Synthesis, Anti-proliferative activity, and molecular docking study of new series of 1,3-5-Triazine schiff base derivatives

Article abstract of DOI:10.3390/molecules25184065

Based on the use of s-triazine as a scaffold, we report here a new series of s-triazine Schiff base derivatives and their anti-proliferative activity against two cancer cell lines: human breast carcinoma (MCF-7), and colon cancer (HCT-116) compared with tamoxifen as a reference compound. Several derivatives exhibited growth inhibition activity in the sub-micromolar range. The results reveal that the s-triazine Schiff base derivatives showed varied activities and that the substituents on the s-triazine core have a great effect on the anti-proliferative activity. Compounds with a piperidino and benzylamino substituent on the s-triazine moiety 4b and 4c were most effective in both cell lines compared to the reference compound used. In addition, compound 4b has a para chlorine atom on the benzylidine residue, demonstrating the most potent activity with IC₅₀ values of 3.29 and 3.64 μM in MCF-7 and HCT-116, respectively. These results indicate that in general, the nature of the substituents on the triazine core and the type of substituent on the benzilyldene ring significantly influenced the anti-proliferative activity. The results obtained from the anti-proliferative activity and the molecular docking study indicate that s-triazine-hydrazone derivatives may be an excellent scaffold for the development of new anti-cancer agents.

Full text of DOI:10.3390/molecules25184065

molecules
Article
Synthesis, Anti-proliferative Activity, and Molecular
Docking Study of New Series of 1,3-5-Triazine Schiff
Base Derivatives
Hessa H. Al Rasheed 1,*, Azizah M. Malebari 2, Kholood A. Dahlous 1, Darren Fayne 3
and Ayman El-Faham 1,4,
*
1
Department of Chemistry, College of Science, King Saud University P.O. Box 2455, Riyadh 11451,
Saudi Arabia; kdahloos@ksu.edu.sa
2
3
4
Department of Pharmaceutical Chemistry, College of Pharmacy, King Abdulaziz University,
Jeddah 21589, Saudi Arabia; amelibary@kau.edu.sa
Molecular Design Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute,
Trinity College Dublin, Dublin 2, Ireland; fayned@tcd.ie
Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia,
Alexandria 12321, Egypt
*
Correspondence: halbahli@ksu.edu.sa (H.H.A.R.);
aelfaham@ksu.edu.sa or aymanel_faham@hotmail.com (A.E.-F.); Tel.: +00-9661-1467-3195 (A.E-F.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Jóhannes Reynisson FRSC and Diego Muñoz-Torrero
Received: 12 August 2020; Accepted: 4 September 2020; Published: 5 September 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Based on the use of s-triazine as a scaffold, we report here a new series of s-triazine Schiff
base derivatives and their anti-proliferative activity against two cancer cell lines: human breast
carcinoma (MCF-7), and colon cancer (HCT-116) compared with tamoxifen as a reference compound.
Several derivatives exhibited growth inhibition activity in the sub-micromolar range. The results
reveal that the s-triazine Schiff base derivatives showed varied activities and that the substituents on
the s-triazine core have a great effect on the anti-proliferative activity. Compounds with a piperidino
and benzylamino substituent on the s-triazine moiety 4b and 4c were most effective in both cell lines
compared to the reference compound used. In addition, compound 4b has a para chlorine atom on the
benzylidine residue, demonstrating the most potent activity with IC50 values of 3.29 and 3.64 µM in
MCF-7 and HCT-116, respectively. These results indicate that in general, the nature of the substituents
on the triazine core and the type of substituent on the benzilyldene ring significantly influenced the
anti-proliferative activity. The results obtained from the anti-proliferative activity and the molecular
docking study indicate that s-triazine-hydrazone derivatives may be an excellent scaffold for the
development of new anti-cancer agents.
Keywords: s-triazine; schiff base; MCF-7; HCT-116; apoptosis; molecular docking
1. Introduction
After cardiovascular diseases, cancer is the second most common cause of death globally, and its
occurrence is predicted to increase dramatically in the near future. The high occurrence and death
ratio of cancer is because there are more than 270 types of cancer that have a great tendency to resist
chemotherapeutics and few cancers are diagnosed in their early stages. For all these reasons, research is
essential for cancer treatments providing more effective and less toxic agents [1–4].
1,3,5-Triazine (s-triazine) is one of the most fascinating chemical cores in medicinal chemistry
applications due to the broad range of biological activities such as anti-microbial [
5], antifungal [6],
antimalarial [7], carbonic anhydrase inhibitors [8–12] and anti-cancer agents [13–17].
Molecules 2020, 25, 4065; doi:10.3390/molecules25184065
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4065
2 of 14
On the other hand, Schiff bases are compounds formed by a condensation reaction between
hydrazine or primary amines and carbonyl compounds to form azomethine type compounds (CH=NH-)
and are considered to be important derivatives with biological, medicinal, clinical, pharmacological and
analytical applications [18–21]. In addition, they are popular ligand precursors due to their versatility
and ease of preparation.
Schiff bases containing s-triazine core showed a wide diversity of biological activities such
as anti-cancer, antimycobacterial, antibacterial, antidepressant, analgesic properties and enzyme
inhibitors [22
estrogen receptor alpha (ER
–
26]. Moreover, 1,3,5-triazine derivatives have been shown to bind to and modulate
) and estrogen receptor beta (ER ) [27 28]. Very recently, Lu et al.,
α
β
,
reported the synthesis of 1,3,5-triazine-based derivatives as selective estrogen receptor degraders
(SERDs). Most of these compounds showed anti-proliferative activities in MCF-7 cells [29].
In continuation of our studies on s-triazine-hydrazone derivatives and their anti-cancer activity
against breast cancer cells (MCF-7) and colon cancer (HCT-116) [30–32], herein we report a new series
of Schiff bases encompassing s-triazine core and benzylidene moieties with different substituents
(Figure 1). The compounds’ anti-proliferative activities against breast cancer MCF-7 and colon
carcinoma HCT-116 cell lines were evaluated. In addition, molecular docking of the compound series
within the hypothesized biological target (ERα) will be discussed.
Figure 1. Structure of the novel synthesized s-triazine compounds.
2. Results and Discussion
2.1. Chemistry
The target compounds 4a-r were synthesized as illustrated in Scheme 1 following the reported
methods [31–34]. The hydrazine derivatives 2 were reacted with p-substituted benzaldehyde derivatives
3a-c in the presence of acetic acid (AcOH) and using ethanol as a solvent to give the target compounds
4a-r (Scheme 1). The structures of 4a-r were established by elemental analysis, Fourier-transform infrared
spectroscopy (FTIR), 1H-NMR, and 13C-NMR spectra (Supplementary information, Figures S1–S18).

