Synthesis, biological evaluation, and: In silico studies of novel chalcone: In pyrazoline-based 1,3,5-triazines as potential anticancer agents

Article abstract of DOI:10.1039/d0ra06799g

A novel series of triazin-chalcones (7,8)a-g and triazin-N-(3,5-dichlorophenyl)pyrazolines (9,10)a-g were synthesized and evaluated for their anticancer activity against nine different cancer strains. Triazine ketones 5 and 6 were synthesized from the cyanuric chloride 1 by using stepwise nucleophilic substitution of the chlorine atom. These ketones were subsequently subjected to a Claisen-Schmidt condensation reaction with aromatic aldehydes affording chalcones (7,8)a-g. Then, N-(3,5-dichlorophenyl)pyrazolines (9,10)a-g were obtained by cyclocondensation reactions of the respective chalcones (7,8)a-g with 3,5-dichlorophenylhydrazine. Among all the evaluated compounds, chalcones 7d,g and 8g exhibited more potent in vitro anticancer activity, with outstanding GI50 values ranging from 0.422 to 14.9 μM and LC50 values ranging from 5.08 μM to >100 μM. In silico studies, for both ligand- and structure-based, were executed to explore the inhibitory nature of chalcones and triazine derivatives. The results suggested that the evaluated compounds could act as modulators of the human thymidylate synthase enzyme. This journal is

Full text of DOI:10.1039/d0ra06799g

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Synthesis, biological evaluation, and in silico
studies of novel chalcone- and pyrazoline-based
Cite this: RSC Adv., 2020, 10, 34114
1,3,5-triazines as potential anticancer agents†
Leydi M. Moreno,a Jairo Quiroga,ab Rodrigo Abonia,ab Antonino Lauria,c
d
ab
Annamaria Martorana,c Henry Insuasty
and Braulio Insuasty
*
A novel series of triazin-chalcones (7,8)a–g and triazin-N-(3,5-dichlorophenyl)pyrazolines (9,10)a–g were
synthesized and evaluated for their anticancer activity against nine different cancer strains. Triazine ketones
5 and 6 were synthesized from the cyanuric chloride 1 by using stepwise nucleophilic substitution of the
chlorine atom. These ketones were subsequently subjected to a Claisen–Schmidt condensation reaction
with aromatic aldehydes affording chalcones (7,8)a–g. Then, N-(3,5-dichlorophenyl)pyrazolines (9,10)a–
g were obtained by cyclocondensation reactions of the respective chalcones (7,8)a–g with 3,5-
dichlorophenylhydrazine. Among all the evaluated compounds, chalcones 7d,g and 8g exhibited more
potent in vitro anticancer activity, with outstanding GI50 values ranging from 0.422 to 14.9 mM and LC50
values ranging from 5.08 mM to >100 mM. In silico studies, for both ligand- and structure-based, were
executed to explore the inhibitory nature of chalcones and triazine derivatives. The results suggested
that the evaluated compounds could act as modulators of the human thymidylate synthase enzyme.
Received 6th August 2020
Accepted 9th September 2020
DOI: 10.1039/d0ra06799g
rsc.li/rsc-advances
rapamycin (PI3Ka/mTOR),11 carbonic anhydrase (CA),12–14
human topoisomerase IIa,15 dihydrofolate reductase (hDHFR)16
and tubulin polymerization17 (Fig. 1).
1. Introduction
Heterocycles constitute a structural scaffold in a wide range of
drugs and have occupied a prominent place in medicinal
chemistry due to their ability to imitate and interact with bio-
logical molecules thus exhibiting remarkable pharmacological
properties.1–4 The utility of heterocyclic rings, such as triazines
and pyrazolines in different areas of medicine, particularly as
potential anticancer drugs continue to be investigated.5–8
The 1,3,5-triazine is a versatile ring that can act as a scaffold
to carry three functionalized branches at 2,4 and 6-positions
and this property allows to easily modulate the physicochemical
and biological properties of these derivatives.5 The anticancer
activity mechanisms of these systems can be associated with
different targets. Thus, 1,3,5-triazine-2-carbohydrazides exhibi-
ted Rad6B inhibitory activity,9 while macrocyclic pyrazolo[1,5-a]
[1,3,5]triazines showed potent inhibition of CK2 protein
kinase.10 Other mechanisms of action are related to the inhi-
bition of phosphatidylinositol 3-kinase a/mammalian target of
Similarly, pyrazoline rings are quite promising fragments
due their anticancer properties. Pyrazoline hybrids with
heterocyclic rings such as imidazopyridine,8 thiazole,18
triazine,19,20 4-thiazolidinone-indole21 and dihydroquinolone22
have been reported as potential anticancer agents. Pyrazoline
rings can be easily obtained by a cyclocondensation reaction of
chalcones with hydrazine derivatives.23 Chalcones have also
shown marked biological activity as anticancer agents,24–26 that
allows comparative studies of antiproliferative activity to be
carried out between the precursor chalcones and the pyrazolinic
derivatives and at the same time expand the mosaic of
compounds evaluated.
The enzyme thymidylate synthase (TS) is an E2F1-regulated
enzyme that is essential for DNA synthesis and repair. TS and
mRNA levels are elevated in many human cancers, and high TS
levels have been correlated with poor prognosis in patients with
colorectal, breast, cervical, bladder, kidney, and non-small cell
lung cancers.27 Inhibition of TS causing cells incapable of
undergoing accurate DNA replication, ultimately leading to cell
death.28,29 Owing to their important role in cell, TS represent
a natural target for anticancer therapies.
aHeterocyclic Compounds Research Group, Department of Chemistry, Universidad del
Valle, A.A. 25360 Cali, Colombia. E-mail: braulio.insuasty@correounivalle.edu.co
bCenter for Bioinformatics and Photonics-CIBioFI, A.A. 25360 Cali, Colombia
cDipartimento di Scienze
e Tecnologie Biologiche Chimiche e Farmaceutiche
In a preliminary work we reported the synthesis and anti-
cancer activity of chalcone- and pyrazoline-based 1,3,5-
triazines.20 Several of these tested molecules showed low toxicity
and outstanding antiproliferative activity against a wide range
of cancer cell lines with GI50 values in the range of 0.569–16.6
`
“STEBICEF”, Universita di Palermo, Viale delle Scienze Ed. 17, I-90128 Palermo, Italy
dHeterocyclic Compounds Research Group, Department of Chemistry, Universidad de
˜
Narino, A.A. 1175 Pasto, Colombia
† Electronic supplementary information (ESI) available: Table S1, spectra data
and Fig. S1. See DOI: 10.1039/d0ra06799g
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Fig. 1 Triazine derivatives with different anticancer activity mechanisms.
mM. A raw SAR analysis showed that N-(3,5-dichlorophenyl) in excellent yields. These precursors were characterized by FTIR,
pyrazolines and chalcones were the most active molecules. 1H-NMR, 13C-NMR, and mass spectrometry (see Experimental
According to the previously described, we focused on the section).
promising trisubstituted-1,3,5-triazinic systems and decided to
(E)-1-(4-((4-((2-hydroxyethyl)amino)-6-morpholino-1,3,5-
synthesize and evaluate a new serie of N-(3,5-dichlorophenyl) triazin-2-yl)amino)phenyl)-3-(substituted)chalcones 7a–g and
pyrazoline and chalcone based 1,3,5-triazines as anticancer (E)-1-(4-((4-((4-chlorophenyl)amino)-6-((2-hydroxyethyl)amino)-
agents. Additionally, in silico studies were done to help under- 1,3,5-triazin-2-yl)amino)phenyl)-3-(substituted)chalcones 8a–g
stand the possible mode of action of the new active compounds. were obtained from triazine-ketones 5 and 6, respectively, by
Claisen–Schmidt condensation reaction with substituted
benzaldehydes in the presence of 20% KOH solution in EtOH
2. Results and discussion
2.1. Chemistry
(Scheme 2). The structures of chalcones 7a–g and 8a–g were
conrmed by spectroscopic techniques (FTIR, 1H-NMR, 13C-
NMR and mass spectrometry). These compounds showed
wide FTIR absorption bands in the range of 3277–3315 cmꢀ1
assigned to O–H groups. The IR spectra also showed absorption
bands at 1641–1651, 1560–1587 and 1506–1558 cmꢀ1 assigned
to C]O, C]N and C]C functionalities, respectively. In the 1H-
NMR spectrum of chalcone 7e, for example, the signal of the
vinylic proton Hb of the a,b-unsaturated system appears as
The target compound 9a–g and 10a–g were obtained by a multi-
stage synthesis as described in Schemes 1 and 2. Initially, 2,4,6-
trichloro-1,3,5-triazine 1 was submitted to consecutive nucleo-
philic substitution reactions: rst with 4-aminoacetophenone,
then with morpholine or 4-chloroaniline and nally with etha-
nolamine, under the conditions described in Scheme 1, to
afford the respective 2,4,6-trisubstituted-1,3,5-triazines 5 and 6
Scheme 1 Synthetic pathway for triazine derivatives 5 and 6. Reagents and conditions: (i) 4-aminoacetophenone; acetone, ꢀ5–0 ꢁC, 5 h; (ii)
morpholine, acetone, ꢀ5–0 ꢁC, 6 h; (iii) 4-chloroaniline, dioxane, room temperature, 12 h; (iv) ethanolamine, dioxane, reflux, 8h.
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Scheme 2 Synthesis of N-(3,5-dichlorophenyl)pyrazolines 9a–g and 10a–g and their precursor chalcones 7a–g and 8a–g. Reagents and
conditions: (i) benzaldehyde–R2, 20% KOH, ethanol, 3–8 h; (ii) 3,5-dichlorophenylhydrazine hydrochloride, ethanol, reflux, 12 h.
a doublet at 7.68 ppm with a coupling constant of 3J ¼ 15.6 Hz,
Reaction of the synthesized chalcones 7a–g and 8a–g with
which agrees with a E-conguration. The signal of other vinyl 3,5-dichlorophenylhydrazine hydrochloride in ethanol under
proton Ha appears overlapped with signals of aromatic protons reux afforded N-(3,5-dichlorophenyl)pyrazolines 9a–g and 10a–
at 7.89–7.99 ppm. In the 13C-NMR spectrum the signals of a and g (Scheme 2), respectively, in racemic mixtures. Taking as
b carbon atoms were observed at 118.4 and 141.4 ppm, example compound 9e, in the 1H-NMR spectrum, the formation
respectively. The mass spectrum shows molecular ion peak at of the pyrazoline ring is conrmed by the appearance of two
m/z 480 and with a isotopic prole of 12 : 4 ([M]+ : [M + 2]+), double doublets at 3.15 ppm (with 2JAM ¼ 17.7 Hz and 3JAX ¼ 4.4
characteristic of a compound bearing one chlorine atom in its Hz), and at 3.92 ppm (with 2JAM ¼ 17.7 Hz, and 3JMX ¼ 11.7 Hz)
structure.
corresponding to protons on the diastereotopic center C-4,
Table 1 Mean growth, %GI and lethality values for the most sensitive cell lines displayed by the tested compounds against 60 NCI human cancer
cell lines at 10 mM
Compound
Mean growth
Most sensitive cell line
Growth inhibition percent (%GI)a/lethalityb
5
6
97.18
89.48
81.57
95.32
87.49
17.60
77.12
79.95
34.86
71.26
77.30
68.77
69.89
58.28
67.99
42.92
82.34
93.57
87.26
50.41
84.61
74.76
72.39
95.13
96.74
90.95
74.43
95.42
94.58
99.72
SNB-75 (CNS cancer)
SR (leukemia)
SR (leukemia)
33.36
35.27
87.24
27.15
70.68
ꢀ31.70b,c
90.27
88.77
ꢀ51.34b,c
91.45
78.50
91.94
84.74
ꢀ78.88a
98.22
ꢀ85.59b,c
45.95
33.41
45.02
94.92
43.41
67.01
80.30
29.35
31.73
35.19
65.16
35.59
38.64
26.04
7a
7b
7c
7d
7e
UACC-62 (melanoma)
MCF7 (breast cancer)
UACC-62 (melanoma)
MCF7 (breast cancer)
MCF7 (breast cancer)
U251 (CNS cancer)
HCT-15 (colon cancer)
MCF7 (breast cancer)
RPMI-8226 (leukemia)
MOLT-4 (leukemia)
HCT-116 (colon cancer)
RPMI-8226 (leukemia)
HCT-116 (colon cancer)
UACC-62 (melanoma)
MCF7 (breast cancer)
MCF7 (breast cancer)
RXF 393 (renal cancer)
MCF7 (breast cancer)
786-0 (renal cancer)
RXF 393 (renal cancer)
HS 578T (breast cancer)
HS 578T (breast cancer)
UO-31 (renal cancer)
MOLT-4 (leukemia)
7f
7g
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
9g
10a
10b
10c
10d
10e
10f
10g
HS 578T (breast cancer)
HS 578T (breast cancer)
HS 578T (breast cancer)
a
b
%GI (growth inhibition percentage) ¼ 100 ꢀ GP (growth percentage). Negative values mean lethality of the respective cancer cell line.
c
Compounds with the most relevant inhibitory activity against all cancer cell lines in terms of their mean values.
