Changing location of the
substituents to ortho- or meta-position in the phenylamino
moiety dramatically decreased potency of compounds. The GI50
values for these subgroups have a 2–3 order difference. For
example, relocation of the methoxy group from the ortho- to
para-position of the ring B resulted in a 200-fold increase in the
antiproliferative activity (13 vs. 19). Even greater improvement
in the activity was achieved when methyl or chloro substituents
changed their location at the ring B from meta- to para-position
leading to compounds 1300–2000-fold more potent than their
regioisomers (14 vs. 16, 15 vs. 18). At the same time, it appeared
that for the trimethoxyphenyl-substituted series (10–21) an
increase in size of the R2 group in para-position from the most
potent compound with a methyl group (18) decreased the
activity. Nevertheless, most of the triazines combining trime-
thoxyphenyl as the ring A and para-substituted phenylamino
moieties as the ring B possessed activity comparable or higher
than that of reference drugs methotrexate and nilotinib. These
compounds also demonstrated good antiproliferative activity
against SKBR-3 cells. The most active 6,N2-diaryl-1,3,5-triazine-
2,4-diamine identied in the series was compound 18, which
was 10-fold more active than methotrexate and 40-fold more
potent than nilotinib against MDA-MB231 breast cancer cells.
This compound (18) and its analogue 16, with the chloro
substituent instead of the para-methyl group in the ring B, were
selected for further experiments to better understand processes
underlying antiproliferative effects of these compounds.
To assess predictive power of the earlier developed 3D-QSAR
model, we compared experimental and predicted pGI50 values,
calculated using the 3D-QSAR model (Table 4). The residual
error values for the rst series of compounds (1–9) were rather
acceptable viz. without extreme differences between the exper-
imental and predicted values. However, a large discrepancy
between the predicted and experimental values was observed
for many trimethoxyphenyl-substituted compounds. These
compounds, especially those with the R2 group in para-position
of the ring B (16–21), appeared to be much more potent than it
was predicted by the model. These ndings indicated a limita-
tion of the earlier prepared 3D-QSAR model,16 which seemed to
be valid for compounds with monosubstituted phenyl rings and
should be used with a caution for more complex structures.
2.3.2. Time-dependent cytotoxicity. To further evaluate
cytotoxicity of the prepared compound against cancer cells, time-
dependent cell viability experiments were carried out with the
selected most active compounds 16 and 18 using MDA-MB231
breast cancer cell line. The MDA-MB231 cell viability was
assessed aer the exposure of the cells to compounds 16 or 18 for
12, 24, 48, and 72 h at concentrations ranging from 0.2 nM to
125 nM. The GI50 values were estimated when treatment with the
highest concentration (125 nM) of tested compounds resulted in
more than 80% of cell growth inhibition (Table 5).
Cell viabilitya (%)
Compound R1
R2
MDA-MB231 SKBR-3 MCF-7
1
2
3
4
5
6
7
8
3-F
4-Cl
4-CF3
4-CF3O
4-Me2N
4-Me
2-MeO 49
84
85
83
86
55
80
76
67
89
37
73
80
57
58
43
48
43
37
48
45
42
88
100
96
95
86
51
95
85
99
62
98
100
100
96
86
52
48
62
52
51
49
4-Me
2-Cl
4-Cl
2-Cl
2-Cl
4-Cl
H
29
49
34
28
36
51
28
32
14
34
42
4-Me
4-MeO
4-MeO
3,4,5-(MeO)3
3,4,5-(MeO)3 2-F
3,4,5-(MeO)3 2-Cl
9
4-Me
H
10
11
12
13
14
15
16
17
18
19
20
21
3,4,5-(MeO)3 2-MeO 16
3,4,5-(MeO)3 3-Cl
3,4,5-(MeO)3 3-Me
3,4,5-(MeO)3 4-Cl
3,4,5-(MeO)3 4-Br
3,4,5-(MeO)3 4-Me
3,4,5-(MeO)3 4-MeO 20
3,4,5-(MeO)3 4-CF3O 20
3,4,5-(MeO)3 4-iPr
40
14
20
24
14
9
a
MTT method, cells incubated with corresponding compounds (10 mM)
for 72 h. Values are mean of three independent experiments.
one point concentration (10 mM) for preliminary assessment of
their antiproliferative potential (Table 2). Percentage cell
viability was calculated 72 h aer treatment with compounds. In
general, triple negative breast cancer cells (MDA-MB231) were
more responsive than hormone positive breast cancer cells
(SKBR-3 and MCF-7) to the treatment with the compounds.
These results are similar to the trend observed earlier for their
structural analogues.16
Since all compounds 1–21 demonstrated signicant anti-
proliferative activity against MDA-MB231 cells at the screening
concentration, they were further tested at concentration
ranging from 0.00002 mM to 20 mM to estimate their 50% growth
inhibitory concentrations (GI50) against breast cancer cells
(Table 3). Nilotinib and methotrexate were used as positive
controls. For compounds active at the screening concentration
against SKBR-3 and MCF-7 cells, concentration-dependent
response was also evaluated and the corresponding GI50
values were estimated.
Compounds 1–21 were also tested against MCF-10A normal
breast cells to evaluate their selectivity towards cancer cells.
None of the compounds showed signicant inhibition of the
normal breast cell growth at the compound concentration of 20
mM.
The prepared 6,N2-diaryl-1,3,5-triazine-2,4-diamines 1–21
possessed specic cytotoxicity against triple negative MDA-
MB231 breast cancer cells with GI50 values ranging widely.
However, the most intriguing results were obtained for
compounds 10–21 with the 3,4,5-trimethoxyphenyl moiety as
Compound 18 possessed higher antiproliferative activity
than 16 against MDA-MB231 cells for all duration of observa-
tions. For both compounds, the cytotoxic effect developed
gradually and no signicant inhibition of the cell growth was
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Table 3 Cytotoxicitya of 6,N2-diaryl-1,3,5-triazine-2,4-diamines (1–21)
GI50 ꢀ SDb (mM)
Compound
R1
R2
MDA-MB231
SKBR-3
MCF-7
MCF-10A
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
3-F
4-Cl
4-CF3
4-CF3O
4-Me2N
4-Me
4-Me
4-MeO
2-MeO
4-Me
2-Cl
4-Cl
2-Cl
2-Cl
4-Cl
H
4-Me
H
2-F
2-Cl
2-MeO
3-Cl
3-Me
4-Cl
4-Br
4-Me
4-MeO
4-CF3O
4-iPr
17.3 ꢀ 0.6
13.8 ꢀ 1.9
13.7 ꢀ 0.6
16.7 ꢀ 1.2
0.1 ꢀ 0.001
3.8 ꢀ 0.4
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
ND
ND
17.7 ꢀ 1.4
>20
0.4 ꢀ 0.04
>20
>20
>20
10.7 ꢀ 1.0
9.6 ꢀ 0.2
>20
8.4 ꢀ 0.3
19.6 ꢀ 0.9
14.2 ꢀ 1.7
>20
>20
>20
>20
>20
>20
>20
4-MeO
6.1 ꢀ 0.6
>20
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
9.7 ꢀ 0.6
17.2 ꢀ 0.4
>20
>20
7.9 ꢀ 0.5
11.3 ꢀ 1.1
2.1 ꢀ 0.2
14.0 ꢀ 1.6
16.7 ꢀ 1.4
6.0 ꢀ 0.1
0.3 ꢀ 0.04
0.17 ꢀ 0.01
0.21 ꢀ 0.01
0.27 ꢀ 0.02
5.0 ꢀ 0.35
1.1 ꢀ 0.05
ND
9.1 ꢀ 1.1
2.2 ꢀ 0.2
0.007 ꢀ 0.00001
0.008 ꢀ 0.0005
0.001 ꢀ 0.00001
0.01 ꢀ 0.001
1.5 ꢀ 0.1
12.5 ꢀ 0.2
17
18
19
20
>20
>20
>20
>20
10.7 ꢀ 1.1
5.8 ꢀ 0.5
ND
21
0.04 ꢀ 0.002
0.01 ꢀ 0.001
0.04 ꢀ 0.001
Methotrexatec
Nilotinibc
9.60 ꢀ 0.5
a
b
c
MTT method, cells incubated with compounds for 72 h, experiments performed in triplicates. Standard deviation of mean values. Positive
control.
detected 12 h aer the treatment. However, compound 18 24 h (GI50 ¼ 4 nM) and even more aer the total exposure for
started showing activity in nanomolar concentrations (GI50 ¼ 5 72 h (GI50 ¼ 1 nM). A similar time-dependent pattern was
nM) at 24 h with an increase in the potency over the following observed for compound 16.
