Synthesis and characterization of new series of 1,3-5-Triazine hydrazone derivatives with promising antiproliferative activity

Article abstract of DOI:10.3390/molecules25112708

A new series of s-triazine hydrazone derivatives was prepared based on the reaction of 6-hydrazino-2,4-disubstituted-s-triazine with p-substituted benzaldehyde derivatives using a straightforward synthetic pathway. The antiproliferative activity of all synthesized compounds was evaluated against two human cancer cell lines; breast cancer MCF-7 and colon carcinoma HCT-116 using MTT assay. Among all, 11 compounds have shown strong to moderate antiproliferative activity with IC50 values in the range 1.01–18.20 μM in MCF-7 and 0.97–19.51 μM in HCT-116. The best results were obtained with 4,4’-(6-(2-(pyridin-2-ylmethylene)hydrazinyl)-1,3,5-triazine-2,4-diyl) dimorpholine 11 (IC50 = 1.0 μM and 0.98 μM in MCF-7 and HCT-116 cell lines, respectively). The substituents on the s-triazine core as well as the substituent at the benzylidene moiety have a great effect on the antiproliferative activity. Whereas compounds containing dimorpholino-striazine derivatives 8a–e showed more potent antiproliferative in MCF-7 compared to their analogs 7a–f (compounds containing two-piperidine rings), compounds containing one piperidine and one morpholine ring 9a–f showed better IC50 values in the range 10.4–22.2 μM. On the other hand, compounds containing two-piperidine rings 7a–f showed more potent antiproliferative in HCT-116 (IC50 values in the range 8.8–19.5 μM) than their analogs 8a–e and 9a–f.

Full text of DOI:10.3390/molecules25112708

molecules
Article
Synthesis and Characterization of New Series of
1,3-5-Triazine Hydrazone Derivatives with
Promising Antiproliferative Activity
Hessa H. Al Rasheed 1,*, Azizah M. Malebari 2, Kholood A. Dahlous 1 and Ayman El-Faham 1,3,
*
1
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451,
Saudi Arabia; kdahloos@KSU.EDU.SA
Department of Pharmaceutical Chemistry, College of Pharmacy, King Abdulaziz University, P.O. Box 80260,
Jeddah 21589, Saudi Arabia; amelibary@kau.edu.sa
Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia,
Alexandria 12321, Egypt
2
3
*
Correspondence: halbahli@ksu.edu.sa (H.H.A.R.); aelfaham@ksu.edu.sa or
aymanel_faham@hotmail.com (A.E.-F.); Tel.: +96-61-1467-3195 (A.E-F.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Margherita Brindisi
Received: 10 May 2020; Accepted: 5 June 2020; Published: 11 June 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A new series of s-triazine hydrazone derivatives was prepared based on the reaction
of 6-hydrazino-2,4-disubstituted-s-triazine with p-substituted benzaldehyde derivatives using
a straightforward synthetic pathway. The antiproliferative activity of all synthesized compounds was
evaluated against two human cancer cell lines; breast cancer MCF-7 and colon carcinoma HCT-116
using MTT assay. Among all, 11 compounds have shown strong to moderate antiproliferative activity
with IC50 values in the range 1.01–18.20
results were obtained with 4,4’-(6-(2-(pyridin-2-ylmethylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)
dimorpholine 11 (IC50 = 1.0 M and 0.98 M in MCF-7 and HCT-116 cell lines, respectively).
µM in MCF-7 and 0.97–19.51 µM in HCT-116. The best
µ
µ
The substituents on the s-triazine core as well as the substituent at the benzylidene moiety have
a great effect on the antiproliferative activity. Whereas compounds containing dimorpholino-s-triazine
derivatives 8a–e showed more potent antiproliferative in MCF-7 compared to their analogs 7a–f
(compounds containing two-piperidine rings), compounds containing one piperidine and one
morpholine ring 9a–f showed better IC50 values in the range 10.4–22.2 µM. On the other hand,
compounds containing two-piperidine rings 7a–f showed more potent antiproliferative in HCT-116
(IC50 values in the range 8.8–19.5 µM) than their analogs 8a–e and 9a–f.
Keywords: s-Triazine; hydrazone derivatives; antiproliferative activity; MCF-7; HCT-116
1. Introduction
s-Triazine (1,3,5-triazine) is a heterocyclic compound, which is quite stable and its derivatives
have wide practical applications in numerous fields. The most important reagent for obtaining these
derivatives is cyanuric chloride (2,4-6-trichloro-1,3,5-triazine). This compound has provoked significant
consideration due to its low cost, commercial availability, and considered as an ideal starting material
in stepwise nucleophilic substitution for the synthesis of symmetric and nonsymmetric s-triazine
derivatives at different temperatures [1–3].