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Table 2 Antiproliferative activity (GI50a and LC50b) displayed by compounds 7d,g and 8g against 60 human cancer cell linesc compared with the
standard drug 5-FU
Compounds
5-FU NS 18893d
7d
7g
8g
a
b
Panel name
Cell name
GI50
LC50
GI50
LC50
GI50
LC50
GI50
LC50
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
0.935
1.79
0.783
1.19
0.612
0.422
2.18
2.83
2.72
1.61
2.15
2.03
2.18
1.58
1.59
1.8
>100
>100
>100
>100
—
>100
>100
>100
>100
>100
>100
>100
>100
7.64
>100
—
6.03
6.91
>100
—
—
—
>100
>100
6.11
—
6.41
—
2.41
2.92
2.33
2.45
2.07
3.1
2.74
2.44
2.22
2.48
2.32
2.55
2.63
2.15
1.85
2.09
2.34
1.43
1.89
2
>100
>100
>100
>100
>100
>100
>100
32
78.6
>100
>100
29.4
36.2
9.18
57.9
7.94
19.2
5.23
19.5
11.6
6.05
8.57
40.6
31.5
5.7
2.75
12.2
2.83
3.56
1.56
1.95
3.48
3.41
8.55
5.39
4.29
10.7
14.9
4
>100
>100
>100
>100
>100
>100
40.2
41.8
50.5
>100
62.7
64.1
>100
36.9
59.4
41.7
28.5
5.41
26.2
43.2
12.7
14.4
52.2
38.3
23.6
30.6
18.8
6.27
58.3
37.4
35.8
51
42.8
62.7
96.5
44.1
40.2
28
39.3
43.4
>100
98.8
87.8
16.6
35.5
41.2
43.4
9.5
10.00
2.51
3.98
0.32
0.05
0.03
0.20
63.10
0.40
79.43
50.12
0.32
0.20
0.06
7.94
0.16
0.05
0.25
0.10
0.16
0.20
1.00
1.58
0.25
0.06
3.98
1.00
0.25
0.05
1.00
0.08
63.10
1.00
0.50
3.98
0.50
1.26
0.02
3.98
10.00
1.58
0.32
19.95
0.79
0.40
0.32
0.08
2.51
0.50
1.26
1.58
2.51
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-75
U251
7.82
4.8
Colon cancer
1.8
1.3
1.43
1.7
2.93
1.48
2.08
2.32
2.13
2.17
5.05
3.98
2.39
2.05
2.18
1.66
13.9
3.5
25.12
>100
>100
>100
>100
>100
>100
>100
>100
>100
1.66
1.81
1.88
3.03
1.63
1.07
1.38
1.48
1.45
1.79
1.66
1.71
1.78
1.76
1.44
1.54
1.9
1.8
1.92
2.68
2.67
1.64
1.84
1.63
1.67
2.91
2.01
2.23
8.66
3.25
3.16
3.98
2.76
2
1.96
3.05
1.74
3.25
2.87
3.82
1.65
1.4
CNS cancer
Melanoma
28.6
7.02
5.79
32.6
9.08
16.8
48.3
34
LOX IMVI
MALME-3M
M14
79.43
—
—
—
8.14
—
>100
>100
>100
>100
>100
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
UO-31
PC-3
DU-145
3.46
11.7
5.88
14
6.12
—
6.67
>100
>100
—
>100
>100
>100
>100
—
>100
>100
>100
7.14
>100
—
32
79.43
70.1
30.2
9.77
7.34
54.2
9.07
86.5
>100
>100
5.82
35.6
8.03
69.6
6.53
21.6
5.08
>100
9.56
10.1
6.32
4.92
2.75
4.14
5.93
5.66
5.58
13.4
2.25
1.67
4.81
5.12
1.82
5.53
1.75
2.99
6.08
>100
>100
>100
Ovarian cancer
Renal cancer
1.69
1.63
2.02
2.2
50.12
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.32
3.05
1.76
1.32
1.95
2.09
1.46
1.78
1.24
2.14
1.35
1.86
2.54
1.63
2.22
1.31
2.24
2.19
41.9
12.5
>100
43.2
Prostate cancer
>100
—
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Table 2 (Contd.)
Compounds
5-FU NS 18893d
7d
7g
8g
a
b
Panel name
Cell name
GI50
LC50
GI50
LC50
GI50
LC50
GI50
0.40
0.08
6.31
10.00
10.00
7.94
LC50
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
1.17
2.2
1.65
1.35
1.8
6.55
1.25
2.69
2.61
1.56
2.28
1.8
79.4
>100
>100
6.11
>100
8.3
1.49
4.21
2.82
3.07
3.4
53.7
40.8
>100
30.5
>100
64.8
>100
>100
>100
>100
>100
>100
>100
>100
6.06
>100
>100
MDA-MB-468
1.34
5.54
a
GI50 was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the
drug incubation, determined at ve concentration levels (100, 10, 1.0, 0.1, and 0.01 mM). Italics entries are the most relevant GI50 values of each
b
compound and bold entries are GI50 values (of our compounds) lower than GI50 values of 5-FU. LC50 is a parameter of cytotoxicity that reects
c
the molar concentration needed to kill 50% of the cells. Data obtained from NCI's in vitro disease-oriented human cancer cell lines screen in
d
mM. The values of activity against human cancer cell lines displayed by 5-FU correspond to that reported by. Please visit: https://
dtp.cancer.gov/dtpstandard/cancerscreeningdata/index.jsp.
while the H-5 proton is observed as a double doublet at
Based on the mean growth data reported in Table 1 and
3
3
5.63 ppm (with JMX ¼ 11.7 Hz and JAX ¼ 4.4 Hz), conrming Fig. S1,† it can be observed that:
the existence of an AMX coupling system in the pyrazoline ring.
ꢂ The chalcones with substituent R1 ¼ 4-chloroaniline 8a–g
In the 13C-NMR, the absence of the carbonyl carbon signal and showed better activity than the respective pyrazolines 10a–g.
the appearance of signals at 43.3 and 61.6 ppm corresponding This pattern of behavior was not observed in all cases when the
to C-4 and C-5, respectively, also conrmed the formation of substituent was R1 ¼ morpholine.
pyrazolic ring. In general, mass spectra of N-(3,5-dichlor-
ꢂ Pyrazolines with substituent R1 ¼ morpholine showed
ophenyl)pyrazolines 9a–g and 10a–g show well-dened molec- better activity than those with substituent R1 ¼ 4-chloroaniline.
ular ion peaks.
ꢂ The presence of the 3,4,5-(OCH3)3 (R2 ¼ d) and CF3 (R2 ¼ g)
It must be highlighted that the synthetic pathway used to groups favor the anticancer properties in both chalcones and
obtain the triazine derivatives was efficient, the formation of by- pyrazolines, with the exception of chalcone 8d and pyrazoline
products was not observed, and high yields were achieved. The 10g.
purity was veried by thin-layer chromatography (TLC), mass
spectrometry and proton NMR spectroscopy.
ꢂ The chalcone 7d with substituent R1 ¼ morpholine and R2
¼ 3,4,5-(OCH3)3 was the leading structure among the series of
synthesized compounds, with the best anticancer property.
Due to compounds 7d,g and 8g exhibited the broadest
spectrum and the highest inhibitory activity among the 30
evaluated compounds against all nine panels of human cancer
cell lines at one-dose assay, they were subjected to evaluation at
ve concentrations of dilution (i.e. 100, 10, 1.0, 0.1 and 0.01
mM), in order to determine their antiproliferative activity (GI50
and LC50) (Table 2). Chalcone 7d showed GI50 values in the
range of 0.422–3.05 mM and LC50 values of 6.03 to >100 mM,
being the SR cell line (leukemia, GI50 ¼ 0.422 mM and LC50 > 100
mM) the most sensitive strain. Compound 7g exhibited GI50
values in the range of 1.25–8.66 mM and LC50 values of 5.08 to
>100 mM, being the MCF7 cell line (breast cancer, GI50 ¼ 1.25
mM) the most sensitive strain, while compound 8g showed GI50
values in the range of 1.48–14.9 mM and LC50 values of 5.41 to
>100 mM, being specially effective against the HCT-116 cell line
(colon cancer) with GI50 ¼ 1.48 mM. The best cytotoxicity value
2.2. Anticancer activity evaluation
The in vitro anticancer activities of all the synthesized triazine
derivatives 5, 6, (7–10)a–g were evaluated by the National Cancer
Institute (NCI). Initially, compounds were subjected to prelim-
inary screening at one-dose (10 mM) against 60 human cancer
cell lines consisting of nine tumor panels, which include
leukemia, lung, colon, melanoma, renal, prostate, CNS, ovarian,
and breast cancer cell lines and the results are reported as NCI
mean graphs. According to the data analysis of the one-dose
mean graphs low GP (growth percent) values indicates better
growth inhibition percentages (%GI) (i.e. %GI ¼ 100 ꢀ GP). The
negative GP values correspond to lethal activity, hence, more
negative values represent higher activity of the assayed
compound. Additionally, low mean values represent better
activity and is used as a criteria for further studies. Indeed,
mean values # 50 indicate that the compound is active.
According to the higher (%GI and lethality) and lower mean
values for one-dose criteria, among the evaluated componds,
chalcones 7d, 7g and 8g were actives and displayed the better %
GI, lethality and mean values than the remaining assayed
compounds, Table 1.
was shown by compound 7g against UO-31 (renal cancer, LC50
¼
5.08 mM). Should be noted that compounds 7d,g and 8g showed
better antiproliferative activity than 5-uorouracil (5-FU) (stan-
dard drug) in several cancer cell lines (Table 2, bold entries).
Additionally, mean GI50 values (per panel) of chalcones 7d,g
and 8g in comparison with the standard anticancer agent 5-
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the lowest mean GI50 values for compounds 7g and 8g were
obtained for renal (1.80 mM) and colon (2.56 mM) cancer panels,
respectively. These nding indicates that compound 7d might
be used as promising lead molecule for discovering a new class
of anticancer agents.
2.3. In silico insights
The antiproliferative activities showed by the chalcone and
pyrazoline derivatives, lead us to investigate on the suitable
biological target that can play a crucial role in the biological
response. With this aim molecular modeling protocols, well
established in previous works,30–33 were applied by using
a mixed ligand- and structure-based approach.
The features of the ligand-based approaches supported by
molecular descriptors allowed the evaluation of the topological,
thermodynamic, and charge-related characteristics of the
ligands. Thus, two complementary standpoints in the evalua-
tion of the binding capability (ligand- and structure-based)
covered all the interaction aspects in the ligand–target complex.
In the rst step the structures were submitted to the web-
server DRUDIT (DRUgs Discovery Tools), an open access
virtual screening platform, recently developed by us (https://
www.drudit.com) based on molecular descriptors,34 useful in
Fig. 2 Comparison of the mean GI50 values, per panel, displayed by
chalcones 7d,g, 8g and the standard drug 5-fluorouracil (5-FU) against
the 60 human cancer cell lines.
uorouracil (5-FU) were determined and drawn in Fig. 2 for
a better understanding. Based on these data, chalcone 7d was
more active against all panels of cancer cell lines than
compounds 7g and 8g, exhibiting lower activity in only two
panels of cancer cell lines (i.e. colon and CNS) compared to the
standard drug 5-FU. Remarkably, chalcone 7d exhibited the
lowest mean GI50 value against leukemia panel (0.96 mM), while
Table 3 Biotarget DRUDIT Affinity Scores (DAS) for the studied molecules
Serine
Thymidylate threonine
Voltage gated sodium Proto-oncogene
Tyrosine
Epidermal growth
factor receptor
Biological target/
cmd
channel subunit
tyrosine protein kinase protein
Nociceptin
receptor
synthase
protein kinase4 Nav1-5
Src
kinase ZAP-70 EGFR
5
6
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
0.778
0.822
0.882
0.894
0.89
0.804
0.882
0.896
0.886
0.874
0.858
0.898
0.84
0.856
0.888
0.878
0.794
0.8
0.786
0.762
0.788
0.808
0.818
0.776
0.768
0.77
0.762
0.796
0.846
0.852
0.84
0.812
0.81
0.898
0.89
0.84
0.712
0.742
0.886
0.894
0.898
0.85
0.888
0.854
0.776
0.83
0.858
0.872
0.782
0.798
0.8
0.724
0.804
0.788
0.818
0.756
0.788
0.792
0.7
0.702
0.786
0.87
0.716
0.748
0.854
0.88
0.858
0.736
0.834
0.824
0.754
0.8
0.832
0.83
0.734
0.794
0.778
0.74
0.792
0.794
0.798
0.716
0.772
0.802
0.734
0.738
0.748
0.746
0.682
0.724
0.744
0.678
0.773
0.808
0.896
0.9
0.87
0.9
0.89
0.874
0.824
0.892
0.874
0.798
0.858
0.876
0.884
0.834
0.832
0.868
0.778
0.816
0.782
0.744
0.668
0.778
0.804
0.646
0.712
0.678
0.68
0.754
0.848
0.834
0.768
0.848
0.866
0.856
0.774
0.832
0.836
0.774
0.822
0.822
0.83
0.79
0.82
0.826
0.79
0.822
0.812
0.804
0.764
0.788
0.808
0.756
0.811
0.768
0.882
0.888
0.84
0.818
0.83
0.84
0.78
0.766
0.804
0.738
0.81
0.802
0.814
0.748
0.778
0.804
0.736
0.708
0.714
0.714
0.692
0.704
0.722
0.678
0.790
0.794
0.872
0.876
0.764
0.846
0.854
0.864
0.782
0.802
0.826
0.724
0.816
0.824
0.81
0.718
0.784
0.81
9g
0.724
0.728
0.728
0.722
0.652
0.694
0.718
0.62
10a
10b
10c
10d
10e
10f
10g
Average
0.754
0.728
0.76
0.704
0.732
0.746
0.616
0.788
0.746
0.756
0.78
0.77
0.825
0.604
0.66
0.69
0.574
0.775
0.790
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the biological targets database implemented in the tool. The
DAS values of the molecules under investigation against the
best biological targets are reported in Table 3.
The obtained results showed as the molecules under inves-
tigation have a quite good affinity against thymidylate synthase
(TS) (DAS averaged 0.825, which reaches 0.87 for the derivatives
7 and 8), thus to get information on which molecular features
are involved in the binding, structure-based studies were
performed.
The monomer of TS consists of an a/b-fold containing 7a-
helices and 10b-strands, arranged in three layers: a six stranded
mixed b-sheet, a long a-helix across the sheet anked by two
shorter helices, and a mixed layer containing two antiparallel
two stranded b-sheets and the remaining four helices. The large
b-sheets from the monomers stack against each other to form
the dimer interface (Fig. 3). The dimer has two active sites, one
within each monomer. In this study, the monomer was extrac-
ted from the high-resolution crystal structure of TS (PDB ID:
5X5Q)35 downloaded from the Protein Databank (https://
www.rcsb.org).36
Fig. 3 Thymidylate synthase dimer (from PDB ID 5X5Q).
the identication of suitable biological targets for small
molecules.
