Design and synthesis of mono-and di-pyrazolyl-s-triazine derivatives, their anticancer profile in human cancer cell lines, and in vivo toxicity in zebrafish embryos

Article abstract of DOI:10.1016/j.bioorg.2019.03.063

s-Triazine is considered a privileged structure, as it is found in several FDA-approved drugs. In the framework of our ongoing medicinal chemistry project based on the use of s-triazine as a scaffold, we synthesized a series of mono- and di-pyrazolyl-s-triazine derivatives and tested them against four human cancer cell lines, namely Human breast carcinoma (MCF 7 and MDA-MB-231), hepatocellular carcinoma (HepG2), colorectal carcinoma (LoVo), and leukemia (K562). The cell viability assay revealed that most of the s-triazine compounds induced cytotoxicity in all four types of human cancer cell lines, however, compounds 4a, and 6g, both of them have a piperidine moiety in their structure were most effective. These two compounds affected the cell viability of cancer cells, with IC₅₀ values within the range between 5 to 9 μM. The cell cycle analysis showed that 4a and 6g induced S and G2/M phase cell cycle arrest in K562 cells. This could be the mechanism by which these molecules induced cytotoxicity in tested cancer cells. The prepared compounds were tested in zebrafish embryos to evaluate in vivo and developmental toxicity of the pyrazolyl-s-triazine derivatives in animals. None of the derivatives were lethal in the concentration range tested.

Full text of DOI:10.1016/j.bioorg.2019.03.063

BioorganicChemistry87(2019)457–464
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of mono-and di-pyrazolyl-s-triazine derivatives, their
anticancer profile in human cancer cell lines, and in vivo toxicity in zebrafish
embryos
Muhammad Farooqa, Anamika Sharmab, Zainab Almarhoonc, Abudalla Al-Dhfyand,e
,
⁎
Ayman El-Fahamc,f, , Nael Abu Tahaa, Mohammad A.M. Wadaana, Beatriz G. de la Torreb,g
Fernando Albericiob,c,h,i,j
,
a Bioproducts Research Chair, College of Science, Department of Zoology, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
b Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, University Road, Westville, Durban 4001, South Africa
c Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
d Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, P. O. Box 2457, Riyadh, Saudi Arabia
e Stem Cell & Tissue Re-Engineering, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia
f Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Alexandria 21321, Egypt
g KRISP, College of Health Sciences, University of KwaZulu-Natal, Westville, Durban 4001, South Africa
h School of Chemistry and Physics, University of KwaZulu-Natal, University Road, Westville, Durban 4001, South Africa
i Department of Organic Chemistry, University of Barcelona, Martí i Franqués 1-11, Barcelona 08028, Spain
j CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, Barcelona 08028, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
s-Triazine is considered a privileged structure, as it is found in several FDA-approved drugs. In the framework of
our ongoing medicinal chemistry project based on the use of s-triazine as a scaffold, we synthesized a series of
mono- and di-pyrazolyl-s-triazine derivatives and tested them against four human cancer cell lines, namely
Human breast carcinoma (MCF 7 and MDA-MB-231), hepatocellular carcinoma (HepG2), colorectal carcinoma
(LoVo), and leukemia (K562). The cell viability assay revealed that most of the s-triazine compounds induced
cytotoxicity in all four types of human cancer cell lines, however, compounds 4a, and 6g, both of them have a
piperidine moiety in their structure were most effective. These two compounds affected the cell viability of
cancer cells, with IC50 values within the range between 5 to 9 µM. The cell cycle analysis showed that 4a and 6g
induced S and G2/M phase cell cycle arrest in K562 cells. This could be the mechanism by which these molecules
induced cytotoxicity in tested cancer cells. The prepared compounds were tested in zebrafish embryos to
evaluate in vivo and developmental toxicity of the pyrazolyl-s-triazine derivatives in animals. None of the de-
rivatives were lethal in the concentration range tested.
s-Triazine
Pyrazole
Anticancer activity
Zebrafish embryos
1. Introduction
positive acute myeloid leukemia in 2017 [2].