Table 4 Antiproliferative activities obtained experimentally and predicted for 6,N2-diaryl-1,3,5-triazine-2,4-diamines (1–21) by the 3D-QSAR
modela
R1
R2
Experimental pGI50
Predicted pGI50
Residual errord
b
c
Compound
1
2
3
4
5
6
7
8
3-F
4-Cl
4-CF3
4-CF3O
4-Me2N
4-Me
4-Me
4-MeO
2-MeO
4-Me
2-Cl
4-Cl
2-Cl
2-Cl
4-Cl
H
4-Me
H
2-F
2-Cl
2-MeO
3-Cl
3-Me
4-Cl
4-Br
4-Me
4-MeO
4-CF3O
4-iPr
4.76
4.86
4.86
4.78
7.00
5.42
5.02
5.08
5.21
5.01
5.10
4.95
5.69
5.04
5.66
8.15
8.10
8.70
8.00
5.83
7.40
5.54
5.51
5.22
4.95
5.79
5.65
5.51
6.58
5.41
5.58
4.85
5.10
4.78
4.62
4.49
4.95
4.45
4.40
4.79
4.49
5.01
ꢁ0.78
ꢁ0.64
ꢁ0.35
ꢁ0.18
1.21
ꢁ0.23
ꢁ0.49
ꢁ1.50
ꢁ0.19
ꢁ0.56
0.25
9
4-MeO
10
11
12
13
14
15
16
17
18
19
20
21
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
3,4,5-(MeO)3
ꢁ0.15
0.91
0.42
1.17
3.20
3.65
4.30
3.21
1.34
2.39
a
b
c
QSAR model reported earlier.16 Experimental pGI50 calculated as pGI50 ¼ ꢁlog 10 ꢂ GI50
Difference between the predicted and experimental pGI50 values.
.
pGI50 values predicted by the QSAR model.
d
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Time-dependent cytotoxic effecta of the most active
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Table
5
MDA-MB231 cells were treated with compounds 16 and 18
and incubated for 24 h prior to the observation of changes in
cell morphology. The selected representative images of uo-
rescence microscopy are presented in Fig. 3: live cells emit green
color (white arrow) because of AO intercalation with DNA and
apoptotic cells appear reddish-orange (red arrow) by inter-
calating PI to the DNA because of altered membrane perme-
ability. Mid-stage apoptosis is evident by the presence of cells
with nuclear chromatic condensation (blue arrow), cell bleb-
bing (purple arrow), and multi-nucleated cells (yellow arrow).
2.3.4. Live cell imaging. To visualize morphological
changes in the cells in real time, live cell imaging of MDA-
MB231 cells treated with compound 18 was carried out. The
cells were stained with AO and PI and treated with 18 (10 mM).
The pictures were taken aer every 10 minutes for 4 h and
intercalated into video (see ESI†). The video clearly shows the
morphological changes of the cells initiated by 18 at different
times, like formation of multinucleation, chromatin conden-
sation, cell blebbing and apoptotic bodies. The death of the
breast cancer cells (MDA-MB231) treated with compound 18
was also evident from turning of live cells (green color) to dead
cells (red color). These observations suggest that compound 18
realizes its cytotoxic activity by inducing apoptosis in MDA-
MB231 cells.
compounds (16 and 18) against MDA-MB231 breast cancer cell line
GI50 ꢀ SD (nM) or growth inhibition at 125 nM
Compound
12 h
24 h
48 h
72 h
16
18
17%b
15%b
61%b
5 ꢀ 0.1
75%b
4 ꢀ 0.3
7 ꢀ 0.6
1 ꢀ 0.02
a
MTT method, values are the mean ꢀ SD, all experiments performed at
b
least three times. Percentage cell growth inhibition at 125 nM
concentration of test compounds.
These results suggest that the antiproliferative effect of
compounds 16 and 18 develop gradually and without an
immediate toxic effect on the cells. A negligible cytotoxicity 12 h
aer the treatment suggests that the compounds are less likely
to cause cell necrosis and probably induce apoptosis. To further
test this assumption, we performed uorescent microscopy
experiments assessing effects of compounds 16 and 18 on the
morphology of MDA-MB231 breast cancer cell.
2.3.3. Acridine orange and propidium iodide double
staining experiments. Aer the determination of cytotoxicity by
the MTT assay, morphological changes of MDA-MB231 cells
treated with most active compounds 16 and 18 were studied using
uorescence microscopy. The acridine orange (AO) and propi-
dium iodide (PI) double staining method was used to determine
morphological features of apoptotic cells (chromatin condensa-
tion, cell blebbing and apoptotic bodies). AO emits green light by
intercalating the DNA of the live and dead cells, while PI emits red
uorescence by intercalating the DNA of dead cells only.20
2.4. Prediction of ADME properties
In the design of biologically active agents, optimization of lead
compounds and selection of drug candidates, in silico evalua-
tion of absorption, distribution, metabolism and elimination
(ADME) of compounds has become a common practice.21
Fig. 3 AO/PI double staining of MDA-MB231 cells with signs of apoptosis 24 h after the treatment with compounds. (A) Cells treated with
a vehicle, 1% DMSO, negative control; (B) cells treated with compound 16 (125 nM); (C) cells treated with compound 16 with (250 nM); (D) cells
treated with methotrexate (10 nM), positive control; (E) cells treated with compound 18 IC20 (2.5 nM); (F) cells treated with compound 18 IC50 (5
nM). Images were taken with a fluorescence microscope at 400ꢂ. White arrow points to live cells, red arrow shows apoptotic cells, blue arrow
points to chromatin condensation, purple arrow shows cell blebbing and yellow arrow indicates multi-nucleated cells.