In particular, by the Biotarget Predictor tool, the DRUDIT
Affinity Score (DAS) is assigned to each input structure versus
With the aim to conrm the ligand-based results, to inves-
tigate the structural interactions, and to predict the binding
poses within the human thymidylate synthase binding site, all
the synthesized molecules were processed by Induced Fit
Docking (IFD) calculations.
The IFD results on chalcone- and pyrazoline-based 1,3,5-
triazine derivatives conrmed the ligand-based evidence. In
particular, the most active compounds (7d,g and 8g) showed
Table 4 IFD results for precursors 5, 6, chalcones (7,8)a–g and N-
(3,5-dichlorophenyl)pyrazolines (9,10)a–g
Docking XP
Cmd
score
IFD score
5
6
7a
7b
7c
7d
7e
7f
7g
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
9g
ꢀ9.485
ꢀ589.383
ꢀ591.632
ꢀ594.712
ꢀ597.86
ꢀ12.496
ꢀ9.645
ꢀ13.681
ꢀ13.301
ꢀ12.161
ꢀ12.382
ꢀ10.889
ꢀ12.672
ꢀ12.765
ꢀ13.461
ꢀ11.157
ꢀ11.701
ꢀ12.406
ꢀ12.018
ꢀ15.116
ꢀ12.401
ꢀ11.993
ꢀ11.898
ꢀ9.545
ꢀ595.368
ꢀ598.947
ꢀ596.397
ꢀ594.444
ꢀ597.106
ꢀ592.985
ꢀ598.765
ꢀ597.44
ꢀ596.742
ꢀ594.546
ꢀ593.787
ꢀ598.53
ꢀ597.581
ꢀ597.761
ꢀ597.398
ꢀ593.173
ꢀ601.736
ꢀ599.526
ꢀ598.997
ꢀ600.72
ꢀ14.838
ꢀ13.206
ꢀ12.101
ꢀ13.657
ꢀ9.9
10a
10b
10c
10d
10e
10f
10g
Raltitrexed
ꢀ600.609
ꢀ599.967
ꢀ589.16
ꢀ15.252
ꢀ11.00
ꢀ11.842
ꢀ13.633
ꢀ11.598
ꢀ13.828
ꢀ598.052
ꢀ598.584
ꢀ591.941
ꢀ584.621
Fig. 4 Amino acid maps representations of the best scored derivatives
(7d,g and 8g).
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4. Experimental section
4.1. General information
Reagents and solvents used were obtained from commercial
sources and used without further purication. Melting points
were measured using a Stuart SMP10 melting point device
(Cole-Parmer Ltd, Stone, Staffordshire, UK) and are uncor-
rected. FTIR spectra were obtained with a IRAffinity-1 spectro-
photometer (Shimadzu, Columbia, MD, USA). The 1H- and 13C-
NMR spectra were run on a DPX 400 spectrometer (Bruker,
Billerica, MA, USA) operating at 400 and 100 MHz respectively,
using DMSO-d6 as solvent and TMS as internal standard. The
mass spectra were obtained on a Shimadzu-GCMS-QP2010
spectrometer (Shimadzu, Kyoto, Honshu, Japan) operating at
70 eV. The elemental analyses were obtained using an Agilent
CHNS elemental analyzer (Thermo Fischer Scientic Inc.,
Madison, WI, USA) and the values are within ꢃ0.4% of the
theoretical values. Thin layer chromatography (TLC) were per-
formed on 0.2 mm pre-coated aluminium plates of silica gel 60
F254 (Merck, Darmstadt, Hesse, Germany).
Fig. 5 Left: superposition of 8g (white) and raltitrexed (green). Right:
stacking interaction between 8g (green) and UMP (white).
excellent docking scores, comparable to reference compound
raltitrexed (Table 4). The derivatives 9e and 10c showed the
higher scores, although the in wet results and the ligand based
outputs not classied them as TS modulators. It is conceivable
that, although the molecular structures well t TS active site,
other chemical–physical properties do not contribute to overall
affinity.
The analysis of the amino acid maps of the most active
compounds (7d,g and 8g), conrmed as they interact with the
pivotal amino acid residues (Arg50, Leu 108, Asn112, Leu192,
Leu221, Phe225, and Met311), which have a central role in the
catalytic enzyme process of the TS (Fig. 4).
As seen from 3D docking representation of the 8g derivative
the core occupies the same binding site as does the reference
ligand (Fig. 5, le) and makes stacking interaction with
pyrimidine ring of UMP (Fig. 5, right). This stacking interaction
is crucial and has been conserved in all the thymidylate syn-
thases for with crystal structures that have been solved in
complex with the cofactor and the inhibitor.
The overall results are in agreement with the hypothesis that
the antiproliferative activities are related to the capability of
these structures to modulate the TS, although, they could
involve other biological targets as suggested from the ligand-
based results (Table 3).
4.2. Chemistry
4.2.1. General procedure for synthesis of 1-(4-((4,6-
dichloro-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-one (2). Using
a methodology similar to that reported by Kathiriya37 and
coworkers, the synthesis of precursor 2 was carried out as
follows: a mixture of 2,4,6-trichloro-1,3,5-triazine 1 (0.027 mol, 1
equiv.) and 4-aminoacetophenone (0.027 mol, 1 equiv.) in
acetone (25 mL) was stirred at ꢀ5–0 ꢁC for 5 h. The mixture was
neutralized with 20% Na2CO3. The content was poured onto
crushed ice, ltered and washed with water.
4.2.2. General procedure for synthesis of 1-(4-((4-chloro-6-
morpholino-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-one (3). A
mixture of ketone 2 (0.014 mmol, 1.2 equiv.) and morpholine
(0.012 mol, 1 equiv.) in acetone (25 mL) was stirred at ꢀ5–0 ꢁC
for 6 h. The mixture was neutralized with 20% Na2CO3. The
content was poured onto crushed ice, ltered and washed with
water. No further purication was required.
4.2.2.1. 1-(4-((4-Chloro-6-morpholino-1,3,5-triazin-2-yl)
amino)phenyl)ethan-1-one (3). White solid; 70% yield; mp 218–
3. Conclusions
220 C. FT-IR (ATR) n (cmꢀ1) 3316 (N–H), 3117 (]C–H), 1659
ꢁ
1
In this study, novel chalcone- and pyrazoline-based 1,3,5- (C]O), 1578 and 1516 (C]N and C]C). H-NMR (400 MHz,
triazines were synthesized using a versatile methodology, DMSO-d6) d ppm 2.50 (s, 3H, CH3), 3.44–3.81 (m, 8H, CH2), 7.76
achieving high yields and purity. These triazine derivatives were (d, J ¼ 8.8 Hz, 2H, Ar–H), 7.90 (d, J ¼ 8.8 Hz, 2H, Ar–H), 10.36
evaluated for their anticancer effects on nine panels of 60 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 26.4 (CH3),
human cancer cell lines. Compounds 7d,g and 8g exhibited 43.4 (CH2), 59.15 (CH2), 119.1 (CH), 129.3 (CH), 131.1 (Cq),
signicant antiproliferative activity on all cancer panels being 143.5 (Cq), 143.7 (Cq), 165.5 (Cq), 167.9 (Cq), 196.4 (Cq). MS (70
specially effective against the SR leukemia cell line, MCF7 breast eV) m/z (%): 333 (11), 282 (23), 267 (100), 170 (26), 145 (44), 90
cancer cell line and HCT-116 colon cancer cell line, respectively, (32). Anal. calcd C15H16ClN5O2: C, 53.98; H, 4.83; N, 20.98;
and furthermore showed better GI50 values than 5-FU against found: C, 54.01; H, 4.86; N, 20.70.
several cell lines. Compound 7d exhibited the lowest mean GI50
4.2.3. General procedure for synthesis of 1-(4-((4-chloro-6-
against all panels of cancer cell lines and showed excellent ((4-chlorophenyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)ethan-
docking score, comparable to reference compound raltitrexed. 1-one (4). A mixture of ketone 2 (0.011 mol, 1 equiv.) and 4-
According to these results, chalcone 7d (R1 ¼ morpholine, R2 ¼ chloroaniline (0.011 mol, 1 equiv.) in dioxane (25 mL) was
3,4,5-(OCH3)3) could be considered as promising leads for stirred at room temperature for 12 h. The mixture was
further development of more potent anticancer agents.
neutralized with 20% Na2CO3. The content was poured onto
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crushed ice, ltered and washed with water. No further puri- [M+] : [M + 2]+ (65/23), 367 (98), 353 (92), 296 (36), 176 (42), 145
cation was required. (45), 57 (38), 43 (100). Anal. calcd C19H19ClN6O2: C, 57.22; H,
4.2.3.1. 1-(4-((4-Chloro-6-((4-chlorophenyl)amino)-1,3,5-triazin- 4.80; N, 21.07; found: C, 57.19; H, 4.83; N, 21.10.
2-yl)amino)phenyl)ethan-1-one (4). White solid; 80% yield; mp 4.2.6. General procedure for synthesis of 1-(4-((4-((2-
258–260 ꢁC. FT-IR (ATR) n (cmꢀ1) 3267 (N–H), 3101 (]C–H), 1666 hydroxyethyl)amino)-6-morpholino-1,3,5-triazin-2-yl)amino)
(C]O), 1566 and 1512 (C]N and C]C). 1H-NMR (400 MHz, phenyl)chalcones (7a–g). A mixture of ketone 5 (1 equiv.), the
DMSO-d6) d ppm 2.54 (s, 3H, CH3), 7.42 (d, J ¼ 8.8 Hz, 2H, Ar–H), respective benzaldehyde (1.2 equiv.) and potassium hydroxide
7.62–7.89 (m, 4H, Ar–H), 7.93 (d, J ¼ 8.8 Hz, 2H, Ar–H), 10.48 (bs, (100 mg) in ethanol (2.5 mL) was stirred at room temperature
2H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 26.5 (CH3), 119.8 for 5 h. The solid formed was ltered and washed with ethanol.
(CH), 122.5 (CH), 127.4 (Cq), 128.5 (CH), 129.2 (CH), 131.6 (Cq), No further purication was required.
137.3 (Cq), 143.0 (Cq), 163.8 (Cq), 168.3 (Cq), 168.4 (Cq), 196.5
4.2.6.1. (E)-1-(4-((4-((2-Hydroxyethyl)amino)-6-morpholino-1,3,5-
(Cq). MS (70 eV) m/z (%): 373 : 375 : 377 [M+] : [M + 2]+ : [M + 4]+ triazin-2-yl)amino)phenyl)-3-phenylprop-2-en-1-one (7a). Yellow
(90/59/11), 358 (75), 222 (10), 179 (16), 145 (100), 90 (27). Anal. solid. 75% yield; mp 175–177 ꢁC. FT-IR (ATR): n (cmꢀ1) 3400 (N–H),
calcd C17H13Cl2N5O: C, 54.56; H, 3.50; N, 18.71; found: C, 54.01; 3315 (O–H), 3120 (]C–H), 1657 (C]O), 1576 y 1533 (C]N and
1
H, 3.55; N, 18.74.
C]C). H-NMR (400 MHz, DMSO-d6) d ppm 3.31–3.47 (m, 2H,
4.2.4. General procedure for synthesis of 1-(4-((4-((2- CH2), 3.49–3.57 (m, 2H, CH2), 3.59–3.67 (m, 4H, CH2), 3.68–3.76
hydroxyethyl)amino)-6-morpholino-1,3,5-triazin-2-yl)amino)
(m, 4H, CH2), 4.76 (s, 1H, OH), 6.99 (t, J ¼ 5.0 Hz, 1H, NH), 7.42–
phenyl)ethan-1-one (5). A mixture of disubstituted triazine 3 7.48 (m, 3H, Ar–H), 7.70 (d, J ¼ 15.6 Hz, 1H, CH), 7.87 (d, J ¼
(0.006 mol, 1 equiv.) and ethanolamine (0.018 mol, 3 equiv.) in 4.8 Hz, 2H, Ar–H), 7.91–8.01 (m, 3H, Ar–H, CH), 8.11 (t, J ¼ 10.0 Hz,
dioxane (20 mL) was stirred at reux for 8 h. The mixture was 2H, Ar–H), 9.53 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm
neutralized with 20% Na2CO3. The content was poured onto 43.0 (CH2), 43.4 (CH2), 60.0 (CH2), 66.1 (CH2), 114.6 (Cq), 118.4
crushed ice, ltered and washed with water. No further purica- (CH), 122.1 (CH), 128.8 (CH), 128.9 (CH), 129.8 (Cq), 129.9 (CH),
tion was required.
4.2.4.1. 1-(4-((4-((2-Hydroxyethyl)amino)-6-morpholino-1,3,5-
130.4 (CH), 134.9 (Cq), 142.9 (CH), 164.1 (Cq), 164.8 (Cq), 165.6
(Cq), 187.3 (Cq). MS (70 eV) m/z (%): 446 [M+] (19), 416 (17), 401
triazin-2-yl)amino)phenyl)ethan-1-one (5). White solid. 76% yield; (12), 131 (27), 103 (37), 69 (60), 55 (86). Anal. calcd C24H26N6O3: C,
mp 191–193 ꢁC. FT-IR (ATR): n (cmꢀ1) 3350 (N–H), 3279 (O–H), 64.56; H, 5.87; N, 18.82; found: C, 64.60; H, 5.90; N, 18.79.
3093 (]C–H), 1668 (C]O), 1583 and 1537 (C]N and C]C).