The s-triazine scaffold affords the basis for the design and synthesis of various biologically
active compounds with widespread applications in medicinal chemistry [4–11]. On the other hand,
many researchers made notable progress on the design, synthesis, and evaluation of numerous
s-triazine derivatives with great and promising results for the further development of new antitumor
agents [12–20].
Molecules 2020, 25, 2708; doi:10.3390/molecules25112708
www.mdpi.com/journal/molecules

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Recently, a series of s-triazine hydrazino derivatives
I
(Figure 1) was reported as selective
inhibitors of the mammalian target of rapamycin (mTOR) [21]. The results showed that the phenolic
hydroxyl group has a critical effect, while its analogs methoxy derivative showed a dramatic loss in the
activity [21]. Later, another series of s-triazine hydrazone derivatives was reported with their particular
target inhibitors to both epidermal growth factor (EGFR) and mutant epidermal growth factor tyrosine
kinase receptors (EGFR TKs) [22]. Of these derivatives, the fluoro derivative II (Figure 1) showed
the most potent activity against epidermal growth factor receptor (EGFR). Moreover, it exhibited
considerable antiproliferative activity against A549, A431, and NCIH1975 cell lines.
Figure 1. Structure of the s-triazine hydrazone derivative with potent anticancer activity.
In a recent study [23], we reported the synthesis and evaluation of the antiproliferative activities of
several s-triazine hydrazone derivatives III (Figure 1). The results showed that the substituents on the
s-triazine ring especially, methoxy group and piperidine ring conferred greater selectivity for human
liver cancer cell lines (HepG2), while the presence of the morpholine and piperidine ring exhibited
greater selectivity for adenocarcinomic human alveolar basal epithelial cells (A549) with a reasonable
inhibitory effect on HepG2 cells [23].
Later, we reported another series of s-triazine hydrazone derivatives that encompasses s-triazine
and (2 or 4)-hydroxylbenzylidene derivatives IV and
V (Figure 1) with their anticancer activity [24].
The results revealed that the position of the hydroxyl group on the benzylidine ring as well as the
substituent on the s-triazine moiety have a great effect on the anticancer activity against breast cancer
cells (MCF-7) and colon cancer (HCT-116).
Based on the reported results by our group and others, s-triazine, morpholine, and piperidine
specifically serve as templates of a number of clinically used drugs [25], we report here the synthesis of
a small library of compounds bearing the s-triazine, morpholine and piperidine core with benzylidene
derivatives through the hydrazone linkage. The antiproliferative activity of this new series against
breast cancer MCF-7 and colon carcinoma HCT-116 cell lines were evaluated as well.
2. Results and Discussion
2.1. Chemistry
The target products 7a–f, 8a–e, 9a–f, and 10–12 were synthesized in three steps as follows.
As a first step, cyanuric chloride was reacted with different amines according to the reported
method [26] to afford 2-chloro-4,6-disubstituted-s-triazine derivatives in good yields and purities.

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The spectral data were in good agreement with the reported ones in literature [26]. As a second
step, 2-chloro-4,6-disubstituted-s-triazine derivatives reacted with hydrazine hydrate in ethanol
following the reported method [26,27] to afford the hydrazino derivatives 2–4 as a white
solid in good yields and purities which have been used directly into the next step. Finally,
6-hydrazino-2,4-disubstituted-s-triazine derivatives 2–4 were reacted with p-substituted benzaldehyde
derivatives 5a–f or 2-pyridinecarboxaldehyde
6
in the presence of the catalytic amount of acetic acid
8a–e 9a–f, and 10–12 (Scheme 1,
(AcOH) and ethanol as solvent to give the target products 7a–f
Table 1). The structure of the target products was confirmed by elemental analysis, FTIR, 1H-NMR,
and 13C-NMR spectra, (Supplementary Materials, Figures S1–S16).
,
,
Scheme 1. Synthetic route for preparation of the target products.
Table 1. Structure of compounds 7a–f, 8a–e, 9a–f, and 10–12 as shown in Scheme 1.
Cpd No.
R1
R2
X
Cpd No.
