Di- and tri-substituted s-triazine derivatives: Synthesis, characterization, anticancer activity in human breast-cancer cell lines, and developmental toxicity in zebrafish embryos

Article abstract of DOI:10.1016/j.bioorg.2019.103397

Here we report on a small library based on a 4-aminobenzonitile-s-triazine moiety. We used a straightforward orthogonal synthetic pathway to prepare di- and tri-substituted s-triazine derivatives, whose basic structure was modified. The newly synthesized compounds were fully characterized by ¹H NMR, ¹³C NMR and elemental analysis. They showed strong anticancer activity against two human breast cancer cell lines (MIDA-MB-231 and MCF-7), with IC₅₀ values less than 1 μM. These s-triazine compounds were generally more selective towards hormone receptor-positive breast cancer cell line MCF-7 than the triple negative MDA-MB-231 cell line. Zebrafish embryos were used to test the developmental toxicity of the target compounds in vivo. The phenotype of embryos treated with the derivatives resembled that of those treated with estrogen disruptors. This observation strongly supports the notion that that these compounds induce their anticancer activity in human breast cancer cells via targeting the estrogen and progesterone receptors.

Full text of DOI:10.1016/j.bioorg.2019.103397

Journal Pre-proofs
Di- and tri-substituted s-triazine derivatives: Synthesis, characterization, anti-
cancer activity in human breast-cancer cell lines and developmental toxicity in
zebrafish embryos
Ayman El-Faham, Muhammad Farooq, Zainab Almarhoon, Rakia Abd
Alhameed, Mohammad A.M. Wadaan, Beatriz G. de la Torre, Fernando
Albericio
PII:
S0045-2068(19)31245-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.103397
YBIOO 103397
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
31 July 2019
15 September 2019
22 October 2019
Please cite this article as: A. El-Faham, M. Farooq, Z. Almarhoon, R. Abd Alhameed, M.A.M. Wadaan, B.G. de
la Torre, F. Albericio, Di- and tri-substituted s-triazine derivatives: Synthesis, characterization, anticancer activity
in human breast-cancer cell lines and developmental toxicity in zebrafish embryos, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.103397
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Di- and tri-substituted s-triazine derivatives: Synthesis, characterization,
anticancer activity in human breast-cancer cell lines and developmental
toxicity in zebrafish embryos
Ayman El-Faham,a,b,* Muhammad Farooq, c Zainab Almarhoon,a Rakia Abd Alhameed,a
Mohammad A.M. Wadaan,c Beatriz G. de la Torre,d Fernando Albericioa,e,f,*
aDepartment of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
bDepartment of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Alexandria 21321, Egypt
cBioproducts Research Chair, College of Science, Department of Zoology, King Saud University, P.O.
Box 2455, Riyadh 11451, Saudi Arabia
dKRISP, College of Health Sciences, University of KwaZulu-Natal, Westville, Durban 4001, South Africa
eSchool of Chemistry and Physics, University of KwaZulu-Natal, University Road, Westville, Durban 4001, South
Africa
fCIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, and Department of Organic
Chemistry, University of Barcelona, Martí i Franqués 1-11, Barcelona 08028, Spain
Abstract: Here we report on a small library based on a 4-aminobenzonitile-s-triazine moiety. We
used a straightforward orthogonal synthetic pathway to prepare di- and tri-substituted s-triazine
derivatives, whose basic structure was modified. The newly synthesized compounds were fully
1
characterized by H-NMR, 13C-NMR and elemental analysis. They showed strong anticancer
activity against two human breast cancer cell lines (MIDA-MB-231 and MCF-7), with IC50 values
less than 1 µM. These s-triazine compounds were generally more selective towards hormone
receptor-positive breast cancer cell line MCF-7 than the triple negative MDA-MB-231 cell line.
Zebrafish embryos were used to test the developmental toxicity of the target compounds in vivo.
The phenotype of embryos treated with the derivatives resembled that of those treated with
estrogen disruptors. This observation strongly supports the notion that that these compounds
induce their anticancer activity in human breast cancer cells via targeting the estrogen and
progesterone receptors.
Keywords: 4-aminobenzonitile-s-triazine, di- trisubstituted s-triazine, anticancer activity,
zebrafish embryos
1