4.2.6.2. (E)-1-(4-((4-((2-Hydroxyethyl)amino)-6-morpholino-1,3,5-
1H-NMR (400 MHz, DMSO-d6) d ppm 2.52 (s, 3H, CH3), 3.32– triazin-2-yl)amino)phenyl)-3-(p-tolyl)prop-2-en-1-one (7b). Yellow
3.42 (m, 2H, CH2), 3.50–3.57 (m, 2H, CH2), 3.57–3.63 (m, 4H, solid. 68% yield; mp 185–191 ꢁC. FT-IR (ATR): n (cmꢀ1) 3419 (N–H),
CH2), 3.66–3.74 (m, 4H, CH2), 4.80 (s, 1H, OH), 6.88 (bs, 1H, 3291 (O–H), 3110 (]C–H), 1651 (C]O), 1585 y 1537 (C]N and
NH), 7.83–7.90 (m, 4H, Ar–H), 9.39 (bs, 1H, NH). 13C-NMR (100 C]C). H-NMR (400 MHz, DMSO-d6) d ppm 2.35 (s, 3H, CH3),
1
MHz, DMSO-d6) d ppm 26.5 (CH3), 43.0 (CH2), 43.6 (CH2), 60.2 3.26–3.47 (m, 2H, CH2), 3.54 (q, J ¼ 6.1 Hz, 2H, CH2), 3.59–3.66 (m,
(CH2), 66.2 (CH2), 118.6 (CH), 129.5 (CH), 130.1 (Cq), 145.4 (Cq), 4H, CH2), 3.67–3.75 (m, 4H, CH2), 4.74 (s, 1H, OH), 6.98 (t, J ¼
165.0 (Cq), 165.9 (Cq), 166.1 (Cq), 196.9 (Cq). MS (70 eV) m/z (%): 5.6 Hz, 1H, NH), 7.26 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.67 (d, J ¼ 15.2 Hz,
358 [M+] (32), 328 (59), 313 (43), 69 (36), 55 (54), 43 (100). Anal. 1H, CH), 7.77 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.85–7.98 (m, 3H, Ar–H,
calcd C17H22N6O3: C, 56.97; H, 6.19; N, 23.45; found: C, 57.00; CH), 8.10 (t, J ¼ 9.8 Hz, 2H, Ar–H), 9.51 (bs, 1H, NH). 13C-NMR (100
H, 6.25; N, 23.60.
MHz, DMSO-d6) d ppm 21.1 (CH3), 42.9 (CH2), 43.4 (CH2), 59.9
4.2.5. General procedure for synthesis of 1-(4-((4-((4- (CH2), 66.1 (CH2), 118.4 (CH), 121.0 (CH), 128.8 (CH), 128.9 (Cq),
chlorophenyl)amino)-6-((2-hydroxyethyl)amino)-1,3,5-triazin-2- 129.2 (Cq), 129.6 (CH), 129.8 (CH), 132.2 (Cq), 140.4 (Cq), 143.0
yl)amino)phenyl)ethan-1-one (6). A mixture of disubstituted (CH), 164.6 (Cq), 164.8 (Cq), 165.9 (Cq), 187.3 (Cq). MS (70 eV) m/z
triazine 4 (0.005 mol, 1 equiv.) and ethanolamine (0.016 mol, 3 (%): 460 [M+] (12), 430 (10), 145 (23), 117 (15), 69 (58), 55 (78), 43
equiv.) in dioxane (20 mL) was stirred at reux for 8 h. The (100). Anal. calcd C25H28N6O3: C, 65.20; H, 6.13; N, 18.25; found: C,
mixture was neutralized with 20% Na2CO3. The content was 65.18; H, 6.18; N, 18.19.
poured onto crushed ice, ltered and washed with water. No
further purication was required.
4.2.5.1. 1-(4-((4-((4-Chlorophenyl)amino)-6-((2-hydroxyethyl)
4.2.6.3. (E)-1-(4-((4-((2-Hydroxyethyl)amino)-6-morpholino-1,3,5-
triazin-2-yl)amino)phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (7c).
Yellow solid. 83% yield; mp 179–183 ꢁC. FT-IR (ATR): n (cmꢀ1) 3479
amino)-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-one (6). White (N–H), 3277 (O–H), 3201 (]C–H), 1649 (C]O), 1571 y 1506 (C]N
solid. 78% yield; mp 160–163 ꢁC. FT-IR (ATR): n (cmꢀ1) 3398 (N– and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 3.38–3.41 (m, 2H,
H), 3284 (O–H), 3113 (]C–H), 1666 (C]O), 1589 y 1566 (C]N CH2), 3.53 (q, J ¼ 6.2 Hz, 2H, CH2), 3.60–3.67 (m, 4H, CH2), 3.67–
and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 2.54 (s, 3H, 3.75 (m, 4H, CH2), 3.81 (s, 3H, OCH3), 4.76 (s, 1H, OH), 6.94–7.04
CH3), 3.45 (bs, 2H, CH2), 3.59 (t, J ¼ 5.6 Hz, 2H, CH2), 4.12 (s, (m, 3H, NH, Ar–H), 7.67 (d, J ¼ 15.6 Hz, 1H, CH), 7.77–7.86 (m, 3H,
1H, OH), 7.39 (d, J ¼ 8.2 Hz, 2H, Ar–H), 7.75 (d, J ¼ 8.2 Hz, 2H, Ar–H, CH), 7.94 (d, J ¼ 7.2 Hz, 2H, Ar–H), 8.09 (t, J ¼ 9.8 Hz, 2H,
Ar–H), 7.84–7.94 (m, 4H, Ar–H), 8.24 (bs, 1H, NH), 10.15–10.55 Ar–H), 9.50 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm
(m, 2H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 26.5 (CH3), 43.0 (CH2), 43.3 (CH2), 55.4 (CH3), 59.9 (CH2), 66.0 (CH2), 114.4
43.3 (CH2), 59.4 (CH2), 119.9 (CH), 122.4 (CH), 122.9 (Cq), 128.5 (CH), 118.4 (CH), 119.6 (CH), 127.5 (Cq), 129.6 (CH), 130.6 (CH),
(CH), 129.2 (CH), 131.4 (Cq), 131.5 (Cq), 143.2 (Cq), 158.7 (Cq), 142.8 (CH), 161.2 (Cq), 164.1 (Cq), 164.2 (Cq), 164.8 (Cq), 165.9
161.0 (Cq), 161.2 (Cq), 196.5 (Cq). MS (70 eV) m/z (%): 398 : 400 (Cq), 165.9 (Cq), 187.2 (Cq). MS (70 eV) m/z (%): 476 [M+] (6), 446
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(5), 161 (7), 98 (12), 83 (19), 69 (40), 55 (61), 43 (100). Anal. calcd (cmꢀ1) 3415 (N–H), 3277 (O–H), 3211 (]C–H), 1651 (C]O), 1577 y
C25H28N6O4: C, 63.01; H, 5.92; N, 17.64; found: C, 62.99; H, 5.93; N, 1535 (C]N and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 3.42–
17.60.
3.20 (m, 2H, CH2), 3.48–3.56 (m, 2H, CH2), 3.59–3.66 (m, 4H, CH2),
4.2.6.4. (E)-1-(4-((4-((2-Hydroxyethyl)amino)-6-morpholino-1,3,5- 3.69–3.76 (m, 4H, CH2), 4.80 (s, 1H, OH), 7.04 (bs, 1H, NH), 7.75 (d,
triazin-2-yl)amino)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(7d). Yellow solid. 87% yield; mp 218–224 ꢁC. FT-IR (ATR): n (cmꢀ1
J ¼ 14.0 Hz, 1H, CH), 7.80 (d, J ¼ 8.4 Hz, 2H, Ar–H), 8.02–7.92 (m,
2H, Ar–H), 8.19–8.07 (m, 5H, Ar–H, CH), 9.58 (bs, 1H, NH). 13C-
)
3354 (N–H), 3267 (O–H), 3100 (]C–H), 1649 (C]O), 1587 y 1537 NMR (100 MHz, DMSO-d6) d ppm 43.0 (CH2), 43.4 (CH2), 59.9
(C]N and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 3.36–3.40 (CH2), 66.1 (CH2), 118.5 (CH), 124.1 (q, 1JCF ¼ 245.9 Hz, CF3), 124.9
(m, 2H, CH2), 3.53 (q, J ¼ 5.7 Hz, 2H CH2), 3.60–3.67 (m, 4H, CH2), (CH), 125.8 (CH), 129.5 (q, 3JCF ¼ 16.7 Hz, (CH)), 130.1 (CH), 139.0
3.68–3.75 (m, 7H, CH2, OCH3), 3.87 (s, 6H, OCH3), 4.73 (s, 1H, OH), (Cq), 139.7 (Cq), 141.0 (CH), 145.8 (Cq), 146.2 (Cq), 164.0 (Cq),
6.98 (t, J ¼ 6.0 Hz, 1H, NH), 7.2 (s, 2H, Ar–H), 7.66 (d, J ¼ 15.2 Hz, 164.2 (Cq), 165.3 (q, 2JCF ¼ 24.6 Hz, Cq), 187.3 (Cq). MS (70 eV) m/z
1H, CH), 7.82–8.02 (m, 3H, Ar–H, CH), 8.13 (t, J ¼ 9.6 Hz, 2H, Ar– (%): 514 (6), 484 (8), 469 (6), 199 (8), 145 (13), 69 (58), 55 (83). Anal.
H), 9.52 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 42.9 calcd C25H25F3N6O3: C, 58.36; H, 4.90; N, 16.33; found: C, 58.40; H,
(CH2), 43.4 (CH2), 56.2 (CH3), 59.8 (CH2), 60.2 (CH3), 66.1 (CH2), 5.00; N, 16.30.
106.4 (CH), 118.4 (CH), 121.2 (CH), 129.7 (CH), 129.8 (CH), 130.5
4.2.7. General procedure for synthesis of 1-(4-((4-((4-
(Cq), 139.5 (Cq), 143.3 (Cq), 145.4 (Cq), 153.1 (Cq), 165.6 (Cq), chlorophenyl)amino)-6-((2-hydroxyethyl)amino)-1,3,5-triazin-2-
165.7 (Cq), 165.8 (Cq), 187.2 (Cq). MS (70 eV) m/z (%): 536 [M+] (15), yl)amino)phenyl)chalcones (8a–g). A mixture of ketone 6 (1
505 (10), 491 (8), 475 (6), 461 (11), 145 (18), 69 (58), 55 (71). Anal. equiv.), the respective benzaldehyde (1.2 equiv.) and potassium
calcd C27H32N6O6: C, 60.47; H, 5.98; N, 15.68; found: C, 60.44; H, hydroxide (100 mg) in ethanol (2.5 mL) was stirred at room
6.01; N, 15.66.
temperature for 5 h. The solid formed was ltered and washed
with ethanol. No further purication was required.
4.2.7.1. (E)-1-(4-((4-((4-Chlorophenyl)amino)-6-((2-hydroxyethyl)
4.2.6.5. (E)-3-(4-Chlorophenyl)-1-(4-((4-((2-hydroxyethyl)amino)-
6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)prop-2-en-1-one (7e).
Yellow solid. 92% yield; mp 200–205 ꢁC. FT-IR (ATR): n (cmꢀ1) 3400 amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-phenylprop-2-en-1-one (8a).
(N–H), 3294 (O–H), 3100 (]C–H), 1657 (C]O), 1572 y 1541 (C]N Yellow solid. 88% yield; mp 192–206 ꢁC. FT-IR (ATR): n (cmꢀ1) 3400
and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 3.38–3.40 (m, 2H, (N–H), 3292 (O–H), 3119 (]C–H), 1637 (C]O), 1572 y 1558 (C]N
CH2), 3.53 (q, J ¼ 6.3 Hz, 2H, CH2), 3.60–3.66 (m, 4H, CH2), 3.76– and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 3.36–3.48 (m, 2H,
3.67 (m, 4H, CH2), 4.76 (s, 1H, OH), 7.00 (t, J ¼ 5.9 Hz, 1H, NH), CH2), 3.53–3.63 (m, 2H, CH2), 4.78 (s, 1H, OH), 7.17 (bs, 1H, NH),
7.52 (d, J ¼ 8.4 Hz, 2H, Ar–H), 7.68 (d, J ¼ 15.6 Hz, 1H, CH), 7.89– 7.28–7.34 (m, 2H, Ar–H), 7.46 (d, J ¼ 5.2 Hz, 2H, Ar–H), 7.71 (d, J ¼
7.99 (m, 5H, Ar–H, CH), 8.11 (t, J ¼ 10.0 Hz, 2H, Ar–H), 9.54 (bs, 15.6 Hz, 1H, CH), 7.81–7.89 (m, 4H, Ar–H, CH), 7.90–7.98 (m, 2H,
1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 42.9 (CH2), 43.4 Ar–H), 8.03 (d, J ¼ 8.4 Hz, 2H, Ar–H), 8.11 (t, J ¼ 8.4 Hz, 2H, Ar–H),
(CH2), 60.0 (CH2), 66.0 (CH2), 118.4 (CH), 122.9 (Cq), 129.0 (CH), 9.33 (bs, 1H, NH), 9.57 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-
129.8 (CH), 129.9 (CH), 130.5 (CH), 130.9 (Cq), 133.9 (Cq), 134.9 d6) d ppm 43.0 (CH2), 59.8 (CH2), 118.8 (CH), 121.4 (CH), 122.2
(Cq), 141.4 (CH), 164.1 (Cq), 164.6 (Cq), 165.9 (Cq), 187.1 (Cq). MS (CH), 125.3 (Cq), 125.4 (Cq), 128.2 (CH), 128.7 (CH), 128.9 (CH),
(70 eV) m/z (%): 480 : 482 [M+] : [M + 2]+ (12/4), 449 (14), 437 (12), 129.6 (CH), 130.4 (CH), 134.0 (Cq), 134.9 (Cq), 139.2 (Cq), 142.9
165 (16), 145 (16), 137 (18), 102 (17), 69 (61), 55 (92). Anal. calcd (CH), 163.8 (Cq), 164.0 (Cq), 165.7 (Cq), 187.4 (Cq). MS (70 eV) m/z
C24H25ClN6O3: C, 59.94; H, 5.24; N, 17.47; found: C, 60.01; H, (%): 486 : 488 [M+] : [M + 2]+ (50/24), 456 (49), 443 (43), 178 (48),
5.19; N, 17.53.
145 (44), 131 (65), 103 (100), 77 (39). Anal. calcd C26H23ClN6O2: C,
64.13; H, 4.76; N, 17.26; found: C, 64.17; H, 4.80; N, 17.23.