R1
R2
X
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
piperidine
piperidine
piperidine
piperidine
piperidine
piperidine
morpholine morpholine
morpholine morpholine
morpholine morpholine
morpholine morpholine
morpholine morpholine
piperidine
piperidine
piperidine
piperidine
piperidine
piperidine
H
Cl
Br
OH
F
OCH3
H
Cl
Br
OH
F
9a
9b
9c
9d
9e
9f
10
11
12
morpholine piperidine
morpholine piperidine
morpholine piperidine
morpholine piperidine
morpholine piperidine
morpholine piperidine OCH3
piperidine piperidine
morpholine morpholine
morpholine piperidine
H
Cl
Br
OH
F
2.2. Biology
2.2.1. Antiproliferative Activity
The antiproliferative activity of the synthesized s-triazine derivatives 7a–f, 8a–e, 9a–f, and 10–12
were studied in human breast cancer (MCF-7) and colon carcinoma (HCT-116). Most of the s-triazine
hydrazone derivatives affected the cell viability of the two cancer cell lines, as determined by the
MTT cell viability assay. The results revealed that the most effective compound was 11 (compound

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containing two morpholine rings on the s-triazine core and pyridine at the hydrazone terminal) which
affected the cell viability of the two cancer cell lines with IC50 values 1.0 M and 0.98 M in MCF-7
µ
µ
and HCT-116, respectively, Figure 2). Its analogs compound 10 (compound containing two-piperidine
rings) and 12 (compound containing piperidine and morpholine ring) showed less potent activities
as shown in Table 2. Therefore, the substituent on the s-triazine ring has a great effect on the activity
of the tested compounds. Compounds containing two morpholine rings 8a–e showed more potent
antiproliferative activity in MCF-7 with IC50 values in the range 13.4–29.3 µM compared to their
analogous containing two piperidine rings 7a–f (IC50 values in the range 11.5–39.9 µM).
100
MCF-7 cells (IC50=1.01 μM)
HCT-116 cells (IC50=0.97 μM)
50
0
-8
-7
-6
-5
-4
Log10 concentration (M)
Figure 2. Antiproliferative effect of compound 11 in MCF-7 and HCT-116 cell lines. Cells were grown
in 96-well plates and treated with compound 11 at 0.05–100 M for 72 h. Cell viability was expressed as
µ
a 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.
Table 2. Antiproliferative activities of compounds 7a–f, 8a–e, 9a–f, and 10–12 in MCF-7 and HCT-116
cell lines.
Compound No.
IC50 (µM) MCF-7
IC50 (µM) HCT-116
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
17.5 ± 5.9
23.5 ± 8.0
39.8 ± 12.9
18.2 ± 5.8
11.5 ± 3.3
39.9 ± 12.7
29.3 ± 9.3
17.6 ± 5.7
23.2 ± 7.3
14.0 ± 4.8
13.4 ± 4.0
22.2 ± 7.9
21.9 ± 6.0
22.5 ± 3.9
10.4 ± 3.1
13.9 ± 4.7
14.2 ± 4.5
32.8 ± 10.4
1.0 ± 0.3
12.6 ± 4.6
10.8 ± 3.5
10.9 ± 3.6
19.5 ± 6.2
14.0 ± 4.4
8.8 ± 2.5
>50
19.2 ± 6.2
38.7 ± 12.4
29.9 ± 9.5
18.3 ± 5.7
44.2 ± 14.4
30.0 ± 9.6
28.2 ± 9.0
25.4 ± 8.2
22.0 ± 7.1
23.4 ± 7.4
>50
10
11
12
0.98 ± 0.3
17.7 ± 5.5
30.4 ± 9.9
Compounds containing one morpholine and one piperidine ring 9a–f showed better IC50 values
in the range 10.4–22.2 M. These data are in good agreement with our previously reported related
s-triazine derivatives [24 27]. Interestingly, compounds containing two-piperidine rings 7a–f affected
the cell viability of the HCT-116 (IC50 values in the range 8.8–19.5 M) more than their analogs 8a–e
M) and 9a–f (IC50 values in the range 22–42.2 M) as shown
µ
,
µ
(IC50 values in the range 18.3 > 50
µ
µ
in Table 2.

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On the other hand, the substituent on the benzylidene moiety has also a great impact on the
antiproliferative activity. As shown in Table 2, p-fluoro benzylidene derivatives 7e 8e, and 9e
derivatives affected the cell viability of both cell lines in lower micromolar range compared to their
analogs containing p-chloro (7b 8b, and 9b) or p-bromo (7c 8c, and 9c) benzylidene derivatives (Table 2).
Thus, the presence of fluorine atoms significantly improves the pharmacological and physicochemical
properties as the metabolic stability, lipophilicity as well as ligand binding [28 29], these results also
agreed with the reported results by Bai et al. [22]. The replacement of fluorine atoms in compounds 7e
8e, and 9e with by hydroxy group as in compounds 7d 8d, and 9d showed also good antiproliferative
activities against MCF-7 cell line with IC50 = 18.2, 14, and 10.4 M, respectively (Table 2). These results
,
,
,
,
,
,
µ
agreed with the reported results which indicated that the phenolic hydroxyl group acts as a hydrogen
donor which improves the anticancer activity [21,24].
3. Experimental Section
3.1. Materials and Methods
All solvents and reagents purchased from commercial suppliers and used without further
1
purification. H NMR and 13C NMR spectra were recorded on a JEOL 400, 600 MHz spectrometer
(JEOL, Ltd., Tokyo, Japan). Elemental analyses performed on Perkin-Elmer 2400 elemental analyzer
(Perkin-Elmer, Inc.940 Winter Street, Waltham, MA, USA). Melting points were recorded on a Mel-Temp
apparatus Sigma-Aldrich (Chemie GmbH, 82,024 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).