1. Introduction
Human diseases pose a major challenge for the pharmaceutical industry. In this context,
chemistry laboratories worldwide are channeling efforts not only into synthesizing new types of
molecules but also into modifying existing drugs in order to enhance activity and decrease toxicity.
1,3,5-Triazine (s-triazine) has drawn significant attention in organic chemistry due to its
ease of handling and low cost of the starting material (cyanuric chloride)[1-8]. The s-triazine
derivatives reported in the literature have been synthesized using methods that take advantage of
the differences in the reactivity of the three chlorines in cyanuric chloride under temperature
control [9, 10].
The 2,4,6-Trisubstituted s-triazine scaffold has played a key role in the medicinal chemistry
field. Indeed, a number of s-triazine-based compounds show valuable biological activities [11-15].
In this regard, s-triazine derivatives bearing a 4-minobenzonitrile moiety show enhanced
antimicrobial activity [16] and improved antitubercular properties over other chemical moieties,
in addition to exerting strong anticancer activity [17]. Recent studies have confirmed that several
s-triazine derivatives with a morpholine, piperidine and/or piperazine ring have significant
biologically activity against M. tuberculosis strain H37Rv [18]. In addition, our group has reported
that several trisubstituted s-triazine compounds show promising biological activity as anticancer
agents [19-21].
In the drug discovery phase, in addition to addressing the biological activity of a target
compound, attention should also be given to its potential toxicity. In this regard, the zebrafish has
emerged as a valuable model in which to evaluate the potential toxicity and biological activity of
new compounds [22]. The experiments conducted in early-stage zebrafish embryos (up to 120
hours post fertilization) do not require the approval of Institutional Animal Care and Use
Committees (IACUCs) [23]. Therefore, a large number of zebrafish embryos can be used for in-
depth statistical analysis, which would otherwise not be possible in other animal models due to
restrictions on the numbers used for experimental purposes.
The potential positive effects of s-triazine and the imperative need to identify new chemical
entities to feed drug discovery programs led us to use a straightforward orthogonal synthetic
pathway to synthesize a small library based on 4-aminobenzonitrile s-triazine. This synthetic
approach was used to prepare di- and tri-substituted s-triazine derivatives The newly synthesized
compounds were then tested in two types of human breast cancer cell lines, namely MIDA-MB-
2

231 and MCF-7. Furthermore, their developmental toxicity in vivo was also evaluated using
zebrafish embryos.
2. Results and discussion
2.1.
Chemistry
In the first step of the the synthesis, cyanuric chloride (TCT, 1) was reacted with 4-
aminobenzonitrile 2 at 0oC for 2 h using acetone-water medium to afford product 3 in excellent
yield and purity. Next, 3 was reacted with various amines using THF as solvent and K2CO3 as HCl
scavengers to afford product 4a-c in high yield and purity, as determined from their spectral data
(Scheme 1).
Cl
NH2
Cl
NC
N
N
Acetone-water
0 oC, 2 h
N
N
+
Cl
N
Cl
N
H
N
Cl
1
CN
3
MeOH
0 oC, 1 h
2
NaHCO3
RH/THF/K2CO3
rt 24 h
Cl
Cl
NC
N
N
N
N
N
N
R
Cl
N
5
OMe
H
4a-c
NH2
4a
4b
4c
R = piperidine;
R = morpholine;
RH/THF/K2CO3
rt 24 h
R = diethylamine;
CN
R
N
Cl
RH/THF/K2CO3
reflux 18 h
NC
NC
N
N
N
N
N
H
OMe
N
N
OMe
H
6
7a-i
7a,
7b, 7c
R = pyrrolidine;
R = piperidine;
R = morpholine;
7d,
7e,
7f
R = diethylamine;
R = N-methylpiperazine;
R = ethanolamine;
7g,
7h,
7i
R = 4-methoxyaniline;
R = aniline;
R = 4-bromoaniline;
Scheme 1. Synthesis of di-and tri-substituted 1,3,5-triazine derivatives
3

Cl
N
b
NC
a
2
N
N
3
6
4
b
N
N
5
1
a
H
Fig. 1. Structure of 4c
1
The H NMR spectrum for compound 4c (Figure 1, Supporting information Figure S3) as a
prototype showed two multiple peaks at  1.09–1.18 and 3.51–3.59 ppm, related to 2CH3 and 2CH2
for the ethyl residue, respectively, a doublet at  7.74 ppm, related to two aromatic protons (2Ha),
a doublet at  7.88 ppm, related to the other two aromatic protons (2Hb), and a broad singlet at
10.46 ppm, corresponding to the NH. The 13C NMR spectrum for 4c showed four peaks at  12.8,
12.9, 41.5,41.9, related to the diethylamino group (CH3, and CH2 , respectively) and absorption
peaks at  104.1(C4), 119.2 (CN), 119.7(C2,6), 133.1 (C3,5), 143.5(C1), 163.3 (C-Cl), 168.3 (C-
NH), and 175.5 (C-N) ppm.
In case of the methoxy derivatives 6 and 7a-i, and to assure a high yield and purity from
product 6, TCT 1 was reacted with methanol in the presence of NaHCO3 following the reported
method [24], affording product 5. Next, 5 was reacted with 4-aminobenzolnitrile 2 using the same
conditions as described above to afford product 6 in high yield and purity. Compound 6 was then
reacted with different amines in THF as solvent in the presence of K2CO3 as HCl scavengers under
refluxing conditions for 18 h to afford product 7a-i in high yield and purity. The spectral data and
elemental analyses confirmed all the structures of the prepared compounds.
N
N
b'
a'
b'
a'
b
3
6
a
NC
a
N
N
4
b
2
5
N
N
OMe
1
H
Fig. 2. Structure of 7d
1
The H NMR for compound 7d (Figure 2, Supporting information, Figure S6) as a prototype
showed a singlet at  2.19 ppm (NCH3), two broad singlets at  2.34 and 3.78 ppm, related the
eight protons corresponding to 4CH2 (Ha' and Hb', respectively) piperazine residue, a singlet at 
4

