Synthesis of new 1,3,5-triazine-based 2-pyrazolines as potential anticancer agents

Article abstract of DOI:10.3390/molecules23081956

A new series of 1,3,5-triazine-containing 2-pyrazoline derivatives (8–11)a–g was synthesized by cyclocondensation reactions of [(4,6-bis((2-hydroxyethyl)amino)-1,3, 5-triazin-2-yl)amine]chalcones 7a–g with hydrazine hydrate and derivatives. Chalcones 7a–g were obtained by Claisen-Schmidt condensation between aromatic aldehydes and triazinic derivative 5, which was synthesized in high yield by a microwave-assisted reaction. Seventeen of the synthesized compounds were selected and tested by the US National Cancer Institute (NCI) for their anticancer activity against 58 different human tumor cell lines. Compounds 7g and 10d,e,g showed important GI₅₀ values ranging from 0.569 to 16.6 μM and LC₅₀ values ranging from 5.15 to >100 μM.

Full text of DOI:10.3390/molecules23081956

molecules
Article
Synthesis of New 1,3,5-Triazine-Based 2-Pyrazolines
as Potential Anticancer Agents
Leydi M. Moreno 1, Jairo Quiroga 1,2 ID , Rodrigo Abonia 1,2, Jonathan Ramírez-Prada 1 and
Braulio Insuasty 1,2,
*
1
Heterocyclic Compounds Research Group, Department of Chemistry, Universidad del Valle,
A.A. 25360 Cali, Colombia; leydi.moreno@correounivalle.edu.co (L.M.M.);
jairo.quiroga@correounivalle.edu.co (J.Q.); rodrigo.abonia@correounivalle.edu.co (R.A.);
jonathan.ramirez@correounivalle.edu.co (J.R.-P.)
Centre for Bioinformatics and Photonics-CIBioFI, Calle 13 No. 100-00, Edificio 320, No. 1069,
A.A. 25360 Cali, Colombia
2
*
Correspondence: braulio.insuasty@correounivalle.edu.co; Tel.: +57-315-484-6665; Fax: +57-2339-3248
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 28 June 2018; Accepted: 26 July 2018; Published: 6 August 2018
Abstract: A new series of 1,3,5-triazine-containing 2-pyrazoline derivatives (
8–11)a–g
was synthesized
by cyclocondensation reactions of [(4,6-bis((2-hydroxyethyl)amino)-1,3, 5-triazin-2-yl)amine]chalcones
7a with hydrazine hydrate and derivatives. Chalcones 7a were obtained by Claisen-Schmidt
condensation between aromatic aldehydes and triazinic derivative , which was synthesized in high
–g
–g
5
yield by a microwave-assisted reaction. Seventeen of the synthesized compounds were selected and
tested by the US National Cancer Institute (NCI) for their anticancer activity against 58 different
human tumor cell lines. Compounds 7g and 10d,e,g showed important GI50 values ranging from
0.569 to 16.6 µM and LC50 values ranging from 5.15 to >100 µM.
Keywords: 1,3,5-triazines; chalcones; 2-pyrazolones; Claisen–Schmidt reaction; cyclocondensation
reactions; microwave irradiation; anticancer activity
1. Introduction
Cancer continue being one of the main causes of death worldwide [1]. Chemotherapy is the
most common procedure used for its treatment, despite of its known side-effects. For that reason,
many researchers are focusing on the design of new more selective anticancer agents, able to combat
the resistance of cancer cells and hence, able to reduce their secondary clinical drawbacks. One strategy
to obtain new anticancer therapeutic agents is the combination of two or more biologically active
molecules/pharmacophores in only one structure (i.e., molecular hybridization), which by synergistic
effects between them, could afford biologically and pharmacologically improved therapeutic entities [2,3].
For example, triazine-pyrazoline-based hybrids have been synthesized and reported to possess important
biological activities, particularly, anticancer activity [4,5].
The 1,3,5-triazine scaffold has been extensively studied due to its wide range of biological activities,
such as antiviral [
and specially anticancer properties [13
altretamine ( ) [16], decitabine (II) [17] and azacitidine (III) [18] are used as antineoplastic drugs for
6
], antibacterial [
7–9
], antifungal [10], anti-inflammatory [11], anti-Alzheimer’s [12
]
–
15]. Currently, some 1,3,5-triazinic compounds such as
I
treating ovarian cancer, acute myeloid leukemia and chronic myelomonocytic leukemia, respectively
(Figure 1A).
Molecules 2018, 23, 1956; doi:10.3390/molecules23081956
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1956
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Figure 1.
(A) Structures of some triazine-based antineoplastic drugs. (B) Pyrazolinic derivative with
anticancer activity.
On the other hand, 2-pyrazoline derivatives also have shown important anticancer properties [19
The anticancer mechanisms of these compounds can be associated with their ability to arrest the G0/G1
and G2/M cell cycle phases [21 23], and inhibit heat shock proteins [24], cyclin-dependent kinase [25
,20].
–
]
and P-glycoprotein [26]. For example, pyrazolinic compound IV (Figure 1B), showed effective growth
inhibition of lung cancer cell lines by inducing G2/M phase arrest [21].
Pyrazoline rings can be obtained by a cyclocondensation reaction of
α,β-unsaturated ketones
(chalcones) with hydrazine derivatives. Their synthetic precursors (i.e., chalcones) have also shown
marked biological activity as anticancer [27
agents [31].
,28], antiviral [29], antimalarial [30] and antioxidant
Recently, we have reported the synthesis and anticancer activity of some novel pyrazolines
and their chalcone-precursors with outstanding GI50 values in the range of 0.13 to 0.99 M [32],
0.28 to 11.7 M [33] and 0.04 to 11.4 M [34], when they were subjected to in vitro assays. Thus,
as a continuation of our current studies on the synthetic utility of chalcones and derivatives [32 34],
µ
µ
µ
–
and based on the proven anticancer properties of triazinic and pyrazolinic derivatives, we report here
the synthesis from chalcone derivatives of 1,3,5-triazine-2-pyrazoline hybrids and their subsequent
evaluation (along with their triazino-ketone and chalcone precursors), by the US National Cancer
Institute (NCI) against 58 different human tumor cell lines.
2. Results and Discussion
2.1. Chemistry
For this study, we initially synthetized 1-(4-((4,6-bis((2-hydroxyethyl)amino)-1,3,5-triazin-2-yl)
amino)phenyl)ethan-1-one
2,4,6-trichloro-1,3,5-triazine (
prepared in 83% yield, from 2,4,6-trichloro-1,3,5-triazine (
previously reported methodology [35]. Subsequently, compound
in dioxane under microwave irradiation for 5 min to give the compound
to Claisen–Schmidt condensation with aldehydes 6a afforded the corresponding 1,3,5-triazinic
chalcones 7a–g in good yields and high purity (Scheme 1).
The structural elucidation of compounds 7a was performed by analysis of their spectroscopic
(
1
5
)
as the precursor of 1,3,5-triazinic chalcones 7a
) as starting material. The amino-1,3,5-triazine derivative
) and 4-aminoacetophenone ( ) using a
was treated with ethanolamine (4)
, which was subjected
–
g
using
3 was
1
2
3
5
–
g
–
g
data (FTIR, 1H-NMR, 13C-NMR and mass spectrometry). We discuss here the spectroscopic data of
compound 7a as representative of this series. The IR spectrum of compound 7a shows absorption
bands at 3362, 3287 and 1645 cm−1 corresponding to stretching vibration of N–H, O–H and C=O
bonds, respectively. In the 1H-NMR spectrum the signals of the methylene groups protons appeared
at 3.33–3.39 and 3.48–3.59 ppm, respectively. Two doublets assigned to both vinylic protons of the
α
,
β
-unsaturated moiety, with a coupling constant of 3J = 15.6 Hz are observed at 7.70 and 7.94 ppm;
this coupling constant corroborates the E-configuration of the carbon-carbon double bond. The mass