2,4,6-Tris-1-aziridinyl-s-triazine (Tetramine, Fig. 1) was the first s-
The search for new agents with therapeutic efficiency is one of the
major concern in medicinal chemistry. However, the potential devel-
opment of resistance or tolerance to these compounds over time, par-
ticularly in the context of the treatment of recurrent diseases such as
cancer, seriously limits their clinical use [1].
triazine derivative to demonstrate anticancer activity, which can be
easily synthesized from 2,4,6-trichlorotriazine (TCT, Fig. 1) [3]. In this
regard, the broad availability of TCT, as well as its high reactivity, has
led many anticancer medicinal chemistry programs to replace the Cl
(aziridinio in tetramine) by (alkyl) amino, alkoxy, aryl or sulfonamide
[4].
1,3,5-Triazine (s-triazine) can be considered a privileged structure
since several drugs approved by the corresponding agencies are based
on its structure. An example of such a drug is Enasidenib (Idhifa)
(Fig. 1), which was approved by the FDA for the treatment of the IDH2-
On the other hand, the attachment of pyrazolyl rings to the s-tria-
zine moiety has been extensively studied and reported to exert antic-
ancer activity [5]. For example, Brzozowski and F. Sączewski [6]
⁎ Corresponding author.
E-mail address: aelfaham@ksu.edu.sa (A. El-Faham).
https://doi.org/10.1016/j.bioorg.2019.03.063
Received 29 January 2019; Received in revised form 16 March 2019; Accepted 23 March 2019
Availableonline25March2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

M. Farooq, et al.
BioorganicChemistry87(2019)457–464
Fig. 1. Selected triazine derivatives.
reported the syntheses and antitumor activities of 4-(3,5,5-trimethyl-2-
pyrazolino)-1,3,5-triazine derivatives I and II against a panel of 60
tumor cell lines at the National Cancer Institute (NCI). More recently,
Insuasty and co-workers have reported a family of 4,6-bis(2-hydro-
xyethyl)amino)-1,3,5-triazin-2-yl derivatives III with a good anticancer
profile (Fig. 1) [5e].
methylbenzylamine or methoxy) at 0–5 °C to afford the products 2a-e
(2,4-dichloro-6-substituted-s-triazine derivatives). The second chlorine
atom was replaced by a second amine at room temperature for 24 h,
yielding 2-chloro-4,6-disubstitued-s-triazine derivatives. The di-chloro
and mono-chloro derivatives were then treated with hydrazine hydrate
(80%) in ethanol for 2 h under ultrasonic irradiation (US) [10] to afford
the hydrazine derivatives 3a-e and 5a-g, respectively. Compounds 3a-e
and 5a-g were then reacted with acetylacetone following the reported
methods [7d,9] to render the target products 4a-e and 6a-g, respec-
tively, as shown in Scheme 1.
In the framework of our ongoing medicinal chemistry project based
on the use of s-triazine as a scaffold [7] for anticancer drug develop-
ment, series of mono- and di-pyrazolyl-s-triazine derivatives were syn-
thesized and tested against four types of common human cancer cells,
namely breast carcinoma (MCF-7 and MDA-MB-231 cells), liver carci-
noma (HepG2 cells), colorectal carcinoma (LoVo cells) and leukemia
(K562 cells). The cell cycle analysis of pyrazolyl-s-triazine derivatives
treated K562 cells was carried out to determine the potential me-
chanism of action of these compounds in human cancer cells.
To assess the potential in vivo toxicity of synthetic compounds which
are intended to be use for the treatment of humans or animals, they
must first be tested in a suitable animal model prior to clinical trials.
Similarly, the developmental toxicity of the compounds must also be
tested to evaluate any harmful effect on fetal growth. In this context,
Zebrafish provide an ideal in vivo system which not only serve to
evaluate the in vivo toxicity of the synthesized compounds but also
helpful to predict the developmental toxicity [8]. In this regard, pyr-
azolyl-s-triazine derivatives were screened using zebrafish embryos to
evaluate their potential in vivo and developmental toxicity.
2.2. Triazine compounds induced cytotoxicity in human cancer cells
The anticancer activity of the synthesized triazine derivatives
(Fig. 2) were studied in human breast cancer (MCF-7 and MDA-MB-
231), human liver carcinoma (HepG2), human colorectal carcinoma
(LoVo), and human leukemia (K562) cell lines.