4.2.7.2. (E)-1-(4-((4-((4-Chlorophenyl)amino)-6-((2-hydroxyethyl)
4.2.6.6. (E)-3-(4-Fluorophenyl)-1-(4-((4-((2-hydroxyethyl)amino)-
6-morpholino-1,3,5-triazin-2-yl)amino)phenyl)prop-2-en-1-one
Yellow solid. 80% yield; mp 198–204 ꢁC. FT-IR (ATR): n (cmꢀ1) 3400 amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(p-tolyl)prop-2-en-1-one
(7f).
(N–H), 3278 (O–H), 3100 (]C–H), 1649 (C]O), 1579 y 1541 (C]N (8b). Yellow solid. 85% yield; mp 219–224 ꢁC. FT-IR (ATR): n (cmꢀ1
and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 3.31–3.38 (m, 2H, 3543 (N–H), 3294 (O–H), 3119 (]C–H), 1616 (C]O), 1583 y 1531
CH2), 3.48–3.58 (m, 2H, CH2), 3.60–3.67 (m, 4H, CH2), 3.68–3.76 (C]N and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 2.35 (s, 3H,
(m, 4H, CH2), 4.77 (s, 1H, OH), 7.00 (t, J ¼ 4.8 Hz, 1H, NH), 7.30 (t, CH3), 3.41–3.48 (m, 2H, CH2), 3.54–3.62 (m, 2H, CH2), 4.70 (bs, 1H,
JHF ¼ 8.8 Hz, 2H, Ar–H), 7.70 (d, J ¼ 15.6 Hz, 1H, CH), 7.84–8.01 OH), 7.13 (bs, 1H, NH), 7.35–7.22 (m, 4H, Ar–H), 7.68 (d, J ¼
(m, 5H, Ar–H, CH), 8.11 (t, J ¼ 10.2 Hz, 2H, Ar–H), 9.53 (bs, 1H, 15.6 Hz, 1H, CH), 7.76 (d, J ¼ 7.6 Hz, 2H, Ar–H), 7.81–7.91 (m, 3H,
NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 42.9 (CH2), 43.4 (CH2), Ar–H, CH), 8.00 (d, J ¼ 8.4 Hz, 2H, Ar–H), 8.09 (t, J ¼ 7.2 Hz, 2H,
60.1 (CH2), 66.0 (CH2), 115.9 (d, 2JCF ¼ 22.0 Hz, (CH)), 118.4 (CH), Ar–H), not observed NH. 13C-NMR (100 MHz, DMSO-d6) d ppm
122.1 (CH), 129.8 (CH), 131.1 (d, 3JCF ¼ 9.0 Hz, (CH)), 131.6 (d, 4JCF 21.1 (CH3), 43.1 (CH2), 59.9 (CH2), 118.8 (CH), 121.2 (CH), 121.4
¼ 3.1 Hz, Cq), 139.2 (Cq), 141.6 (CH), 162.1 (Cq), 164.6 (Cq), 165.7 (CH), 125.3 (Cq), 128.2 (CH), 128.8 (CH), 129.6 (CH), 129.6 (CH),
(Cq), 167.0 (Cq), 173.8 (d, 1JC–F ¼ 241.6 Hz, Cq), 187.2 (Cq). MS (70 130.7 (Cq), 132.2 (Cq), 139.3 (Cq), 140.4 (Cq), 143.0 (CH), 145.4
eV) m/z (%): 464 (1), 256 (1), 185 (1), 97 (8), 73 (33), 69 (57), 55 (56). (Cq), 163.9 (Cq), 164.1 (Cq), 165.8 (Cq), 187.4 (Cq). MS (70 eV) m/z
Anal. calcd. C24H25FN6O3: C, 62.06; H, 5.43; N, 18.09; found: C, (%): 500 : 502 [M+] : [M + 2]+ (5/3), 367 (10), 353 (9), 149 (12), 119
)
62.10; H, 5.38; N, 18.09.
(22), 91 (33), 81 (31). Anal. calcd C27H25ClN6O2: C, 64.73; H, 5.03; N,
4.2.6.7. (E)-1-(4-((4-((2-Hydroxyethyl)amino)-6-morpholino-1,3,5- 16.78; found: C, 64.70; H, 4.99; N, 16.80.
triazin-2-yl)amino)phenyl)-3-(4-(triuoromethyl)phenyl)prop-2-en-1-
one (7g). Yellow solid. 76% yield; mp 227–230 C. FT-IR (ATR): n amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(4-methoxyphenyl)prop-2-
4.2.7.3. (E)-1-(4-((4-((4-Chlorophenyl)amino)-6-((2-hydroxyethyl)
ꢁ
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1529 (C]N and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 3.38–
3.52 (m, 2H, CH2), 3.52–3.64 (m, 2H, CH2), 4.74 (s, 1H, OH), 7.17
(bs, 1H, NH), 7.26–7.36 (m, 4H, Ar–H), 7.71 (d, J ¼ 15.6 Hz, 1H,
CH), 7.82–7.88 (m, 2H, Ar–H), 7.88–7.99 (m, 3H, Ar–H, CH), 8.03 (d,
J ¼ 8.4 Hz, 2H, Ar–H), 8.12 (t, J ¼ 8.8 Hz, 2H, Ar–H), 9.33 (bs, 1H,
NH), 9.57 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 43.0
(CH2), 59.9 (CH2), 115.9 (d, 2JCF ¼ 21.8 Hz, (CH)), 118.7 (CH), 121.4
(CH), 122.1 (CH), 125.3 (Cq), 128.2 (CH), 129.7 (CH), 130.6 (Cq),
en-1-one (8c). Yellow solid. 75% yield; mp 210–214 ꢁC. FT-IR (ATR):
n (cmꢀ1) 3400 (N–H), 3277 (O–H), 3117 (]C–H), 1637 (C]O), 1576
y 1558 (C]N and C]C). H-NMR (400 MHz, DMSO-d6) d ppm
1
3.38–3.46 (m, 2H, CH2), 3.54–3.62 (m, 2H, CH2), 3.82 (s, 3H, OCH3),
4.78 (bs, 1H, OH), 7.01 (d, J ¼ 8.4 Hz, 2H, Ar–H), 7.16 (bs, 1H, NH),
7.31 (t, J ¼ 6.0 Hz, 2H, Ar–H), 7.68 (d, J ¼ 15.6 Hz, 1H, CH), 7.81–
7.89 (m, 5H, Ar–H, CH), 8.01 (d, J ¼ 8.4 Hz, 2H, Ar–H), 8.09 (t, J ¼
8.2 Hz, 2H, Ar–H), 9.32 (bs, 1H, NH), 9.55 (bs, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6) d ppm 43.0 (CH2), 55.4 (CH3), 59.9 (CH2),
114.4 (CH), 118.8 (CH), 119.7 (CH), 121.4 (CH), 125.3 (Cq), 127.5
(Cq), 128.2 (CH), 129.5 (CH), 130.6 (CH), 130.9 (Cq), 139.3 (Cq),
142.9 (CH), 145.0 (Cq), 161.2 (Cq), 163.9 (Cq), 164.1 (Cq), 165.8
(Cq), 187.3 (Cq). MS (70 eV) m/z (%): 516 : 518 [M+] : [M + 2]+ (100/
50), 486 (57), 473 (50), 356 (52), 236 (39), 161 (20), 121 (17), 77 (20).
Anal. calcd C27H25ClN6O3: C, 62.73; H, 4.87; N, 16.26; found: C,
62.70; H, 4.90; N, 16.29.
4.2.7.4. (E)-1-(4-((4-((4-Chlorophenyl)amino)-6-((2-hydroxyethyl)
amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(3,4,5-trimethoxyphenyl)
prop-2-en-1-one (8d). Yellow solid. 91% yield; mp 242–246 ꢁC. FT-
IR (ATR): n (cmꢀ1) 3564 (N–H), 3290 (O–H), 3098 (]C–H), 1649
(C]O), 1560 y 1516 (C]N and C]C). 1H-NMR (400 MHz, DMSO-
d6) d ppm 3.65–3.52 (m, 4H), 3.72 (s, 3H, OCH3), 3.87 (s, 6H,
OCH3), 4.79 (bs, 1H, OH), 7.16 (bs, 1H, NH), 7.22 (s, 2H, Ar–H),
7.31 (t, J ¼ 6.0 Hz, 2H, Ar–H), 7.66 (d, J ¼ 15.6 Hz, 1H, CH), 7.93–
7.76 (m, 3H, Ar–H), 8.03 (d, J ¼ 7.6 Hz, 2H, Ar–H), 8.13 (t, J ¼
8.2 Hz, 2H, Ar–H), 9.32 (bs, 1H, NH), 9.57 (bs, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6) d ppm 43.0 (CH2), 56.2 (CH3), 59.9 (CH2),
60.2 (CH3), 106.4 (CH), 107.1 (CH), 118.8 (CH), 121.5 (CH), 125.3
(Cq), 128.2 (CH), 129.7 (CH), 130.5 (Cq), 130.8 (Cq), 139.2 (Cq),
139.6 (Cq), 143.4 (CH), 152.4 (Cq), 153.2 (Cq), 163.9 (Cq), 164.1
(Cq), 165.8 (Cq), 187.4 (Cq). MS (70 eV) m/z (%): 576 : 578
[M+] : [M + 2]+ (94/46), 561 (25), 545 (27), 501 (21), 356 (21), 195
(44), 98 (40), 84 (96). Anal. calcd C29H29ClN6O5: C, 60.36; H,
5.07; N, 14.56; found: C, 60.40; H, 5.03; N, 14.49.
3
4
131.1 (d, JCF ¼ 8.6 Hz, (CH)), 131.6 (d, JCF ¼ 2.9 Hz, Cq), 139.2
(Cq), 141.7 (CH), 145.2 (Cq), 163.1 (d, 1JCF ¼ 201.6 Hz, Cq), 163.8
(Cq), 164.5 (Cq), 165.7 (Cq), 187.3 (Cq). MS (70 eV) m/z (%):
504 : 506 [M+] : [M + 2]+ (74/32), 474 (65), 461 (60), 313 (71), 264
(81), 236 (100), 98 (78), 84 (67). Anal. calcd C26H22ClFN6O2: C,
61.84; H, 4.39; N, 16.64; found: C, 61.86; H, 4.42; N, 16.70.
4.2.7.7. (E)-1-(4-((4-((4-Chlorophenyl)amino)-6-((2-hydroxyethyl)
amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(4-(triuoromethyl)phenyl)
prop-2-en-1-one (8g). Yellow solid. 83% yield; mp 235–240 ꢁC. FT-IR
(ATR): n (cmꢀ1) 3441 (N–H), 3281 (O–H), 3119 (]C–H), 1643 (C]
1
O), 1574 y 1528 (C]N and C]C). H-NMR (400 MHz, DMSO-d6)
d ppm 3.38–3.49 (m, 2H, CH2), 3.53–3.63 (m, 2H, CH2), 4.74 (s, 1H,
OH), 7.18 (bs, 1H, NH), 7.25–7.37 (m, 2H, Ar–H), 7.76 (d, J ¼
16.0 Hz, 1H, CH), 7.81 (d, J ¼ 8.0 Hz, 2H, Ar–H), 7.83–7.89 (m, 2H,
Ar–H), 7.98–8.19 (m, 7H, Ar–H), 9.33 (bs, 1H, NH), 9.59 (bs, 1H,
NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 43.0 (CH2), 59.8 (CH2),
118.7 (CH), 121.3 (CH), 124.1 (q, 1JCF ¼ 272.2 Hz, CF3), 124.9 (CH),
4
125.3 (Cq), 125.4 (Cq), 125.7 (q, JCF ¼ 3.0 Hz, (CH)), 128.2 (CH),
129.3 (CH), 129.8 (q, 3JCF ¼ 18.6 Hz, (CH)), 130.3 (Cq), 138.9 (Cq),
2
139.2 (Cq), 139.2 (Cq), 140.9 (CH), 145.4 (Cq), 163.9 (q, JCF
¼
23.2 Hz, Cq), 165.7 (Cq), 187.2 (Cq). MS (70 eV) m/z (%): 554 : 556
[M+] : [M + 2]+ (100/37), 535 (12), 524 (93), 511 (93), 452 (15), 199 (9),
127 (12), 98 (9). Anal. calcd C27H22ClF3N6O2: C, 58.44; H, 4.00; N,
15.14; found: C, 58.39; H, 4.09; N, 15.10.
4.2.8. General procedure for the synthesis of 2-((4-((4-(1-
(3,5-dichlorophenyl)-5-(aryl)-4,5-dihydro-1H-pyrazol-3-yl)
phenyl)amino)-6-morpholino-1,3,5-triazin-2-yl)amino)ethanol
(9a–g). A mixture of chalcone 7 (1 equiv.) and 3,5-dichlor-
ophenylhydrazine hydrochloride (3 equiv.) in ethanol (2.5 mL)
was subjected to reux for 12 h. The solid formed was ltered
and washed with ethanol. No further purication was required.
4.2.8.1. 2-((4-((4-(1-(3,5-Dichlorophenyl)-5-phenyl-4,5-dihydro-
1H-pyrazol-3-yl)phenyl)amino)-6-morpholino-1,3,5-triazin-2-yl)
amino)ethan-1-ol (9a). Yellow solid. 78% yield; mp 279–281 ꢁC. FT-
IR (ATR): n (cmꢀ1) 3416 (N–H), 3202 (O–H), 3090 (]C–H), 1626 y
1599 (C]N and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 3.16
(dd, J ¼ 17.4, 4.6 Hz, 1H, H-4), 3.38–3.49 (m, 2H, CH2), 3.52–3.60
(m, 2H, CH2), 3.64–3.72 (m, 5H, CH2, OH), 3.74–3.84 (m, 4H, CH2),
3.94 (dd, J ¼ 17.4, 11.8 Hz, 1H, H-4), 5.59 (dd, J ¼ 11.8, 4.2 Hz, 1H,
H-5), 6.80 (s, 1H, Ar–H), 6.93 (s, 2H, Ar–H), 7.23–7.31 (m, 3H, Ar–H),
7.32–7.40 (m, 2H, Ar–H), 7.63–7.82 (m, 4H, Ar–H), 8.30 (bs, 1H,
NH), 10.59 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 43.2
(CH2), 44.1 (CH2), 44.3 (CH2), 59.3 (CH2), 62.4 (CH), 65.7 (CH2),
110.8 (CH), 116.8 (Cq), 116.9 (CH), 120.3 (CH), 121.0 (Cq), 125.7
(CH), 126.8 (Cq), 126.9 (CH), 127.8 (CH), 129.2 (CH), 134.4 (Cq),
141.4 (Cq), 145.1 (Cq), 145.7 (Cq), 148.0 (Cq), 149.7 (Cq). MS (70 eV)
m/z (%): 604 : 606 : 608 [M+] : [M + 2]+ : [M + 4]+ (100/70/15), 588 (4),
574 (9), 527 (8), 310 (10), 287 (8), 252 (8). Anal. calcd C30H30Cl2N8O2:
C, 59.51; H, 4.99; N, 18.51; found: C, 60.00; H, 4.89; N, 18.53.