Molecules 2020, 25, 4065
3 of 14
Scheme 1. Synthetic route for preparation of s-triazine Schiff base derivatives.
2.2. Biology
2.2.1. Anti-Proliferative Activity and Induced Apoptosis
s-Triazine-hydrazone derivatives 4a-r were screened for their anti-proliferative activity against two
representative human cancer cell lines; breast cancer MCF-7 and colon cancer HCT-116 and compared
with tamoxifen [35] as a reference compound using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. Based on the data illustrated in Table 1 (Supporting information, Method S1),
the s-triazine-hydrazone derivatives displayed varied anti-proliferative activity, with better efficacy
on MCF-7 than HCT-116 cells. Generally, the nature of the substituents on the triazine ring
significantly influenced the biological activity. s-Triazine-hydrazone derivatives 4m, 4n, and 4o
(containing morpholine and phenylamine on the triazine core) showed potent anti-proliferative
in MCF-7 and HCT-116 cells with IC50 values of 7.93, 6.10, and 6.58
respectively. Interestingly, replacement of the morpholine ring in compounds 4m
group to furnished compounds 4p maintained good to moderate anti-proliferative in MCF-7 with
IC50 values in the range of 3.98–10.44 M. To examine the significance of the phenylamino substituent
on the triazine ring, a related series of benzylamino substituent analogs including compounds 4a
(containing piperidine on the triazine core) and compounds 4g (containing morpholine on the triazine
core) were next evaluated. Compounds 4b and 4c, with a piperidine ring, showed more potent
anti-proliferative activity with, IC50 values of 3.29 and 4.63 M in MCF-7 cell lines and 3.64 and 5.60
in HCT-116 cell lines, respectively compared to their corresponding analogs with a morpholine ring in
compounds 4g (IC50 values in range of 16.44–24.46 M and 8.19–14.01 M in MCF-7 and HCT-116,
respectively) and compared with tamoxifen as a reference compound (IC50 values of 5.12 and 26.41
µM and 5.10, 4.54, 10.41
µM,
–o
with a methoxy
–r
µ
–c
–i
µ
µM
–i
µ
µ
µM
in MCF-7 and HCT-116 cells, respectively) as shown in Table 1. These results could reflect the importance
of a piperidine substituent on the triazine ring as a less polar group for optimum activity in cancer
cells. These results are consistent with our previously reported results, where the piperidine derivatives
showed better activity compared to their corresponding morpholine analogs [33,34,36].
Conversely, replacement of the phenylamino substituent on the triazine moiety in compounds
4m–o with a small group as a methoxy group (compounds 4d–f and 4j–l) significantly affected the
anti-proliferative activity with a 2.5–5.5 and 3.7–12.5-fold loss in the potency on MCF-7 and HCT-116
cells, respectively. For example, replacement of phenylamino in morpholine substituted triazine
derivative 4n with a methoxy group in 4j resulted in a marked decrease in the activity in both cell lines:
IC50 values for 4n were 6.10 and 4.54 µM vs. IC50 values for 4k were 14.08 and 42.34 µM in MCF-7 and
HCT-116 cells, respectively.

Molecules 2020, 25, 4065
4 of 14
However, it was observed that methoxy s-triazine-hydrazone compounds 4p
–
r
with phenyl
amine moiety, exhibited better anti-proliferative activity compared to their analogs 4d
–f
(piperidine derivatives) and derivatives 4j (morpholine derivatives) as shown in Table 1. For example,
–
l
4p vs. 4d and 4j, exhibited significant activity against both tested cell lines. These results agreed with
the reported data, where aromatic amines can help to enhance biological activity [37,38].
Table 1. Anti-proliferative effects of 4a–q series compounds in MCF-7 and HCT-116 cells.
IC50 (µM) a
MCF-7
IC50 (µM)a
HCT-116
Compound no.
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
11.35 ± 1.81
3.29 ± 0.83
4.63 ± 0.40
28.62 ± 2.80
33.90 ± 0.94
18.49 ± 1.03
24.46 ± 1.83
16.44 ± 1.13
22.20 ± 2.59
18.59 ± 1.62
14.08 ± 0.06
26.91 ± 1.20
7.93 ± 0.77
6.10 ± 0.42
6.58 ± 0.65
3.98 ± 0.22
9.37 ± 1.74
10.44 ± 0.43
5.12 ± 0.36
12.45 ± 1.16
3.64 ± 1.58
5.60 ± 1.97
>50
>50
>50
14.01 ± 1.61
8.19 ± 0.22
12.22 ± 0.34
24.10 ± 3.94
42.34 ± 4.74
37.29 ± 0.34
5.10 ± 1.01
4.54 ± 0.41
10.71± 2.91
8.45 ± 0.24
6.26 ± 0.49
7.57 ± 1.34
26.41 ± 4.11
4q
4r
Tamoxifen b
aIC50 values are half maximal inhibitory concentrations required to block the growth stimulation of cells.
Values represent the mean for three experiments performed in triplicate; bThe IC50 value obtained for CA-4
(5.12 µM for MCF-7 and 26.41 µM for HCT-116) are in good agreement with reported values [39,40].
With the exception of compounds 4d–f and 4j–l, the introduction of electron-withdrawing groups
such as chloro and bromo substituent on the benzylidene ring with different substituted s-triazine
motifs demonstrated good anti-cancer activities against both cell lines in the low micromolar range
compared to its corresponding unsubstituted analogs. Derivatives with piperidino and benzylamino
substituent on the triazine moiety 4b (chloro substituent) and 4c (bromo substituent) showed the most
potent activities in this series in both cell lines as shown in Table 1 and Figures 2 and 3.
MCF-7 (IC50= 3.29 μM)
HCT-116 (IC50= 3.64 μM)
100
50
0
-7
-6
-5
-4
Log10 concentration (M)
Figure 2. Anti-proliferative effect of 4b in MCF-7 and HCT-116 cells. Cells were grown in 96-well
plates and treated with 4b at 0.1 M to 100 M 0.05–100 M for 72 h. Cell viability was expressed as a
µ
µ
µ
percentage of vehicle control [ethanol 0.1% (v/v)] treated cells. The values represent the mean
for three independent experiments performed in triplicate.
±
S.E.M.

Molecules 2020, 25, 4065
5 of 14
MCF-7 (IC50= 4.63 μM)
HCT-116 (IC50= 5.60 μM)
100
50
0
-7
-6
-5
-4
Log10 concentration (M)
Figure 3. Anti-proliferative effect of 4c in MCF-7 and HCT-116 cells. Cells were grown in 96-well plates
and treated with 4c at 0.1 M to 100 M for 72 h. Cell viability was expressed as a percentage of vehicle
µ
µ
control [ethanol 0.1% (v/v)] treated cells. The values represent the mean
experiments performed in triplicate.
±
S.E.M. for three independent
Finally, s-triazine-hydrazone derivatives that encompass benzylamino and piperidino rings on
the s-triazine core can be envisaged as interesting novel molecules with promising anti-proliferative
activities in MCF-7 and HCT-116 cell lines.
Based on the obtained results, we examined apoptosis in MCF-7 cells after treatment with the
most active compound 4b. The percentage of early and late apoptotic cells was determined by double
staining with Annexin V and PI via flow cytometry. Positioning of quadrants on dot plots was
designated, and living cells (Annexin V
cells (Annexin V+/PI+) and necrotic cells (Annexin V
prove that the MCF-7 cells were incubated with 4b at a concentration of 6
population of viable cells and increasing the percentage of apoptotic cells. The percentage of early and
−
/PI−
), early apoptotic cells (Annexin V+/PI
/PI+) were identified. The data shown in Figure 4
M for 24 h decreased the
−
), late apoptotic
−
µ
late apoptotic cells together of 4b increased after 24 h to 21.81% and 6.93%, respectively at 6 µM when
compared to the control cells (2.23%) as shown in Figure 4.
Figure 4. Flow cytometric analysis (Annexin V-FTIC/PI assay) of MCF-7 cells exposed for 24 h to 4b
) (approximately 2x IC50 value), respectively. = untreated control, and vehicle control (DMSO).
(C
A
B
The represented dot plots showing percentage of viable, early apoptotic, late apoptotic, and necrotic cells.
These results show the ability of 4b to induce apoptosis in cancer cells and may deliver an excellent
scaffold for the development of anti-cancer drug candidates.