Most of the triazine compounds affected the cell viability of the four
cancer cell lines, as determined by the MTT cell viability assay (Table 1
and Fig. 3). The most effective molecules were 4a, and 6g, which af-
fected the cell viability of tested cancer cell lines with IC50, values
within the range between 5 and 9 µM. Of note, both compounds contain
piperidinyl moiety in their structure. Interestingly, and although pi-
peridine and morpholine can be considered to be from the same family
in some aspects of their synthetic chemistry, the presence of the mor-
pholine moiety was detrimental for anticancer activity. Thus, com-
pounds containing morpholine in the absence of piperidine were in-
active (4b, 6c, 6f). This observation was confirmed by the very low
activity of compound 6d in breast cancer cells (IC50 values of 48.7 µM
in MDA-MB-231 and 33 µM in MCF7 cells) and the counteraction of the
negative effect of morpholine by the presence of piperidine. The pre-
sence of benzylamine was not as beneficial to anticancer activity as that
of piperidine (4c vs 4a), but its presence helps to keep some activity in
the presence of piperidine (6a), which is as expected slightly superior to
6b (benzylamine and morpholine). The methoxy derivative showed no
anticancer activity (4e).
2. Results and discussion
2.1. Chemistry
The mono- and di-pyrazolyl-s-triazine derivatives were synthesized
following the reported method, where the hydrazinyl-s-triazine was
reacted with acetylacetone in the presence of triethylamine and DMF as
solvent [7d,9]. The initial nucleophilic substitution of the first chlorine
atom of cyanuric chloride 1 was achieved with great regioselectivity as
the reaction is a temperature-controlled process. Thus, the first chlorine
atom was replaced by amine (morpholine, piperidine, benzylamine, N-
458

M. Farooq, et al.
BioorganicChemistry87(2019)457–464
Scheme 1. Synthetic route for the synthesis of triazine derivatives.
2.3. Cell cycle distribution activity of compounds 4a and 6g
were performed. As shown in Table 2 and Fig. 5, most of them induced
developmental arrest in treated zebrafish embryos. The activity of these
compounds in zebrafish embryos was analogous to their activity in
human cancer cells. Compounds 4a and 6g were the most active in
zebrafish embryos and they caused a significant arrest in the develop-
ment of the embryos at a minimum concentration between 0.5 and
1 µM (Table 2), with 6g being the most effective. Control (DMSO 0.5%
V/V) embryos developed normally as observed at 24 hpf; however, the
development of embryos treated with compounds 4a and 6g was sig-
nificantly delayed and arrested (Fig. 5; B and C). The treated embryos
exhibited severe developmental delay after continuous exposure of 4a
and 6g for another 24 h when compared to control (Fig. 5 E and F). The
compounds were not lethal to the embryos in the concentration range
tested, and when the treated embryos were moved to compound-free
water, they grew normally without any sign of developmental arrest.
In general, it is believe that the chemically synthesize compounds
contain some level of toxicity as compared to natural products, and
these kind of compounds, even though produce very good results ob-
tained from in vitro systems (cell lines), fail in clinical trials if they are
To gain insight into the mechanism underlying anti-proliferation
capacity, the effect of the most active compounds on cell cycle dis-
tribution was investigated by fluorescence-activated cell sorting (FACS)
analysis. K562 cells were exposed to 10 and 6 µM of the compounds 4a
and 6g respectively for 24 h. The analysis showed the accumulation of S
phase, from 20% to 27% and 26.98% respectively. This accumulation
was also accompanied by a compensatory decrease in G1 to phase cells
form from 47.22% to 23% and 33.55%, respectively (Fig. 4). Com-
pounds 4a and 6g caused a minor accumulation of cells at G2/M phase
arrest, from 24% in untreated cells to 29.98% and 31%, respectively.
These results suggested that these two compounds inhibited cell pro-
liferation via S and G2/M phase arrest.
2.4. In vivo toxicity testing in zebrafish embryos
To evaluate the safety profile of these newly synthesized triazine
compounds in animals, drug toxicity assays in zebrafish (Danio rerio)
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M. Farooq, et al.
BioorganicChemistry87(2019)457–464
Fig. 2. Structure of the target compounds.
Table 1
not tested in suitable animal model system and hence discarded or
approved as potential drugs. Testing new compounds in suitable animal
models is therefore important to save efforts and capital. The zebrafish
has emerged as a highly effective tool to test the synthesized com-
pounds for potential toxicity. Moreover, zebrafish assays can also in-
dicate possible developmental toxicity for the foetus.
The cytotoxicity profile of pyrazole compounds in human cancer cell lines.