3.2. General Method for the Synthesis of 1,3,5-Triazine-Hydrazone Derivatives
2-Hydrazino-4,6-disubstituted-1,3,5-triazine derivatives 2–4 were synthesized first following the
synthetic strategy and methods reported by our group and others [20
–24]. The target products were
synthesized as follows: Aldehyde derivatives 5a–f or (10 mmol) in ethanol (10 mL) were added
6
to a solution of 2–4 in ethanol (20 mL) containing 2–3 drops of acetic acid at room temperature.
After complete addition the reaction mixture refluxed for 4–6 h, and the progress of the reaction was
followed by thin-layer chromatography (TLC) using ethyl acetate–hexane 2:1. The reaction left to cool
down to room temperature and the solid product separated by filtration, washed with cooled ethanol,
and then dried at room temperature.
The characterization of compounds 8a–d studied herein previously reported by our group [30].
The characterization of the remaining compounds described below.
3.2.1. 2-(2-Benzylidenehydrazinyl)-4,6-di(piperidin-1-yl)-1,3,5-triazine, 7a
White solid in yield 82%; mp 240–242 ◦C; IR (KBr, cm−1): 3280 (NH), 1574 (C=N), 1504,1446 (C=C);
1H NMR (DMSO-d6):
δ
= 1.45 (s, 4H, 2CH2), 1.56 (s, 2H, CH2), 3.67 (s, 12H, 4 NCH2-, 2 OCH2
7.29–7.38 (m, 3H, 1H-3,4,5), 7.57 (d, 4H, J = 6.5 Hz, 2H-2,6), 8.02 (s, 1H, CH), 10.61 (s, 1H, NH)
ppm; 13C NMR (DMSO-d6):
= 24.4, 25.4, 43.4, 126.2, 128.7, 128.8, 135.2, 141.0, 164.2, 164.5 ppm.
−
),
δ
Anal. Calcd for C20H27N7 (365.48): C, 65.73; H, 7.45; N, 26.83. Found C, 65.87; H, 7.59; N, 26.98.
3.2.2. 2-(2-(4-Chlorobenzylidene)hydrazinyl)-4,6-di(piperidin-1-yl)-1,3,5-triazine, 7b
White solid in yield 94%; mp 254–256 ◦C; IR (KBr, cm−1): 3284 (NH), 1579 (C=N), 1505,1444 (C=C);
1H NMR (DMSO-d6):
δ
= 1.47 (s, 8H, 4CH2), 1.59 (s, 4H, 2CH2), 3.71 (s, 12H, 4 NCH2-, 2 OCH2-),
7.45(d, 2H, J = 8.4, Ar), 7.62 (d, 2H, J = 8.8, Ar), 8.03 (s, 1H, CH), 10.73 (s, 1H, NH) ppm; 13C NMR
(DMSO-d6): = 24.4, 25.4, 43.4, 127.8, 128.8, 133.1, 134.2, 139.6, 164.1, 164.5 ppm. Anal. Calc. for
C20H26ClN7 (399.92): C, 60.07; H, 6.55; N, 24.52. Found C, 60.23; H, 6.62; N, 24.71.
δ

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3.2.3. 2-(2-(4-Bromobenzylidene)hydrazinyl)-4,6-di(piperidin-1-yl)-1,3,5-triazine, 7c
White solid in yield 95%; mp 253–255 ◦C; IR (KBr, cm−1): 3284 (NH), 1578 (C=N), 1503,1445 (C=C);
1H NMR (DMSO-d6):
= 1.47 (s, 8H, 4CH2), 1.59 (s, 4H, 2CH2), 3.70 (s, 8H, 4CH2N), 7.54–7.60 (m,
4H, Ar), 8.02 (s, 1H, CH), 10.73 (s, 1H, NH) ppm; 13C NMR (DMSO-d6):
= 24.4, 25.4, 43.5, 121.9,
δ
δ
128.1, 131.7, 134.5, 139.8, 164.2, 164.5 ppm. Anal. Calc. for C20H26BrN7 (444.37): C, 54.06; H, 5.90; N,
22.06. Found C, 54.21; H, 5.98; N, 22.23.
3.2.4. 4-((2-(4,6-Di(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol, 7d
White solid in yield 97%; mp 150–152 ◦C; IR (KBr, cm−1): 3399(OH), 3145(NH), 1570(C=N), 1513,
1443(C=C);1H NMR (DMSO-d6):
δ
= 1.47 (s, 8H, 4CH2), 1.58 (s, 4H, 2CH2), 3.68 (s, 8H, 4CH2N),
6.78 (d, 2H, J = 8.0 Hz, Ar), 7.43 (d, 2H, J = 8.0 Hz, Ar), 7.94 (s, 1H, CH), 9.73 (s, 1H, OH), 10.40 (s, 1H,
NH) ppm; 13C NMR (DMSO-d6):
= 24.4, 25.5, 43.5, 115.6, 126.3, 127.9, 141.6, 158.4, 164.2, 164.6 ppm.