Molecules 2018, 23, 1956
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spectrum shows a molecular ion peak at m/z 420, which is in agreement with the structure proposed
for 7a.
Scheme 1. Synthesis of the novel 1,3,5-triazinic chalcones 7a–g.
The synthesis of target 2-pyrazolines
cyclocondensation reactions under different experimental conditions (Scheme 2). Thus, the N-acetyl
pyrazolines 8a and N-formyl pyrazolines 9a were obtained by reaction of chalcones 7a
8(a–g)–11(a–g) was performed through 1,2-dinucleophilic
–
g
–
g
–g
with hydrazine monohydrate and their subsequent functionalization with acetic anhydride and
formic acid, respectively, under stirring in ethanol at room temperature for 2.5–3.5 h. On the
other hand, the treatment of chalcones 7a–g with 3,5-dichlorophenyl hydrazine and 4-chlorophenyl
hydrazine under reflux in ethanol for 2 h afforded the N-3,5-dichlorophenylpyrazolines 10a
N-4-chlorophenylpyrazolines 11a–g, respectively, Scheme 2.
–g and the
Scheme 2.
Synthesis of N-acetyl-8a–g, N-formyl-9a–g, N-3,5-dichlorophenyl-10a–g and
N-4-chlorophenyl-11a–g pyrazolines.

Molecules 2018, 23, 1956
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The obtained pyrazolinic compounds
8
(
a
–
g
)–11
(
a
–
g
) showed wide FT-IR absorption bands in the
range of 3228–3309 cm−1 assigned to O–H groups. The IR spectra also showed absorption bands at
1514–1604 and 1562–1651 cm−1 assigned to C=C and C=N functionalities, respectively. In the 1H-NMR
spectrum of N-3,5-dichlorophenylpyrazoline 10e, for example, the protons on the diastereotopic
center C-4, of the pyrazoline ring appears as two double doublets at
δ
3
3.17 ppm (with 2JAM = 17.6 Hz
3
2
and JAX = 4.8 Hz), and at
δ
3.93 ppm (with JAM = 17.6 Hz, and JMX = 12.0 Hz), while the H-5
3
3
proton is observed as a double doublet at
δ
5.63 ppm (with JMX = 12.0 Hz and JAX = 4.8 Hz),
confirming the existence of an AMX coupling system in the pyrazoline ring. The mass spectrum
showed molecular ion peak at m/z 612 (in agreement with the expected mass), and a 100:81:32:5
([M]+:[M + 2]+:[M + 4]+:[M + 6]+), isotopic profile, respectively, characteristic of a compound bearing
three chlorine atoms in its structure.
2.2. Anticancer Activity
The two-stage screening process started with the selection of seventeen of the obtained compounds
(i.e., 5, 7f,g, 8c,e,f,g, 9e,f,g, 10a,d,e,g, 11b,c,e) by the Drug Evaluation Branch of National Cancer
Institute (NCI-USA) through the COMPARE program [36].
The selected compounds were subjected to a primary in vitro evaluation against 58 cell lines at a
single dose of 10 µM during an incubation time of 48 h, (it is referred to as one-dose assay). The 58 cell
panel is derived from nine different cancer strains: leukemia, lung, melanoma, colon, CNS, ovary,
renal, breast and prostate cancers. The output from the one-dose screening was reported as a mean
graph of the growth percent (GP) of the treated cells supplied by the NCI for analysis; the values are
illustrated in Table 1. The mean graph is drawn with the GP data corresponding to the growth relative
to the no-drug control, and relative to the time zero number of cells. This graph allows detection of
both growth inhibition (GI) (i.e., GP values between 0 and 100) and lethality (i.e., negative values).
For example, a GP value of 100 means no growth inhibition. A GP value of 30 would mean 70% growth
inhibition. A GP value of 0 means no net growth over the course of the experiment. A GP value of
−30 would mean 30% lethality and a GP value of −100 means all cells are dead.
Table 1. Percentages of mean growth and growth inhibition of NCI human cancer cell lines treated
with selected compounds (5, 7f,g, 8c,e–g, 9e–g, 10d,e,g, 11b,c,e) at one-dose of 10 µM.
Growth Inhibition (GI)
Compound
Mean Growth (%)
Most Sensitive Cell Line
of Most Sensitive Cell
Line (%) a
5
7f
101.03
76.18
T-47D (Breast)
HCT-116 (Colon)
14.43
86.27
SR (Leukemia)
HCT-116 (Colon)
U251 (CNS)
LOX (Melanoma)
T-47D (Breast)
−2.01
−25.44
−21.24
−44.16
30.83
7g
48.43
8c
8e
8f
8g
9e
9f
97.95
98.23
97.92
95.06
97.45
99.59
100.63
T-47D (Breast)
T-47D (Breast)
T-47D (Breast)
33.81
32.18
34.82
22.64
17.64
20.02
A549/ATCC (Non-Small Cell Lung)
T-47D (Breast)
NCI-H522 (Non-Small Cell Lung)
9g

Molecules 2018, 23, 1956
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Table 1. Cont.
Growth Inhibition (GI)
Compound
10a
Mean Growth (%)
Most Sensitive Cell Line
of Most Sensitive Cell
Line (%) a
45.23
LOX IMVI (Melanoma)
HT29 (Colon)
−36.95
−47.03
−61.30
−60.24
−66.26
−69.97
−68.27
−53.44
−55.82
−57.77
−6.50
SF-539 (CNS)
LOX IMVI (Melanoma)
MALME-3M (Melanoma)
SK-MEL-28 (Melanoma)
SF-295 (CNS)
LOX IMVI (Melanoma)
SK-MEL-28 (Melanoma)
RXF 393 (Renal)
CCRF-CEM (Leukemia)
SF-295 (CNS)
LOX IMVI (Melanoma)
RXF 393 (Renal)
3.94
10d
10e
10g
9.09
−15.49
−48.52
−27.23
−12.62
64.68
33.33
11b
11c
11e
a
58.24
75.54
55.01
RXF 393 (Renal)
RXF 393 (Renal)
RXF 393 (Renal)
−26.15
Negative values of growth inhibition indicate that compound causes the death of the respective cancer cell.
The background color highlight the compounds with the most relevant growth inhibition values.
As shown in Table 1, compounds 7g and 10d
against diverse cell lines, at lethality level, because of their larger number of negative growth inhibition
values. Compounds 10a and 11b also showed remarkable anticancer behavior against a single cell
line with lethality values of 36.95% (LOX IMVI, melanoma), 12.62% (RXF 393, renal) and 26.15%
,e,g displayed an outstanding anticancer behavior
,e
−
−
−
(RXF 393, renal), respectively. Compounds 7f and 11c displayed moderate GI values against HCT-116
of renal cancer cell line (i.e., 86.27%) and RXF 393 also of renal cancer cell line (i.e., 64.68%), respectively.
While the remaining compounds showed less activity with GI values in the range of 14.43–34.82.
This primary assay showed that compounds 7g and 10d,e,g satisfied the predetermined threshold
inhibition criteria, therefore they were considered active. For that, these compounds were subjected to
a second in vitro screening in order to determine their cytostatic activity (GI50 and LC50), against the
full 58 cells panel at five concentrations of 10-fold dilution (i.e., 100, 10, 1.0, 0.1 and 0.01 µM) (it is
referred to as five-dose assay). The test consisted of a 48 h continuous drug exposure protocol by
using sulforhodamine B (SRB) protein assay to estimate cell growth. More details of this evaluation
method and the complementary information which is encoded by the activity pattern over all cell lines
have been published elsewhere [37–39]. The GI50 value (growth inhibitory activity) corresponds to the
molar concentration of the compounds required to inhibit 50% of the growth of cell lines (relative to
untreated cells), and LC50 value (cytotoxic activity) is the molar concentration needed to kill 50% of
the cells at the end of the incubation period of 48 h [40].
As shown in Table 2, compounds 7g and 10d
cell lines, some of them lower than 1.00 M. Chalcone 7g showed GI50 values in the range of 1.54–15.2
and LC50 values of 5.76 to >100 M, being UO–31 (renal, GI50 = 1.54 M and LC50 = 19.5 µM) the most
sensitive strain. The best cytotoxicity value was shown against LOX IMVI (melanoma, LC50 = 5.76).
Compound 10d showed GI50 values in the range of 0.569–16.6 M and LC50 values of 5.22–81.2 M,
being RXF 393 (renal, GI50 = 0.569 M) and HS 578T (breast, GI50 = 0.644
The best cytotoxicity value was shown against 786-0 (Renal, LC50 = 5.22
GI50 values in the range of 1.35–2.48
showed GI50 values in the range of 1.18–2.58
10e and 10g) were found especially effective against SF-539 (CNS) with GI50 = 1.35 and 1.18
,e,g showed outstanding values of GI50 against several
µ
µM
µ
µ
µ
µ
µ
µM) the most sensitive strains.
µ
M). Compound 10e showed
M, while compound 10g
M and LC50 values of 5.16 to >100 M. Both compounds
M,
µ
M and LC50 values of 5.15 to >100 µ
µ
µ
(
µ