Molecules 2020, 25, 4065
6 of 14
2.2.2. Molecular Docking Study
Estrogen actions are mainly mediated through two nuclear estrogen receptor (ER) subtypes namely
and ER 41 42]. In Western blot analyses, the antibody against ER detected a single sharp
band related to the expected molecular weight of 72 kD in all MCF-7 subclones. In agreement with
Western blot analyses, a high expression of the ER mRNA was detected in MCF-7 [43]. Accordingly,
ERα
β
[
,
α
α
an in-depth binding pose analysis on all compounds with ERα was performed.
The top docked pose of each compound was analyzed and mapping to the X-ray structure ligands
binding poses was considered (Supporting information, Method S2). The triazine derivatives 4a
were scored following docking, in order of preference. This ranking broadly maps to the cellular
efficacy biological data, as the 4a series are the most potent compounds while the 4j and 4d series
are the least potent and 4p–q have moderate potency.
Analysis of the predicted binding poses assisted to illustrate the potency trends. The most potent
compounds (4b and 4c) had near identical scores ( 7.8387 and 7.8355 respectively). As shown
–r
–
c
–
l
–f
−
−
in Figure 5a and b, the halogenated benzylidene ring mapped to the tertiary amine group of
4-hydroxytamoxifen (4-OHT), the triazine ring mapped to the 4-OHT A ring, the benzyl ring mapped to
the 4-OHT B ring and the piperidine ring mapped to the 4-OHT C ring. Binding was primarily driven
by hydrophobic and van der Waals interactions due to the benzyl and piperidine rings occupying
apolar sub-pockets in the ERα binding site. The benzyl group was in a sub-pocket comprising of
Leu349, Ala350, Leu391, and Phe404. The group did not make interactions with the polar Glu353 and
Arg394 amino acids deeper in the sub-pocket, which will be a future route for compound optimization.
The piperidine ring occupied a sub-pocket encompassing Val418, Ile424, Leu525, and Leu346. The amine
of the benzylidene chain can make a hydrogen bond donor interaction with a co-crystallized water
molecule but did not make a hydrogen bond with the important Asp351 residue. The halogens are
2.91 Å from the backbone nitrogen atom of Cys530 so may make an attractive electrostatic interaction.
Conversely, the worst ranked docked series, for example, 4l (Figure 5c) was unable to recapitulate
the favorable interactions formed by the benzyl group of the 4a
–
c
series. The more polar morpholine
series as it was in a hydrophobic
ring in 4g series is also not preferred to the piperidine ring of the 4a–c
–
i
sub-pocket. Due to the smaller substituent sizes, the docked compound was located lower in the
binding site so the triazine group is not overlaid on the 4-OHT A ring. Similar binding poses were
found for the piperidine and methoxy on the triazine series 4d–f (i.e., 4f, Figure 5d), in that the
methoxy group occupies one of the hydrophobic sub-pockets and the compound is located lower in
the binding site.
In summary, the results obtained from molecular docking are consistent with the results obtained
from the anti-proliferative activity screening, where the most active series are compounds with
piperidine and benzylamine on the triazine core.

Molecules 2020, 25, 4065
7 of 14
a
b
c
Figure 5. Best ranked docked poses of (
d
a)
4c, (
b
)
4b, (c) 4l and (d) 4f in ERα overlaid on the 4-OHT
X-ray structure 3ERT generated with the Molecular Operating Environment (MOE) 2019 software.
Carbon atoms are illustrated in grey for 4-OHT, orange for the docked compounds and light green
for the protein (oxygen atoms—red; nitrogen—dark blue; sulfur—yellow; bromine—dark red and
chlorine—dark green). ERα and associated water molecules are in light green.
3. Materials and Methods
All reagents and solvents purchased from commercial suppliers and used without further
purification. 1H-NMR and 13C-NMR spectra were recorded on a JEOL 400 MHz spectrometer
(JEOL, Ltd., Tokyo, Japan), and chemical shift (δ) values were expressed in ppm. Elemental analyses
performed on Perkin–Elmer 2400 elemental analyzer (PerkinElmer, Inc.940 Winter Street, Waltham,
MA, USA). Melting points were recorded on a Mel-Temp apparatus Sigma-Aldrich Chemie GmbH,
82024 Taufkirchen, Germany) in an open capillary and are uncorrected. Fourier transform infrared
spectroscopy (FTIR) recorded on Shimadzu 8201 PC FTIR spectrophotometer (Shimadzu, Ltd., Kyoto,
Japan). The reaction was follow-up and checks of the purity using thin layer liquid chromatograph
(TLC) on silica gel-protected aluminum sheets (Type 60 GF254, Merck).

Molecules 2020, 25, 4065
8 of 14
3.1. General Method for the Synthesis of 1,3,5-triazine Schiff Base Derivatives
2-Hydrazino-4,6-disubstituted-1,3,5-triazine derivatives
reported methods [26,32–34,36] and used directly without further purification into the next step.
Hydrazine derivatives were reacted with aldehyde derivatives 3a (10 mmol) in ethanol
2 were synthesized first following the
2
–c
(30 mL) in the presence of 2–3 drops of acetic acid at room temperature. The reaction mixture was
refluxed for 4–6 h, and the progress of the reaction was follow by TLC using ethyl acetate-hexane
2:1. After completion of the reaction, the mixture left to cool down to room temperature and the solid
product was collected by filtration, washed with cooled ethanol, and then dried at room temperature.
N-benzyl-4-(2-benzylidenehydrazinyl)-6-(piperidin-1-yl)-1,3,5-triazin-2-amine (4a). the product was
obtained as a white solid in yield 83%; mp 148–150 ◦C; IR (KBr, cm−1): 3241 (NH), 1662 (C=N), 1590,
1545 (C=C); 1H-NMR (400 MHz, DMSO-d6):
δ = 1.40 (brs, 4H, 2CH2), 1.55 (brs, 2H, CH2), 3.65 (brs, 4H,
2 NCH2-), 4.39–4.45 (m, 2H, CH2-NH), 7.16–7.36 (m, 8H, Ar), 7.57 (s, 2H, Ar), 8.03 (s, 1H, CH),
10.57 (brs, 1H, NH) ), 10.64 (brs, 1H, NH) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 24.9, 25.9, 43.4,
56.6, 126.7, 126.9, 127.9, 128.6, 129.2, 129.4, 135.7, 141.3, 141.9, 164.8, 166.5 ppm. Anal. Calcd for
C22H25N7 (387.48): C, 68.19; H, 6.50; N, 25.30. Found C, 68.32; H, 6.66; N, 25.50.
N-benzyl-4-(2-(4-chlorobenzylidene)hydrazinyl)-6-(piperidin-1-yl)-1,3,5-triazin-2-amine (4b). the product
was obtained as a white solid in yield 88%; mp 200–202 ◦C; IR (KBr, cm−1): 3376 (NH), 1592 (C=N),
1516,1442 (C=C); 1H-NMR (400 MHz, DMSO-d6): δ = 1.40 (brs, 4H, 2CH2), 1.55 (brs, 2H, CH2),
3.64 (brs, 4H, 2 NCH2-), 4.38–4.45 (m, 2H, CH2-NH), 7.16 (t, 1H, J = 7.0 Hz, Ar), 7.23–7.29 (m, 4H, Ar),
7.42 (d, 2H, J = 8.5 Hz, Ar), 7.58 (d, 2H, J = 8.0 Hz, Ar), 8.01 (s, 1H, CH), 10.66 (brs, 1H, NH),
10.71 (s, 1H, NH) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 24.9, 25.9, 43.5, 56.6, 126.9, 127.9, 128.4,
128.6, 129.3, 133.7, 134.7, 140.5, 141.2, 164.6, 166.4 ppm. Anal. Calc. for C22H24ClN7 (421.93): C, 62.63;
H, 5.73; N, 23.24. Found C, 62.54; H, 5.68; N, 23.45.
N-benzyl-4-(2-(4-bromobenzylidene)hydrazinyl)-6-(piperidin-1-yl)-1,3,5-triazin-2-amine (4c). the product was
obtained as a white solid in yield 84%; mp 195–197 ◦C; IR (KBr, cm−1): 3351 (NH), 1656 (C=N),
1548,1514 (C=C); 1H-NMR (400 MHz, DMSO-d6): δ = 1.40 (brs, 4H, 2CH2), 1.55 (brs, 2H, CH2),
3.65 (brs, 4H, 2 NCH2-), 4.40–4.45 (m, 2H, CH2-NH), 7.14 (t, 1H, J = 7.0 Hz, Ar), 7.23–7.29 (m, 4H, Ar),
7.52–7.57 (m, 4H, Ar), 8.00 (s, 1H, CH), 10.72–10.77 (m, 2H, 2NH)) ppm; 13C-NMR (100 MHz, DMSO-d6):
δ = 24.9, 25.9, 43.7, 44.1.9, 122.4, 127.0, 127.9, 128.6, 132.2, 134.9, 140.5, 140.9, 164.8, 166.7 ppm. Anal.
Calc. for C22H24BrN7 (466.38): C, 56.66; H, 5.19; N, 21.02. Found C, 56.83; H, 5.41; N, 21.27.
2-(2-benzylidenehydrazinyl)-4-methoxy-6-(piperidin-1-yl)-1,3,5-triazine (4d). the product was obtained as a
white solid in yield 81%; mp 221–223 ◦C; IR (KBr, cm−1): 3219 (NH), 1591 (C=N), 1542, 1513 (C=C);
1H-NMR (400 MHz, DMSO-d6):
δ = 1.47 (brs, 4H, 2CH2), 1.58 (brs, 2H, CH2), 3.70 (brs, 4H, 2 NCH2-),
3.79 (s, 3H, OCH3), 7.31–7.39 (m, 3H, Ar), 7.61 (d, 2H, J = 8.25 Hz, Ar), 8.07 (s, 1H, CH), 11.08
(s, 1H, NH) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 24.8, 25.9, 44.3, 54.1, 127.0, 129.3, 129.8, 135.5,
143.3, 165.3, 171.5 ppm. Anal. Calc. for C16H20N6O (312.37): C, 61.52; H, 6.45; N, 26.90. Found C,
61.71; H, 6.56; N, 27.03.
2-(2-(4-chlorobenzylidene)hydrazinyl)-4-methoxy-6-(piperidin-1-yl)-1,3,5-triazine (4e). the product was
obtained as a white solid in yield 86%; mp 123–125 ◦C; IR (KBr, cm−1): 3219 (NH), 1590 (C=N), 1540,
1462 (C=C);1H-NMR (400 MHz, DMSO-d6):
δ = 1.47 (brs, 4H, 2CH2), 1.58 (brs, 2H, CH2), 3.69 (brs, 4H,
2 NCH2-), 3.79 (s, 3H, OCH3), 7.43 (d, 2H, J = 7.5 Hz, Ar), 7.62 (d, 2H, J = 6.5 Hz, Ar), 8.05 (s, 1H, CH),
11.15 (s, 1H, NH) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 24.7, 25.9, 43.7, 54.1, 128.6, 129.4, 129.6,
130.5, 1341, 134.3, 142.0, 165.8, 171.4 ppm. Anal. Calc. for C16H19ClN6O (346.81): C, 55.41; H, 5.52; N,
24.23. Found C, 55.62; H, 5.68; N, 24.44.