4.2.7.5. (E)-3-(4-Chlorophenyl)-1-(4-((4-((4-chlorophenyl)
amino)-6-((2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)
prop-2-en-1-one (8e). Yellow solid. 93% yield; mp 200–204 ꢁC. FT-
IR (ATR): n (cmꢀ1) 3400 (N–H), 3279 (O–H), 3117 (]C–H), 1641
(C]O), 1577 y 1529 (C]N and C]C). 1H-NMR (400 MHz, DMSO-
d6) d ppm 3.38–3.47 (m, 2H, CH2), 3.54–3.61 (m, 2H, CH2), 4.77 (s,
1H, OH), 7.17 (bs, 1H, NH), 7.31 (t, J ¼ 7.5 Hz, 2H, Ar–H), 7.51 (d, J
¼ 8.4 Hz, 2H, Ar–H), 7.68 (d, J ¼ 15.6 Hz, 1H, CH), 7.82–7.89 (m,
2H, Ar–H), 7.92 (d, J ¼ 8.4 Hz, 2H, Ar–H), 7.94–8.05 (m, 3H, Ar–H,
CH), 8.11 (t, J ¼ 8.8 Hz, 2H, Ar–H), 9.41 (bs, 2H, NH). 13C-NMR
(100 MHz, DMSO-d6) d ppm 43.0 (CH2), 59.9 (CH2), 118.7 (CH),
121.4 (CH), 123.0 (CH), 125.3 (Cq), 128.1 (CH), 128.9 (CH), 129.7
(CH), 130.5 (CH), 133.9 (Cq), 134.8 (Cq), 139.2 (Cq), 141.4 (CH),
143.6 (Cq), 145.3 (Cq), 163.8 (Cq), 164.0 (Cq), 165.8 (Cq), 187.2
(Cq). MS (70 eV) m/z (%): 520 : 522 : 524 [M+] : [M + 2]+ : [M + 4]+
(20/14/4), 491 (19), 477 (23), 475 (15), 367 (11), 339 (12), 178 (20),
145 (25), 135 (32). Anal. calcd C26H22Cl2N6O2: C, 59.89; H, 4.25; N,
16.12; found: C, 60.01; H, 4.25; N, 16.16.
4.2.7.6. (E)-1-(4-((4-((4-Chlorophenyl)amino)-6-((2-hydroxyethyl)
amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(4-uorophenyl)prop-2-en-
1-one (8f). Yellow solid. 85% yield; mp 196–200 ꢁC. FT-IR (ATR): n
(cmꢀ1) 3412 (N–H), 3279 (O–H), 3117 (]C–H), 1641 (C]O), 1576 y
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4.2.8.2. 2-((4-((4-(1-(3,5-Dichlorophenyl)-5-(p-tolyl)-4,5-dihydro-
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[M+] : [M + 2]+ : [M + 4]+ (100/72/15), 677 (6), 527 (49), 310 (15), 280
(7), 252 (14), 179 (15), 83 (27). Anal. calcd C33H36Cl2N8O5: C,
56.98; H, 5.22; N, 16.11; found: C, 57.01; H, 5.20; N, 16.15.
4.2.8.5. 2-((4-((4-(5-(4-Chlorophenyl)-1-(3,5-dichlorophenyl)-4,5-
dihydro-1H-pyrazol-3-yl)phenyl)amino)-6-morpholino-1,3,5-triazin-2-
1H-pyrazol-3-yl)phenyl)amino)-6-morpholino-1,3,5-triazin-2-yl)
amino)ethan-1-ol (9b). Yellow solid. 83% yield; mp 262–265 ꢁC.
FT-IR (ATR): n (cmꢀ1) 3454 (N–H), 3248 (O–H), 3088 (]C–H),
1626 y 1599 (C]N and C]C). 1H-NMR (400 MHz, DMSO-d6)
d ppm 2.24 (s, 3H, CH3), 3.10 (dd, J ¼ 17.2, 3.8 Hz, 1H, H-4), 3.38–
3.49 (m, 2H, CH2), 3.50–3.72 (m, 7H, CH2, OH), 3.82–3.74 (m, 4H,
CH2), 3.89 (dd, J ¼ 17.2, 11.6 Hz, 1H, H-4), 5.51 (dd, J ¼ 11.6,
3.8 Hz, 1H, H-5), 6.77 (s, 1H, Ar–H), 6.91 (s, 2H, Ar–H), 7.18–7.09
(m, 4H, Ar–H), 7.62–7.80 (m, 4H, Ar–H), 8.36 (bs, 1H, NH), 10.65
(bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 20.7 (CH3),
43.1 (CH2), 43.3 (CH2), 44.4 (CH2), 59.1 (CH2), 62.3 (CH), 65.8
(CH2), 110.9 (CH), 116.9 (CH), 116.9 (Cq), 120.5 (CH), 121.2 (Cq),
125.8 (CH), 127.0 (CH), 127.3 (Cq), 129.8 (CH), 134.5 (Cq), 137.1
(Cq), 138.5 (Cq), 139.6 (Cq), 145.8 (Cq), 148.8 (Cq), 149.8 (Cq). MS
(70 eV) m/z (%): 618 : 620 : 622 [M+] : [M + 2]+ : [M + 4]+ (100/68/
14), 587 (8), 527 (14), 310 (12), 294 (7), 252 (6), 117 (9), 91 (9).
Anal. calcd C31H32Cl2N8O2: C, 60.10; H, 5.21; N, 18.09; found: C,
60.15; H, 5.18; N, 18.10.
4.2.8.3. 2-((4-((4-(1-(3,5-Dichlorophenyl)-5-(4-methoxyphenyl)-
4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-6-morpholino-1,3,5-
triazin-2-yl)amino)ethan-1-ol (9c). Yellow solid. 75% yield; mp
275–278 ꢁC. FT-IR (ATR): n (cmꢀ1) 3327 (N–H), 3252 (O–H), 3094
(]C–H), 1661 y 1582 (C]N and C]C). 1H-NMR (400 MHz,
DMSO-d6) d ppm 3.12 (dd, J ¼ 17.2, 3.7 Hz, 1H, H-4), 3.44 (d, J ¼
18.5 Hz, 2H, CH2), 3.50–3.60 (m, 2H, CH2), 3.61–3.74 (m, 7H,
CH2, OCH3), 3.75–3.82 (m, 4H, CH2), 3.89 (dd, J ¼ 17.2, 11.9 Hz,
1H, H-4), 4.50 (bs, 1H, OH), 5.52 (dd, J ¼ 11.9, 3.7 Hz, 1H, H-5),
6.79 (s, 1H, Ar–H), 6.82–6.98 (m, 4H, Ar–H), 7.18 (d, J ¼ 8.2 Hz,
2H, Ar–H), 7.65–7.85 (m, 4H Ar–H), 8.46 (bs, 1H, NH), 10.76 (bs,
1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 43.0 (CH2), 43.2
(CH2), 44.4 (CH2), 55.0 (OCH3), 59.0 (CH2), 61.9 (CH), 65.7
(CH2), 110.8 (CH), 114.5 (CH), 116.8 (CH), 120.3 (CH), 120.4
(Cq), 121.0 (Cq), 126.7 (Cq), 126.9 (CH), 127.0 (CH), 127.2 (Cq),
133.2 (Cq), 134.3 (Cq), 145.7 (Cq), 149.6 (Cq), 149.7 (Cq), 158.7
(Cq). MS (70 eV) m/z (%): 634 : 636 : 638 [M+] : [M + 2]+ : [M + 4]+
(100/70/15), 604 (5), 527 (39), 310 (12), 280 (7), 252 (6), 134 (14),
121 (14). Anal. calcd C31H32Cl2N8O3: C, 58.59; H, 5.08; N, 17.63;
found: C, 58.62; H, 5.12; N, 17.59.
ꢁ
yl)amino)ethan-1-ol (9e). Yellow solid. 91% yield; mp 230–234 C.
FT-IR (ATR): n (cmꢀ1) 3520 (N–H), 3273 (O–H), 3105 (]C–H), 1653
y 1582 (C]N and C]C). 1H-NMR (400 MHz, DMSO-d6) d ppm 3.15
(dd, J ¼ 17.7, 4.4 Hz, 1H, H-4), 3.44 (d, J ¼ 18.5 Hz, 2H, CH2), 3.50–
3.60 (m, 2H, CH2), 3.62–3.74 (m, 4H, CH2), 3.74–3.83 (m, 5H, CH2,
OH), 3.92 (dd, J ¼ 17.7, 11.7 Hz, 1H, H-4), 5.63 (dd, J ¼ 11.7, 4.4 Hz,
1H, H-5), 6.80 (s, 1H, Ar–H), 6.92 (s, 2H, Ar–H), 7.27 (d, J ¼ 8.4 Hz,
2H, Ar–H), 7.42 (d, J ¼ 8.4 Hz, 2H, Ar–H), 7.63–7.81 (m, 4H Ar–H),
8.48 (bs, 1H, NH), 10.77 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-
d6) d ppm 43.0 (CH2), 43.3 (CH2), 44.4 (CH2), 59.0 (CH2), 61.6 (CH),
65.7 (CH2), 110.9 (CH), 117.1 (CH), 117.1 (Cq), 120.4 (Cq), 120.4
(CH), 121.0 (Cq), 124.0 (Cq), 127.0 (CH), 127.8 (CH), 129.2 (CH),
132.3 (Cq), 134.5 (Cq), 140.3 (Cq), 145.5 (Cq), 149.5 (Cq), 149.9
(Cq). MS (70 eV) m/z (%): 638 : 640 : 642 : 644 [M+] : [M + 2]+ : [M +
4]+ : [M + 6]+ (49/41/14/3), 607 (6), 527 (4), 310 (10), 239 (7), 185 (7),
123 (26), 97 (40). Anal. calcd C30H29Cl3N8O2: C, 56.30; H, 4.57; N,
17.51; found: C, 56.20; H, 4.60; N, 17.48.
4.2.8.6. 2-((4-((4-(1-(3,5-Dichlorophenyl)-5-(4-uorophenyl)-4,5-
dihydro-1H-pyrazol-3-yl)phenyl)amino)-6-morpholino-1,3,5-triazin-
2-yl)amino)ethan-1-ol (9f). Yellow solid. 86% yield; mp 249–252 ꢁC.
FT-IR (ATR): n (cmꢀ1) 3535 (N–H), 3244 (O–H), 3113 (]C–H),
1653 y 1587 (C]N and C]C). 1H-NMR (400 MHz, DMSO-d6)
d ppm 3.16 (dd, J ¼ 17.4, 3.5 Hz, 1H, H-4), 3.44 (d, J ¼ 18.4 Hz, 2H,
CH2), 3.51–3.61 (m, 2H, CH2), 3.63–3.72 (m, 4H, CH2), 3.75–3.84
(m, 4H, CH2), 4.06 (bs, 1H, OH), 3.92 (dd, J ¼ 17.4, 12.1 Hz, 1H, H-
4), 5.62 (dd, J ¼ 12.1, 3.5 Hz, 1H, H-5), 6.80 (s, 1H, Ar–H), 6.93 (s,
2H, Ar–H), 7.18 (t, J ¼ 8.2 Hz, 2H, Ar–H), 7.25–7.35 (m, 2H, Ar–H),
7.70 (t, J ¼ 11.2 Hz, 2H, Ar–H), 7.78 (d, J ¼ 8.4 Hz, 2H, Ar–H), 8.50
(bs, 1H, NH), 10.80 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
d ppm 43.1 (CH2), 43.3 (CH2), 44.4 (CH2), 59.0 (CH2), 61.6 (CH),
65.7 (CH2), 110.9 (CH), 116.0 (d, 2JCF ¼ 21.5 Hz, (CH)), 117.0 (CH),
3
117.1 (Cq), 120.4 (CH), 120.9 (Cq), 127.0 (CH), 127.9 (d, JCF
¼
8.3 Hz, (CH)), 134.4 (Cq), 137.5 (d, 4JCF ¼ 3.2 Hz, Cq), 138.4 (Cq),
1
145.6 (Cq), 149.3 (Cq), 149.7 (Cq), 149.8 (Cq), 161.5 (d, JC–F
¼
244.0 Hz, Cq). MS (70 eV) m/z (%): 622 : 624 : 626 [M+] : [M +
2]+ : [M + 4]+ (100/71/15), 592 (11), 527 (10), 310 (16), 266 (11),
124.69 (33). Anal. calcd C30H29Cl2FN8O2: C, 57.79; H, 4.69; N,
17.97; found: C, 57.90; H, 4.72; N, 18.00.