Molecules 2018, 23, 1956
6 of 20
respectively. Compound 10e showed the best cytotoxicity value against SF–539 (CNS, LC50 = 5.15
while compound 10g showed the best cytotoxicity value against ACHN (renal, LC50 = 5.16 µM).
µM),
Table 2. In vitro testing expressed as growth inhibition GI50 and lethal concentration LC50 of cancer
cell lines for compounds 7g and 10e,d,g a.
Compounds
Panel Cell Line
7g
10d
10e
10g
c
b
LC50
GI50
LC50
Leukemia
32.8
GI50
LC50
GI50
LC50
GI50
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI–8226
SR
3.24
2.97
2.84
2.68
2.12
2.30
>100
49.6
2.53
1.50
1.47
1.00
1.15
1.29
2.11
1.86
1.73
1.52
1.91
1.45
59.1
7.93
8.07
11.9
15.2
7.55
2.17
2.58
2.31
1.56
1.82
1.48
>100
>100
>100
>100
>100
>100
7.88
8.68
18.7
26.3
15.5
77.0
61.7
>100
>100
Non–small cell lung
A549/ATCC
EKVX
HOP-62
2.91
3.49
2.42
5.28
2.62
3.85
2.33
3.23
2.15
55.1
54.6
91.8
78.6
>100
61.9
34.4
45.2
>100
1.38
2.03
——
1.46
3.85
2.02
3.01
1.77
2.25
7.55
23.0
——
26.6
44.6
15.2
31.3
8.15
30.6
1.96
2.04
——
2.04
2.45
1.72
2.46
1.89
2.03
10.3
12.5
——
9.60
>100
6.73
25.6
7.10
11.9
1.92
1.89
——
——
2.41
1.71
——
2.06
——
——
>100
——
——
>100
——
>100
——
——
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
2.05
3.25
1.79
3.07
2.50
2.01
1.97
8.65
51.3
8.50
41.2
>100
8.61
9.17
2.70
2.14
1.28
1.50
1.52
1.53
1.80
30.5
12.7
5.61
6.51
6.97
6.73
8.00
1.91
1.96
1.59
1.48
2.04
1.85
2.03
7.75
6.33
5.86
5.29
>100
8.64
10.2
——
1.64
1.55
1.51
1.87
1.52
——
——
——
——
——
>100
——
——
KM12
SW-620
CNS
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
2.80
3.20
1.68
1.79
2.31
1.58
80.8
40.7
6.79
——
97.9
——
1.52
1.16
1.20
1.72
1.05
1.25
12.3
7.10
5.51
9.84
8.90
6.34
1.86
1.47
1.35
2.07
1.51
1.51
7.72
5.49
5.15
8.22
6.19
6.12
1.78
1.36
1.18
2.26
1.32
1.53
——
5.37
——
>100
——
——
Melanoma
5.98
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
1.56
4.95
3.96
3.82
——
3.45
3.12
10.3
5.76
50.3
66.2
37.9
——
38.1
32.8
53.1
1.60
1.96
1.58
1.55
15.5
1.53
1.70
1.86
1.65
2.10
1.69
1.64
2.13
1.61
1.81
1.95
5.49
7.28
6.20
5.88
9.79
5.66
6.31
7.34
1.56
1.85
1.65
1.54
——
1.46
1.72
——
5.66
——
——
——
——
——
5.62
7.51
6.65
6.80
53.7
6.44
6.58
8.05
——
Ovarian
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
3.00
2.02
4.29
2.85
3.31
15.2
4.00
>100
8.70
84.8
43.2
>100
80.7
39.8
2.02
1.62
1.63
2.15
2.01
3.37
11.1
13.6
6.07
8.21
27.4
21.5
38.3
48.1
2.48
1.75
2.00
1.96
2.08
2.60
2.31
>100
5.83
18.8
9.98
12.4
>100
>100
2.00
1.54
1.52
1.55
——
2.04
——
——
——
——
——
>100
>100
——

Molecules 2018, 23, 1956
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Table 2. Cont.
Compounds
Panel Cell Line
7g
10d
10e
10g
c
b
LC50
GI50
LC50
Renal
5.22
GI50
LC50
GI50
LC50
GI50
786-0
A498
ACHN
RXF 393
SN12C
TK-10
3.12
8.85
4.38
1.60
2.71
2.93
1.54
75.1
46.1
39.5
7.42
1.29
16.6
2.58
0.569
1.75
2.01
2.08
1.64
1.73
1.55
1.44
1.82
1.89
1.63
6.53
7.37
6.47
5.86
6.62
7.33
6.12
1.51
——
1.37
1.46
1.69
——
1.55
——
——
5.16
——
——
——
5.50
55.0
36.2
7.73
8.33
9.85
28.7
>100
38.4
19.5
UO-31
Prostate
PC-3
DU-145
3.29
4.03
49.8
37.9
1.04
1.84
14.4
9.18
1.62
1.67
13.3
6.22
1.60
1.64
——
——
Breast
MCF7
1.66
55.5
53.5
>100
50.6
>100
24.8
1.26
1.45
0.644
1.67
1.18
1.68
6.82
8.14
81.2
14.9
34.7
14.2
1.42
1.71
2.22
1.77
1.50
1.92
7.76
6.81
>100
8.18
10.2
7.43
1.27
1.63
2.12
1.80
——
2.18
>100
——
>100
——
>100
——
MDA-MB-231/ATCC 3.29
HS 578T
BT-549
T-47D
5.44
2.53
2.51
2.02
MDA-MB-468
a
Data obtained from NCI’s in vitro disease–oriented human cancer cell lines screen in µ
M. b 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 five concentration levels (100, 10, 1.0, 0.1, and 0.01
µ
M). c LC50 is a
parameter of cytotoxicity that reflects the molar concentration needed to kill 50% of the cells. The background color
highlight the most relevant GI50 values of each compound.
A raw SAR analysis (from the preliminary one-dose and five-dose data) shows that N-acetyl
and N-formyl structures
while structures 10 were the most active ones, followed by pyrazolines 11 and chalcones
remarkable that the presence of halogen atoms (Cl and F), in most cases, increased the activity of
their structures. In consequence, the halogenated structures 7g 10a and 11b were the
8
and
9
, respectively, were the less actives of the whole evaluated series,
7. It is
,
f,
,d
,e,
g
,c,e
most active compounds of their corresponding series. Finally, it seems that the presence of the
N-(3,5-dicholorophenyl) substituent in the pyrazoline ring of compounds 10 is very convenient for
improving their relative activity in comparison with the N-(4-cholorophenyl) substituent in pyrazolines
11. This suggestion is supported by the fact that three pyrazolines 10 (i.e., 10d,e,g) were promoted
by the NCI to five-dose assays (due to their outstanding inhibitory values at one-dose), against none
pyrazoline 11 (see Tables 1 and 2). The above findings and in particular the high activity displayed
by structures 7g and 10d,e,g will be used as reference for further structural-reforming experiments
addressed to find the most potent as possible chalcone or pyrazoline-triazine lead hybrid for the
developing of potential anticancer agents based in our synthesized compounds 7 or 10.
3. Experimental Section
3.1. General Information
Reagents and solvents used were obtained from commercial sources and used without further
purification. Melting points were measured using a Stuart SMP10 melting point device (Cole-Parmer
Ltd., Stone, Staffordshire, UK) and are uncorrected. FTIR spectra were obtained with a IRAffinity-1
spectrophotometer (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
Scientific Inc., Madison, WI, USA) and the values are within
±
0.4% of the theoretical values. Thin layer