3.84 ppm (OCH3), two doublets at  7.70 and 7.90 ppm, related to four aromatic protons (Ha, and
Hb, respectively), and a singlet at  10.02 ppm, related to the NH. The 13C NMR spectrum for 7d
showed the expected peaks at  42.8(N-CH3), 45.7(Ca'), 54.8 (Cb'), 54.2 (OCH3), 103.3 (C-CN),
119.3 (CN), 119.4(C2,6), 132.9(C3,5), 144.2(C1), 165.2 (C-Cl), 170.6 (C-NH), and 175.5 (C-N)
ppm.
2.2.
Biology
2.2.1. The triazine derivatives markedly perturbed the proliferation of breast cancer cell lines
s-Triazine-based compounds show promising anticancer activity [16,17, 25-27]. Here we
performed some structural modifications in order to enhance this activity. Accordingly, the
compounds synthesized here were based on an active substituent 4-aminbenzonitrile as first
substituent, attached to s-triazine to generate two distinct series 4a-c and 7a-i (Scheme 1). To allow
comparison of the activity of different nucleophiles, such as methoxy, acyclic amine, aliphatic
and aromatic cyclic amine, in human breast cancer cells MCF-7 and MDA-MB-231, the type of
substituent in the two series was selected on the basis of the reported structure-reactivity
relationship [3,28,29] [30].
The anti-cancer activity of derivatives 4a-c and 7a-7i was tested in two human breast
cancer cell lines: the MDA-MB-231 cell line, a triple negative breast cancer— meaning it does
not express any kind of hormone receptors, and the MCF-7 cell line, which has estrogen and
progesterone receptors.
The results of the MTT cell proliferation assay indicated that these compounds inhibited
the proliferation of both cancer cell lines at minimum IC50 values. However, they showed much
stronger activity in MCF-7 than MDA-MB- 231 cells (Table 1 and Figures 3-6). The most
effective compounds were 7g and 7i, with IC50 values of 0.77 ± 0.01 and 0.1 ± 0.01 µM in the
MCF-7 cell line and 8.43 ± 0.01 and 14.28 ± 0.01 µM in the MDA-MB-231 cell line, respectively.
Moderate anticancer activity (IC50 values between 1 and 5 µM) was shown by 4c, 7b, and 7f,
which had IC50 values of 1.3 ± 0.01, 4.291 ± 0.02, and 3.71 ± 0.04 µM in the MCF-7 cell line, and
25.60 ± 0.00, 7.80 ± 0.03, and 6.49 ± 0.04 µM, respectively in the MDA-MB-231 cell line.
However, neither 7d or 7e showed activity in either cancer cell line.
A similar type of anticancer activity and selectivity towards MCF-7 cells has also been
described for s-triazine-bearing benzimidazole and benzothiazole derivatives. Those compounds,
5

showing IC50 values between 5–15 µM, impaired the proliferation of MCF7 cells [31]. In contrast,
the triazine compounds reported herein, with IC50 values of less than µM, showed greater
cytotoxicity towards MCF-7 cells.
Table 1. Cell proliferation assay of the triazine derivatives in two human breast cancer cell lines
IC50 values (µM)*
Compd.
3
MDA-MB-231
MCF-7
23.11 ± 0.01
6.84 ± 0.03
6
19.08 ± 0.01
7.26 ± 0.03
10.61 ± 0.01
25.60 ± 0.00
21.30 ± 0.00
7.80 ± 0.03
10.79 ± 0.01
Not Active
Not Active
6.49 ± 0.04
8.43 ± 0.01
Not Active
14.28 ± 0.01
20.41 ± 0.0
10.12 ± 0.03
11.02 ± 0.03
1.3 ± 0.01
13.38 ± 0.02
4.29 ± 0.02
14.92 ± 0.02
Not Active
Not Active
3.71 ± 0.04
0.77 ± 0.01
9.44 ± 0.05
0.1 ± 0.01
4a
4b
4c
7a
7b
7c
7d
7e
7f
7g
7h
7i
* These values are the mean of three replicates ± standard deviation.
6

Fig. 3. Line graph showing the effect of compound 3 on the survival of MCF-7 and MDA-MB-
231 human breast cancer cells
Fig. 4. Line graph showing the effect of compound 6 on the survival of MCF-7 and MDA-MB-
231 human breast cancer cells
7

Fig. 5. Line graph showing the effect of compounds 4a-c on the survival of MCF-7 and MDA-
MB-231 human breast cancer cells
8

Fig. 6. Line graph showing the effect of a compound from series 7a-i on the survival of MCF-7
and MDA-MB-231 human breast cancer cells
9