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Table 6 Selected ADME properties of 6,N2-diaryl-1,3,5-triaizne-2,4-diamines (1–21)a
Donor
HBd
Accpt
HBe
QP log P
QPP
Compound
MWb
SASAc
o/wf
Cacog
#Metabh
Percent human oral absorptioni
1
2
3
4
5
6
7
8
311.32
311.77
365.75
381.74
340.81
311.77
311.77
293.33
307.35
353.38
371.37
387.83
383.41
387.83
367.41
387.83
432.28
367.41
383.41
437.38
395.46
663.94
659.67
642.85
696.37
709.92
664.90
668.19
644.11
666.66
735.08
742.37
755.90
766.44
759.21
766.57
759.20
764.15
758.05
750.27
803.46
800.44
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4.75
4.00
4.00
4.00
5.00
4.00
4.00
4.75
4.75
6.25
6.25
6.25
7.00
6.25
6.25
6.25
6.25
6.25
7.00
6.25
6.25
3.50
3.86
4.06
4.56
4.03
3.89
3.87
3.17
3.44
3.43
3.67
3.93
3.34
3.91
3.72
3.91
3.99
3.71
3.44
4.78
4.27
849.24
784.19
922.41
800.65
903.51
924.51
783.98
789.12
787.51
825.33
887.43
969.42
893.13
822.03
824.23
822.03
821.52
822.74
821.39
802.60
818.46
2
1
1
1
2
2
1
2
2
4
4
4
5
4
5
3
3
4
4
4
4
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
9
10
11
12
13
14
15
16
17
18
19
20
21
a
b
2
Calculated using QikProp 4.3 module of the Schrodinger soware. Molecular weight. c Total solvent accessible surface area in A using a probe
˚
¨
d
˚
with a 1.4 A radius, range 95% of drugs (300.0–1000.0). Estimated number of hydrogen bonds that would be donated by the solute to water
molecules in an aqueous solution, range 95% of drugs (0.0–6.0). Estimated number of hydrogen bonds that would be accepted by the solute
e
f
from water molecules in an aqueous solution, range 95% of drugs (2.0–20.0). Predicted log of the octanol/water partition coefficient, range
95% of drugs (ꢁ2–6.5). Caco-2 cell permeability in nm sꢁ1, range 95% of drugs (<25 poor, >500 great). Caco-2 cells are a model for the gut
g
blood barrier, non-active transport. Number of likely metabolic reactions; range 95% of drugs (1–8). i Human oral absorption predicted on the
h
basis of a quantitative multiple linear regression model. 0 to 100% scale (>85% high).
¨
QikProp (version 4.3) module of the Schrodinger soware was three breast cancer cell lines and it was found that triple
used to predict the molecular properties inuencing critical negative breast cancer cells (MDA-MB231) were signicantly
pharmacokinetic parameters of compounds 1–21 (Table 6). more sensitive to the treatment with the prepared compounds.
Parameters like octanol/water partition coefficient (QP log P, o/ Some 6,N2-diaryl-1,3,5-triazine-2,4-diamines demonstrated
w) and aqueous solubility (QP log S) are important for the good antiproliferative activity against SKBR-3 cells, but MCF-7
prediction of drug absorption, transport and distribution in the cells were generally resistant to the treatments with these
body. These parameters calculated for 1–12 have values similar compounds.
to those, which are typical for commonly used drugs. Steric and
Some of the synthesized compounds demonstrated even
molecular surface descriptors i.e., total solvent accessible area greater activity against MDA-MB231 cells than it was predicted
(SASA) and its hydrophobic (FOSA) and hydrophilic (FISA) by the 3D-QSAR model. The 3D-QSAR model limitation might
components were also calculated and found to be within the originate from multiple targets responsible for the activity of
95% range of values for known drugs. Lipinski's rule of ve has 6,N2-diaryl-1,3,5-triazine-2,4-diamines and hence requires
been oen used as a rst lter for the prediction the drug-like further investigations. The discrepancy between the predicted
properties of compounds.22 None of the prepared compounds values and the experimental data was particularly evident for
violate Lipinski's rule of ve. The complete absorption and N2-aryl-6-(3,4,5-trimethoxyphenyl)-1,3,5-triazine-2,4-diamines
absence of effects on CNS were predicted for compounds 1–21. 16–21 possessing para-substituted phenyl ring B. The most
Overall, all evaluated compounds were predicted to possess active compound in the series also belongs to this group:
ADME properties favorable for potential agents targeting breast compound 18 inhibited triple negative MDA-MB231 breast
cancer cells. More detailed ADME prole for the compounds cancer cell growth with GI50 value of 1 nM. Importantly, the
predicted by QikProp module is available in ESI.†
prepared compounds demonstrated no cytotoxicity towards
non-cancerous MCF-10A breast cells. The cytotoxic evaluation at
different time intervals for the most active compounds 16 and
18 showed that these compounds possessed a concentration-
and time-dependent cytotoxic effect on MDA-MB231 breast
cancer cells. Morphological features observed by the uorescent
microscopy and live cell imaging aer the AO/PI double staining
3. Conclusions
We synthesized a library of novel 6,N2-diaryl-1,3,5-triazine-2,4-
diamines designed using the 3D-QSAR data from the previous
report.16 Their antiproliferative activity was evaluated against
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suggested that the tested compounds induced apoptosis in (75 MHz, DMSO-d6): d 20.3 (CH3), 120.1 (C-200 and C-600), 128.4
MDA-MB231 cells. All compounds, including 18, were predicted (C-30 and C-50), 128.8 (C-300 and C-500), 129.4 (C-20 and C-60), 130.9
to have ADME proles favorable for potential antiproliferative (C-100), 135.7 (C-10), 136.1 (C-40), 137.2 (C-400), 164.5 (C-4), 167.1
agents targeting breast cancer.
(C-6), 169.1 (C-2). Anal. calcd for C16H14ClN5: C, 61.64; H,
4.53; N, 22.46. Found: C, 61.54; H, 4.50; N, 22.29.
4.2.3. N2-(2-Chlorophenyl)-6-(4-(triuoromethyl)phenyl)-
1,3,5-triazine-2,4-diamine (3). Yield 38%. Mp 165–167 ꢃC
4. Experimental
4.1. General
1
(EtOH). H NMR (300 MHz, DMSO-d6): d 7.23 (2H, brs, NH2),
7.23 (1H, ddd, J ¼ 1.6 Hz, J ¼ 7.7 Hz, J ¼ 7.7 Hz, H-400), 7.38 (1H,
ddd, J ¼ 1.3 Hz, J ¼ 7.7 Hz, J ¼ 7.7 Hz, H-500), 7.53 (1H, dd, J ¼
1.4 Hz, J ¼ 8.0 Hz, H-300), 7.80 (1H, dd, J ¼ 1.6 Hz, J ¼ 8.0 Hz, H-
600), 7.88 (2H, d, J ¼ 8.3 Hz, H-30 and H-50), 8.45 (2H, d, J ¼ 8.1 Hz,
H-20 and H-60), 8.97 (1H, s, NH); 13C NMR (75 MHz, DMSO-d6):
d 120.5 (q, 1JCF ¼ 273.6 Hz, CF3), 125.2 (q, 3JCF ¼ 3.5 Hz, C-30 and
C-50), 126.2 (C-600), 127.3 (C-200), 127.5 (C-500), 128.4 (C-20 and C-
Melting points (uncorrected) were determined using a Stuart™
SMP40 automatic melting point apparatus. 1H and 13C NMR
spectra were recorded on a Bruker Fourier NMR spectrometer
(300 MHz) using DMSO-d6 as a solvent and TMS as an internal
reference. Microwave-assisted reactions were carried out in the
closed vessel focused single mode using a Discover SP micro-
wave synthesizer (CEM, USA) monitoring reaction temperature
by the equipped IR sensor.
2
60), 128.5 (C-400), 129.4 (C-300), 131.1 (q, JCF ¼ 31.8, C-40), 135.6
(C-100), 140.5 (C-10), 165.3 (C-4), 167.3 (C-6), 169.0 (C-2). Anal.
calcd for C16H11ClF3N5: C, 52.54; H, 3.03; N, 19.15. Found: C,
52.33; H, 2.95; N, 18.98.