Molecules 2018, 23, 1956
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chromatography (TLC) were performed on 0.2 mm pre-coated aluminium plates of silica gel 60 F254
(Merck, Darmstadt, Hesse, Germany).
3.2. Chemistry
3.2.1. Synthesis of 1-(4-((4,6-Dichloro-1,3,5-triazin-2-yl)amino)phenyl)ethan-1-one (3)
Using a methodology similar to that reported by Kathiriya and coworkers [33], the synthesis of
precursor
4-aminoacetophenone (
3
was carried out as follows: Aamixture of 2,4,6-trichloro-1,3,5-t◦riazine (
1, 1 mmol) and
5–0 C for 5 h. The mixture
2
, 1 mmol) in acetone (25 mL) was stirred at
−
was neutralized witH-Na2CO3 20%. The content was poured onto crushed ice, filtered and washed
with water.
3.2.2. Synthesis of 4-[(4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino]acetophenone (5)
A mixture of ketone
3 (0.71 mmol) and ethanolamine (4, 2.84 mmol) in dioxane (1.5 mL) was
subjected to microwave irradiation for 5 min, at 70 ◦C, 50 PSI and 200 W. Then, the reaction mixture
was treated with crushed ice. The solid formed was filtered and washed with water. Purification of
product
Beige solid; 86% yield; m.p. 175–177 ◦C. FT-IR (ATR) v (cm−1) 3367 (N–H), 3290 (O–H), 3107 (=C–H),
1664 (C=O), 1597 and 1539 (C=N and C=C). 1H-NMR (400 MHz, DMSO-d6)
ppm 2.50 (s, 3H, CH3),
5 was performed by column chromatography employing a mixture CHCl3/EtOH (7:1) as eluent.
δ
3.31–3.39 (m, 4H, CH2), 3.47–3.56 (m, 4H, CH2), 4.67 (s, 2H, OH), 6.72 (bs, 1H, NH), 6.84 (bs, 1H, NH),
7.84 (d, J = 8.0 Hz, 2H, Ar–H), 7.95 (d, J = 8.0 Hz, 2H, Ar–H), 9.32 (bs, 1H, NH). 13C-NMR (100 MHz,
DMSO-d6)
δ ppm 26.4 (CH3), 42.8 (CH2), 60.1 (CH2), 118.1, 123.6 (Cq), 129.1, 135.9 (Cq), 145.7 (Cq),
145.8 (Cq), 196.1 (Cq). MS (70 eV) m/z (%): 332 [M+] (55), 302 (99), 289 (100), 245 (81), 186 (59), 134 (89).
Anal. Calcd. C15H20N6O3: C, 54.21; H, 6.07; N, 25.29; Found: C, 54.28; H, 5.98; N, 25.31.
3.2.3. General Procedure for the Synthesis of
(4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)aminochalcones 7a–g
A mixture of acetophenone
5 (3 mmol), the respective benzaldehyde 6a–g (3.0 mmol) and
potassium hydroxide (100 mg) in ethanol (7 mL) was stirred at room temperature for 5 h. The solid
formed was filtered and washed with ethanol. No further purification was required.
(E)-1-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-phenylprop-2-en-1-one (7a).
◦
Yellow solid; 80% yield; m.p. 167–169 C. FT-IR (ATR) v (cm−1) 3362 (N–H), 3287 (O–H), 3110 (=C–H),
1645 (C=O), 1575 and 1514 (C=N and C=C). 1H-NMR (400 MHz, DMSO-d6)
δ ppm 3.33–3.39 (m, 4H,
CH2), 3.48–3.59 (m, 4H, CH2), 4.72 (s, 2H, OH), 6.74 (bs, 1H, NH), 6.87 (bs, 1H, NH), 7.51–7.41 (m, 3H,
Ar–H), 7.70 (d, J = 15.6 Hz, 1H, CH), 7.84–7.90 (m, 2H, Ar–H), 7.94 (d, J = 15.6 Hz, 1H, CH) 8.01 (d,
J = 8.9 Hz, 2H, Ar–H), 8.04–8.14 (m, 2H, Ar–H), 9.40 (t, J = 39.23 Hz, 1H, NH). 13C-NMR (100 MHz,
DMSO-d6)
δ ppm 42.9 (CH2), 60.1 (CH2), 118.3, 122.2, 128.7, 128.9, 129.6, 129.7 (Cq), 130.1 (Cq),
130.2 (Cq), 130.3, 134.9 (Cq), 142.8, 145.7 (Cq), 145.8 (Cq), 187.3 (Cq). MS (70 eV) m/z (%): 420 [M+]
(66), 390 (100), 377 (61), 333 (46), 131 (56), 43 (42). Anal. Calcd. C22H24N6O3: C, 62.84; H, 5.75; N, 19.99;
Found: C, 62.80; H, 5.80; N, 20.04.
(E)-1-(4-((4,6-bis((2-Hydroxyethyl)amin◦o)-1,3,5-triazin-2-yl)amino)phenyl)-3-(p-tolyl)prop-2-en-1-one (7b).
Yellow solid; 70% yield; m.p. 195–197 C. FT-IR (ATR) v (cm−1) 3383 (N–H), 3283 (O–H), 3103 (=C–H),
1635 (C=O), 1568 and 1508 (C=N and C=C). 1H-NMR (400 MHz, DMSO-d6)
δ ppm 2.35 (s, 3H, CH3),
3.29–3.40 (m, 4H, CH2), 3.48–3.58 (m, 4H, CH2), 4.70 (s, 2H, OH), 6.73 (bs, 1H, NH), 6.86 (bs, 1H,
NH), 7.27 (d, J = 8.0 Hz, 2H, Ar–H), 7.67 (d, J = 15.5 Hz, 1H, CH), 7.76 (d, J = 8.0 Hz, 2H, Ar–H),
7.88 (d, J = 15.5 Hz, 1H, CH), 8.00 (d, J = 8.9 Hz, 2H, Ar–H), 8.03–8.11 (m, 2H, Ar–H), 9.32 (bs, 1H,
NH). 13C-NMR (100 MHz, DMSO-d6)
δ
ppm 21.1 (CH3), 42.9 (CH2), 60.1 (CH2), 118.3, 121.1, 128.7,
129.5, 129.6 (Cq), 130.3 (Cq), 132.2 (Cq), 135.9 (Cq), 140.3 (Cq), 142.8, 145.6 (Cq), 163.9(Cq), 165.8 (Cq),