4.2.8.4. 2-((4-((4-(1-(3,5-Dichlorophenyl)-5-(3,4,5-trimethox-
yphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-6-morpholino-
1,3,5-triazin-2-yl)amino)ethan-1-ol (9d). Yellow solid. 80% yield;
mp 239–242 C. FT-IR (ATR): n (cmꢀ1) 3410 (N–H), 3242 (O–H),
ꢁ
3098 (]C–H), 1659 y 1583 (C]N and C]C). 1H-NMR (400 MHz,
DMSO-d6) d ppm 3.20 (dd, J ¼ 17.7, 6.2 Hz, 1H, H-4), 3.36–3.43
(m, 2H, CH2), 3.49–3.54 (m, 2H, CH2), 3.59–3.66 (m, 7H, OCH3,
CH2), 3.67–3.73 (m, 10H, OCH3, CH2), 3.90 (dd, J ¼ 17.7, 11.7 Hz,
1H, H-4), 4.67 (s, 1H, OH), 5.39 (dd, J ¼ 11.7, 6.2 Hz, 1H, H-5),
6.60 (s, 2H, Ar–H), 6.76–6.89 (m, 2H, Ar–H, NH), 6.96 (s, 2H,
Ar–H), 7.68 (t, J ¼ 7.2 Hz, 2H, Ar–H), 7.83 (d, J ¼ 8.7 Hz, 2H, Ar–H),
9.24 (bs 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 42.9
(CH2), 43.0 (CH2), 43.3 (CH2), 56.0 (OCH3), 59.9 (CH2), 60.0
(OCH3), 63.0 (CH), 66.0 (CH2), 103.0 (CH), 109.7 (Cq), 110.9 (CH),
116.1 (CH), 119.1 (CH), 122.5 (Cq), 126.8 (CH), 131.2 (Cq), 134.4
(Cq), 136.8 (Cq), 137.4 (Cq), 149.0 (Cq), 150.5 (Cq), 153.2 (Cq),
153.4 (Cq), 158.7 (Cq). MS (70 eV) m/z (%): 694 : 696 : 698
4.2.8.7. 2-((4-((4-(1-(3,5-Dichlorophenyl)-5-(4-(triuoromethyl)
phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-6-morpholino-
1,3,5-triazin-2-yl)amino)ethan-1-ol (9g). Yellow solid. 84% yield;
mp 258–263 C. FT-IR (ATR): n (cmꢀ1) 3445 (N–H), 3242 (O–H),
ꢁ
3105 (]C–H), 1653 y 1584 (C]N and C]C). 1H-NMR (400 MHz,
DMSO-d6) d ppm 3.21 (dd, J ¼ 17.8, 4.2 Hz, 1H, H-4), 3.44 (d, J ¼
16.4 Hz, 2H, CH2), 3.50–3.60 (m, 2H, CH2), 3.62–3.73 (m, 5H, CH2,
OH), 3.74–3.85 (m, 4H, CH2), 3.96 (dd, J ¼ 17.8, 11.8 Hz, 1H, H-4),
5.75 (dd, J ¼ 11.8, 4.2 Hz, 1H, H-5), 6.82 (s, 1H, Ar–H), 6.93 (s, 2H,
Ar–H), 7.48 (d J ¼ 8.4 Hz, 2H, Ar–H), 7.62–7.86 (m, 6H, Ar–H), 8.40
(bs, 1H, NH), 10.72 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
d ppm 42.9 (CH2), 44.1 (CH2), 44.4 (CH2), 59.0 (CH2), 61.7 (CH),
65.8 (CH2), 110.8 (CH), 110.9 (Cq), 113.0 (Cq), 114.1 (Cq), 117.2
(CH), 118.2 (Cq), 120.3 (Cq), 120.4 (Cq), 120.9 (CH), 121.0 (q, 1JCF
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1
¼ 244.51 Hz, CF3), 125.5 (Cq), 126.2 (d, 3JCF ¼ 5.7 Hz, (CH)), 126.8 H), 3188 (]C–H), 1634 y 1585 (C]N and C]C). H-NMR (400
4
2
(CH), 127.0 (d, JCF ¼ 1.2 Hz, (CH)), 134.6 (Cq), 145.8 (d, JCF
¼
MHz, DMSO-d6) d ppm 3.14 (dd, J ¼ 17.0, 4.3 Hz, 1H, H-4), 3.42–
46.76 Hz, Cq), 150.0 (Cq). MS (70 eV) m/z (%): 672 : 674 : 676 3.50 (m, 2H, CH2), 3.55–3.63 (m, 2H, CH2), 3.71 (s, 3H, OCH3),
[M+] : [M + 2]+ : [M + 4]+ (100/66/13), 642 (12), 515 (7), 310 (14), 252 3.76–3.97 (m, 2H, H-4, OH), 5.52 (dd, J ¼ 11.6, 4.3 Hz, 1H, H-5),
(8), 124 (25), 69 (32). Anal. calcd C31H29Cl2F3N8O2: C, 55.28; H, 6.78 (s, 1H, Ar–H), 6.86–6.98 (m, 4H, Ar–H), 7.19 (d, J ¼ 8.4, 2H,
4.34; N, 16.64; found: C, 55.32; H, 4.29; N, 16.58.
Ar–H), 7.36–7.44 (m, 2H, Ar–H), 7.65–7.85 (m, 6H, Ar–H), 8.59 (bs,
4.2.9. General procedure for the synthesis of 2-((4-((4- 1H, NH), 10.50 (bs, 1H, NH), 10.76 (bs, 1H, NH). 13C-NMR (100
chlorophenyl)amino)-6-((4-(1-(3,5-dichlorophenyl)-5-(aryl)-4,5-
MHz, DMSO-d6) d ppm 43.2 (CH2), 43.4 (CH2), 55.1 (OCH3), 59.2
dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-triazin-2-yl)
(CH2), 62.0 (CH), 110.9 (CH), 114.5 (CH), 116.8 (CH), 120.8 (CH),
amino)etanol (10a–g). A mixture of chalcone 8 (1 equiv.) and 121.3 (Cq), 122.7 (Cq), 123.3 (CH), 126.7 (CH), 126.8 (Cq), 127.1
3,5-dichlorophenylhydrazine hydrochloride (3 equiv.) in (CH), 128.5 (Cq), 128.6 (CH), 133.3 (Cq), 134.4 (Cq), 136.6 (CH),
ethanol (2.5 mL) was subjected to reux for 12 h. The solid 142.1 (Cq), 145.8 (Cq), 149.8 (Cq), 152.2 (Cq), 158.7 (Cq). MS (70 eV)
formed was ltered and washed with ethanol. No further puri- m/z (%): 674 : 676 : 678 : 680 [M+] : [M + 2]+ : [M + 4]+ : [M + 6]+ (30/
cation was required.
29/11/2), 658 (9), 640 (8), 567 (13), 350 (12), 124 (25), 84 (100). Anal.
calcd C33H29Cl3N8O2: C, 58.63; H, 4.32; N, 16.58; found: C, 58.66;
H, 4.32; N, 16.60.
4.2.9.1. 2-((4-((4-Chlorophenyl)amino)-6-((4-(1-(3,5-dichlor-
ophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-
triazin-2-yl)amino)ethan-1-ol (10a). Yellow solid. 84% yield; mp 222–
4.2.9.4. 2-((4-((4-Chlorophenyl)amino)-6-((4-(1-(3,5-dichlor-
224 ꢁC. FT-IR (ATR): n (cmꢀ1) 3419 (N–H), 3319 (O–H), 3113 (]C– ophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)
H), 1622 y 1583 (C]N and C]C). 1H-NMR (400 MHz, DMSO-d6) phenyl)amino)-1,3,5-triazin-2-yl)amino)ethan-1-ol (10d). Yellow
d ppm 3.18 (dd, J ¼ 17.5, 4.6 Hz, 1H, H-4), 3.32–3.49 (m, 2H, CH2), solid. 86% yield; mp 226–229 ꢁC. FT-IR (ATR): n (cmꢀ1) 3500 (N–
3.57–3.62 (m, 2H, CH2), 3.75–4.12 (m, 2H, H-4, OH), 5.58 (dd, J ¼ H), 3192 (O–H), 3074 (]C–H), 1636 y 1583 (C]N and C]C). 1H-
11.9, 4.6 Hz, 1H, H-5), 6.79 (s, 1H, Ar–H), 6.93 (s, 2H, Ar–H), 7.24– NMR (400 MHz, DMSO-d6) d ppm 3.24 (dd, J ¼ 17.4, 5.8 Hz, 1H,
7.31 (m, 3H, Ar–H), 7.33–7.44 (m, 4H, Ar–H), 7.68–7.83 (m, 6H, Ar– H-4), 3.40–3.50 (m, 2H, CH2), 3.56–3.62 (m, 2H, CH2), 3.64 (s,
H), 8.50 (bs, 1H, NH), 10.43 (bs, 1H, NH), 10.65 (bs, 1H, NH). 13C- 3H, OCH3), 3.71 (s, 6H, OCH3), 3.79–3.99 (m, 2H, H-4, OH), 5.43
NMR (100 MHz, DMSO-d6) d ppm 43.2 (CH2), 43.4 (CH2), 59.2 (dd, J ¼ 12.2, 5.8 Hz, 1H, H-5), 6.62 (s, 2H, Ar–H), 6.83 (s, 1H, Ar–
(CH2), 62.5 (CH), 110.8 (CH), 116.9 (CH), 120.7 (CH), 121.2 (CH), H), 6.97 (s, 2H, Ar–H), 7.40 (d, J ¼ 6.8 Hz, 2H, Ar–H), 7.68–7.86
122.7 (Cq), 123.2 (Cq), 123.3 (Cq), 125.8 (CH), 126.6 (Cq), 126.7 (m, 6H, Ar–H), 8.53 (bs, 1H, NH), 10.46 (bs, 1H, NH), 10.68 (s,
(CH), 127.0 (Cq), 127.8 (CH), 128.6 (CH), 129.2 (CH), 134.4 (Cq), 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 43.3 (CH2), 43.4
136.8 (Cq), 137.2 (Cq), 141.4 (Cq), 145.8 (Cq), 149.8 (Cq). MS (70 eV) (CH2), 56.0 (OCH3), 59.2 (CH2), 60.0 (OCH3), 63.2 (CH), 103.1
m/z (%): 644 : 646 : 648 : 650 [M+] : [M + 2]+ : [M + 4]+ : [M + 6]+ (CH), 111.0 (CH), 117.1 (CH), 120.7 (CH), 120.8 (Cq), 121.2 (Cq),
(95/100/38/5), 628 (49), 567 (12), 350 (17), 221 (19), 178 (17), 124 122.7 (Cq), 123.1 (CH), 123.3 (Cq), 126.7 (Cq), 126.8 (CH), 126.9
(24), 91 (30). Anal. calcd C32H27Cl3N8O: C, 59.50; H, 4.21; N, 17.35; (Cq), 128.6 (CH), 134.4 (Cq), 136.8 (Cq), 137.3 (Cq), 139.1 (Cq),
found: C, 59.48; H, 4.27; N, 17.40.
4.2.9.2. 2-((4-((4-Chlorophenyl)amino)-6-((4-(1-(3,5-dichlor-
ophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-
146.2 (Cq), 150.1 (Cq), 153.4 (Cq). MS (70 eV) m/z (%):
734 : 736 : 738 : 740 [M+] : [M + 2]+ : [M + 4]+ : [M + 6]+ (18/15/6/
1), 656 (18), 567 (9), 363 (12), 221 (18), 152 (15), 118 (18). Anal.
triazin-2-yl)amino)ethan-1-ol (10b). Yellow solid. 90% yield; mp calcd C35H33Cl3N8O4: C, 57.11; H, 4.52; N, 15.22; found: C,
277–279 C. FT-IR (ATR): n (cmꢀ1) 3311 (N–H), 3252 (O–H), 3190 57.15; H, 4.55; N, 15.32.
ꢁ
(]C–H), 1629 y 1583 (C]N and C]C). 1H-NMR (400 MHz,
4.2.9.5. 2-((4-((4-(5-(4-Chlorophenyl)-1-(3,5-dichlorophenyl)-4,5-
DMSO-d6) d ppm 2.25 (s, 3H, CH3), 3.14 (dd, J ¼ 17.1, 4.4 Hz, 1H, dihydro-1H-pyrazol-3-yl)phenyl)amino)-6-((4-chlorophenyl)amino)-
H-4), 3.40–3.50 (m, 2H, CH2), 3.56–3.62 (m, 2H, CH2), 3.85–4.00 1,3,5-triazin-2-yl)amino)ethan-1-ol (10e). Yellow solid. 95% yield;
(m, 2H, H-4, OH), 5.53 (dd, J ¼ 12.1, 4.4 Hz, 1H, H-5), 6.79 (s, 1H, mp 277–279 C. FT-IR (ATR): n (cmꢀ1) 3309 (N–H), 3200 (O–H),
ꢁ
Ar–H), 6.93 (d, J ¼ 1.6 Hz, 2H, Ar–H), 7.13–7.17 (m, 4H, Ar–H), 3192 (]C–H), 1632 y 1583 (C]N and C]C). 1H-NMR (400 MHz,
7.35–7.45 (m, 2H, Ar–H), 7.67–7.83 (m, 6H, Ar–H), 8.47 (bs, 1H, DMSO-d6) d ppm 3.18 (dd, J ¼ 16.5, 4.0 Hz, 1H, H-4), 3.41–3.51 (m,
NH), 10.41 (bs, 1H, NH), 10.62 (bs, 1H, NH). 13C-NMR (100 MHz, 2H, CH2), 3.57–3.63 (m, 2H, CH2), 3.74–4.07 (m, 2H, H-4, OH), 5.63
DMSO-d6) d ppm 20.7 (CH3), 43.2 (CH2), 43.4 (CH2), 59.2 (CH2), (dd, J ¼ 12.5, 4.0 Hz, 1H, H-5), 6.81 (s, 1H, Ar–H), 6.92 (s, 2H, Ar–
62.3 (CH), 110.8 (CH), 116.8 (CH), 120.0 (Cq), 120.8 (CH), 122.7 H), 7.29 (d, J ¼ 8.4 Hz, 2H, Ar–H), 7.36–7.45 (m, 4H, Ar–H), 7.66–
(Cq), 123.2 (CH), 125.7 (CH), 125.9 (Cq), 126.7 (CH), 127.1 (Cq), 7.86 (m, 6H, Ar–H), 8.54 (bs, 1H, NH), 10.46 (bs, 1H, NH), 10.69 (bs,
127.6 (Cq), 128.5 (CH), 128.6 (Cq), 129.7 (CH), 134.4 (Cq), 134.5 1H, NH). 13C-NMR (100 MHz, DMSO-d6) d ppm 43.0 (CH2), 43.4
(Cq), 137.0 (Cq), 138.5 (Cq), 145.8 (Cq), 149.8 (Cq). MS (70 eV) m/z (CH2), 59.2 (CH2), 61.7 (CH), 110.9 (CH), 117.1 (CH), 120.7 (CH),
(%): 658 : 660 : 662 : 664 [M+] : [M + 2]+ : [M + 4]+ : [M + 6]+ (100/ 121.2 (Cq), 121.3 (Cq), 122.7 (Cq), 123.1 (CH), 123.6 (Cq), 125.9
98/37/5), 642 (23), 557 (36), 350 (31), 178 (27), 117 (33), 91 (34). (Cq), 126.8 (CH), 126.9 (Cq), 127.8 (CH), 128.6 (CH), 128.9 (Cq),
Anal. calcd C33H29Cl3N8O: C, 60.05; H, 4.43; N, 16.98; found: C, 129.2 (CH), 132.3 (Cq), 134.5 (Cq), 140.4 (Cq), 145.6 (Cq), 149.9
59.98; H, 4.40; N, 17.01.