Molecules 2020, 25, 4065
9 of 14
2-(2-(4-bromobenzylidene)hydrazinyl)-4-methoxy-6-(piperidin-1-yl)-1,3,5-triazine (4f). the product was
obtained as a yellow solid in yield 85%; mp 138–140 ◦C; IR (KBr, cm−1): 3217 (NH), 1590 (C=N), 1541,
1462 (C=C); 1H-NMR (400 MHz, DMSO-d6):
δ
= 1.47 (brs, 4H, 2CH2), 1.58 (brs, 2H, CH2), 3.69 (brs, 4H,
2CH2N), 3.79 (s, 3H, OCH3), 7.54–7.59 (m, 4H, Ar), 8.03 (s, 1H, CH), 11.16 (s, 1H, NH) ppm; 13C-NMR
(100 MHz, DMSO-d6):
δ = 24.7, 25.9, 43.7, 54.1, 122.9, 128.8, 130.7, 132.3, 134.6, 142.1, 165.8, 171.4 ppm.
Anal. Calc. for C16H19BrN6O (391.27): C, 49.12; H, 4.89; N, 21.48. Found C, 49.33; H, 4.97; N, 21.67.
4,40-(6-(2-(4-fluorobenzylidene)hydrazinyl)-1,3,5-triazine-2,4-diyl)di morpholine(4g). the product was
obtained as a white solid in yield 74%; mp 140–142 ◦C; IR (KBr, cm−1): 3253 (NH), 1662 (C=N), 1619,
1510 (C=C); 1H-NMR (400 MHz, DMSO-d6): δ = 3.54 (brs, 4H, 2 OCH2-), 3.65 (brs, 4H, 2 NCH2-),
4.42–4.48 (m, 2H, CH2-NH), 7.16 (t, 1H, J = 7.25 Hz, Ar), 7.24–7.31 (m, 4H, Ar), 7.36 (t, 3H, J = 7.5 Hz,
Ar), 7.59 (d, 2H, J = 7.5 Hz, Ar), 8.06 (s, 1H, CH), 10.73 (s, 1H, NH) ppm;13C-NMR (100 MHz, DMSO-d6):
δ = 43.2, 43.6, 65.9, 126.4, 126.5, 127.3, 128.1, 128.7, 128.9, 135.1, 140.5, 141.6, 164.2, 164.7, 164.9ppm.
Anal. Calc. for: C21H23N7O (389.45): C, 64.76; H, 5.95; N, 25.18. Found C, 64.91; H, 6.03; N, 25.33.
N-benzyl-4-(2-(4-chlorobenzylidene)hydrazinyl)-6-morpholino-1,3,5-triazin-2-amine (4h). the product was
obtained as a yellow solid in yield 77%; mp 260–262 ◦C; IR (KBr, cm-1): 3272 (NH), 1658 (C=N), 1613,
1513 (C=C); 1H-NMR (400 MHz, DMSO-d6): δ = 3.58 (brs, 4H, 2 OCH2-), 3.71 (brs, 4H, 2 NCH2-),
4.47–4.51 (m, 2H, CH2-NH), 7.26–7.33 (m, 3H, Ar), 7.44 (d, 2H, J = 9.0 Hz, Ar), 7.54 (d, 2H, J = 8.5 Hz,
Ar), 7.86 (d, 2H, J = 8.5 Hz, Ar), 8.11 (s, 1H, CH), 10.81 (brs, 1H, NH) ppm; 13C-NMR (100 MHz,
DMSO-d6):
δ 43.3, 44.1, 66.6, 127.6, 128.0, 128.8, 129.3, 129.6, 130.6, 133.1, 136.6, 140.6, 161.4, 164.2 ppm.
Anal. Calc. for: C21H22ClN7O (423.90): C, 59.50; H, 5.23; N, 23.13. Found C, 59.67; H, 5.34; N, 23.31.
N-benzyl-4-(2-(4-bromobenzylidene)hydrazinyl)-6-morpholino-1,3,5-triazin-2-amine (4i). the product was
obtained as a yellow solid in yield 75%; mp 256–258 ◦C; IR (KBr, cm-1): 3424(NH), 1657(C=N),
1613,1513(C=C);1H-NMR (400 MHz, DMSO-d6): δ = 3.57 (brs, 4H, 2 OCH2-), 3.69 (brs, 4H, 2 NCH2-),
4.48 (brs, 2H, CH2-NH), 7.18–7.32 (m, 5H, Ar), 7.56–7.58 (m, 4H, Ar), 8.08 (s, 1H, CH), 10.81 (s, 1H, NH)
ppm; 13C-NMR (100 MHz, DMSO- d6): δ 43.2, 44.1, 66.4, 127.1, 127.5, 128.7, 128.9, 130.7, 131.9, 132.6,
134.2, 137.3, 140.9, 161.3, 164.8 ppm. Anal. Calc. for: C21H22BrN7O (468.35): C, 53.85; H, 4.73; N, 20.93.
Found C, 53.61; H, 4.62; N, 21.14.
4-(4-(2-benzylidenehydrazinyl)-6-methoxy-1,3,5-triazin-2-yl)morpholine (4j). the product was obtained as a
white solid in yield 74%; mp 144–146 ◦C; IR (KBr, cm−1): 3353(NH), 1677(C=N), 1583,1537(C=C);1H-NMR
(400 MHz, DMSO-d6):
δ = 3.60 (brs, 4H, 2 OCH2-), 3.70 (brs, 4H, 2 NCH2-), 3.80 (s, 3H, OCH3), 7.32–7.39
(m, 3H, Ar), 7.62 (d, 2H, J = 6.5 Hz, Ar), 8.08 (s, 1H, CH), 11.17 (s, 1H, NH) ppm; 13C-NMR (100 MHz,
DMSO- d6): δ 43.4, 53.7, 65.9, 126.6, 128.7, 129.3, 134.7, 143.2, 165.3, 165.7 ppm. Anal. Calc. for:
C15H18N6O2 (314.34): C, 57.31; H, 5.77; N, 26.74. Found C, 57.55; H, 5.89; N, 26.93.
4-(4-(2-(4-chlorobenzylidene)hydrazinyl)-6-methoxy-1,3,5-triazin-2-yl)morpholine (4k). the product was
obtained as a white solid in yield 77%; mp 240–242 ◦C; IR (KBr, cm-1): 3220(NH), 1588(C=N),
1542,1460(C=C); 1H-NMR (400 MHz, DMSO-d6):
δ = 3.60 (brs, 4H, 2 OCH2-), 3.69 (brs, 4H, 2 NCH2-),
3.80 (s, 3H, OCH3), 7.44 (dd, 2H, J = 6.5, 2.5 Hz, Ar), 7.63 (dd, 2H, J = 6.5, 2.5 Hz, Ar), 8.06 (s, 1H, CH),
11.24 (s, 1H, NH) ppm; 13C-NMR (100 MHz, DMSO- d6): δ 43.4, 54.2, 66.4, 128.7, 129.4, 130.6, 134.2,
142.4, 161.6, 165.8 ppm. Anal. Calc. for: C15H17ClN6O2 (348.79): C, 51.65; H, 4.91; N, 24.09. Found C,
51.82; H, 4.83; N, 24.31.
4-(4-(2-(4-bromobenzylidene)hydrazinyl)-6-methoxy-1,3,5-triazin-2-yl)morpholine (4l). the product was
obtained as a white solid in yield 76%; mp 234–236 ◦C; IR (KBr, cm-1): 3228(NH), 1584(C=N),
1538,1462(C=C); 1H-NMR (400 MHz, DMSO-d6): δ = 3.64 (d, 4H, J = 4.4 Hz, 2 OCH2-), 3.73 (brs, 4H,
2 NCH2-), 3.83 (s, 3H, OCH3), 7.59 (s, 4H, Ar), 8.07 (s, 1H, CH), 11.26 (s, 1H, NH) ppm; 13C-NMR (100
MHz, DMSO-d6): δ 43.4, 53.7, 65.9, 122.4, 128.3, 131.7, 134.0, 141.9, 164.8, 166.4 ppm. Anal. Calc. for:
C15H17BrN6O2 (393.24): C, 45.81; H, 4.36; N, 21.37. Found C, 45.99; H, 4.51; N, 21.59.