Molecules 2018, 23, 1956
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187.2 (Cq). MS (70 eV) m/z (%): 434 [M+] (84), 404 (100), 347 (38), 145 (36), 69 (18), 43 (23). Anal. Calcd.
C23H26N6O3: C, 63.58; H, 6.03; N, 19.34; Found: C, 63.51; H, 6.08; N, 19.39.
(E)-1-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(4-methoxyphenyl)prop-2-en
-1-one (7c). Yellow solid; 62% yield; m.p. 177–179 ◦C. FT-IR (ATR) v (cm−1) 3396 (N–H), 3262 (O–H),
3190 (=C–H), 1635 (C=O), 1606 (C=N), 1577 (C=C) and 1167 (C–O). 1H-NMR (400 MHz, DMSO-d6)
δ
ppm 3.30–3.42 (m, 4H, CH2), 3.47–3.58 (m, 4H, CH2), 3.82 (s, 3H, OCH3), 4.71 (s, 2H, OH), 6.85 (bs, 1H,
NH), 6.72 (bs, 1H, NH), 7.01 (d, J = 8.6 Hz, 2H, Ar–H), 7.67 (d, J = 15.5 Hz, 1H, CH), 7.75–7.86 (m, 3H,
CH, Ar–H), 7.99 (d, J = 8.8 Hz, 2H, Ar–H), 8.06 (d, J = 8.8 Hz, 2H, Ar–H), 9.35 (bs, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6) δ ppm 42.9 (CH2), 55.4 (CH3), 60.1 (CH2), 114.4, 118.3, 119.7, 127.5 (Cq), 129.5,
130.4 (Cq), 130.6, 135.9 (Cq), 142.7, 145.6 (Cq), 161.1 (Cq), 165.9 (Cq), 187.3 (Cq). MS (70 eV) m/z (%):
450 [M+] (89), 420 (100), 363 (36), 161 (64), 133 (40), 43 (39). Anal. Calcd. C23H26N6O4: C, 61.32; H, 5.82;
N, 18.66; Found: C, 61.30; H, 5.81; N, 18.70.
(E)-1-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(3,4,5-trimethoxyphenyl)-prop-2
-en-1-one (7d). Yellow solid; 57% yield; m.p. 197–200 C. FT-IR (ATR) v (cm−1) 3433 (N–H), 3302 (O–H),
◦
3105 (=C–H), 1645 (C=O), 1573br (C=N, C=C), 1124 (C–O). 1H-NMR (400 MHz, DMSO-d6)
δ ppm
3.32–3.40 (m, 4H, CH2), 3.48–3.59 (m, 4H, CH2), 3.71 (s, 3H, OCH3), 3.87 (s, 6H, OCH3), 4.69 (s, 2H,
OH), 6.72 (bs, 1H, NH), 6.85 (bs, 1H, NH), 7.21 (s, 2H, Ar–H), 7.65 (d, J = 15.4 Hz, 1H, CH), 7.89 (d,
J = 15.4 Hz, 1H, CH), 8.02 (d, J = 8.6 Hz, 2H, Ar–H), 8.09 (d, J = 8.6 Hz, 2H, Ar–H), 9.38 (t, J = 35.9 Hz,
1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 42.9 (CH2), 56.1 (OCH3), 60.1 (CH2), 60.2 (OCH3),
106.4, 118.3, 121.4, 129.6 (Cq), 130.5, 135.9 (Cq), 139.5, 143.2 (Cq), 143.3 (Cq), 145.6 (Cq), 145.7 (Cq),
153.1 (Cq), 187.2 (Cq). MS (70 eV) m/z (%): 510 [M+] (21), 313 (24), 236 (21), 97 (50), 58 (100), 43 (95).
Anal. Calcd. C25H30N6O6: C, 58.81; H, 5.92; N, 16.46; Found: C, 58.87; H, 6.01; N, 16.45.
(E)-1-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-tria◦zin-2-yl)amino)phenyl)-3-(4-chlorophenyl)prop-2-en-1-one
(
7e). Yellow solid; 87% yield; m.p. 193–195 C. FT-IR (ATR) v (cm−1) 3383 (N–H), 3285 (O–H),
1
3100 (=C–H), 1651 (C=O), 1568 and 1510 (C=N and C=C). H-NMR (400 MHz, DMSO-d6)
δ ppm
3.28–3.42 (m, 4H, CH2), 3.48–3.58 (m, 4H, CH2), 4.70 (s, 2H, OH), 6.73 (bs, 1H, NH), 6.87 (bs, 1H, NH),
7.52 (d, J = 8.4 Hz, 2H, Ar–H), 7.68 (d, J = 15.6 Hz, 1H, CH), 7.88–7.96 (m, 3H, CH, Ar–H), 8.01 (d,
J = 8.6 Hz, 2H, Ar–H), 8.08 (d, J = 8.6 Hz, 2H, Ar–H), 9.39 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ
ppm 42.9 (CH2), 60.0 (CH2), 118.3, 122.9, 128.9, 129.7, 130.0 (Cq), 130.1 (Cq), 130.4, 133.9 (Cq),
134.8 (Cq), 135.9 (Cq), 141.3, 145.8 (Cq), 187.1 (Cq). MS (70 eV) m/z (%): 454:456 [M+]:[M + 2]+ (34/11),
454 (62), 367 (36), 137 (37), 55 (81), 43 (100). Anal. Calcd. C22H23ClN6O3: C, 58.09; H, 5.10; N, 18.47;
Found: C, 58.02; H, 5.15; N, 18.49.
(E)-1-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(4-fluorophenyl)prop-2-en-1-one
◦
(
7f). Yellow solid; 88% yield; m.p. 176–178 C. FT-IR (ATR) v (cm−1) 3373 (N–H), 3283 (O–H),
1
3105 (=C–H), 1651 (C=O), 1575 and 1504 (C=N and C=C). H-NMR (400 MHz, DMSO-d6)
δ ppm
3.32–3.42 (m, 4H, CH2), 3.48–3.57 (m, 4H, CH2), 4.73 (s, 2H, OH), 6.73 (bs, 1H, NH), 6.87 (bs, 1H,
NH), 7.25–7.33 (m, 2H, Ar–H), 7.70 (d, J = 15.6 Hz, 1H, CH), 7.90 (d, J = 15.6 Hz, 1H, CH), 7.96 (dd,
J = 8.3 and JHF = 6.0 Hz, 2H, Ar–H), 8.01 (d, J = 9.0 Hz, 2H, Ar–H), 8.08 (d, J = 9.0 Hz, 2H, Ar–H),
9.38 (t, J = 36.9 Hz, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 42.8 (CH2), 60.0 (CH2), 115.8 (d,
2JCF = 21.6 Hz), 118.3, 122.1, 129.7, 130.1 (Cq), 131.0 (d, 3JCF = 8.5 Hz), 131.6 (d, 4JCF = 2.9 Hz, Cq),
131.6 (Cq), 141.5, 145.7 (Cq), 163.3 (d, 1JCF = 248.7 Hz, C–F), 163.9 (Cq), 187.2 (Cq). MS (70 eV) m/z (%):
438 [M+] (46), 408 (86), 395 (56), 302 (71), 149 (58), 43 (100). Anal. Cald. C22H23FN6O3: C, 60.27; H, 5.29;
N, 19.17; Found: C, 60.23; H, 5.27; N, 19.20.
(E)-1-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(4-(trifluoromethyl)phenyl)-prop
◦
-2-en-1-one (7g). Yellow solid; 71% yield; m.p. 193–196 C. FT-IR (ATR) v (cm−1) 3390 (N–H),
3317 (O–H), 3110 (=C–H), 1661 (C=O), 1568 and 1512 (C=N and C=C). 1H-NMR (400 MHz, DMSO-d6)
δ
ppm 3.28–3.41 (m, 4H, CH2), 3.49–3.57 (m, 4H, CH2), 4.73 (s, 2H, OH), 6.74 (bs, 1H, NH), 6.88 (bs,
1H, NH), 7.74 (d, 1H, J = 15.6 Hz, 1H, CH), 7.80 (d, 2H, J = 8.8 Hz, Ar–H), 8.02 (d, J = 8.8 Hz, 2H,