4.2. General method for the synthesis of 6,N2-diaryl-1,3,5-
triazine-2,4-diamines (1–21)
4.2.4. N2-(4-Chlorophenyl)-6-(4-(triuoromethoxy)phenyl)-
The microwave irradiation parameters optimized earlier19 for 1,3,5-triazine-2,4-diamine (4). Yield 55%. Mp 201–203 ꢃC
1
the synthesis of 6,N2-diaryl-1,3,5-triazine-2,4-diamines were (EtOH). H NMR (300 MHz, DMSO-d6): d 7.27 (2H, brs, NH2),
5
applied for the preparation of 1–21. To a solution of cyano- 7.36 (2H, d, J ¼ 8.9 Hz, H-300 and H-500), 7.51 (2H, dd, JHF
¼
guanidine (0.21 g, 2.5 mmol), a substituted benzaldehyde (2.5 0.9 Hz, 3JHH ¼ 9.0 Hz, H-30 and H-50), 7.89 (2H, d, J ¼ 8.9 Hz, H-
mmol), and an aniline (2.5 mmol) in EtOH (2 mL) in a 10 mL 200 and H-600), 8.43 (2H, d, J ¼ 8.9 Hz, H-20 and H-60), 9.75 (1H, s,
seamless pressure vial, conc. HCl (0.21 mL, 2.5 mmol) was NH); 13C NMR (75 MHz, DMSO-d6): d 120.0 (q, 1JCF ¼ 257.0 Hz,
added. The reaction mixture was heated at 140 ꢃC for 50 min by OCF3), 120.5 (C-30 and C-50), 121.4 (C-200 and C-600), 125.7 (C-100),
irradiation in the Discover SP (CEM) microwave reactor oper- 128.2 (C-300 and C-500), 129.9 (C-20 and C-60), 135.7 (C-10), 138.8 (C-
ating at maximal microwave power up to 150 W. Then, an aq. 400), 150.6 (q, 3JCF ¼ 1.7 Hz, C-40), 164.5 (C-4), 167.1 (C-6), 169.1
solution of NaOH (5 N, 1 mL) was added to the reaction mixture (C-2). Anal. calcd for C16H11ClF3N5O: C, 50.34; H, 2.90; N, 18.35.
ꢃ
and heating was continued for another 15 min at 140 C. Aer Found: C, 50.22; H, 3.02; N, 18.26.
cooling, the precipitated product was ltered, washed with
4.2.5. N2-(2-Chlorophenyl)-6-(4-(dimethylamino)phenyl)-
water and recrystallized from suitable solvents (EtOH, aq. EtOH, 1,3,5-triazine-2,4-diamine (5). Yield 15%. Mp 191–193 ꢃC
1
or MeCN) specied below. Yields of products 1–21 are reported (EtOH). H NMR (300 MHz, DMSO-d6): d 2.99 (6H, s, N(CH3)2),
as overall isolated yields for the one-pot procedure.
6.74 (2H, d, J ¼ 9.1 Hz, H-30 and H-50), 6.89 (2H, brs, NH2), 7.16
(1H, ddd, J ¼ 1.6 Hz, J ¼ 7.7 Hz, J ¼ 7.7 Hz, H-400), 7.36 (1H, ddd,
4.2.1. 6-(3-Fluorophenyl)-N2-(2-methoxyphenyl)-1,3,5-
triazine-2,4-diamine (1). Yield 33%. Mp 140–142 ꢃC (EtOH/ J ¼ 1.2 Hz, J ¼ 7.8 Hz, J ¼ 7.8 Hz, H-500), 7.50 (1H, dd, J ¼ 1.5 Hz, J
water). 1H NMR (300 MHz, DMSO-d6): d 3.87 (OCH3), 6.95– ¼ 8.0 Hz, H-300), 7.96 (1H, dd, J ¼ 1.5 Hz, J ¼ 8.1 Hz, H-600), 8.13
7.10 (3H, m, H-300, H-400 and H-500), 7.22 (2H, brs, NH2), 7.40 (1H, (2H, d, J ¼ 9.2 Hz, H-20 and H-60), 8.43 (1H, s, NH); 13C NMR (75
dddd, 4JHH ¼ 0.8 Hz, 4JHH ¼ 2.6 Hz, 3JHH ¼ 8.5 Hz, 3JHF ¼ 8.4 Hz, MHz, DMSO-d6): d 39.6 (N(CH3)2), 110.9 (C-30 and C-50), 123.2 (C-
H-40), 7.56 (1H, ddd, 4JHF ¼ 6.0 Hz, 3JHH ¼ 8.0 Hz, 3JHH ¼ 8.0 Hz, 10), 125.2 (C-600), 126.2 (C-200), 126.9 (C-400), 127.2 (C-500), 129.2 (C-
4
4
3
H-50), 8.00 (1H, ddd, JHH ¼ 1.3 Hz, JHH ¼ 2.6 Hz, JHF
¼
20 and C-60), 129.2 (C-300), 135.9 (C-100), 152.5 (C-40), 164.8 (C-4),
10.6 Hz, H-20), 8.12 (1H, s, NH), 8.13–8.17 (2H, m, H-600 and H- 167.0 (C-6), 170.2 (C-2). Anal. calcd for C17H17ClN6: C, 59.91;
13
60); C NMR (75 MHz, DMSO-d6): d 55.7 (OCH3), 111.0 (C-300), H, 5.03; N, 24.66. Found: C, 59.79; H, 4.96; N, 24.47.
2
2
114.0 (d, JCF ¼ 23.1 Hz, C-20), 118.2 (d, JCF ¼ 21.8 Hz, C-40),
4.2.6. N2-(2-Chlorophenyl)-6-(4-methylphenyl)-1,3,5-
1
120.2 (C-500), 122.3 (C-600), 123.7 (d, 4JCF ¼ 2.2 Hz, C-60), 123.8 (C- triazine-2,4-diamine (6). Yield 40%. Mp 146–148 ꢃC (EtOH). H
3
3
400), 127.6 (C-100), 130.3 (d, JCF ¼ 8.2 Hz, C-50), 139.2 (d, JCF
¼
NMR (300 MHz, DMSO-d6): d 2.37 (3H, s, CH3), 7.07 (2H, brs,
7.5 Hz, C-10), 149.9 (C-200), 162.1 (d, 1JCF ¼ 242.9 Hz, C-30), 164.6 NH2), 7.20 (1H, ddd, J ¼ 1.6 Hz, J ¼ 7.8 Hz, J ¼ 7.6 Hz, H-400), 7.29
4
(C-2), 167.2 (C-4), 169.0 (d, JCF ¼ 3.0 Hz, C-6). Anal. calcd for (2H, d, J ¼ 8.0 Hz, H-30 and H-50), 7.37 (1H, ddd, J ¼ 1.4 Hz, J ¼
C
16H14FN5O: C, 61.73; H, 4.53; N, 22.50. Found: C, 61.65; H, 7.7 Hz, J ¼ 7.9 Hz, H-500), 7.52 (1H, dd, J ¼ 1.4 Hz, J ¼ 8.0 Hz, H-
4.77; N, 22.26.