2.2.2. The s- triazine derivatives caused significant degree of developmental toxicity in zebrafish
embryos
For preliminary toxicity tests, the zebrafish is a powerful drug screening model due to its
rapid embryonic development, transparent embryos and feasibility of testing compounds in a small
volume (due to small size of embryo) [32-39].
The zebrafish embryo toxicity screening assays revealed that most of the triazine
derivatives were toxic. In this regard, at concentrations over 5 µM, all the derivatives caused 100%
lethality (Table 2 and 3). However, as in the cancer cell lines, 7e and 7d showed no activity in
these assays.
Compounds 3 and 6 showed a moderate level of toxicity in zebrafish embryos. The LD50
value (concentration at which half the treated embryos died) for 3 was 20 ± 0.25 µM, while for 6
it was 15± 0.20 µM. Zebrafish embryos treated with a sub-lethal concentration (below the LD50
values) of 3 showed developmental abnormalities. As shown in the photomicrograph in Table 2,
the embryos treated with 3 were smaller than control embryos, thereby indicating that the
compound impaired proliferation and development. Embryos treated with 6 showed cardiac edema
and hypertrophy at concentrations ≥ 3 µM, as shown in the photomicrograph in Table 2.
Compounds 7e and 7d showed no activity and were well tolerated by the zebrafish
embryos. Indeed, no mortalities or embryonic abnormalities were observed even at a concentration
of 40 µM.
The remaining compounds showed a range of teratogenic effects at sub-lethal
concentrations and the severity of the abnormalities directly correlated with the concentration of
the compounds used. The results of the zebrafish screening assays with compounds 4a-c and 7a-i
are summarized in Table 2 and 3, respectively. Almost all the compounds tested caused cardiac
hypertrophy (larger heart), as shown in the accompanying photomicrograph in Table 2 and 3.
The effect of the s-triazine derivatives was consistent with the effects of treating zebrafish
embryos with estrogen disruptors, such as bisphenol A [40,41]. The cardiac hypertrophy, cardiac
edema, and bending of the tail by s-triazine derivatives observed in this study also resemble the
phenotype of zebrafish embryo exposed to benomyl [42].
10

Table 2. The bioactivity of compounds 3, 4a-c and 6 in zebrafish embryos
Cl
NC
N
N
N
N
R
H
Comp
ID
R
Concentration *
Representative
micrograph of zebrafish
embryos
1 µM
3 µM
5 µM
Control
Control
3
Cl
Not Toxic
Not Toxic
Not Toxic
The bodies of treated
embryos were much
smaller than those of
controls and they showed
cardiac edema.
6
Not active
Not Active
Not toxic
OMe
The treated embryos had
cardiac edema and cardiac
hypertrophy
4a
Not Toxic
Toxic
Toxic
3 µM-treated embryos
N
100%
100%
mortality
mortality
The embryos treated with
0.5 µM had short bodies,
smaller heads, kinked tails,
cardiac hypertrophy and
severe developmental delay
11

4b
4c
Not Active
Not Active
Not Toxic
N
N
O
3
µM-treated embryos
showed cardiac edema and
cardiac hypertrophy
Same as 4a
Not Toxic
Toxic
100%
Toxic
100%
mortality
mortality
* Three biological replicates were done for each treatment, using at least 30 embryos in each
treatment
Table 3. The bioactivity of compounds 7a-i in zebrafish embryos
R
NC
N
N
N
N
OMe
H
Compd
ID
R
Concentration used *
PHENOTYPE
0.5 µM
1 µM
3 µM
Control
7a
-
-
-
Not toxic
Not toxic
Toxic
100%
mortality
N
3 µM-treated embryos
showed severe cardiac
hypertrophy and
cardiac edema
7b
Not toxic
Toxic
Toxic
3
µM-treated
O
N
embryos
100% mortality
100%
mortality
had short bodies,
smaller heads, kinked
tails and cardiac
hypertrophy
12

7c
Not toxic
Not toxic
Toxic
N
100%
mortality
Cardiac hypertrophy
7d
7e
Not active
Not active
Not active
Not active
Not active
Not active
N
N
OH
HN
7f
Not toxic
Not toxic
Toxic
100% mortality
Toxic
100%
mortality
Same as 7b
Same as 7b
N
7g
Toxic
Toxic
100% mortality
100%
mortality
HN
7h
7i
Not toxic
Not toxic
Not toxic
Toxic
100%
mortality
Same as 7a
Same as 7b
Br
HN
Toxic
Toxic
OMe
100% mortality
100%
mortality
HN
* Three biological replicates for each treatment were done, using at least 30 embryos in each
treatment
13

3. Conclusion
The newly synthesized s-triazine derivatives reported herein showed strong anticancer activity
against hormone receptor-positive human breast cancer cell line MCF-7 and triple receptor
negative breast cancer cell line MDA-MB-231. In this regard, compounds 7i and 7g had IC50
values of less than 1 µM. Moreover, these compounds showed selective cytotoxicity in MCF-7
cells. Most of the trisubstituted s-triazine derivative 7 series showed higher activity than the
disubstituted 4 series. While Compound 4c showed high selectivity for MCF-7 cells (IC50 1.3 ±
0.01). The combination between the methoxy group, 4-aminobenzonotrile and aniline ring in the
triazine core showed the highest activity, with 7g and 7i showing IC50 values of 0.77 ± 0.01 and
0.1 ± 0.01 µM for the MCF-7 cell line and 8.43 ± 0.01 and 14.28 ± 0.01 µM for the MDA-MB-
231 cell line, respectively. These two compounds have two aniline moieties, and it appears that
the presence of electron-donating substituents (OCH3) (7i) is relevant for the activity. Compound
7h with a bromoaniline moiety showed much less activity, presumably due to both the electron-
withdrawing effect and the hindrance of the Br. Compared with the other derivatives, which had
only one aniline, in addition to the 4-aminobenzonitrile group; IC50 values for 7b and 7f were 4.29
± 0.02 and 3.71 ± 0.04 µM in the MCF-7 cell line, respectively, and 7.80 ± 0.03 and 6.49 ± 0.04
µM, respectively in the MDA-MB-231 cell line. In contrast, 7d and 7e were completely inactive
in both cell lines. In addition the combination between the methoxy and the morpholino group
beside the 4-aminobenzonitrile derivative behaves in the same manner, where it showed higher
activity than the piperidino analogs, in agreement with previous reported data [20].
The zebrafish embryo screening assays revealed that compounds from series 4a-c and 7a-i induced
cardiac hypertrophy. The effect of the compounds reported herein correlate with published reports
of zebrafish embryos treated with estrogen disruptors such as bisphenol A [40,41]. The cardiac
hypertrophy, cardiac edema, and bending of the tail induced by s-triazine derivatives reported here
are also similar to the phenotype of zebrafish embryos exposed to benomyl [42].
Overall, given that 7i was the most active compound and showed toxicity only at 1 µM, thus having
a therapeutic window of 10, it deserves further attention as a potential HIT.
Finally, this work has demonstrated once again that the triazine core is a privileged structure that
should be considered in medicinal chemistry programs.
4. Experimental
14