Molecules 2018, 23, 1956
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Ar–H), 8.05–8.14 (m, 5H, Ar–H, CH), 9.41 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ
ppm 42.9
(CH2), 60.1 (CH2), 118.4, 122.8 (Cq), 124.9, 125.7 (q, JCF = 3.4 Hz), 128.3 (q, 1JCF = 241.2 Hz, CF3), 129.3,
129.9 (unresolved q), 130.0 (unresolved q, Cq), 136.0 (Cq), 139.0 (Cq), 140.8 (Cq), 146.0 (Cq), 187.1 (Cq).
MS (70 eV) m/z (%): 488 [M+] (28), 458 (65), 445 (41), 97 (36), 69 (62), 57 (69), 43 (100). Anal. Calcd.
C23H23F3N6O3: C, 56.55; H, 4.75; N, 17.21; Found: C, 56.53; H, 4.71; N, 17.19.
3.2.4. General Procedure for the Synthesis of 1-(3-(4-((4,6-bis((2-Hydroxyethyl)
amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)ethanones 8a–g
A mixture of chalcone
7 (0.48 mmol) and hydrazine monohydrate (4.80 mmol) in ethanol (1.0 mL)
was stirred at room temperature for 30 min. Subsequently, acetic anhydride was added (1.5 mL) and
the solution was stirred at room temperature for 3 h. The solid obtained was filtered and washed
with water. Purification of products was performed by column chromatography employing a mixture
CH2Cl2/EtOH (20:1) as eluent.
1-(3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1
-yl)ethanone (8a). Beige solid; 56% yield; m.p. 137–139 ◦C. FT-IR (ATR) v (cm−1) Not observed (N–H),
3288 (O–H), 3105 (=C–H) 1612 (C=O), 1587 and 1560 (C=N and C=C). 1H-NMR (400 MHz, DMSO-d6)
δ
ppm 2.29 (s, 3H, CH3), 3.09 (dd, J = 17.9, 4.4 Hz, 1H, H-4), 3.30–3.38 (m, 4H, CH2), 3.47–3.55 (m,
4H, CH2), 3.81 (dd, J = 17.9, 11.8 Hz, 1H, H-4), 4.66 (s, 2H, OH), 5.51 (dd, J = 11.8, 4.4 Hz, 1H, H-5),
6.65 (bs, 1H, NH), 6.78 (bs, 1H, NH), 7.17 (d, J = 7.3 Hz, 2H, Ar–H), 7.24 (t, J = 7.3 Hz, 1H, Ar–H),
7.32 (t, J = 7.3 Hz, 2H, Ar–H), 7.64 (d, J = 8.7 Hz, 2H, Ar–H), 7.90 (d, J = 8.7 Hz, 2H, Ar–H), 9.17 (bs,
1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 21.7 (CH3), 42.2 (CH2), 42.8 (CH2), 59.2, 60.1 (CH2),
118.9, 125.4, 127.0, 127.1, 128.6, 128.7 (Cq), 135.9 (Cq), 142.6 (Cq), 142.9 (Cq), 143.0 (Cq), 154.2 (Cq),
167.1 (Cq). MS (70 eV) m/z (%): 476 [M+] (49), 446 (6), 434 (12), 405 (16), 55 (17), 43 (100). Anal. Calcd.
C24H28N8O3: C, 60.49; H, 5.92; N, 23.51; Found: C, 60.44; H, 5.93; N, 22.98.
1-(3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-
◦
1-yl)ethanone (8b). Beige solid; 67% yield; m.p. 133–135 C. FT-IR (ATR) v (cm−1) Not observed
(N–H), 3288 (O–H), 3106 (=C–H), 1611 (C=O), 1562 and 1514 (C=N and C=C). 1H-NMR (400 MHz,
DMSO-d6)
δ ppm 2.25 (s, 3H, CH3), 2.28 (s, 3H, CH3), 3.06 (dd, J = 17.9, 4.2 Hz, 1H, H-4), 3.23–3.39 (m,
4H, CH2), 3.46–3.57 (m, 4H, CH2), 3.78 (dd, J = 17.9, 11.7 Hz, 1H, H-4), 4.74 (bs, 2H, OH), 5.46 (dd,
J = 11.7, 4.2 Hz, 1H, H-5), 6.65 (bs, 1H, NH), 6.78 (bs, 1H, NH), 7.05 (d, J = 8.0 Hz, 2H, Ar–H), 7.12 (d,
J = 8.0 Hz, 2H, Ar–H), 7.63 (d, J = 8.6 Hz, 2H, Ar–H), 7.90 (d, J = 8.6 Hz, 2H, Ar–H), 9.17 (bs, 1H,
NH). 13C- NMR (100 MHz, DMSO-d6)
δ ppm 21.1 (CH3), 22.2 (CH3), 42.6 (CH2), 43.3 (CH2), 59.5,
60.6 (CH2), 119.4, 124.0 (Cq), 125.9, 127.5, 129.6, 136.4 (Cq), 136.8 (Cq), 140.1 (Cq), 143.3 (Cq), 143.4 (Cq),
154.7 (Cq), 167.6 (Cq). MS (70 eV) m/z (%): 490 [M+] (100), 448 (29), 419 (26), 208 (5), 43 (60). Anal.
Calcd. C25H30N8O3: C, 61.21; H, 6.16; N, 22.84; Found: C, 61.26; H, 6.20; N, 22.79.
1-(3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(4-methoxyphenyl)-4,5-dihydro-
1H-pyrazol-1-yl)ethanone (8c). Beige solid; yield 62%; m.p. 136–138 ◦C. FT-IR (ATR) v (cm−1
)
3312 (N–H), 3296 (O–H), 3103 (=C–H), 1611 (C=O), 1587 (C=N), 1560 (C=C) and 1240 (C–O). 1H-NMR
(400 MHz, DMSO-d6) ppm 2.27 (s, 3H, CH3), 3.07 (dd, J = 17.9, 3.9 Hz, 1H, H-4), 3.26–3.40 (m, 4H,
δ
CH2), 3.47–3.56 (m, 4H, CH2), 3.71 (s, 3H, OCH3), 3.77 (dd, J = 17.9, 11.6 Hz, 1H, H-4), 4.69 (s, 2H, OH),
5.45 (dd, J = 11.6, 3.9 Hz, 1H, H-5), 6.65 (bs, 1H, NH), 6.78 (bs, 1H, NH), 6.87 (d, J = 8.4 Hz, 2H, Ar–H),
7.10 (d, J = 8.4 Hz, 2H, Ar–H), 7.64 (d, J = 8.5 Hz, 2H, Ar–H), 7.90 (d, J = 8.5 Hz, 2H, Ar–H), 9.17 (bs,
1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 21.8 (CH3), 42.1 (CH2), 42.9 (CH2), 55.1 (CH3), 58.7,
60.1 (CH2), 114.0, 119.0, 123.6 (Cq), 126.8, 127.1, 134.7 (Cq), 136.0 (Cq), 142.9 (Cq), 143.0 (Cq) 154.3 (Cq),
158.4 (Cq), 167.1 (Cq). MS (70 eV) m/z (%): 506 [M+] (100), 464 (28), 435 (22), 316 (12), 134 (14), 43 (70).
Anal. Calcd. C25H30N8O4: C, 59.28; H, 5.97; N, 22.12; Found: C, 59.24; H, 6.01; N, 22.14.
1-(3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (8d). Beige solid; 75% yield; m.p. 134–136 ◦C. FT-IR (ATR) v (cm−1
)