δ
Anal. Calc. for C20H27N7O (381.47): C, 62.97; H, 7.13; N, 25.70. Found C, 63.12; H, 7.02; N, 25.89.
3.2.5. 2-(2-(4-Fluorobenzylidene)hydrazinyl)-4,6-di(piperidin-1-yl)-1,3,5-triazine, 7e
White solid in yield 94%; mp 251–253 ◦C; IR (KBr, cm−1): 3222 (NH), 1564 (C=N), 1498, 1444 (C=C);
1H NMR (DMSO-d6):
δ
= 1.44 (s, 8H, 4CH2), 1.56 (s, 4H, 2CH2), 3.67 (s, 8H, 4CH2N), 7.20 (t, 2H,
J = 9.0 Hz Ar), 7.62 (t.d, 2H, J = 7.3, 3.0 Hz, Ar), 8.01 (s, 1H, CH), 10.61 (s, 1H, NH) ppm; 13C NMR
(DMSO-d6): = 24.6, 25.8, 44.0, 116.0, 128.5, 132.0, 140.2, 161.1, 164.2, 164.8 ppm. Anal. Calc. for
δ
C20H26FN7 (383.47): C, 62.64; H, 6.83; N, 25.57. Found C, 62.88; H, 6.98; N, 25.34.
3.2.6. 2-(2-(4-Methoxybenzylidene)hydrazinyl)-4,6-di(piperidin-1-yl)-1,3,5-triazine, 7f
White solid in yield 90%; mp 230–232 ◦C; IR (KBr, cm−1): 3286 (NH), 1578 (C=N), 1494,1448 (C=C);
1H NMR (DMSO-d6):
δ
= 1.47 (s, 8H, 4CH2), 1.59 (s, 4H, 2CH2), 3.69 (s, 8H, 4CH2N), 3.77 (s, 3H, OCH3),
6.96 (d, 2H, J = 8.8, Ar), 7.55 (d, 2H, J = 8.8, Ar), 7.99 (s, 1H, CH), 10.50 (s, 1H, NH) ppm; 13C NMR
(DMSO-d6): = 24.4, 25.5, 43.4, 55.2, 114.2, 127.7, 127.9, 141.0, 159.9, 164.1, 164.4 ppm. Anal. Calc. for
δ
C21H29N7O (395.50): C, 63.77; H, 7.39; N, 24.79. Found C, 63.91; H, 7.51; N, 24.96.
3.2.7. 4,4’-(6-(2-(4-Fluorobenzylidene)hydrazinyl)-1,3,5-triazine-2,4-diyl)dimorpholine, 8e
White solid in yield 88%; mp 213–215 ◦C; IR (KBr, cm−1): 3237 (NH), 1564 (C=N), 1520, 1441 (C=C);
1H NMR (DMSO-d6):
δ
= 3.54–3.74 (m, 16H, 4 N-CH2-CH2-O), 7.20–7.23(m, 2H, Ar), 7.63–7.67 (m,
2H, Ar), 8.10 (s, 1H, CH), 10.77 (s, 1H, NH) ppm; 13C NMR (DMSO-d6):
δ
= 43.2, 66.0, 115.6, 128.4,
132.1, 140.6, 161.5, 164.3, 164.8 ppm. Anal. Calc. for: C18H22FN7O2 (387.41): C, 55.80; H, 5.72; N,
25.31. Found C, 56.01; H, 5.89; N, 25.95.
3.2.8. 4-(4-(2-Benzylidenehydrazinyl)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)morpholine, 9a
White solid in yield 75%; mp 176–178 ◦C; IR (KBr, cm−1): 3224(NH), 1536(C=N),
1485,1441(C=C);1H NMR (DMSO-d6):
δ
= 1.44 (s, 4H, 2CH2), 1.56 (s, 2H, CH2), 3.53–3.67 (m, 12H,
4CH2N, 2 OCH2-), 7.28–7.38 (m, 3H, Ar), 7.59(d, 2H, J = 8.0, Ar), 8.03 (s, 1H, CH), 10.68 (s, 1H, NH) ppm;
13C NMR (DMSO-d6):
24.4, 25.4, 43.2, 43.5, 66.1, 126.3, 128.7, 128.9, 135.1, 141.3, 164.2, 164.9 ppm.
δ
Anal. Calc. for: C19H25N7O (367.45): C, 62.10; H, 6.86; N, 26.68. Found C, 62.29; H, 6.94; N, 26.79.