4.1.
Chemistry
Material and Methods: All reagents and solvents were obtained from commercial suppliers and
1
13
used without further purification, H-NMR and C-NMR were recorded on a JEOL 400 MHz
spectrometer, the chemical shifts (δ) are referred in terms of ppm and J -coupling constants are
given in Hz. Elemental analysis was carried out on an Elmer 2400 Elemental Analyzer. All melting
points for the prepared compounds were measured on a Gallenkamp melting point apparatus in
open glass capillaries and are uncorrected.
4.1.1. Synthesis of 4-((4,6-dichloro-1,3,5-triazin-2-yl)amino)benzonitrile , 3 [25]
Cl
NC
N
N
N
N
Cl
H
A solution of 4-aminobenzonitrile 2 (1.84 g, 0.01 mol) was added portion-wise to a mixture of
cyanuric chloride 1 (1.85 g, 0.01 mol) and anhydrous potassium carbonate (K2CO3) (1.38 g, 0.01
mol) in 25 ml acetone at 0–5 oC over 15 min. After the addition, the reaction mixture was stirred
for 4 h at the same temperature. TLC using MeOH/Chloroform (2:8) was used to monitor the
progress of the reaction. After completion of the reaction, the reaction mixture was poured onto
crushed ice. The solid product obtained was filtered, washed with distilled water, dried, and
purified or used directly for the next step. White solid; Yield 92%; m.p. 288-290oC [Lit. [25] 300oC
1
(dec.)]; H-NMR (DMSO-d6):  11.50 (s, 1H, –NH proton of s-triazine to aminobenzonitrile
13
linkage), 7.86–7.78 (m, 4H, Ar–H aromatic proton); C-NMR (DMSO- d6):  106.84 (–CN of
aminobenzonitrile moiety), 141.99–119.32 (6C, Ar–C of aromatic), 164.37 (C–NH of s-triazine to
aminobenzonitrile linkage), 173.81 (C–Cl of s-triazine).
4.1.2. General method for the synthesis of 4-chloro-6-subsitited (1,3,5-triazin-2-
yl)aminobenzonitrile
A solution of the amine (10 mmole) in 50 mL THF was added dropwise to a solution of 4,6-
dichloro (1,3,5-triazin-2-yl)aminobenzonitrile (10 mmol) and K2CO3 (10 mmol) in 50 mL THF
over 5 min. The reaction mixture was stirred at rt for 24 h and then THF was concentrated under
vacuum and excess ice water was added. The product was obtained as a white solid. It was then
filtered, washed with water and dried. The crude product was recrystallized from ethylacetate.
4.1.2.1.
4-((4-Chloro-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)amino)benzonitrile, 4a
15

Cl
N
NC
N
N
N
N
H
White solid; Yield 85%; m.p. 164-165oC; 1H-NMR (DMSO-d6) : 1.53-1.62 (m, 6H, 3CH2), 3.69-
3.74 (m, 4H, 2CH2), 7.75(d, 2H, J = 8.8 Hz, Ar), 7.82 (d, 2H, J = 8.8 Hz, Ar), 10.44(br.s., 1H, NH)
13
ppm; C-NMR(DMSO-d6):  23.9, 25.3, 66.9, 104.2,119.2, 119.8, 133.1, 143.4, 163.4, 168.8,
175.5 ppm. Anal.Calc for C15H15ClN6 (314.78): C, 57.24; H, 11.26; N, 26.70. Found: C, 57.45;
H, 11.41; N, 26.86.
4.1.2.2.
4-((4-Chloro-6-morpholino-1,3,5-triazin-2-yl)amino)benzonitrile, 4b
Cl
NC
N
N
N
N
N
H
O
White solid; Yield 84%; m.p. 238-240oC; 1H-NMR (DMSO-d6) : 3.64 (t, 4H, J = 8.0 Hz, CH2-
N-CH2), 3.74 (t, 4H, J = 8.0 Hz, CH2-O-CH2), 7.75(d, 2H, J = 8.8 Hz, Ar), 7.84 (d, 2H, J = 8.8
Hz, Ar), 10.53(br.s., 1H, NH) ppm; 13C-NMR(DMSO-d6):  43.9, 65.7, 104.4 (CN), 119.1, 119.5,
120.1, 132.9, 133.3, 143.2, 163.5, 163.9, 168.6, 175.5 ppm. Anal.Calc for C14H13ClN6O (316.75):
C, 53.09; H, 11.19; N, 26.53. Found: C, 53.25; H, 11.33; N, 26.87.
4.1.2.3.
4-((4-Chloro-6-(diethylamino)-1,3,5-triazin-2-yl)amino)benzonitrile, 4c
Cl
NC
N
N
N
N
N
H
White solid;Yield 88%; m.p. 142-144 oC; 1H-NMR (DMSO-d6) : 1.09-1.18 (m 6H, 2CH3), 3.51-
3.59 (m, 4H, 2CH2), 7.74(d, 2H, J = 8.8 Hz, Ar), 7.88 (d, 2H, J = 8.8 Hz, Ar), 10.46(s, 1H, NH)
ppm; 13C-NMR(DMSO-d6):  12.8, 12.9,41.5,41.9, 104.1, 119.2, 119.7, 133.1, 143.5, 163.3,
168.3, 175.5 ppm. Anal.Calc for C14H15ClN6 (302.77): C, 55.54; H, 4.99; N, 27.76. Found: C,
55.78; H, 5.13; N, 27.99.
4.1.2.4.
Synthesis of 4-chloro-6-methoxy(1,3,5-triazin-2-yl)amino)benzonitrile, 6
16