Compounds
Anti-cancer activity in human carcinoma cells lines
IC50 values (µM)
MCF7
MDA-MB-231
HepG2
LoVo
K-562
The developmental arrest observed upon treatment of zebrafish
embryos with pyrazole derivatives described herein could be due to cell
cycle arrest. Previous studies analysing the cell cycle content during
normal embryonic development in zebrafish has revealed a prolonged S
phase during the first cleavage (1.5 h) and then G1, G2 and M phase
accumulation during the transition from blastula (4–5 h) to shield (6 h)
stage [11] Any intervention in embryonic development at this time
would mean that a transition from S phase to G2 or M has been re-
tarded. A similar kind of developmental delay has also been reported
for other types of triazine derivatives in zebrafish screening assays [12].
The pyrazole derivatives, particularly 4a and 6g, caused significant
developmental arrest in zebrafish embryos, given this observation, we
propose, that the embryonic developmental delay in zebrafish upon
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
6f
7.5
NA*
19
14
17.5
18.5
13.6
8.2
6.1
9.8
NA
27
41.1
11.8
5.6
15.2
16.2
NA
12.5
NA
23.5
38
50
NA
30
NA
NA
NA
10.5
8.8
14.9
5.9
14.5
37.3
NA
43
54
NA
48.7
NA
NA
15
11.9
9.4
40.8
9.6
33
31.8
NA
NA
NA
5
NA
NA
10.8
21.2
NA
NA
6g
8.4
5.9
* NA = Not Active.
460

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BioorganicChemistry87(2019)457–464
Fig. 3. Line graph showing the anticancer profile of triazine compounds in the following cell lines: (A) MCF-7; (B) MDA-MB-231; (C) HepG2; (D) LoVo; and (E) K562.
treatment with pyrazole derivatives is most likely due to cell cycle ar-
rest and this notion is also supported by the observation that these
pyrazole derivatives exerted their effect on cancer cells via S and G2/M
phase arrest. Therefore, cell cycle arrest emerges as the possible mode
of action of these compounds both in in vitro (cancer cells) and in vivo
(zebrafish embryos).
these compounds, 4a and 6g (Fig. 6) were the most effective at in-
hibiting the cell survival of breast carcinoma, hepatocellular carcinoma,
colorectal carcinoma, and leukemia cell lines, with an IC50 within the
range of 5–9 µM. These two compounds also caused a significant delay
in the embryonic development of zebrafish. The cell cycle analysis in
K562 cells suggested that these pyrazole derivatives exert their action
by causing S and G2/M phase cell cycle arrest. The activity of these
compounds could be attributed to the presence of piperidinyl moieties
in their structure, since the compounds with piperidinyl moieties (4a
and 6g) were the most active (Fig. 6).
3. Conclusions
The pyrazole derivatives described herein showed moderate to
strong cytotoxicity in four types of human cancer cell lines. Among
Given the demonstrated activity of the pyrazole derivatives against
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M. Farooq, et al.
BioorganicChemistry87(2019)457–464
Fig. 4. K562 cells were treated with compounds 4a and 6g and analysed at 0 h, and 24 h by DNA flow cytometry. Histograms show the number of cells per channel
(vertical axis) vs. DNA content (horizontal axis). The values indicate the percentage of cells in the relevant phases of the cell cycle.
Table 2
acetate-hexane (4:6) was used as eluent.
The comparative in vivo toxicity profile of triazine compounds in zebrafish
The substituted s-triazine compounds (2a-e, 3a-e, 4a-e, 5a-g and
6a-g) were synthesized following the strategies and methods already
reported by our group [7d] and others [13], as shown in Scheme 1. The
characterization of some of the compounds (2a-e, 3a-e, 4a-c, and 6a-e)
studied herein has already been reported by our group [7d] while the
characterization of the remaining compounds is described below.
embryos.
Compound
Concentration (µM)*
Level of developmental arrest in zebrafish
embryos**
+++
4a
4b
4c
4d
4e
6a
6b
6c
6d
6e
6f
1
NA
1.5
NA
NA
2.5
2.0
12
−
++
+
4.1.1. 2,4-Bis(3,5-dimethyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)-1,3,5-
−
+
triazine, 4a [7d]
+
+
NA
NA
NA
0.5
−
+
−
+++
6g
NA = Not Active.
* Concentration at which developmental arrest was induced.
** The observation is based on at least three biological replications and using
clutches of embryos from different parents.