300), 7.87 (1H, dd, J ¼ 1.5 Hz, J ¼ 8.1 Hz, H-600), 8.18 (2H, d, J ¼
4.2.2. 6-(4-Chlorophenyl)-N2-(4-methylphenyl)-1,3,5-
8.2 Hz, H-20 and H-60), 8.71 (1H, s, NH); 13C NMR (75 MHz,
triazine-2,4-diamine (2). Yield 45%. Mp 179–181 ꢃC (MeCN). 1H DMSO-d6): d 21.0 (CH3), 125.7 (C-600), 126.9 (C-400), 127.2 (C-200),
NMR (300 MHz, DMSO-d6): d 2.27 (3H, s, CH3), 7.11 (2H, d, J ¼ 127.8 (C-500, C-30 and C-50), 128.8 (C-20 and C-60), 129.3 (C-300),
8.3 Hz, H-300 and H-500), 7.12 (2H, brs, NH2), 7.58 (2H, d, J ¼ 133.8 (C-10), 135.7 (C-100), 141.2 (C-40), 165.1 (C-4), 167.2 (C-6),
8.6 Hz, H-30 and H-50), 7.68 (2H, d, J ¼ 8.3 Hz, H-200 and H-600), 170.2 (C-2). Anal. calcd for C16H14ClN5: C, 61.64; H, 4.53; N,
8.29 (2H, d, J ¼ 8.6 Hz, H-20 and H-60), 9.46 (1H, s, NH); 13C NMR 22.46. Found: C, 61.53; H, 4.35; N, 22.28.
25524 | RSC Adv., 2020, 10, 25517–25528
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4.2.7. N2-(4-Chlorophenyl)-6-(4-methylphenyl)-1,3,5-
RSC Advances
10.8 Hz, C-100), 131.9 (C-10), 140.3 (C-40), 152.5 (C-30 and C-50),
155.3 (d, 1JCF ¼ 245.9 Hz, C-200), 165.0 (C-4), 167.3 (C-6), 169.6 (C-
2). Anal. calcd for C18H19FN5O3: C, 58.22; H, 4.89; N, 18.86.
Found: C, 58.05; H, 4.79; N, 18.68.
triazine-2,4-diamine (7). Yield 26%. Mp 202–204 ꢃC (MeCN). 1H
NMR (300 MHz, DMSO-d6): d 2.08 (3H, s, CH3), 7.14 (2H, brs,
NH2), 7.32 (2H, d, J ¼ 8.6 Hz, H-30 and H-50), 7.35 (2H, d, J ¼
9.0 Hz, H-300 and H-500), 7.90 (2H, d, J ¼ 8.9 Hz, H-200 and H-600),
8.23 (2H, d, J ¼ 8.2 Hz, H-20 and H-60), 9.65 (1H, s, NH); 13C NMR
(75 MHz, DMSO-d6): d 21.0 (CH3), 121.2 (C-200 and C-600), 125.4
(C-100), 127.8 (C-30 and C-50), 128.2 (C-300 and C-500), 128.8 (C-20
and C-60), 133.9 (C-10), 139.0 (C-400), 141.3 (C-40), 164.4 (C-4),
167.0 (C-6), 170.2 (C-2). Anal. calcd for C16H14ClN5: C, 61.64;
H, 4.53; N, 22.46. Found: C, 61.49; H, 4.47; N, 22.32.
4.2.12. N2-(2-Chlorophenyl)-6-(3,4,5-trimethoxyphenyl)-
1,3,5-triazine-2,4-diamine (12). Yield 43%. Mp 176–178 ꢃC
(EtOH). 1H NMR (300 MHz, DMSO-d6): d 3.75 (3H, p-OCH3), 3.84
(6H, m-(OCH3)2), 7.11 (2H, brs, NH2), 7.19 (1H, ddd, J ¼ 1.6 Hz, J
¼ 7.6 Hz, J ¼ 7.8 Hz, H-400), 7.36 (1H, ddd, J ¼ 1.3 Hz, J ¼ 7.6 Hz, J
¼ 7.8 Hz, H-500), 7.51 (1H, dd, J ¼ 1.4 Hz, J ¼ 8.0 Hz, H-300), 7.64
(2H, s, H-20 and H-60), 7.87 (1H, dd, J ¼ 1.5 Hz, J ¼ 8.1 Hz, H-600),
8.80 (1H, s, NH); 13C NMR (75 MHz, DMSO-d6): d 55.7 (m-
(OCH3)2), 60.0 (p-OCH3), 105.0 (C-20 and C-60), 125.8 (C-600),
127.1 (C-200 and C-400), 127.9 (C-500), 129.3 (C-300), 131.8 (C-10),
135.8 (C-100), 140.4 (C-40), 152.6 (C-30 and C-50), 165.0 (C-4), 167.3
(C-6), 169.7 (C-2). Anal. calcd for C18H19ClN5O3: C, 55.75; H,
4.68; N, 18.06. Found: C, 55.49; H, 4.57; N, 17.90.
4.2.8. 6-(4-Methoxyphenyl)-N2-phenyl-1,3,5-triazine-2,4-
1
ꢃ
diamine (8). Yield 50%. Mp 190–192 C (EtOH). H NMR (300
MHz, DMSO-d6): d 3.84 (3H, OCH3), 7.00 (1H, t, J ¼ 7.4 Hz, H-400),
7.04 (2H, brs, NH2), 7.07 (2H, d, J ¼ 9.0 Hz, H-30 and H-50), 7.31
(2H, dd, J ¼ 7.5 Hz, J ¼ 8.4 Hz, H-300 and H-500), 7.86 (2H, dd, J ¼
1.1 Hz, J ¼ 8.6 Hz H-200 and H-600), 8.31 (2H, d, J ¼ 9.0 Hz, H-20
and H-60), 9.46 (1H, s, NH); 13C NMR (75 MHz, DMSO-d6): d 55.2
(OCH3), 113.6 (C-30 and C-50), 119.8 (C-200 and C-600), 121.8 (C-100),
128.3 (C-300 and C-500), 129.0 (C-10), 129.5 (C-20 and C-60), 140.0 (C-
400), 161.9 (C-40), 164.5 (C-4), 167.0 (C-6), 169.8 (C-2). Anal. calcd
for C16H15N5O: C, 65.52; H, 5.15; N, 23.88. Found: C, 65.52; H,
5.15; N, 23.88.
4.2.13. N2-(2-Methoxyphenyl)-6-(3,4,5-trimethoxyphenyl)-
1,3,5-triazine-2,4-diamine (13). Yield 49%. Mp 173–175 ꢃC
(EtOH). 1H NMR (300 MHz, DMSO-d6): d 3.75 (3H, p-OCH3), 3.86
(9H, m-(OCH3)2 and o-OCH3), 6.96 (1H, ddd, J ¼ 4.2 Hz, J ¼
4.2 Hz, J ¼ 8.4 Hz, H-400), 7.05–7.07 (2H, m, H-300 and H-500), 7.15
(2H, brs, NH2), 7.67 (2H, s, H-20 and H-60), 8.02 (1H, s, NH), 8.24
(1H, d, J ¼ 7.5 Hz, H-600); 13C NMR (75 MHz, DMSO-d6): d 55.7 (o-
OCH3), 55.7 (m-(OCH3)2), 60.0 (p-OCH3), 105.0 (C-20 and C-60),
110.9 (C-300), 120.1 (C-500), 122.0 (C-600), 123.4 (C-400), 127.8 (C-100),
131.8 (C-10), 140.4 (C-40), 149.6 (C-200), 152.6 (C-30 and C-50), 164.5
(C-4), 167.2 (C-6), 169.6 (C-2). Anal. calcd for C19H21N5O4: C,
59.52; H, 5.52; N, 18.27. Found: C, 59.42; H, 5.39; N, 18.05.