Cl
N
NC
N
N
N
OMe
H
A solution of 4-aminobenzonitrile (10 mmole) in 50 mL acetone was added dropwise, as described
previously [32], to an ice-cold solution of 2,4-dichloro-6-methoxy-1,3,5-triazine 5 (10 mmol) and
NaHCO3 (10 mmol) in 50 mL acetone over 10 min. The reaction mixture was stirred at rt for 24 h
and then acetone was concentrated under vacuum, and excess ice water was added. The solid
product obtained was filtered, washed with water, and dried to afford the product. White solid;
o
1
Yield 87%; m.p. 226-227 C; H-NMR (CDCl3) : 4.06 (s, 3H, OCH3), 7.49 (br.s., 1H, NH),
7.64(d, 2H, J = 8.8 Hz, Ar), 7.72 (d, 2H, J = 8.8 Hz, Ar) ppm; 13C-NMR (CDCl3) : 52.1 (OMe),
102.2 (CN), 120.3, 133.4, 163.5, 172.3 ppm. Anal.Calc for C11H8ClN5O (261.67): C, 50.49; H,
3.08; N, 26.76. Found: C, 50.66; H, 3.19; N, 26.92.
4.1.3. General method for the synthesis of 4-substituted-6-methoxy((1,3,5-triazin-2-yl)
aminobenzonitrile
A solution of 4-amine (10 mmole) in 50 mL acetonitrile was added dropwise to a solution of 4-
chloro-6-methoxy(1,3,5-triazin-2-yl)amino)benzonitrile (10 mmol) and K2CO3 (10 mmol) in 50
mL acetonitrile, and the reaction mixture was stirred under reflux 18 h. Acetonitrile was then
concentrated under vacuum, and excess ice water was added. The product was obtained as white
solid. It was filtered, washed with water and dried. The crude product was recrystallized from
ethylacetate-ethanol (3:1)
4.1.3.1.
4-((4-Methoxy-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)amino)benzonitrile, 7a
N
NC
N
N
N
N
OMe
H
White solid; Yield 82%; m.p. 213-214 oC; 1H-NMR (CDCl3) : 1.59 (br.s., 4H, 2CH2), 1.67 (br.s.,
2H, CH2), 3.78 (br.s. 4H, 2CH2), 3.92(s, 3H, OCH3), 7.57(d, 2H, J = 8.8 Hz, Ar), 7.72 (d, 2H, J =
13
8.8 Hz, Ar), 7.82(br.s., 1H, NH) ppm; C-NMR (CDCl3):  24., 25.7, 44.9, 54.3, 105.2, 119.2,
119.5, 133.0, 143.1, 164.3, 164.9, 170.4 ppm. Anal.Calc for C16H18N6O (310.36): C, 61.92; H,
5.85; N, 27.08. Found: C, 62.15; H, 5.72; N, 27.29.
4.1.3.2.
4-((4-Methoxy-6-morpholino-1,3,5-triazin-2-yl)amino)benzonitrile, 7b
17