White crystals, mp = 141–142 °C, in 78%; IR (KBr, cm−1) 1660,
1614 (C]N), 1595 (C]N, C]C); 1H NMR (CDCl3): δ 1.60 (m, 6H,
3CH2), 2.27 (s, 6H, 2CH3), 2.62(s, 6H, 2CH3), 3.78 (t, 4H, , J = 4.4 Hz,
2CH2-NCH2), 5.97 (s,2H, 2CH) ppm; 13C NMR (CDCl3): δ 13.5(CH2-
CH2-CH2), 15.8(CH2-CH2-CH2), 24.3(CH3), 25.5(CH3), 45.1(CH2-N-
CH2), 110.8(Cb, pyrazole), 143.3(Cc, pyrazole), 151.7(Ca, pyrazole),
163.5(C]N, triazine), 164.7(C]N, triazine) ppm.
+
Mild developmental delay.
++
+++
Moderate developmental delay (few hours).
Severe developmental delay (at least 15–18 h).
four types of human cancer cells and minimal lethality in zebrafish
embryos, we can conclude that the capacity of these compounds as
potent anticancer agents deserves further attention.
4.1.2. N-Benzyl-4,6-bis(3,5-dimethyl-1H-pyrazol-1-yl)-N-methyl-1,3,5-
triazin-2-amine, 4d
4. Experimental section
4.1. Chemistry
Materials and method: Solvents were of analytical reagent grade and
were used without further purification. NMR (1H and 13C) spectra were
recorded on a JEOL 400 MHz spectrometer at room temperature in
CDCl3 and/or DMSO-d6 using internal standard δ = 0 ppm. Elemental
analysis was performed on Perkin-Elmer 2400 elemental analyzer.
Melting points were determined on a Mel-Temp apparatus in an open
capillary tube and are uncorrected. Fourier transform infrared spec-
troscopy (FTIR) spectra were recorded on Shimadzu model IRAffinity-1
Spectrometer from KBr disks. Silica gel-coated TLC plates (Type 60
GF254, Merck) were used to monitor the reaction and check the purity
of the compounds. A mixture of methanol-chloroform (1:9) or ethyl
White crystals, mp 248–250 °C, yield 86%; IR (KBr, cm−1): 1634
(C]N), 1595, 1541 (C]N, C]C); 1H NMR (CDCl3): δ 2.54 (s, 3H,
CH3), 2.57 (s, 3H, CH3), 2.68 (s, 3H, CH3), 2.87 (s, 3H, CH3), 3.28 (s,
3H, CH3), 4.97 (s, 2H, phCH2), 6.31 (s, 1H, CH), 6.37 (s, H, CH),
7.32–7.39 (m, 5H, C6H5) ppm; 13C NMR (CDCl3):
δ 13.7(CH3),
15.7(CH3), 40.1(N-CH3), 45.2(ph-CH2), 111.2 (Cb, pyrazole),
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BioorganicChemistry87(2019)457–464
Fig. 5. Pyrazolyl-s-Triazine derivatives in-
duced developmental delay in zebrafish
embryos. Representative micrograph of
zebrafish embryos at 24–32 hpf and 48 hpf
of control; A & D, and treated with com-
pound 4a; B & E, and 6g; C & F. Control
(mock treated) embryos were around 5 prim
stage (24–32 hpf), while the treated em-
bryos either with 4a or 6g were at 50%
epiboly (shield stage which is usually ob-
served after 6 hpf during normal embryonic
development in zebrafish embryos). The
treated embryos showed marked delay in
development upon continuous exposure of
compounds for another 24 h (compare D
with E &F). Control embryos were at long
pectoral fin stage (48hpf: D), while those
embryos treated with 4a (E) or 6g (F)
showed significant level of developmental
delay and were almost 24 h delay in devel-
opment (by developmental stage criteria) as
compared to control. Abbreviation used hpf:
hours post fertilization.
4.1.4. 4,4′-(6-(3,5-Dimethyl-1H-pyrazol-1-yl)-1,3,5-triazine-2,4-diyl)
N
N
N
N
dimorpholine, 6f
N
N
N
N
N
N
N
N
N
N
N
Off-white solid, mp 152–153 °C, yield 81%; IR (KBr, cm−1): 1643,
1622 (C]N), 1553 (C]N, C]C); 1H NMR (CDCl3): δ 2.27 (s, 3H, CH3),
2.59 (s, 3H, CH3), 3.17 (t, 8H, J = 2.4 Hz, 4CH2), 3.82 (brs, 8H, 4CH2),
5.97 (s, 1H, CH) ppm; 13C NMR (CDCl3): δ 14.0 (CH3), 16.0(CH3), 43.6
(CeNeC, morpholine), 43.9 (CeNeC, morpholine), 66.7 (CeOeC,
morpholine), 66.8 (CeOeC, morpholine), 110.4(Cb, pyrazole),
4a
6g
Fig. 6. Structure of the most reactive compounds.