Molecules 2020, 25, 4065
10 of 14
4-(2-benzylidenehydrazinyl)-6-morpholino-N-phenyl-1,3,5-triazin-2-amine (4m). the product was obtained
as a white solid in yield 84%; mp 244–246 ◦C; IR (KBr, cm−1): 3259(NH), 1608(C=N), 1551,1503(C=C);
1H-NMR (400 MHz, DMSO-d6): δ = 3.65 (d, 4H, J = 4.4 Hz, 2 OCH2-), 3.75 (brs, 4H, 2 NCH2-),
6.95 (t, 1H, J = 7.6 Hz, Ar), 7.27 (t, 2H, J = 8.0 Hz, Ar), 7.36–7.45 (m, 3H, Ar), 7.67 (d, 2H, J = 8.5 Hz, Ar),
7.83 (brs, 2H, Ar), 8.14 (s, 1H, CH), 9.30 (brs, 1H, NH), 10.92 (brs, 1H, NH) ppm; 13C-NMR (100 MHz,
DMSO-d6):
δ 43.2, 66.0, 119.7, 121.6, 126.5, 128.4, 128.7, 129.1, 135.1, 140.3, 142.2, 164.2, 164.8 ppm. Anal.
Calc. for: C20H21N7O (375.43): C, 63.98; H, 5.64; N, 26.12. Found C, 64.08; H, 5.77; N, 26.36.
4-(2-(4-chlorobenzylidene)hydrazinyl)-6-morpholino-N-phenyl-1,3,5-triazin-2-amine (4n). the product was
obtained as a white solid in yield 84%; mp 258–260 ◦C; IR (KBr, cm-1): 3253(NH), 1586(C=N), 1544,
1510 (C=C); 1H-NMR (400 MHz, DMSO-d6): δ = 3.65 (d, 4H, J = 4.4 Hz, 2 OCH2-), 3.75 (brs, 4H, 2
NCH2-), 6.95 (t, 1H, J = 7.6 Hz, Ar), 7.28 (t, 2H, J = 7.8 Hz, Ar), 7.50 (d, 2H, J = 8.0 Hz, Ar), 7.68 (d, 2H,
J = 8.8 Hz, Ar), 7.82 (brs., 2H, Ar), 8.12 (s, 1H, CH), 9.33 (brs., 1H, NH), 10.99 (brs., 1H, NH) ppm;
13C-NMR (100 MHz, DMSO- d6):
δ 43.4, 66.0, 119.8, 121.5, 127.8, 128.1, 128.9, 133.4, 133.9, 140.3, 141.0,
164.1, 164.2, 164.8 ppm. Anal. Calc. for: C20H20ClN7O (409.87): C, 58.61; H, 4.92; N, 23.92. Found C,
58.79; H, 5.04; N, 24.12.
4-(2-(4-bromobenzylidene)hydrazinyl)-6-morpholino-N-phenyl-1,3,5-triazin-2-amine (4o). the product was
obtained as a white solid in yield 85%; mp 259–261 ◦C; IR (KBr, cm−1): 3249 (NH), 1586(C=N), 1547,
1510 (C=C); 1H-NMR (400 MHz, DMSO-d6): δ = 3.65 (d, 4H, J = 4.4 Hz, 2 OCH2-), 3.75 (brs, 4H, 2
NCH2-), 6.94 (t, 1H, J = 7.4 Hz, Ar), 7.27 (t, 2H, J = 8.2 Hz, Ar), 7.62 (brs, 4H, Ar), 7.82 (brs, 2H, Ar), 8.11
(s, 1H, CH), 9.34 (brs, 1H, NH), 10.99 (brs., 1H, NH) ppm; 13C-NMR (100 MHz, DMSO-d6): δ = 43.4,
66.0, 119.8, 121.6, 122.1, 128.3, 131.7, 134.3, 140.3, 141.1, 164.2, 164.8 ppm. Anal. Calc. for C20H20BrN7O
(454.32): C, 52.87; H, 4.44; N, 21.58. Found C, 53.03; H, 4.54; N, 21.79.
4-(2-benzylidenehydrazinyl)-6-methoxy-N-phenyl-1,3,5-triazin-2-amine (4p). the product was obtained as a
white solid in yield 83%; mp 148–150 ◦C; IR (KBr, cm−1): 3254(NH), 1590(C=N), 1506, 1423(C=C);
1H-NMR (400 MHz, DMSO-d6): δ = 3.68 (s, 3H, OCH3), 6.81 (d, 3H, J = 9.5 Hz, Ar), 7.30–7.40 (m, 3H,
Ar), 7.62 (d, 2H, J = 7.5 Hz, Ar), 7.70 (brs, 2H, Ar), 8.11 (s, 1H, CH), 9.05 (brs., 1H, NH), 10.73 (brs, 1H,
NH) ppm; 13C-NMR (100 MHz, DMSO-d6):
δ = 55.1, 113.5, 120.9, 126.3, 128.6,132.9, 133.7, 135.2, 141.7,
154.1, 164.0, 164.2ppm. Anal. Calc. for C17H16N6O (320.35): C, 63.74; H, 5.03; N, 26.23. Found C, 63.93;
H, 5.13; N, 26.47.
4-(2-(4-chlorobenzylidene)hydrazinyl)-6-methoxy-N-phenyl-1,3,5-triazin-2-amine (4q). the product was
obtained as a white solid in yield 81%; mp 178–180 ◦C; IR (KBr, cm-1): 3346(NH), 1649(C=N),
1515,1417(C=C); 1H-NMR (400 MHz, DMSO-d6): δ = 3.68 (s, 3H, OCH3), 6.81 (d, 3H, J = 9.0 Hz, Ar),
7.46 (d, 2H, J = 8.5 Hz, Ar), 7.62–7.69 (m, 4H, Ar), 8.09 (s, 1H, CH), 9.07 (brs, 1H, NH), 10.80 (brs., 1H,
NH) ppm; 13C-NMR (100 MHz, DMSO- d6): δ 55.1, 113.5, 120.9, 127.8, 128.8, 133.2, 133.7, 134.2, 140.3,
154.1, 164.0, 164.2 ppm. Anal. Calc. for: C17H15ClN6O (354.79): C, 57.55; H, 4.26; N, 23.69. Found C,
57.79; H, 4.37; N, 23.91.
3.1.18. 4-(2-(4-bromobenzylidene)hydrazinyl)-6-methoxy-N-phenyl-1,3,5-triazin-2-amine (4r). the product
was obtained as a white solid in yield 77%; mp 173–175 ◦C; IR (KBr, cm-1): 3333(NH), 1655(C=N),
1556,1515(C=C);1H-NMR (400 MHz, DMSO-d6): δ = 3.71 (s, 3H, OCH3), 6.84 (d, 3H, J = 6.5 Hz, Ar),
6.85–7.72 (m, 6H, Ar), 8.11 (s, 1H, CH), 9.10 (brs., 1H, NH), 10.83 (brs, 1H, NH) ppm; 13C-NMR
(100 MHz, DMSO- d6):
δ 55.1, 113.5, 120.9, 121.9, 128.1, 131.7, 133.7, 134.5, 140.4, 154.1, 164.0, 164.2 ppm.
Anal. Calc. for: C17H15BrN6O (399.24): C, 51.14; H, 3.79; N, 21.05. Found C, 51.31; H, 3.92; N, 21.28.
3.2. Biology
3.2.1. In Vitro Anti-Proliferative Assay
The synthesized compounds 4a–r were evaluated for anti-proliferative using the MTT viability
assay of MCF-7 and HCT-116 cell lines and to calculate the relative IC50 values for each compound as
illustrated in the (Supporting information, Method S1).