O
N
NC
N
N
N
N
OMe
H
White solid; Yield 84%; m.p. 216-218 oC; 1H-NMR (CDCl3) : 3.73(br.s., 4H, 2CH2), 3.83(br.s.,
4H, 2CH2), 3.94 (s. 3H, OCH3), 7.56(d, 2H, J = 8.8 Hz, Ar), 7.70 (d, 2H, J = 8.4 Hz, Ar), 10.22(s.,
13
1H, NH) ppm; C-NMR (CDCl3):  44.5, 54.2, 66.6, 105.3, 119.3, 119.6, 122.3, 133.1, 142.7,
164.3, 165.6, 170.4, ppm. Anal.Calc for C15H16N6O2 (312.33): C, 57.68; H, 5.16; N, 26.91. Found:
C, 57.76; H, 5.33; N, 27.13.
4.1.3.3.
4-((4-Methoxy-6-(pyrrolidin-1-yl)-1,3,5-triazin-2-yl)amino)benzonitrile, 7c
N
NC
N
N
N
N
OMe
H
o
1
White solid; Yield 86%; m.p. 223-25 C; H NMR (CDCl3) : 1.96-2.02 (m, 4H, 2CH2), 3.60-
3.64(m, 2H, CH2), 3.95 (s. 4H, OCH3), 7.57(d, J = 8.8, 2H, Ar), 7.76 (d, 2H, J = 8.8 Hz, Ar),
8.67(br.s., 1H, NH) ppm; 13C-NMR (CDCl3):  24.9, 25.0, 46.5, 46.7, 54.2, 105.2, 119.1, 119.4,
132.8, 143.1, 164.3, 164.9, 170.4 ppm. Anal.Calc for C15H16N6O (296.33): C, 60.80; H, 5.44; N,
28.36. Found: C, 60.98; H, 5.65; N, 28.59.
4.1.3.4.
4-((4-Methoxy-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yl)amino)benzonitrile,
7d
N
N
NC
N
N
N
N
OMe
H
o
1
White solid; Yield 87%; m.p. 186-187 C; H-NMR (CDCl3) : 2.19 (s, 3H, NCH3), 2.34 (br.s.,
4H, 2CH2), 3.78 (br.s. 4H, 2CH2), 3.84(s, 3H, OCH3), 7.70(d, 2H, J = 8.8 Hz, Ar), 7.90 (d, 2H, J
13
= 8.8 Hz, Ar), 10.02(s, 1H, NH) ppm; C-NMR (CDCl3):  42.8, 45.7, 54.8,54.2, 103.3, 119.3,
119.4, 132.9, 144.2, 165.0, 165.2, 170.6,175.5 ppm. Anal.Calc for C16H19N7O (325.38): C, 59.06;
H, 5.89; N, 30.13. Found: C, 59.33; H, 6.03; N, 30.43.
18

4.1.3.5.
4-((4-((2-Hydroxyethyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzonitrile,
7e
OH
HN
NC
N
N
N
N
OMe
H
White solid;Yield 80%; m.p. 190-192 oC; 1H-NMR (CDCl3) : 3.37 (m, 2H,), 3.52 (m, 2H, CH2),
3.78 (s, 3H, OCH3), 4.71(m,1H, OH), 7.58-7.72(m, 3H, NH, Ar), 7.99(dd, 2H, J = 8.0, 8.8 Hz,
13
Ar), 9.99 (s, 1H, NH) ppm; C-NMR (CDCl3):  42.9, 53.7, 59.5, 103.1, 119.4, 132.9, 144.4,
164.7, 166.7, 170.4, 175.5 ppm. Anal. Calc for C13H14N6O2 (286.30): C, 54.54; H, 4.93; N, 29.36.
Found: C, 54.79; H, 4.86; N, 29.58.
4.1.3.6.
4-((4-(Diethylamino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzonitrile, 7f
N
NC
N
N
N
N
OMe
H
1
White solid; Yield 89%; m.p. 182-183 oC; H-NMR (CDCl3) : 1.17-1.24(m, 6H, 2CH3), 3.57-
3.64(m, 4H, 2CH2), 3.92 (s, 3H, OCH3), 7.56(d, 2H, J = 6.8 Hz, Ar), 7.70 (d, 2H, J = 6.8 Hz, Ar),
13
7.82(s., 1H, NH) ppm; C-NMR (CDCl3):  13.1, 41.2, 54.2, 66.6, 105.2, 119.3, 132.9, 143.2,
164.1, 165.0, 170.2, ppm. Anal. Calc for C15H18N6O (298.35): C, 60.39; H, 6.08; N, 28.17.
4.1.3.7.
4-((4-Methoxy-6-(phenylamino)-1,3,5-triazin-2-yl)amino)benzonitrile, 7g
HN
NC
N
N
N
N
OMe
H
White solid; Yield 83%; m.p. 256-258 oC; 1H-NMR (DMSO-d6) : 3.91 (s. 3H, OCH3), 7.01(m,
1H, Ar), 7.31-7.37(m, 3H, Ar), 7.72(d, 4H, J = 8.8 Hz, Ar), 7.99 (br.s., 2H, Ar), 9.85 (s, 1H, NH)
13
10.15(s, 1H, NH) ppm; C-NMR (DMSO-d6):  54.2, 103.6, 119.3, 119.7, 120.2, 120.8, 122.9,
128.5, 128.8, 132.9, 139.1, 144.1, 165.2, 170.7, 175.5 ppm. Anal.Calc for C17H14N6O (318.34): C,
64.14; H, 4.43; N, 26.40. Found: C, 64.37; H, 4.66; N, 26.69.
19