3.2.9. 4-(4-(2-(4-Chlorobenzylidene)hydrazinyl)-6-(piperidin-1-yl)-1,3,5-triazin-2- yl)morpholine, 9b
White solid in yield 84%; mp 176–178 ◦C; IR (KBr, cm−1): 3229(NH), 1560(C=N), 1515,1483(C=C);
1H NMR (DMSO-d6):
δ
= 1.44 (s, 4H, 2CH2), 1.56 (s, 2H, CH2), 3.52–3.66 (m, 12H, 4CH2N, 2 OCH2-),
7.42 (d, 2H, J = 6.8, Ar), 7.60 (d, 2H, J = 6.5, Ar), 8.0 (s, 1H, CH), 10.76 (s, 1H, NH) ppm; 13C NMR
(DMSO- d6): 24.4, 25.4, 43.2, 43.5, 66.0, 127.9, 128.8, 133.2, 134.1, 140.0, 164.2, 164.9 ppm. Anal. Calc.
for: C19H24ClN7O (401.89): C, 56.78; H, 6.02; N, 24.40. Found C, 56.97; H, 6.18; N, 24.65.
δ

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3.2.10. 4-(4-(2-(4-Bromobenzylidene)hydrazinyl)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)morpholine, 9c
White solid in yield 72%; mp 236–238 ◦C; IR (KBr, cm−1): 3270(NH), 1578(C=N), 1506,1483(C=C);
1H NMR (DMSO-d6):
δ
= 1.47 (s, 4H, 2CH2), 1.59 (s, 2H, CH2), 3.60–3.69 (m, 12H, 4CH2N, 2 OCH2-),
7.54–7.60 (m, 4H, Ar), 8.02 (s, 1H, CH), 10.79 (s, 1H, NH) ppm; 13C NMR (DMSO- d6):
δ
24.4, 25.4, 43.3,
43.5, 66.0, 121.9, 128.1, 131.5, 134.2, 140.1, 164.2, 164.8 ppm. Anal. Calc. for: C19H24BrN7O (446.34):
C, 51.13; H, 5.42; N, 21.97. Found C, 51.34; H, 5.58; N, 21.78.
3.2.11. 4-((2-(4-Morpholino-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol, 9d
◦
White solid in yield 76%; mp 156–158 C; IR (KBr, cm−1): 3399 (OH), 3145(NH), 1570(C=N),
1515,1442(C=C); 1H NMR (DMSO-d6):
= 1.50 (s, 4H, 2CH2), 1.62 (s, 2H, CH2), 3.59–3.71 (m, 12H,
4CH2N, 2 OCH2-), 6.81(d, 2H, J = 9.0, Ar), 7.47(d, 2H, J = 8.5, Ar), 7.99 (s, 1H, CH), 9.77 (s, 1H, OH),
10.51 (s, 1H, NH) ppm; 13C NMR (DMSO-d6): 24.4, 25.5, 43.2, 43.5, 66.1, 115.6, 126.2, 127.9, 141.9,
δ
δ
158.4, 164.1, 164.9 ppm; Anal. Calc. for: C19H25N7O2 (383.45): C, 59.51; H, 6.57; N, 25.57. Found C,
59.73; H, 6.71; N, 25.86.
3.2.12. 4-(4-(2-(4-Fluorobenzylidene)hydrazinyl)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)morpholine, 9e
White solid in yield 90%; mp 240–242 ◦C; IR (KBr, cm−1): 3219(NH), 1563(C=N), 1518,1440(C=C);
1H NMR (DMSO-d6):
δ
= 1.47 (s, 4H, 2CH2), 1.59 (s, 2H, CH2), 3.60–3.69 (m, 12H, 4CH2N, 2 OCH2-),
7.23(t, 2H, J = 8.8, Ar), 7.66 (t.d, 2H, J = 7.0, 3.2 Hz, Ar), 8.05 (s, 1H, CH), 10.72 (s, 1H, NH) ppm;
13C NMR (DMSO-d6):
24.4, 25.4, 43.2, 43.4, 66.0, 116.0, 128.2, 132.8, 140.2, 161.1, 164.1, 164.9 ppm;
δ
Anal. Calc. for: C19H24FN7O (385.44): C, 59.21; H, 6.28; 25.44. Found C, 59.43; H, 6.40; 25.69.
3.2.13. 4-(4-(2-(4-Methoxybenzylidene)hydrazinyl)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)morpholine, 9f
White solid in yield 76%; mp 175–177 ◦C; IR (KBr, cm−1): 3267(NH), 1556(C=N), 1516, 1486 (C=C);
1H NMR (DMSO-d6):
δ
= 1.44 (s, 4H, 2CH2), 1.56 (s, 2H, CH2), 3.54–3.66 (m, 12H, 4CH2N, 2 OCH2-),
3.74 (s, 3H, OCH3), 6.93(d, 2H, J = 9.5, Ar), 7.53(d, 2H, J = 9.5, Ar), 8.0 (s, 1H, CH), 10.54 (s, 1H, NH)
ppm; 13C NMR (DMSO-d6):
24.4, 25.4, 43.2, 43.4, 55.2, 66.1, 114.2, 127.7, 127.8, 141.3, 159.9, 164.3,
δ
164.9 ppm. Anal. Calc. for: C20H27N7O2 (397.47): C, 60.44; H, 6.85; N, 24.67. Found C, 60.68; H, 6.96;
N, 24.88.