Molecules 2020, 25, 4065
11 of 14
3.2.2. Annexin V/PI Apoptotic Assay:
Apoptotic cell death was detected by flow cytometry using Annexin V and propidium iodide
(PI). MCF-7 cells were seeded in 6 well plated at density of 1
105 cells/mL and treated with vehicle
(0.1% (v/v) DMSO) and compound 4b (6 M) for 24 h. Cells were then harvested and prepared for
×
µ
flow cytometric analysis as illustrated in the (Supporting information, Method S2).
3.2.3. Molecular Docking Study
The 3ERT X-ray structure of hERα co-crystallized with 4-hydroxytamoxifen (4-OHT) [44] was
downloaded from the protein data bank (PDB) website. The crystal structure was prepared using
QuickPrep (minimized to a gradient of 0.001 kcal/mol/Å), Protonate 3D, Residue pKa and Partial
Charges protocols in MOE 2019 [45] with the MMFF94x force field. All compounds were drawn in
ChemBioDraw Ultra 13.0.2.3021 (PerkinElmer), converted to sd files within Pipeline Pilot [46] and
read into a MOE mdb file. For each compound, MMFF94x partial charges were calculated and each
was minimized to a gradient of 0.001 kcal/mol/Å. Default parameters were used for docking except
that 300 poses were sampled for each compound and the top 50 docked poses were retained for
subsequent analysis.
4. Conclusions
In summary, the synthesized compounds in this study showed varied activity against two cell
lines MCF-7 and HCT-116. Generally, the nature of the substituents on the triazine ring significantly
influenced the biological activity. Compounds with piperidine and benzylamine moieties on the triazine
core 4a-c showed the most potent anti-proliferative activity with IC50 values in range of 3.29–11.35
and 3.64 -12.45 M for MCF-7 and HCT-116 cells, respectively compared with tamoxifen as a reference
compound (IC50 values of 5.12 and 26.41 M in MCF-7 and HCT-116 cells, respectively) and their
analogs with substituted morpholine and benzylamine 4g (IC50 values in range of 16.44–24.46
and 8.19 -14.01 M in MCF-7 and HCT-116 cells, respectively). Furthermore, compounds 4m
(morpholine and phenylamine) exhibited better activity compared to their analogous 4p (methoxy
µM
µ
µ
–i
µM
µ
–o
–r
and phenylamine). In addition, the substituent on the benzylidene ring showed great impact on the
anti-proliferative activity, where the chloro derivatives showed better anti-cancer activities compared
to other unsubstituted and bromo substituent analogs. The obtained results are consistent with the
previously reported results, where the presence of piperidine on the s-triazine ring and the presence
of an electronegative atom on the benzylidene increased the compounds activity against the cancer
cells [32–35].
The results demonstrate that derivative 4b can induce apoptosis in MCF-7 cells. Indeed, the results
obtained from the molecular docking agreed with the anti-proliferative activity assay, where the
combination of the piperidino and benzylamino on the s-triazine core is the optimal configuration.
Plans for future optimization of the triazine compound series ER binding affinity include
introducing hydrogen bonding acceptor moieties ortho and/or meta on the halogenated benzylidene
ring and hydrogen bond donating moieties para on the benzyl ring so as to more fully mimic the 4-OHT
binding interactions.
Supplementary Materials: The following are available online: http://www.mdpi.com/1420-3049/25/18/4065/s1
,
Figures S1–S18: represented the spectral data (1H and 13C-NMR spectra) for all prepared compounds.
Author Contributions: The work designed and supervised by A.E.-F.; H.H.A.R., K.A.D. and A.M.M. carried out
synthesis and characterization of the reported compounds; A.M.M. and D.F. carried the biological activity and
computational studies respectively. All authors discussed the results. The first drafts of the manuscript was
prepared by H.H.A. and A.M. All authors have read and agreed to the published version of the manuscript.
Funding: Deanship of Scientific Research at King Saud University, research group no. (RG-1441-365, Saudi Arabia).
Acknowledgments: The authors extend their appreciation to the Deanship of Scientific Research at King Saud
University for funding this work through research group no. (RG-1441-365, Saudi Arabia). The Trinity Biomedical