Molecules 2018, 23, 1956
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3422 (N–H), 3289 (O–H), 3196 (=C–H), 1699 (C=O), 1647(C=N) 1560 (C=C) and 1278 (C–O). 1H-NMR
(400 MHz, DMSO-d6) ppm 2.31 (s, 3H, CH3), 3.12 (dd, J = 17.9, 4.5 Hz, 1H, H-4), 3.29–3.40 (m, 4H,
δ
CH2), 3.46–3.57 (m, 4H, CH2), 3.62 (s, 3H, OCH3), 3.73 (s, 6H, OCH3), 3.79 (dd, J = 17.9 Hz, 11.4 Hz,
1H, H-4), 4.67 (s, 2H, OH), 5.45 (dd, J = 11.4, 4.5 Hz, 1H, H-5), 6.45 (s, 2H, Ar–H), 6.65 (bs, 1H, NH),
6.78 (bs, 1H, NH), 7.63 (d, J = 8.6 Hz, 2H, Ar–H), 7.90 (d, J = 8.6 Hz, 2H, Ar–H), 9.17 (bs, 1H, NH).
13C- NMR (100 MHz, DMSO-d6)
δ ppm 22.0 (CH3), 42.5 (CH2), 43.0 (CH2), 56.1 (CH3), 59.7, 60.1 (CH3),
60.3 (CH2), 102.7, 119.1, 123.8 (Cq), 127.3, 136.1 (Cq), 136.7 (Cq), 138.6 (Cq), 143.1 (Cq), 143.2 (Cq),
153.3 (Cq), 154.4 (Cq), 167.6 (Cq). MS (70 eV) m/z (%): 566 [M+] (41), 524 (8), 496 (13), 481 (9), 251 (9),
43 (100). Anal. Calcd. C27H34N8O6: C, 57.23; H, 6.05; N, 19.78; Found: C, 57.29; H, 6.03; N, 19.80.
1-(3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(4-chlorophenyl)-4,5-dihydro-1H-
pyrazol-1-yl)ethanone (8e). Beige solid; 86% yield; m.p. 174–176 ◦C. FT-IR (ATR) v (cm−1) 3443 (N–H),
3288 (O–H), 3104 (=C–H), 1647 (C=O), 1598 and 1547 (C=N and C=C). 1H-NMR (400 MHz, DMSO-d6)
δ
ppm 2.28 (s, 3H, CH3), 3.10 (dd, J = 18.0, 4.6 Hz, 1H, H-4), 3.28–3.36 (m, 4H, CH2), 3.46–3.55 (m,
4H, CH2), 3.81 (dd, J = 18.0, 11.8 Hz, 1H, H-4), 4.76 (bs, 2H, OH), 5.51 (dd, J = 11.8, 4.6 Hz, 1H,
H-5), 6.65 (bs, 1H, NH), 6.79 (bs, 1H, NH), 7.21 (d, J = 8.5 Hz, 2H, Ar–H), 7.38 (d, J = 8.5 Hz, 2H,
Ar–H), 7.64 (d, J = 8.0 Hz, 2H, Ar–H), 7.90 (d, J = 8.0 Hz, 2H, Ar–H), 9.18 (bs, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6)
δ ppm 21.7 (CH3), 42.0 (CH2), 42.9 (CH2), 58.7, 60.1 (CH2), 118.9, 127.1, 127.5,
128.6, 131.7 (Cq), 136.0 (Cq), 141.5 (Cq), 141.6 (Cq), 154.2 (Cq), 163.9 (Cq), 165.9 (Cq), 167.3 (Cq).
MS (70 eV) m/z (%): 510:512 [M+]:[M + 2]+ (18/6), 468 (46), 440 (100), 55 (22), 43 (39). Anal. Calcd.
C24H27ClN8O3: C, 56.41; H, 5.33; N, 21.93; Found: C, 56.47; H, 5.38; N, 21.99.
1-(3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-
◦
pyrazol-1-yl)ethanone (8f). Beige solid; 55% yield; m.p. 132–134 C. FT-IR (ATR) v (cm−1) Not observed
(N–H), 3292 (O–H), 3108 (=C–H), 1645 (C=O), 1602 and 1568 (C=N and C=C). 1H-NMR (400 MHz,
DMSO-d6)
δ ppm 2.28 (s, 3H, CH3), 3.10 (dd, J = 17.9, 4.4 Hz, 1H, H-4), 3.28–3.38 (m, 4H, CH2),
3.47–3.55 (m, 4H, CH2), 3.80 (dd, J = 17.9, 11.7 Hz, 1H, H-4), 4.66 (s, 2H, OH), 5.52 (dd, J = 11.7, 4.4 Hz,
1H, H-5), 6.65 (bs, 1H, NH), 6.78 (bs, 1H, NH), 7.14 (t, J = 8.5 Hz, 2H, Ar–H), 7.22 (dd, J = 8.5 Hz
and JHF = 5.6 Hz, 2H, Ar–H), 7.64 (d, J = 8.6 Hz, 2H, Ar–H), 7.90 (d, J = 8.6 Hz, 2H, Ar–H), 9.17 (bs,
1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 21.7 (CH3), 42.0 (CH2), 42.8 (CH2), 58.5, 60.1 (CH2),
115.3 (d, 2JCF = 21.4 Hz), 118.9, 127.0 (unresolved d), 127.6 (d, 3JCF = 8.2 Hz), 135.9 (Cq), 138.7 (Cq),
142.9 (Cq), 147.0 (Cq), 154.1 (Cq), 161.2 (d, 1JCF = 242.7 Hz, C–F), 165.9 (Cq), 167.1 (C=O). MS (70 eV)
m/z (%): 494 [M+] (25), 423 (7), 210 (5), 60 (14), 69 (5), 43 (100). Anal. Calcd. C24H27FN8O3: C, 58.29; H,
5.50; N, 22.66; Found: C, 58.35; H, 5.58; N, 22.64.
1-(3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-
dihydro-1H-pyrazol-1-yl)ethanone (8g). Beige solid; 66% yield; m.p. 136–138 ◦C. FT-IR (ATR) v (cm−1
)
3385 (N–H), 3290 (O–H), 3103 (=C–H), 1647 (C=O), 1598 and 1570 (C=N and C=C). 1H-NMR (400 MHz,
DMSO-d6) ppm 2.30 (s, 3H, CH3), 3.14 (dd, J = 18.0, 4.8 Hz, 1H, H-4), 3.26–3.38 (m, 4H, CH2),
δ
3.47–3.55 (m, 4H, CH2), 3.85 (dd, J = 18.0, 11.9 Hz, 1H, H-4), 4.66 (s, 2H, OH), 5.60 (dd, J = 11.9, 4.8 Hz,
1H, H-5), 6.66 (bs, 1H, NH), 6.79 (bs, 1H, NH), 7.41 (d, J = 8.1 Hz, 2H, Ar–H), 7.64 (d, J = 8.1 Hz, 2H,
Ar–H), 7.69 (d, J = 8.5 Hz, 2H, Ar–H), 7.91 (d, J = 8.5 Hz, 2H, Ar–H), 9.18 (bs, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6)
δ ppm 21.7 (CH3), 42.0 (CH2), 42.8 (CH2), 59.0, 60.1 (CH2), 119.0, 122.9 (Cq),
123.4 (Cq), 125.6 (q, 3JCF = 3.6 Hz), 125.7 (q, 1JCF = 237.3 Hz, CF3), 125.8 (Cq), 126.5, 127.2 (unresolved
q), 127.9 (q, 2JCF = 31.8 Hz, Cq), 143.0 (Cq), 147.1 (Cq), 154.3 (Cq), 167.4 (C=O). MS (70 eV) m/z (%):
544 [M+] (96), 513 (20), 473 (21), 458 (10), 43 (100). Anal. Calcd. C25H27F3N8O3: C, 55.14; H, 5.00; N,
20.58; Found: C, 55.18; H, 4.96; N, 20.53.