3.2.14. 2,4-Di(piperidin-1-yl)-6-(2-(pyridin-2-ylmethylene)hydrazinyl)-1,3,5-triazine, 10
White solid in yield 75%; mp 196–198 ◦C; IR (KBr, cm−1): 3223 (NH), 1577(C=N), 1514, 1440 (C=C);
1H NMR (DMSO-d6):
δ
= 1.48 (s, 8H, 4CH2), 1.60 (s, 4H, 2CH2), 3.71 (s, 8H, 4CH2N), 7.29–7.32(m, 2H,
Ar), 7.80–7.88 (m, 2H, Ar), 8.10 (s, 1H, CH), 8.53 (d, 1H, J = 5.2 Hz, Ar), 10.90 (s, 1H, NH) ppm; 13C NMR
(DMSO-d6): = 24.4, 25.4, 43.5, 119.0, 123.1, 136.5, 141.5, 149.3,154.0, 164.1, 164.3 ppm; Anal. Calc. for
δ
C19H26N8 (366.46): C, 62.27; H, 7.15; N, 30.58. Found C, 62.45; H, 7.31; N, 30.81.
3.2.15. 4,4’-(6-(2-(Pyridin-2-ylmethylene)hydrazinyl)-1,3,5-triazine-2,4-diyl)dimorpholine, 11
White solid in yield 92%; mp 203–205 ◦C; IR (KBr, cm−1): 3219(NH), 1568(C=N), 1516, 1435(C=C);
1H NMR (DMSO-d6):
δ
= 3.53–3.67 (m, 16H, 4 N-CH2-CH2-O), 7.44–7.46(m, 2H, Ar), 7.64 (d, 2H,
J = 7.5 Hz, Ar), 8.00 (s, 1H, CH), 8.71 (d, 1H, J = 4.5 Hz, Ar), 11.00 (s, 1H, NH) ppm; 13C NMR
(DMSO-d6): = 43.2, 66.0, 123.9, 125.6, 133.6, 138.1, 148.2, 152.5, 164.5, 164.8 ppm; Anal. Calc. for
δ
C17H22N8O2 (370.41): C, 55.12; H, 5.99; N, 30.25. Found C, 55.36; H, 5.81; N, 30.04.
3.2.16. 4-(4-(Piperidin-1-yl)-6-(2-(pyridin-2-ylmethylene)hydrazinyl)-1,3,5-triazin-2-yl)morpholine, 12
White solid in yield 72%; mp 236–238 ◦C; IR (KBr, cm-1): 3223(NH), 1572(C=N), 1514,1439(C=C);
1H NMR (DMSO-d6):
δ
= 1.45(s, 4H, 2CH2), 1.57 (s, 2H, CH2), 3.54–3.67 (m, 12H, 4CH2N,
2 OCH2-),7.27–7.29 (m, 2H, Ar), 7.76–7.85 (m, 2H, Ar), 8.08 (s, 1H, CH), 8.50 (d, 1H, J = 5.2 Hz,
Ar), 10.94 (s, 1H, NH) ppm; 13C NMR (DMSO-d6):
24.3, 25.4, 43.2, 43.5, 66.0, 119.1, 123.3, 136.6, 141.9,
δ

Molecules 2020, 25, 2708
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149.2,154.0, 164.2, 164.9 ppm; Anal. Calc. for: C18H24N8O (368.44): C, 58.68; H, 6.57; N, 30.41. Found C,
58.91; H, 6.66; N, 30.20.
3.3. Biology
3.3.1. In Vitro Antiproliferative Assay
The target products 7a–f, 8a–e, 9a–f, 10–12 were evaluated for antiproliferative activity using
the MTT (Tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability
assay in human breast cancer (MCF-7) and colon carcinoma (HCT-116) cell lines. Cells were seeded in
triplicate in 96-well plates at a density of 10
DMSO used as vehicle control. Each well was treated with 2
pre-prepared as stock solutions in ethanol to furnish the concentration range of study, 10 nM to 100
and re-incubated for a further 72 h. The culture medium was then removed and the cells washed
×
103 cells/mL in a total volume of 200
L tested compounds, which had been
M,
µL per well. 0.1% of
µ
µ
with 100 µL phosphate-buffered saline (PBS) and 50 µL MTT added, to reach a final concentration
of 1 mg/mL MTT added. Cells incubated for 2 h in darkness at 37 ◦C. At this point, solubilization
was begun through the addition of 200 mL DMSO and the cells maintained at room temperature in
darkness for 20 min to ensure thorough color diffusion before reading the absorbance. Plates were
◦
incubated for 72 h at 37 C + 5% CO2. The MTT (5 mg/mL in PBS) was added and incubated for
another 4 h, the optical density was detected with a microplate reader at 570 nm. Results expressed
as percentage viability relative to vehicle control (100%). Dose–response curves plotted and IC50
values (concentration of drug resulting in 50% reduction in cell survival) were obtained using the
commercial software package Prism (GraphPad Software, Inc., La Jolla, CA, USA). All the experiments
were repeated in at least three independent experiments.