4.2.14. N2-(3-Chlorophenyl)-6-(3,4,5-trimethoxyphenyl)-
1,3,5-triazine-2,4-diamine (14). Yield 52%. Mp 193–195 ꢃC
(EtOH). 1H NMR (300 MHz, DMSO-d6): d 3.76 (3H, p-OCH3), 3.89
(6H, m-(OCH3)2), 7.03 (1H, ddd, J ¼ 0.8 Hz, J ¼ 2.0 Hz J ¼ 8.0 Hz,
H-400), 7.24 (2H, br s, NH2), 7.32 (1H, dd, J ¼ 8.1 Hz, J ¼ 8.1 Hz,
H-500), 7.67 (3H, m, H-600, H-20 and H-60), 8.20 (1H, s, H-200), 9.74
(1H, s, NH); 13C NMR (75 MHz, DMSO-d6): d 55.8 (m-(OCH3)2),
60.1 (p-OCH3), 105.0 (C-20 and C-60), 118.1 (C-600), 119.1 (C-200),
121.4 (C-100), 129.9 (C-500), 131.8 (C-10), 132.8 (C-300), 140.5 (C-40),
141.6 (C-400), 152.6 (C-30 and C-50), 164.4 (C-4), 167.0 (C-6), 169.8
(C-2). Anal. calcd for C18H19ClN5O3: C, 55.75; H, 4.68; N, 18.06.
Found: C, 55.63; H, 4.54; N, 17.86.
4.2.9. 6-(4-Methoxyphenyl)-N2-(4-methylphenyl)-1,3,5-
triazine-2,4-diamine (9). Yield 41%. Mp 186–188 ꢃC (MeCN). 1H
NMR (300 MHz, DMSO-d6): d 2.27 (3H, s, CH3), 3.84 (3H, s,
OCH3), 6.99 (2H, brs, NH2), 7.06 (2H, d, J ¼ 9.0 Hz, H-30 and H-
50), 7.11 (2H, d, J ¼ 8.3 Hz, H-300 and H-500), 7.71 (2H, d, J ¼
8.4 Hz, H-200 and H-600), 8.29 (2H, d, J ¼ 9.0 Hz, H-20 and H-60),
9.35 (1H, s, NH); 13C NMR (75 MHz, DMSO-d6): d 20.3 (CH3),
55.2 (OCH3), 113.5 (C-30 and C-50), 120.0 (C-200 and C-600), 128.7
(C-300 and C-500), 129.1 (C-100), 129.5 (C-20 and C-60), 130.7 (C-10),
137.4 (C-400), 161.9 (C-40), 164.4 (C-4), 167.0 (C-6), 169.7 (C-2).
Anal. calcd for C17H17N5O: C, 66.43; H, 5.58; N, 22.79. Found:
C, 66.32; H, 5.44; N, 22.68.
4.2.10. N2-Phenyl-6-(3,4,5-trimethoxyphenyl)-1,3,5-triazine-
1
ꢃ
2,4-diamine (10). Yield 48%. Mp 111–113 C (EtOH). H NMR
(300 MHz, DMSO-d6): d 3.75 (3H, s, p-OCH3), 3.87 (6H, s, m-
(OCH3)2), 6.99 (1H, t, J ¼ 7.3 Hz, H-400), 7.11 (2H, brs, NH2), 7.30
(2H, dd, J ¼ 7.7 Hz, J ¼ 8.1 Hz, H-300 and H-500), 7.68 (2H, s, H-20
and H-60), 7.85 (2H, dd, J ¼ 0.9 Hz, J ¼ 8.5 Hz, H-200 and H-600),
9.51 (1H, s, NH); 13C NMR (75 MHz, DMSO-d6): d 55.8 (m-
(OCH3)2), 60.0 (p-OCH3), 105.0 (C-200and C-600), 119.9 (C-20 and C-
60), 121.9 (C-10), 128.3 (C-30 and C-50), 132.0 (C-100), 139.9 (C-40),
140.3 (C-400), 152.6 (C-300 and C-500), 164.4 (C-4), 167.1 (C-6), 169.6
(C-2). Anal. calcd for C18H19N5O3: C, 61.18; H, 5.42; N, 19.82.
Found: C, 60.96; H, 5.34; N, 19.67.
4.2.15. N2-(3-Methylphenyl)-6-(3,4,5-trimethoxyphenyl)-
1,3,5-triazine-2,4-diamine (15). Yield 55%. Mp 192–194 ꢃC
(EtOH). 1H NMR (300 MHz, DMSO-d6): d 2.31 (3H, s, CH3), 3.76
(3H, p-OCH3), 3.88 (6H, m-(OCH3)2), 6.82 (1H, d, J ¼ 7.4 Hz, H-
400), 7.11 (2H, br s, NH2), 7.18 (1H, dd, J ¼ 7.8 Hz, J ¼ 7.8 Hz, H-
500), 7.63 (1H, d, J ¼ 8.4 Hz, H-600), 7.71 (2H, s, H-20 and H-60), 7.75
(1H, s, H-200), 9.44 (1H, s, NH); 13C NMR (75 MHz, DMSO-d6):
d 21.3 (CH3), 55.8 (m-(OCH3)2), 60.1 (p-OCH3), 105.0 (C-20 and C-
60), 117.2 (C-600), 120.5 (C-200), 122.7 (C-100), 128.1 (C-500), 132.1 (C-
10), 137.4 (C-400), 139.9 (C-300), 140.4 (C-40), 152.6 (C-30 and C-50),
164.5 (C-4), 167.1 (C-6), 169.6 (C-2). Anal. calcd for C19H21N5O3:
C, 62.11; H, 5.76; N, 19.06. Found: C, 61.93; H, 5.64; N, 18.88.
4.2.16. N2-(4-Chlorophenyl)-6-(3,4,5-trimethoxyphenyl)-
1,3,5-triazine-2,4-diamine (16). Yield 53%. Mp 209–211 ꢃC
4.2.11. N2-(2-Fluorophenyl)-6-(3,4,5-trimethoxyphenyl)-
1,3,5-triazine-2,4-diamine (11). Yield 50%. Mp 177–179 ꢃC
(EtOH). 1H NMR (300 MHz, DMSO-d6): d 3.74 (3H, p-OCH3), 3.84
(6H, m-(OCH3)2), 7.07 (2H, brs, NH2), 7.16–7.29 (3H, m, H-300, H-
400 and H-500), 7.64 (2H, s, H-20 and H-60), 7.78–7.84 (1H, m, H-600),
9.03 (1H, s, NH); 13C NMR (75 MHz, DMSO-d6): d 55.7 (m-
2
(OCH3)2), 60.0 (p-OCH3), 105.0 (C-20 and C-60), 115.4 (d, JCF
¼
¼
¼
3
3
19.4 Hz, C-300), 123.9 (d, JCF ¼ 3.4 Hz, C-400), 125.3 (d, JCF
1
4
2
8.4 Hz, C-600), 126.5 (d, JCF ¼ 1.5 Hz, C-500), 126.7 (d, JCF
(EtOH). H NMR (300 MHz, DMSO-d6): d 3.75 (3H, s, p-OCH3),
3.87 (6H, s, m-(OCH3)2), 7.19 (2H, brs, NH2), 7.35 (2H, d, J ¼
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8.9 Hz, H-300 and H-500), 7.68 (2H, s, H-20 and H-60), 7.90 (2H, d, J (EtOH). 1H NMR (300 MHz, DMSO-d6): d 1.20 (6H, d, J ¼ 6.9 Hz,
¼ 8.9 Hz, H-200 and H-600), 9.67 (1H, s, NH); 13C NMR (75 MHz, CH(CH3)2), 2.85 (1H, sept, J ¼ 6.9 Hz, CH(CH3)2), 3.75 (3H, s, p-
DMSO-d6): d 55.8 (m-(OCH3)2), 60.0 (p-OCH3), 105.0 (C-20 and C- OCH3), 3.87 (6H, s, m-(OCH3)2), 7.06 (2H, brs, NH2), 7.16 (2H, d,
60), 121.2 (C-200 and C-600), 125.5 (C-100), 128.1 (C-300 and C-500), J ¼ 8.6 Hz, H-300 and H-500), 7.68 (2H, s, H-20 and H-60), 7.72 (2H,
131.9 (C-10), 139.0 (C-400), 140.4 (C-40), 152.6 (C-30 and C-50), 164.3 d, J ¼ 8.6 Hz, H-200 and H-600), 9.41 (1H, s, NH); 13C NMR (75
(C-4), 167.0 (C-6), 169.7 (C-2). Anal. calcd for C18H19ClN5O3: C, MHz, DMSO-d6): d 23.9 (CH(CH3)2), 32.7 (CH(CH3)2), 55.7 (m-
55.75; H, 4.68; N, 18.06. Found: C, 55.62; H, 4.55; N, 17.94.