Molecules 2020, 25, 4065
12 of 14
Sciences Institute is supported by a capital infrastructure investment from Cycle 5 of the Irish Higher Education
Authority’s Programme for Research in Third Level Institutions (PRTLI). D.F. thanks the software vendors for
their continuing support of academic research efforts, in particular the contributions of Biovia, the Chemical
Computing Group, and OpenEye Scientific. The support and provisions of Dell Ireland, the Trinity Centre
for High Performance Computing (TCHPC), and the Irish Centre for High-End Computing (ICHEC) are also
gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest
References
1.
Zhou, C.; Min, J.; Liu, Z.; Young, A.; Deshazer, H.; Gao, T.; Chang, Y.T.; Kallenbach, N.R. Synthesis and
biological evaluation of novel 1,3,5-triazinederivatives as antimicrobial agents. Bioorg. Med. Chem. Lett.
2008, 18, 1308–1311. [CrossRef] [PubMed]
2.
Solankee, A.; Kapadia, K.; Ciric, A.; Sokovic, M.; Doytchinova, I.; Geronikaki, A. Synthesis of some
new s-triazine based chalcones and their derivatives as potent antimicrobial agents. Eur. J. Med. Chem.
2010, 45, 510–518. [CrossRef] [PubMed]
3.
4.
Patel, R.V.; Kumari, P.; Chikhalia, K.H. Design, synthesis and antimicrobial screening of s-triazinyl piperazine
and piperidine derivatives. Int. J. Adv. Pharm. Sci. 2010, 1, 395–403.
Kumar, G.V.P.; Srinivasa, R.D.; Pooja, B.; Harika, G.; Kumar, Y.A.; Sadasiva, R.G. An extensive review on
1,2,3 and 1,2,4-triazines scaffold-valuable lead molecules with potent and diverse pharmacological activities.
Der Chem. Sin. 2016, 7, 101–130.
5.
Ma, X.; Tan, S.-T.; Khoo, C.-L.; Sim, H.-M.; Chan, L.-W.; Chui, W.K. Synthesis and antimicrobial activity of
N1-benzylorN1-benzyloxy-1, 6-dihydro-1, 3, 5triazine-2, 4-diamines. Bioorg. Med. Chem. Lett. 2011, 21, 5428–5431.
[CrossRef]
6.
7.
Sharma, A.; Singh, S.; Utreja, D. Recent Advances in Synthesis and antifungal activity of 1,3,5-triazines.
Curr. Org. Synth. 2016, 13, 484–503. [CrossRef]
Manohar, S.; Khan, S.I.; Rawat, D.S. 4-Aminoquinoline-triazine-based hybrids with improved in vitro
antimalarial activity against CQ-sensitive and CQ-resistant strains of plasmodium falciparum. Chem. Biol.
Drug Des. 2013, 81, 625–630. [CrossRef]
8.
9.
Garaj, V.; Puccetti, L.; Fasolis, G.; Winum, J.Y.; Montero, J.L.; Scozzafava, A.; Vullo, D.; Innocenti, A.;
Supuran, C.T. Carbonic anhydrase inhibitors: Synthesis and inhibition of cytosolic/tumor-associated carbonic
anhydrase isozymes I, II, and IX with sulfonamides incorporating 1,2,4-triazine moieties. Bioorg. Med.
Chem. Lett. 2004, 14, 5427–5433. [CrossRef]
Garaj, V.; Puccetti, L.; Fasolis, G.; Winum, J.Y.; Montero, J.L.; Scozzafava, A.; Vullo, D.; Innocenti, A.;
Supuran, C.T. Carbonic anhydrase inhibitors: Novel sulfonamides incorporating 1,3,5-triazine moieties as
inhibitors of the cytosolic and tumour-associated carbonic anhydrase isozymes I, II, and IX. Bioorg. Med.
Chem. Lett. 2005, 15, 3102–3108. [CrossRef]
10. Ceruso, M.; Vullo, D.; Scozzafava, A.; Supuran, C.T. Inhibition of human carbonic anhydrase isoforms I-XIV
with sulfonamides incorporating fluorine and 1,3,5-triazine moieties. Bioorg. Med. Chem. 2013, 21, 6929–6936.
[CrossRef]
11. Carta, F.; Garaj, V.; Maresca, A.; Wagner, J.; Avvaru, B.S.; Robbins, A.H.; Scozzafava, A.; McKenna, R.;
Supuran, C.T. Sulfonamides incorporating 1,3,5-triazine moieties selectively and potently inhibit carbonic
anhydrase transmembrane isoforms IX, XII, and XIV over cytosolic isoforms I and II: Solution and X-ray
crystallographic studies. Bioorg. Med. Chem. 2011, 19, 3105–3119. [CrossRef] [PubMed]
12. Lolaka, N.; Akocaka, S.; Buab, S.; Supuranb, C.T. Design, synthesis and biological evaluation of novel
ureido benzenesulfonamides incorporating 1,3,5-triazine moieties as potent carbonic anhydrase IX inhibitors.
Bioorg. Chem. 2019, 82, 117–122. [CrossRef] [PubMed]
13. Zheng, M.; Xu, C.; Ma, J.; Sun, Y.; Du, F.; Liu, H.; Lin, L.; Li, C.; Ding, J.; Chen, K.; et al. Synthesis and
antitumor evaluation of a novel series of triaminotriazine derivatives. Bioorg. Med. Chem. 2007, 15, 1815–1827.
[CrossRef] [PubMed]
14. Patel, R.V.; Keum, Y.S.; Park, S.W. Medicinal chemistry discoveries among 1,3,5- triazines: Recent advances
(2000–2013) as antimicrobial, anti-TB, anti-HIV and antimalarials. Mini Rev. Med. Chem. 2014, 14, 768–789.
[CrossRef] [PubMed]