4. Conclusions
The s-triazine hydrazone derivatives reported herein were evaluated for their inhibitory effect of
the growth of two human cancer cell lines; breast cancer MCF-7 and colon cancer HCT-116 using MTT
assay. Compound 11 which containing two morpholine rings on the s-triazine core and pyridine at the
hydrazone terminal showed the greatest antiproliferative activity amongst all the tested compounds
(IC50 = 1.0
ring has a great effect on the activity of the tested compounds. This was observed with compound
11 vs. 10 (IC50 = 32.8 M in MCF-7 and >50 M in HCT-116) and 12 (IC50 = 17.7 M in MCF-7 and
30.4 M in HCT-116). This also was noticed in the series of compounds containing two morpholine
rings 8a–e which showed more potent antiproliferative activity in MCF-7 (IC50 values in the range
13.4–29.3 M) compared to their analogous containing two-piperidine rings 7a–f (IC50 values in the
range 11.5–39.9 M). While compounds containing one morpholine and one piperidine rings 9a–f
showed better IC50 values in the range 10.4–22.2 M, these results agreed with the reported in the
literature [24 27]. Interestingly, compounds containing two-piperidine rings 7a–f showed more potent
activity in HCT-116 cell line (IC50 values in the range 8.8–19.5 M) compared with their analogs 8a–e
µM and 0.98 µM in MCF-7 and HCT-116, respectively). The substituent on the s-triazine
µ
µ
µ
µ
µ
µ
µ
,
µ
(IC50 values in the range 18.3 > 50 µM) and 9a–f (IC50 values in the range 22–42.2 µM).
The substituent on the benzylidene moiety also affected the antiproliferative activity,
where derivatives with p-fluoro derivatives (7e
both tested cell lines (IC50 = 11.5, 13.4, 13.9 M in MCF-7 and 14, 18.3, 22
than their analogs p-chloro (7b 8b, and 9b) and p-bromo (7c 8c, and 9c) derivatives. Replacement of
,
8e, and 8e) exhibited more potent activities in
µ
µM in HCT-116, respectively)
,
,
the fluorine atom by hydroxy group showed better anticancer activities compared to its analogous
methoxy group.
Taken together, these results efforts on the synthesis of more new series based on s-triazine
hydrazone derivatives with different groups in progress in our lab, proving the antiproliferative activity
and possible mechanism of action of s-triazine hydrazone derivatives which might be of special interest
in medicinal chemistry.

Molecules 2020, 25, 2708
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Supplementary Materials: The following are available online, Figure S1: 1H-NMR and 13C-NMR for compound 7a,
Figure S2: 1H-NMR and 13C-NMR for compound 7b, Figure S3: 1H-NMR and 13C-NMR for compound 7c,
Figure S4: 1H-NMR and 13C-NMR for compound 7d, Figure S5: 1H-NMR and 13C-NMR for compound 7e,
Figure S6: 1H-NMR and 13C-NMR for compound 7f, Figure S7: 1H-NMR and 13C-NMR for compound 8e,
Figure S8: 1H-NMR and 13C-NMR for compound 9a, Figure S9: 1H-NMR and 13C-NMR for compound 9b,
Figure S10: 1H-NMR and 13C-NMR for compound 9c, Figure S11: 1H-NMR and 13C-NMR for compound 9d,
Figure S12: 1H-NMR and 13C-NMR for compound 9e, Figure S13: 1H-NMR and 13C-NMR for compound 9f,
Figure S14: 1H-NMR and 13C-NMR for compound 10, Figure S15: 1H-NMR and 13C-NMR for compound 11,
Figure S16: 1H-NMR and 13C-NMR for compound 12.
Author Contributions: The work designed and supervised by A.E.-F. Synthesis and characterization of the
reported compounds were carried out by H.H.A.R. and K.A.D. The biological activity was carried by A.M.M.
All authors discussed the results. The first drafts of the manuscript were prepared by H.H.A.R. and the final
version included contributions from all authors. All authors have read and agreed to the published version of
the manuscript.
Funding: Deanship of Scientific Research at King Saud University, Saudi Arabia, research group no. (RG-1441-365).
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).
Conflicts of Interest: The authors declare no conflicts of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).