127.1(C4), 128.6(C3,5), 137.4 (C2,6), 143.9(C1), 144.4(Ca,pyrazole),
152.3(Cc, pyrazole), 159.8 (C]N, triazine), 165.6(C]N, triazine) ppm.
Anal. Calc. for C21H24N8 (388.48): C, 64.93; H, 6.23; N, 28.84; Found:
C, 64.81; H, 6.43; N, 28.99.
143.1(Ca,
pyrazole),
151.3(Cc,
pyrazole),
163.2(C]N,
16H23N7O2
triazine),165.4(C]N, triazine) ppm; Anal. Calc. for
C
(345.41): C, 55.64; H, 6.71; N, 28.39; Found: C, 55.84; H, 6.90; N,
28.55.
4.1.3. 2,4-Bis(3,5-dimethyl-1H-pyrazol-1-yl)-6-methoxy-1,3,5-triazine, 4e
4.1.5. 2-(3,5-Dimethyl-1H-pyrazol-1-yl)-4,6-di(piperidin-1-yl)-1,3,5-
triazine, 6g
White crystals, mp 175–176 °C, yield 87%; IR (KBr, cm−1): 3376
(NH), 1635 (C]N), 1595, 1542 (C]N, C]C); 1H NMR (CDCl3): δ 2.30
(s, 6H, 2CH3), 2.72 (s, 6H, 2CH3), 4.11 (s, 3H, OCH3), 6.05 (s, 2H, 2CH)
ppm; 13C NMR (CDCl3): δ 13.8(CH3),15.8(CH3), 55.7(OCH3), 111.9(Cb,
pyrazole), 144.4(Ca, pyrazole), 153.3(Cc, pyrazole), 164.8 (N-C]N,
triazine), 172.3(eOeC]N, triazine) ppm. Anal. Calc. for C14H17N7
(299.34): C, 56.18; H, 5.72; N, 32.76; Found: C, 56.33; H, 5.91; N,
32.99.
Off-white solid, mp 178–179 °C, in yield 83%; IR (KBr, cm−1): 1643,
1654 (C]N), 1563 (C]N, C]C); 1H NMR (CDCl3): δ 1.67 (brs, 12H,
6CH2), 2.47 (s, 3H, CH3), 2.69 (s, 3H, CH3), 3.87 (brs, 8H, 4CH2), 6.21
(s, 1H, CH) ppm; 13C NMR (CDCl3): δ 12.7 (CH3), 15.3(CH3), 24.2(CH2-
CH2-CH2), 25.5(CH2-CH2-CH2), 45.6(CH2-N-CH2), 112.7(Cb, pyrazole),
145.6(Ca, pyrazole), 151.9(Cc, pyrazole), 155.4 (C]N, triazine),
159.7(C]N, triazine) ppm; Anal. Calc. for C18H27N7 (341.23): C, 63.32;
H, 7.97; N, 28.71; Found: C, 63.13; H, 8.01; N, 28.93.
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BioorganicChemistry87(2019)457–464
4.2. Biological evaluation
and Universities (CTQ2015-67870-P) and the Generalitat de Catalunya
(2017 SGR 1439; Spain).
4.2.1. Material and method
4.2.1.1. Human cancer cell lines. The cancer cell lines used in this study
were purchased from the American Type Culture Collection (American
Type Culture Collection (ATCC) Manassas, VA 20,108 USA). The celll
lines are: (1) K562 human chronic myeloid leukemia cells (ATCC® CCL-
243™); (2) MCF7 human breast adenocarcinoma cells (ATCC® HTB-
22™); (3) MDA-MB-231 human breast adenocarcinoma cells (ATCC®
HTB-26™); (4) HepG2 human hepatocellular carcinoma cells (ATCC®
HB-8065™); and (5) LoVo human colorectal adenocarcinoma cells
(ATCC® CCL-229™).
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Acknowledgements
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The work was funded in part by the following: the International
Scientific Partnership Program ISPP at King Saud University (ISPP#
0061; 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,
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