(OCH3)2), 60.0 (p-OCH3), 105.0 (C-20 and C-60), 120.3 (C-200 and C-
4.2.17. N2-(4-Bromophenyl)-6-(3,4,5-trimethoxyphenyl)-
600), 126.0 (C-300 and C-500), 132.1 (C-10), 137.5 (C-100), 140.3 (C-40),
1,3,5-triazine-2,4-diamine (17). Yield 61%. Mp 217–219 ꢃC 142.1 (C-400), 152.6 (C-30 and C-50), 164.4 (C-4), 167.1 (C-6), 169.5
1
(EtOH). H NMR (300 MHz, DMSO-d6): d 3.76 (3H, s, p-OCH3), (C-2). Anal. calcd for C21H25N5O3: C, 63.78; H, 6.37; N, 17.71.
3.87 (6H, s, m-(OCH3)2), 7.19 (2H, brs, NH2), 7.47 (2H, d, J ¼ Found: C, 63.65; H, 6.22; N, 17.54.
8.9 Hz, H-300 and H-500), 7.68 (2H, s, H-20 and H-60), 7.86 (2H, d, J
¼ 8.9 Hz, H-200 and H-600), 9.68 (1H, s, NH); 13C NMR (75 MHz,
4.3. X-ray structure determination of 20
DMSO-d6): d 55.8 (m-(OCH3)2), 60.0 (p-OCH3), 105.0 (C-20 and C-
60), 113.4 (C-100), 121.7 (C-200 and C-600), 131.0 (C-300 and C-500), Intensity data for a colourless crystal of 20 (0.05 ꢂ 0.09 ꢂ 0.13
131.9 (C-10), 139.4 (C-400), 140.4 (C-40), 152.6 (C-30 and C-50), 164.3 mm) were measured at 100 K on an XtaLAB Synergy Dual Atlas
(C-4), 167.0 (C-6), 169.7 (C-2). Anal. calcd for C18H18BrN5O3: C, diffractometer equipped with a CCD area detector and graphite-
˚
50.01; H, 4.20; N, 16.20. Found: C, 49.85; H, 4.09; N, 16.03.
monochromated Cu Ka radiation (l ¼ 1.54184 A) so that qmax
¼
4.2.18. N2-(4-Methylphenyl)-6-(3,4,5-trimethoxyphenyl)-
67.1ꢃ. Data reduction and empirical absorption corrections,
1,3,5-triazine-2,4-diamine (18). Yield 55%. Mp 208–210 ꢃC based on a multi-scan technique, were applied.23 The structure
(EtOH). 1H NMR (300 MHz, DMSO-d6): d 2.26 (3H, s, CH3), 3.75 was solved by direct methods24 and rened on F2 with aniso-
(3H, s, p-OCH3), 3.86 (6H, s, m-(OCH3)2), 7.07 (2H, brs, NH2), tropic displacement parameters and C-bound H atoms in the
7.10 (2H, d, J ¼ 8.3 Hz, H-300 and H-500), 7.68 (2H, s, H-20 and H- riding model approximation.25 The nitrogen-bound H atoms
60), 7.72 (2H, d, J ¼ 8.3 Hz, H-200 and H-600), 9.41 (1H, s, NH); 13C were rened with a distance restraint N–H ¼ 0.88 ꢀ 0.01 A. A
˚
NMR (75 MHz, DMSO-d6): d 20.3 (CH3), 55.7 (m-(OCH3)2), 60.0 weighting scheme of the form w ¼ 1/[s2(Fo2) + (0.104P)2
+
2
2
(p-OCH3), 105.0 (C-20 and C-60), 120.0 (C-200and C-600), 128.7 (C-300 1.179P] where P ¼ (Fo + 2Fc )/3 was introduced. The nal
and C-500), 130.8 (C-100), 132.1 (C-10), 137.3 (C-400), 140.3 (C-40), renement on 292 parameters yielded R ¼ 0.059 (3044 data with
152.6 (C-30 and C-50), 164.3 (C-4), 167.0 (C-6), 169.5 (C-2). Anal. I $ 2s(I)) and wR2 ¼ 0.176 (all 3357 data). The maximum and
ꢁ3
˚
calcd for C19H21N5O3: C, 62.11; H, 5.76; N, 19.06. Found: C, minimum residual electron density peaks of 1.73 and 0.62 eA
,
˚
61.97; H, 5.65; N, 18.91.
respectively, were located 1.85 and 0.69 A from the C19 and F1
atoms, respectively, that is, in chemically non-sensible posi-
4.2.19. N2-(4-Methoxyphenyl)-6-(3,4,5-trimethoxyphenyl)-
1,3,5-triazine-2,4-diamine (19). Yield 52%. Mp 195–197 ꢃC tions. The molecular structure diagram was generated at the
1
(EtOH). H NMR (300 MHz, DMSO-d6): d 3.74 (3H, s, p-OCH3), 70% probability level by ORTEP for Windows,26 and the packing
3.75 (3H, s, p-OCH3), 3.86 (6H, s, m-(OCH3)2), 6.89 (2H, d, J ¼ diagrams were generated with DIAMOND.27 Additional analysis
9.0 Hz, H-300 and H-500), 7.03 (2H, brs, NH2), 7.68 (2H, s, H-20 and was conducted with PLATON.28
H-60), 7.71 (2H, d, J ¼ 9.1 Hz, H-200 and H-600), 9.34 (1H, s, NH);
ꢀ
ꢃ
Crystal data for C19H18F3N5O4: M ¼ 437.38, triclinic, P1, a ¼
13C NMR (75 MHz, DMSO-d6): d 55.1 (p-OCH3), 55.7 (m-(OCH3)2), 7.2212(2), b ¼ 10.9597(3), c ¼ 13.4202(3) A, a ¼ 104.412(2) , b ¼
˚
3
60.0 (p-OCH3), 104.9 (C-20 and C-60), 113.5 (C-300 and C-500), 121.7 99.253(2) , g ¼ 108.517(2) , V ¼ 941.57(4) A , Z ¼ 2, Dx
¼
ꢃ
ꢃ
˚
(C-200 and C-600), 132.1 (C-10), 132.9 (C-100), 140.2 (C-40), 152.5 (C-30 1.543 g cmꢁ3, F(000) ¼ 452 and m ¼ 1.125 mmꢁ1
.
and C-50), 154.6 (C-400), 164.3 (C-4), 167.1 (C-6), 169.4 (C-2). Anal.
calcd for C19H21N5O4: C, 59.52; H, 5.52; N, 18.27. Found: C,
4.4. In vitro cytotoxicity assay
59.36; H, 5.44; N, 18.12.