Molecules 2020, 25, 4065
13 of 14
15. Liu, B.; Sun, T.; Zhou, Z.; Du, L. A systematic review on antitumor agents with 1,3,5-triazines. Med. Chem.
2015, 5, 131–148. [CrossRef]
16. Cascioferro, S.; Parrino, B.; Spano, V.; Carbone, A.; Montalbano, A.; Barraja, P.; Diana, P.; Cirrincione, G.
1,3,5-Triazines: A promising scaffold, for anticancer drugs development. Eur. J. Med. Chem. 2017, 142, 523–549.
[CrossRef]
17. Srivastava, G.K.; Alonso-Alonso, M.L.; Fernandez-Bueno, I.; Garcia-Gutierrez, M.T.; Rull, F.; Medina, J.;
Coco, R.M.; Pastor, J.C. Comparison between direct contact and extract exposure methods for PFO cytotoxicity
evaluation. Sci. Rep. 2018, 8, 1425. [CrossRef]
18. Desmukh, A.P.; Soni, P.K.; Kankoriya, A.; Halve, A.K.; Dixit, R. 4-Aminoantipyrine: A significant tool for the
synthesis of biologically active Schiff bases and metal complexes. Int. J. Pharm. Sci. Rev. Res. 2015, 34, 162–170.
19. Goel, P.; Kumar, D.; Chandra, S. Schiff’s Base Ligands and their transition metal complexes as Antimicrobial
agents. J. Chem. Biol. Phy. Sci. 2014, 2014, 1946–1964.
20. Ebrahimipour, S.Y.; Khosravan, M.; White, J.; Fekri, S. Preparation, crystal structure, spectroscopic studies,
DFT calculations, antibacterial activities and molecular docking of a tridentate Schiff base ligand and its
cis-MoO2 complex. Appl. Organomet. Chem. 2018, 32, e4233. [CrossRef]
21. Kajal, A.; Bala, S.; Kamboj, S.; Sharma, N.; Saini, V. Schiff bases: A versatile pharmacophore. J. Catal.
2013, 2013, 14. [CrossRef]
22. Uddin, N.; Rashid, F.; Ali, S.; Tirmizi, S.A.; Ahmad, I.; Zaib, S.; Zubir, M.; Diaconescu, P.L.; Tahir, M.N.;
Iqbal, J.; et al. Synthesis, characterization, and anticancer activity of Schiff bases. J. Biomol. Str. Dynam.
2020, 38, 3246–3259. [CrossRef] [PubMed]
23. Hassanpour, S.H.; Dehghani, M. Review of cancer from perspective of molecular. J. Cancer Res. Pract.
2017, 4, 127–129. [CrossRef]
24. Abd-Elzaher, M.M.; Labib, A.A.; Mousa, H.A.; Moustafa, S.A.; Ali, M.M.; El-Rashedy, A.A. Synthesis,
anticancer activity and molecular docking study of Schiff base complexes containing thiazole moiety.
Beni Suef Univ. J. Basic. Appl. Sci. 2016, 5, 85–96. [CrossRef]
25. Menear, K.A.; Gomez, S.; Malagu, K.; Bailey, C.; Blackburn, K.; Cockcroft, X.L.; Sebastian, L. Identification
and optimisation of novel and selective small molecular weight kinase inhibitors of mTOR. Bioorg Med.
Chem. Lett. 2009, 19, 5898–5901. [CrossRef]
26. Bai, F.; Liu, H.; Tong, L.; Zhou, W.; Liu, L.; Zhao, Z.; Liu, X.; Jiang, H.; Wang, X.; Xie, H. Discovery of novel
selective inhibitors for EGFR-T790M/L858R. Bioorg. Med. Chem. Lett. 2012, 22, 1365–1370. [CrossRef]
27. Herke Herke, B.R.; Consler, T.G.; Go, N.; Hale, R.L.; Hohman, D.R.; Jones, S.A.; Lu, A.T.; Moore, L.B.;
Moore, J.T.; Orband-Miller, L.A.; et al. A new series of estrogen receptor modulators that display selectivity
for estrogen receptor. β J. Med. Chem. 2002, 45, 5492–5505.
28. Hale, R.L.; Henke, B.R.; Lambert, M.H.; Lu, A.T.; Spearing, P.K.; Turnbull, P.S. Piperazinyltriazine as Estrogen
Modulators. U.S. Patent 6,943,162 B2, 13 September 2005.
29. Lu, X.; Huang, A.; Xiao, M.; Sun, L.; Mao, J.; Luo, G.; Xiang, H. A new class of 1,3,5-triazine-based selective
estrogen receptor degraders (SERDs): Lead optimization, molecular docking and dynamic simulation.
Bioorg. Chem. 2020, 97, 103666. [CrossRef]
30. El-Faham, A.; Soliman, S.M.; Ghabbour, H.A.; Elnakady, Y.A.; Mohaya, T.A.; Siddiqui, M.R.; Albericio, F.
Ultrasonic promoted synthesis of novel s-triazine-Schiff base derivatives; molecular structure, spectroscopic
studies and their preliminary anti-proliferative activities. J. Mol. Str. 2016, 1125, 121–135. [CrossRef]
31. Barakat, A.; El-Senduny, F.F.; Almarhoon, Z.; Al-Rasheed, H.H.; Badria, F.A.; Al-Majid, A.M.;
El-Faham, A. Synthesis, X-ray crystal structures, and preliminary antiproliferative activities of new
s-triazine-hydroxybenzylidene hydrazone derivatives. J. Chem. 2019, 2019, 10. [CrossRef]
32. Al Rasheed, H.H.; Malebari, A.M.; Dahlous Kh., A.; El-Faham, A. Synthesis and characterization of new series
of 1,3-5-triazine hydrazone derivatives with promising anti-proliferative activity. Molecules 2020, 25, 2708.
[CrossRef] [PubMed]
33. Sharma, A.; Ghabbour, H.; Khan, S.T.; Beatriz, G.; Albericio, F.; El-Faham, A. Novel pyrazolyl-s-triazine
derivatives, molecular structure and antimicrobial activity. J. Mol. Str. 2017, 1145, 244–253. [CrossRef]
34. El-Faham, A.A.; Elnakady, Y. Synthesis, characterization of novel morpholino-1, 3, 5-triazinyl amino acid
Ester derivatives and their anti-proliferation activities. Lett. Org. Chem. 2015, 12, 753–758. [CrossRef]
35. Hassan, F.; Mohammed, G.; El-Hiti, G.A.; Alshanon, A.; Yousif, E. Cytotoxic effects of tamoxifen in breast
cancer cells. J. Unexplored Med. Data 2018, 3, 1–9. [CrossRef]

Molecules 2020, 25, 4065
14 of 14
36. Al Rasheed, H.H.; Dahlous Kh., A.; Sharma, A.; Sholkamy, E.; El-Faham, A.; De La Torre, B.G.; Albericio, F.
Barbiturate- and Thiobarbituarte-based s-triazine hydrazone derivatives with promising antiproliferative
activities. ACS Omega 2020, 5, 15805–15811. [CrossRef]
37. Alaghaz, A.; Bayoumi, H.A. Synthesis, Spectral Properties and Potentiometric Studies on Some Metal Schiff
Base Complexes Derived from 4–chlorophenyl–2–aminothiazole. Int. J. Electrochem. Sci. 2013, 8, 11860–11876.
38. Mojena, M.; Povo-Retana, A.; Gonz
ález-Ramos, S.; Fernández-García, V.; Regadera, J.; Zazpe, A.; Artaiz, I.;
Mart n-Sanz, P.; Ledo, F.; Bosc , L. Benzylamine and Thenylamine derived drugs induce apoptosis and
í
á
reduce proliferation, migration and metastasis formation in melanoma cells. Front. Oncol. 2018, 8, 328.
[CrossRef]
39. Carr, M.; Knox, A.J.; Lloyd, D.G.; Zisterer, D.M.; Meegan, M.J. Development of the
β-lactam type molecular
scaffold for selective estrogen receptor modulator action: Synthesis and cytotoxic effects in MCF-7 breast
α
cancer cells. J. Enzy. Inhib. Med. Chem. 2016, 31, 130–117. [CrossRef]
40. Ma, D.; Tremblay, P.; Mahngar, K.; Akbari-Asl, P.; Collins, J.; Hudlicky, T.; McNulty, J.; Pandey, S.
A novel synthetic C-1 analogue of 7-deoxypancratistatin induces apoptosis in p53 positive and negative
human colorectal cancer cells by targeting the mitochondria: Enhancement of activity by tamoxifen.
Investig. New Drugs 2012, 30, 1012–1027. [CrossRef]
41. Tsai, M.J.; O’Malley, B.W. Molecular mechanisms of action of steroid/thyroid receptor superfamily members.
Annu. Rev. Biochem. 1994, 63, 451–486. [CrossRef]
42. Ogawa, S.; Inoue, S.; Watanabe, T.; Hiroi, H.; Orimo, A.; Hosoi, T.; Ouchi, Y.; Muramatsu, M. The complete
primary structure of human estrogen receptor beta (hER beta) and its heterodimerization with ER alpha
in vivo and in vitro. Biochem. Biophys. Res. Commun. 1998, 243, 122–126. [CrossRef] [PubMed]
43. Filardo, E.J.; Quinn, J.A.; Bland, K.I.; Frackelton, A.R., Jr. Estrogen-induced activation of Erk-1 and Erk-2
requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal
growth factor receptor through release of HB-EGF. Mol. Endocrinol. 2000, 14, 1649–1660. [CrossRef] [PubMed]
44. Shiau, A.K.; Barstad, D.; Loria, P.M.; Cheng, L.; Kushner, P.J.; Agard, D.A.; Greene, G.L. The structural
basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell
1998, 95, 927–937. [CrossRef]
45. Chemical Computing Group. Molecular Operating Environment (MOE); ULC: Montreal, QC, Canada, 2020.
46. BIOVIA, Dassault Systèmes. Available online: http://accelrys.com/products/collaborative-science/biovia-
pipeline-pilot/ (accessed on 20 July 2020).
Sample Availability: Samples of the compounds are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).