4.1.3.8.
4-((4-((4-Bromophenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzonitrile,
7h
Br
HN
NC
N
N
N
N
OMe
H
o
1
White solid; Yield 81%; m.p. 258-260 C; H-NMR (DMSO-d6) : 3.90 (s. 3H, OCH3), 7.50(d,
2H, J = 8.4 Hz, Ar), 7.73 (d, 2H, J = 8.4 Hz, Ar), 7.87-7.96 (m, 4H, AR), 10.0 (s., 1H, NH) 10.22(s.,
1H, NH) ppm; 13C-NMR (DMSO-d6):  54.2, 103.8, 105.2, 114.5,119.4, 119.9, 122.3, 131.4,
133.2, 142.5, 143.9, 165.0, 165.0, 170.4, 175.5 ppm. Anal.Calc for C17H13BrN6O (397.24): C,
51.40; H, 3.30; N, 21.16. Found: C, 51.67; H, 3.43; N, 21.39.
4.1.3.9.
4-((4-Methoxy-6-((4-methoxyphenyl)amino)-1,3,5-triazin-2-yl)amino)
benzonitrile, 7i
OMe
HN
NC
N
N
N
N
OMe
H
White solid; Yield 86%; m.p. 206-208oC; 1H-NMR (DMSO-d6) : 3.73(s, 3H, OCH3), 3.91 (s. 3H,
OCH3), 6.89(d, 2H, J = 8.8 Hz, Ar), 7.57(br.s., 2H, Ar), 7.72(d, 2H, J = 8.8 Hz, Ar), 7.92(br.s.,2H,
Ar), 9.70 (s, 1H, NH) 10.10(s, 1H, NH) ppm; 13C-NMR (DMSO-d6):  54.1, 55.2, 103.5, 113.7,
119.4, 119.6, 131.9, 132.9, 144.2, 155.3, 165.1, 170.6, 175.5 ppm. Anal. Calc for C18H16N6O2
(348.37): C, 62.06; H, 4.63; N, 24.12. Found: C, 62.30; H, 4.77; N, 24.37.
4.2. Biology
4.2.1. Zebrafish embryo treatment.
The zebrafish embryos were obtained by natural pair wise breeding and treated essentially as
described previously [43]. The compounds were dissolved in dimethyl sulfoxide (DMSO) to
prepare a stock concentration of 10 mM. Embryos treated with 0.10% DMSO (v/v) served as
negative controls.
20

4.2.2. Imaging and microscopy.
A Zeiss Observer D1 inverted microscope was used to capture images of live zebrafish
embryos using Zeiss ZEN software.
4.2.3. MTT cell proliferation assays
Human breast adenocarcinoma cells MCF-7 (ATCC® HTB-22™) and MDA-MB-231 (ATCC®
HTB-26™) were from the American Type Culture Collection (American Type Culture Collection
(ATCC) Manassas, VA 20108 USA). The cells were maintained in Dulbecco's Modified Eagle's
Medium DMEM (11965-092 Thermo Fisher supplemented with 10% FCS (Cambrex Bio Science),
100 IU/mL penicillin, 100 mg/mL streptomycin and 2 mmol/L L-glutamine (Sigm-Aldrich). The
cells were supplemented with 10% fetal bovine serum (Lonza) and 1% ABM (GIBCO) and
maintained at37˚C and 5% CO2 in a humidified cell culture incubator.
4.2.4. Cell culture and cell viability assay
The MCF-7 and MDA-MB-231 lineswere grown in 48-well culture plates. They were
allowed to adhere to the plate for 6 h and allowed to grow for 24 h. The cells were treated with
serial dilution of each compound (in triplicate) and treated for 24 h. Cytotoxicity was tested using
a colorimetric MTT cell viability assay, which was performed essentially as described previously
[44].
4.2.5. Statistics
The zebrafish screening assays were done in three biological replications using embryos
from different batches of adult fish each time. Replication was done with at least 30 embryos. The
cell proliferation assay in human breast cancer cell lines was done at five concentrations of the
compounds (0.5, 1, 2, 4, and 10 µM) and triplicate 48-well culture plates. Origin pro 8.5 was used
to calculate the IC50 values and standard deviation in the MTT assay.
Acknowledgements
The work was funded in part by the following: the Deanship of Scientific Research at King Saud
University Research Group no. RGP-234, Saudi Arabia; the National Research Foundation
(NRF) (Blue Sky’s Research Programme # 110960) and the University of KwaZulu-Natal (South
Africa); and the Spanish Ministry of Science, Innovation, and Universities (CTQ2015-67870-P)
and the Generalitat de Catalunya (2017 SGR 1439; Spain).
21

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24

Graphical Abstract
Di- and tri-substituted s-triazine derivatives: Synthesis,characterization anticancer activity
in human breast-cancer cell lines and developmental toxicity in zebrafish embryos
Ayman El-Faham*, Muhammad Farooq , Zainab Almarhoon, Rakia Abd Alhameed, Mohammad
A.M. Wadaan, Beatriz G. de la Torre, Fernando Albericio *
Cl
R
Cl
NC
NC
NC
N
N
N
N
N
N
N
N
OMe
N
N
OMe
N
N
Cl
H
H
H
Cl
NC
N
N
N
N
R
H
Small library based on 4-aminobenzonitile-s-triazine moiety has been synthesized and fully
1
13
characterized by H-NMR, C-NMR and elemental analysis. The reported s-triazine derivatives
had shown strong anticancer activity against two of human breast cancer cell lines (MCF-7 and
MIDA-MB-231) with IC50 values less than 1 µM. General trend that these compounds were more
selective towards hormone receptor positive breast cancer cell line MCF-7 as compared to MDA-
MB-231 cell line. Zebrafish embryos were used to evaluate in vivo and developmental toxicities
of the target compounds.
25

Highlights
 Di-and trisubstituted-s-triazine based on 4-aminobenzonitrile moiety.
 Anticancer profile.

Human breast carcinoma (MCF 7 and MDA-MB-231)
 In vivo toxicity in zebrafish embryos.
26