4.2.20. N2-(4-(Triuoromethoxy)phenyl)-6-(3,4,5-
The synthesized compounds were tested against three breast
trimethoxyphenyl)-1,3,5-triazine-2,4-diamine (20). Yield 56%. tumor cell lines (MDA-MB231, SKBR-3 and MCF-7) and epithelial
1
Mp 179–181 C (EtOH). H NMR (300 MHz, DMSO-d6): d 3.75 breast cell line (MCF-10A) by the MTT colorimetric assay.29,30 All
ꢃ
(3H, s, p-OCH3), 3.87 (6H, s, m-(OCH3)2), 7.20 (2H, brs, NH2), cells were obtained from the American Type Culture Collection.
7.30 (2H, d, J ¼ 8.8 Hz, H-300 and H-500), 7.67 (2H, s, H-20 and H- The cancerous cell lines were grown in Dulbecco's modied eagle
60), 7.95 (2H, d, J ¼ 9.1 Hz, H-200 and H-600), 9.72 (1H, s, NH); 13C medium (DMEM) supplemented with 10% fetal bovine serum
NMR (75 MHz, DMSO-d6): d 55.8 (m-(OCH3)2), 60.1 (p-OCH3), (FBS) and 1% pen-strep antibiotic. The MCF-10A human
105.0 (C-20 and C-60), 120.2 (q, 1JCF ¼ 255.1 Hz, OCF3), 121.1 (C- epithelial breast cell line was grown in the complete mammary
300 and C-500), 121.2 (C-200 and C-600), 131.9 (C-10), 139.2 (C-100), epithelial growth medium containing horse serum 5%, epithelial
140.4 (C-40), 142.7 (q, 3JCF ¼ 1.7 Hz, C-400), 152.6 (C-30 and C-50), growth factor 20 ng mLꢁ1, hydrocortisone 0.5 mg mLꢁ1, cholera
164.4 (C-4), 167.1 (C-6), 169.7 (C-2). Anal. calcd for toxin 100 ng mLꢁ1, insulin 10 mg mLꢁ1, and pen-step antibiotic.31
C
19H18F3N5O4: C, 52.18; H, 4.15; N, 16.01. Found: C, 52.07; H, For the cytotoxic assay, 20 to 75 ꢂ 103 cells per mL (based on the
4.08; N, 15.96.
doubling time for each cell line) were seeded in 96-well plates and
4.2.21. N2-(4-Isopropylphenyl)-6-(3,4,5-trimethoxyphenyl)-
the plates were incubated overnight in a humidied air atmo-
1,3,5-triazine-2,4-diamine (21). Yield 49%. Mp 190–192 ꢃC sphere at 37 ꢃC in 5% CO2 incubator.
25526 | RSC Adv., 2020, 10, 25517–25528
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The cells were then treated with compounds at different dish was then transferred to motorized inverted uorescent
concentrations. Aer 72 h of incubation, the MTT (0.5 mg microscope (Eclipse Ti2-E, Nikon). The pictures were taken every
mLꢁ1) was added to wells, followed by 4 h of incubation. The 10 minutes for 4 h and intercalated into video (see ESI†).
culture medium was then removed and DMSO (100 mL per well)
was added and the absorbance values were measured at 570 nm
using the multi-well Tecan NanoQuant, Innite m200 Pro plate
reader. Growth inhibitory values (GI50) were calculated using
GraphPad Prism 7 (GraphPad Soware, San Diego, USA) by
4.8. QSAR model testing
The previously reported16 3D-QSAR model for 6,N2-1,3,5-
triazine-2,4-diamines against MDA-MB231 breast carcinoma
was applied. The structures of the proposed compounds were
drawn using ChemDraw 15.0 and imported to Discovery Studio
v18 (ref. 32) for the activity prediction. The structures were
prepared for the 3D-QSAR modeling and aligned to minimum
energy using the ‘align small molecules’ protocol, which is
nonlinear regression analysis. Three independent experiments
were carried out and the data was expressed in mean ꢀ stan-
dard deviation (SD). The concentration–response curves used
for the GI50 calculation are available in ESI.†
based on 50% steric and 50% electrostatic elds for alignment
of molecules. The predicted pGI50 values were then obtained by
the ‘calculate molecular properties’ protocol in Discover Studio
using the previously prepared 3D-QSAR model.
4.5. Time-dependent cytotoxicity
MDA-MB231 breast cancer cells were grown in DMEM supple-
mented with 10% FBS and 1% pen-strep antibiotic. The cells
were seeded in 96-well plates (20 ꢂ 103 cells per mL) and the
plates were incubated overnight in a humidied air atmosphere
4.9. ADME properties prediction
at 37 ꢃC in 5% CO2 incubator. The cells were then treated with
QikProp module of the Schrodinger33 was used to predict the
compounds 16 or 18 in concentrations ranging from 0.2 nM to
absorption, distribution, metabolism and excretion (ADME)
properties. The QikProp module predicts the descriptors which
are pharmaceutically signicant to identify the relevant prop-
erties of the organic molecule in relation to the 95% of the
marketed drugs. The molecules were drawn and prepared
(energy minimized and aligned) in Maestro program (v10.1) of
Schrodinger soware suit. QikProp (v4.3) was run with default
options in normal processing mode.
125 nM for 12, 24, 48, and 72 h. Aer specied time for each
experiment, MTT (0.5 mg mLꢁ1) was added to wells, followed by
4 h of incubation. The culture medium was then removed and
DMSO (100 mL per well) was added and the absorbance values
were measured at 570 nm using the multi-well Tecan Nano-
Quant, Innite m200 Pro plate reader. GI50 values were calcu-
lated using GraphPad Prism 7 (GraphPad Soware, San Diego,
USA) by nonlinear regression analysis. All the time-dependent
experiments at different times were carried out using the
same passage of MDA-MB231 cells. Three independent experi-
ments were carried out and the data were expressed in mean ꢀ
standard deviation (SD).
Conflicts of interest
There are no conicts to declare.
4.6. Acridine orange/propidium iodide staining
Acknowledgements
Fluorescence microscopy was used to visualize the apoptosis in
cancer cells with AO/PI staining.20 MDA-MB231 cells were grown
in 6-well plate (5 ꢂ 105 cells per well). The cells were treated
with respective compounds at concentrations equal to esti-
mated GI20 and GI50 values and incubated for 24 h. One of the
six wells was treated with 1% DMSO and served as a negative
control, another well treated with methotrexate served as
a positive control. Aer 24 h of incubation, the wells were
washed with phosphate buffered saline (PBS) three times and
100 mL of AO (100 mg mLꢁ1 in PBS) and 25 mL of PI (100 mg mLꢁ1
in PBS) in 1 mL of media were added to each well. The plate was
then observed under a motorized inverted uorescent micro-
scope (Eclipse Ti2-E, Nikon). Three independent experiments at
each concentration were carried out.
This work is supported by the Ministry of Higher Education,
Malaysia under Fundamental Research Grant Scheme, grant
number FRGS/1/2015/SG01/MUSM/03/1. This work received
a partial support from the School of Pharmacy, Monash
University Malaysia (Bridging Grant 2020). Sunway University
Sdn Bhd is thanked for nancial support of the X-ray crystal-
lography laboratory (Grant No. STR-RCTR-RCCM-001-2019).
Ahmad Junaid thanks Dr David Manallack and Monash
University for the opportunity to carry out part of his research
under the Vice Chancellor's International Intercampus PhD
Mobility Scheme.
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