4.2.9.3. 2-((4-((4-Chlorophenyl)amino)-6-((4-(1-(3,5-dichlor-
ophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)
(Cq). MS (70 eV) m/z (%): 678 : 680 : 682 : 684 : 686 [M+] : [M +
2]+ : [M + 4]+ : [M + 6]+ : [M + 8]+ (70/89/47/11/1), 662 (25), 555 (18),
417 (20), 350 (25), 262 (31), 221 (27), 118 (40). Anal. calcd
amino)-1,3,5-triazin-2-yl)amino)ethan-1-ol (10c). Yellow solid. 95% C32H26Cl4N8O: C, 56.49; H, 3.85; N, 16.47; found: C, 56.52; H,
yield; mp 276–279 ꢁC. FT-IR (ATR): n (cmꢀ1) 3308 (N–H), 3246 (O– 3.87; N, 16.50.
34126 | RSC Adv., 2020, 10, 34114–34129
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4.2.9.6. 2-((4-((4-Chlorophenyl)amino)-6-((4-(1-(3,5-dichlor-
RSC Advances
the time of compound addition, an aliquot of frozen concen-
trate was thawed and diluted to twice the desired nal
maximum test concentration with complete medium contain-
ing 50 mg mLꢀ1 gentamicin. An additional four 10-fold or 1/2 log
serial dilutions were made to provide a total of ve drug
concentrations plus the control. Aliquots of 100 mL of these
different sample dilutions were added to the appropriate
microtiter wells already containing 100 mL of medium, resulting
in the required nal sample concentrations. Aer the tested
compounds were added, the plates were incubated for an
ophenyl)-5-(4-uorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)
amino)-1,3,5-triazin-2-yl)amino)ethan-1-ol (10f). Yellow solid. 77%
yield; mp 208–211 ꢁC. FT-IR (ATR): n (cmꢀ1) 3319 (N–H), 3253 (O–
1
H), 3095 (]C–H), 1620 y 1585 (C]N and C]C). H-NMR (400
MHz, DMSO-d6) d ppm 3.18 (dd, J ¼ 16.8, 4.8 Hz, 1H, H-4), 3.40–
3.50 (m, 2H, CH2), 3.56–3.62 (m, 2H, CH2), 3.86–4.01, (m, 2H, H-4,
OH), 5.62 (dd, J ¼ 12.3, 4.8 Hz, 1H, H-5), 6.81 (s, 1H, Ar–H), 6.93 (d,
J ¼ 1.3 Hz, 2H, Ar–H), 7.19 (t, J ¼ 8.6 Hz, 2H, Ar–H), 7.28–7.35 (m,
2H, Ar–H), 7.36–7.44 (m, 2H, Ar–H), 7.66–7.86 (m, 6H, Ar–H), 8.50
(bs, 1H, NH), 10.43 (bs, 1H, NH), 10.63 (bs, 1H, NH). 13C-NMR (100
MHz, DMSO-d6) d ppm 43.1 (CH2), 43.4 (CH2), 59.2 (CH2), 61.7
(CH), 110.9 (CH), 116.0 (d, 2JCF ¼ 21.5 Hz, (CH)), 117.0 (CH), 120.7
(CH), 120.8 (Cq), 121.3 (Cq), 122.3 (Cq), 122.8 (Cq), 123.2 (CH),
ꢁ
additional 48 h at 37 C, 5% CO2, 95% air, and 100% relative
humidity. For adherent cells, the assay was terminated by the
addition of cold TCA. Cells were xed in situ by the gentle
addition of 50 mL of cold 50% (w/v) TCA (nal concentration,
3
ꢁ
10% TCA) and incubated for 60 min at 4 C. The supernatant
123.5 (Cq), 126.8 (CH), 126.9 (Cq), 127.9 (d, JCF ¼ 7.8 Hz, (CH)),
4
was discarded, and plates were washed ve times with tap water
and air dried. Sulforhodamine B (SRB) solution (100 mL) at 0.4%
(w/v) in 1% acetic acid was added to each well, and plates were
incubated for 10 min at room temperature. Aer staining,
unbound dye was removed by washing ve times with 1% acetic
acid and the plates were air dried. Bound stain was subse-
quently solubilized with 10 mM trizma base, and the absor-
bance was read on an automated plate reader at a wavelength of
515 nm. Using the seven absorbance measurements [time zero
(Tz), control growth in the absence of drug, and test growth in
the presence of drug at the ve concentration levels (Ti)], the
percentage growth was calculated at each of the drug concen-
trations levels. Percentage growth inhibition was calculated as
[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢄ 100 for concentrations for which Ti > Tz,
and [(Ti ꢀ Tz)/Tz] ꢄ 100 for concentrations for which Ti < Tz. Two
dose–response parameters were calculated for each compound.
Growth inhibition of 50% (GI50) was calculated from [(Ti ꢀ Tz)/
(C ꢀ Tz)] ꢄ 100 ¼ 50, which is the drug concentration resulting
in a 50% lower net protein increase in the treated cells
(measured by SRB staining) as compared to the net protein
increase seen in the control cells and the LC50 (concentration of
drug resulting in a 50% reduction in the measured protein at
the end of the drug treatment as compared to that at the
beginning), indicating a net loss of cells; calculated from [(Ti ꢀ
Tz)/Tz] ꢄ 100 ¼ ꢀ50. Values were calculated for each of these
two parameters if the level of activity is reached; however, if the
effect was not reached or was exceeded, the value for that
parameter was expressed as greater or less than the maximum
or minimum concentration tested.38
128.5 (CH), 128.6 (Cq), 134.4 (Cq), 137.6 (d, JCF ¼ 2.5 Hz, Cq),
145.6 (Cq), 149.8 (Cq), 161.5 (d, 1JC–F ¼ 243.7 Hz, Cq). MS (70 eV) m/
z (%): 662 : 664 : 666 : 668 [M+] : [M + 2]+ : [M + 4]+ : [M + 6]+ (65/
51/17/3), 644 (25), 557 (9), 350 (13), 279 (13), 262 (17), 221 (27),
124 (25). Anal. calcd C32H26Cl3FN8O: C, 57.89; H, 3.95; 2.86; N,
16.88; found: C, 57.90; H, 4.00; N, 16.74.
4.2.9.7. 2-((4-((4-Chlorophenyl)amino)-6-((4-(1-(3,5-dichlorophe
nyl)-5-(4-(triuoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)
amino)-1,3,5-triazin-2-yl)amino)ethan-1-ol (10g). Yellow solid. 83%
yield; mp 274–277 ꢁC. FT-IR (ATR): n (cmꢀ1) 3310 (N–H), 3292 (O–
1
H), 3190 (]C–H), 1632 y 1585 (C]N and C]C). H-NMR (400
MHz, DMSO-d6) d ppm 3.17–3.28 (m, 1H, H-4), 3.41–2.51 (m, 2H,
CH2), 3.56–6.63 (m, 2H, CH2), 3.98 (dd, J ¼ 16.6, 12.5 Hz, 1H, H-4),
4.23 (bs, 1H, OH), 5.74 (dd, J ¼ 12.5, 4.8 Hz, 1H, H-5), 6.83 (s, 1H,
Ar–H), 6.94 (s, 2H, Ar–H), 7.35–7.45 (m, 2H, Ar–H), 7.50 (d, J ¼
8.0 Hz, 2H, Ar–H), 7.68–7.84 (m, 8H, Ar–H), 8.48 (bs, 1H, NH), 10.42
(bs, 1H, NH), 10.61 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
d ppm 42.9 (CH2), 43.3 (CH2), 59.2 (CH2), 61.8 (CH), 110.8 (CH),
117.2 (CH), 120.5 (Cq), 120.7 (CH), 121.3 (CH), 123.1 (CH), 123.4
1
4
(Cq), 124.1 (q, JCF ¼ 272.2 Hz, CF3), 126.2 (Cq), 126.5 (d, JCF
¼
3
5.86 Hz, (CH)), 126.8 (d, JCF ¼ 7.00 Hz, (CH)), 127.6 (Cq), 127.9
(Cq), 128.2 (Cq), 128.6 (CH), 134.5 (Cq), 136.8 (Cq), 137.1 (Cq), 145.8
(d, 2JCF ¼ 46.58 Hz, Cq), 148.2 (Cq), 149.9 (Cq). MS (70 eV) m/z (%):
712 : 714 : 716 : 718 [M+] : [M + 2]+ : [M + 4]+ : [M + 6]+ (100/94/35/
5), 696 (11), 682 (12), 557 (10), 350 (23), 279 (19), 178 (20), 124
(29). Anal. calcd C33H26Cl3F3N8O: C, 55.52; H, 3.67; N, 15.69; found:
C, 55.50; H, 3.69; N, 15.74.
4.3. Anticancer activity
The human cancer cell lines of the cancer screening panel were
grown in an RPMI-1640 medium containing 5% fetal bovine
serum and 2 mM L-glutamine. For a typical screening experi-
4.4. Molecular modeling
ment, cells were inoculated into 96-well microtiter plates. Aer
4.4.1. Ligand based studies. The synthetized molecules
ꢁ
cell inoculation, the microtiter plates were incubated at 37 C, were submitted to Biotarget Finder module, available in the
5% CO2, 95% air, and 100% relative humidity for 24 h prior to web-server http://www.drudit.com.34 The tool allows to predict
the addition of the tested compounds. Aer 24 h, two plates of the binding affinity of candidate molecules versus a large data-
each cell line were xed in situ with TCA, to represent base of biological targets. The templates of the biological targets
a measurement of the cell population for each cell line at the are built by using a set of known modulators implemented in
time of sample addition (Tz). The samples were solubilized in the system. The synthetized compounds are submitted to Bio-
dimethyl sulfoxide (DMSO) at 400–fold the desired nal logical Predictor module by using the default parameters, and
maximum test concentration and stored frozen prior to use. At the output results are obtained as DAS (Drudit Affinity Score) for
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RSC Advances
Paper
each structure (see user manual available at http:// GlideScore function, dened as: Gscore ¼ 0.065 ꢄ vdW + 030 ꢄ
www.drudit.com for further details).
Coul + Lipo + Hbond + Metal + BuryP + RotB + Site, where: vdW
is the van der Waals energy term, Coul is the Coulomb energy,
4.4.2. Structure based studies
4.4.2.1. Ligand preparation. The default setting of the Lig- Lipo is a lipophilic contact term which rewards favorable
¨
Prep tool implemented in Schrodinger's soware (version 2017- hydrophobic interactions, Hbond is an H-bonding term, metal
1) has been used to prepare the ligands for docking.39 All is a metal-binding term (where applicable), BuryP is a penalty
possible tautomers and combination of stereoisomers have term applied to buried polar groups, RotB is a penalty for
been generated for pH 7.0 ꢃ 0.4, using the Epik ionization freezing rotatable bonds and site is a term used to describe
method.40 Energy minimization is subsequently done using the favourable polar interactions in the active site.
integrated OPLS 2005 force eld.41
Finally, IFD score (IFD score ¼ 1.0 Glide Gscore + 0.05 prime
ꢀ
4.4.2.2. Protein preparation. The high-resolution (2.79 A) X- energy), which accounts for both protein–ligand interaction
ray structure of Human thymidylate synthase complexed with energy and total energy of the system, is calculated and used to
dUMP and raltitrexed (PDB ID: 5X5Q)35 is downloaded from the rank the IFD poses. The more negative is the IFDscore, the more
Protein Databank.36
favorable is the binding.
The receptor grid preparation has been carried out with
substrate dUMP and without water molecules, to elucidate the
role of dUMP for the binding, by assigning the original ligand
(raltitrexed) as the centroid of the grid box. Protein Preparation
Conflicts of interest
There are no conicts to declare.
¨
Wizard of Schrodinger soware that was employed using the
default settings.42 Bond orders have been assigned and
hydrogen atoms added as well as protonation of the heteroatom Acknowledgements
states using Epik-tool (with the pH set at biologically relevant
The authors thank The Developmental Therapeutics Program
values, i.e. at 7.0 ꢃ 0.4). The H-bond network has been then
optimized. The structure is nally subjected to a restrained
energy minimization step (rmsd of the atom displacement for
(DTP) of the National Cancer Institute of the United States for
performing the anticancer screening of the compounds. This
work was nancially supported by COLCIENCIAS and Uni-
versidad del Valle, Colombia.
˚
terminating the minimization was 0.3 A), using the Optimized
Potentials for Liquid Simulations (OPLS) 2005 force eld.41
4.4.3. Docking validation. Molecular Docking is performed
by Glide program.43–45 The generated 3D conformers of the
ligands are docked into the receptor model using the Extra
Precision (XP) mode as the scoring function. A total of 5 poses
per ligand conformer are included in the post-docking mini-
mization step, and a maximum of 2 docking poses are gener-
ated for each ligand conformer. The proposed docking
procedure was validated by the re-dock of the crystallized ral-
titrexed within the receptor-binding pocket. The results ob-
tained was in good agreement of the experimental poses,
showing a RMSD of 0.79.
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