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3.2.5. General Procedure for the Synthesis of 5-(4-((4,6-bis((2-Hydroxyethyl)
amino)-1,3,5-triazin-2-yl)amino)phenyl)-3-(aryl)-4,5-dihydro-1H-pyrazol-1-carbaldehydes 9a–g
N-Formylpyrazolines 9a–g were synthesized using a similar methodology for obtaining N-acetyl
pyrazolines 8a , but formic acid was used instead of acetic anhydride. The solid obtained was filtered
–
g
and washed with water. No further purification was required.
3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-phenyl-4,5-dihydro-1H-pyrazole-1-
carbaldehyde (9a). White solid; 93% yield; m.p. 163–166 ◦C. FT-IR (ATR) v (cm−1) Not observed (N–H),
3273 (O–H), 3101 (=C–H), 1649 (C=O), 1593 and 1564 (C=N and C=C). 1H-NMR (400 MHz, DMSO-d6)
δ
ppm 3.15 (dd, J = 18.0, 4.7 Hz, 1H, H-4), 3.26–3.40 (m, 4H, CH2), 3.44–3.56 (m, 4H, CH2), 3.68 (bs,
2H, OH), 3.88 (dd, J = 18.0, 11.7 Hz, 1H, H-4), 5.50 (dd, J = 11.7, 4.7 Hz, 1H, H-5), 6.66 (bs, 1H, NH),
6.79 (bs, 1H, NH), 7.30–7.20 (m, 3H, Ar–H), 7.31–7.37 (m, 2H, Ar–H), 7.65 (d, J = 8.8 Hz, 2H, Ar–H),
7.91 (d, J = 8.8 Hz, 2H, Ar–H), 8.86 (s, 1H, CHO), 9.20 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ
ppm 42.2 (CH2), 42.7 (CH2), 58.1, 59.9 (CH2), 118.7, 125.5, 126.9, 127.2, 127.4 (Cq), 128.5, 130.4 (Cq),
135.7 (Cq), 141.2 (Cq), 142.94 (Cq), 156.0 (Cq), 159.2. MS (70 eV) m/z (%): 462 [M+] (100), 434 (64),
406 (19), 91 (36), 69 (39), 43 (64). Anal. Calcd. C23H26N8O3: C, 59.73; H, 5.67; N, 24.23; Found: C, 59.80;
H, 6.00; N, 24.27.
3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazole-1
-carbaldehyde 9b. White solid; 60% yield; m.p. 167–170 ◦C. FT-IR (ATR) v (cm−1) Not observed (N–H),
3269 (O–H), 3108 (=C–H), 1672 (C=O), 1647 and 1593 (C=N and C=C). 1H-NMR (400 MHz, DMSO-d6)
δ
ppm 2.26 (s, 3H, CH3), 3.12 (dd, J = 18.0, 4.6 Hz, 1H, H-4), 3.26–3.39 (m, 4H, CH2), 3.46–3.55 (m,
4H, CH2), 3.85 (dd, J = 18.0, 11.6 Hz, 1H, H-4), 3.93 (bs, 2H, OH), 5.45 (dd, J = 11.6, 4.6 Hz, 1H, H-5),
6.66 (bs, 1H, NH), 6.79 (bs, 1H, NH), 7.06–7.17 (m, 4H, Ar–H), 7.64 (d, J = 8.7 Hz, 2H, Ar–H), 7.90 (d,
J = 8.7 Hz, 2H, Ar–H), 8.84 (s, 1H, CHO), 9.19 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm
20.7 (CH3), 42.4 (CH2), 43.0 (CH2), 58.1, 60.1 (CH2), 118.9, 125.6, 127.2, 129.3, 122.2 (Cq), 122.3 (Cq),
136.7 (Cq), 138.6 (Cq), 143.1 (Cq), 156.1 (Cq), 159.3 (Cq), 163.7. MS (70 eV) m/z (%): 476 [M+] (100),
448 (58), 420 (22), 118 (29), 91 (33), 43 (41). Anal. Calcd. C24H28N8O3: C, 60.49; H, 5.92; N, 23.51; Found:
C, 60.53; H, 6.04; N, 23.53.
3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-
◦
pyrazole-1-carbaldehyde 9c. White solid; 91% yield; m.p. 156–157 C. FT-IR (ATR) v (cm−1) Not observed
1
(N–H), 3228 (O–H), 3101 (=C–H), 1674 (C=O), 1643 (C=N), 1593 (C=C) and 1249 (C–O). H-NMR
(400 MHz, DMSO-d6)
δ ppm 3.14 (dd, J = 18.0, 4.6 Hz, 1H, H-4), 3.27–3.38 (m, 4H, CH2), 3.51–3.55
(m, 4H, CH2), 3.72 (s, 3H, OCH3), 3.84 (dd, J = 18.0, 11.6 Hz, 1H, H-4), 3.99 (s, 2H, OH), 5.44 (dd,
J = 11.6, 4.6 Hz, 1H, H-5), 6.66 (bs, 1H, NH), 6.78 (bs, 1H, NH), 6.89 (d, J = 8.7 Hz, 2H, Ar–H), 7.14 (d,
J = 8.7 Hz, 2H, Ar–H), 7.64 (d, J = 8.8 Hz, 2H, Ar–H), 7.91 (d, J = 8.8 Hz, 2H, Ar–H), 8.83 (s, 1H, CHO),
9.19 (bs, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 42.3 (CH2), 42.8 (CH2), 55.1 (CH3), 57.8,
60.1 (CH2), 114.1, 118.9, 123.2 (Cq), 127.0, 127.2, 127.3 (Cq), 133.5 (Cq), 156.1 (Cq), 158.6 (Cq), 159.3. MS
(70 eV) m/z (%): 492 [M+] (27), 464 (35), 436 (100), 405 (41), 361 (31), 43 (36). Anal. Calcd. C24H28N8O4:
C, 58.53; H, 5.73; N, 22.75; Found: C, 58.50; H, 5.75; N, 22.70.
3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro
-1H-pyrazole-1-carbaldehyde (9d). White solid; 40% yield; m.p. 154–156 ◦C. FT-IR (ATR) v (cm−1) 3483
1
(N–H), 3298 (O–H), 3102 (=C–H), 1662 (C=O), 1583 (C=N), 1562 (C=C) and 1234 (C–O). H-NMR
(400 MHz, DMSO-d6)
δ ppm 3.18 (dd, J = 18.0, 5.1 Hz, 1H, H-4), 3.29–3.44 (m, 4H, CH2), 3.48–3.54 (m,
4H, CH2), 3.63 (s, 3H, OCH3), 3.73 (s, 6H, OCH3), 3.84 (dd, J = 18.0, 11.5 Hz, 1H, H-4), 4.69 (s, 2H, OH),
5.43 (dd, J = 11.5, 5.1 Hz, 1H, H-5), 6.50 (s, 2H, Ar–H), 6.67 (bs, 1H, NH), 6.78 (bs, 1H, NH), 7.64 (d,
J = 8.7 Hz, 2H, Ar–H), 7.90 (d, J = 8.7 Hz, 2H, Ar–H), 8.88 (s, 1H, CHO), 9.19 (bs, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6)
δ ppm 42.4 (CH2), 42.9 (CH2), 55.9 (CH3), 58.6, 60.0 (CH3), 60.1 (CH2), 99.6 (Cq),
102.8, 119.0, 126.0 (Cq), 127.2, 136.0 (Cq), 136.7 (Cq), 137.2 (Cq), 143.0 (Cq), 153.1 (Cq), 156.1 (Cq),

Molecules 2018, 23, 1956
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159.6. MS (70 eV) m/z (%): 552 [M+] (27), 524 (28), 496 (100), 481 (60), 435 (29), 43 (61). Anal. Calcd.
C26H32N8O6: C, 56.51; H, 5.84; N, 20.28; Found: C, 56.53; H, 5.83; N, 20.28.
3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(4-chlorophenyl)-4,5-dihydro-1H-
◦
pyrazole-1-carbaldehyde (9e). White solid; 65% yield; m.p. 165–167 C. FT-IR (ATR) v (cm−1) Not
1
observed (N–H), 3271 (O–H), 3097 (=C–H), 1674 (C=O), 1649 and 1593 (C=N and C=C). H-NMR
(400 MHz, DMSO-d6)
δ ppm 3.17 (dd, J = 18.0, 4.8 Hz, 1H, H-4), 3.27–3.40 (m, 4H, CH2), 3.45–3.56
(m, 6H, CH2, OH), 3.88 (dd, J = 18.0, 11.7 Hz, 1H, H-4), 5.51 (dd, J = 11.7, 4.8 Hz, 1H, H-5), 6.67 (bs,
1H, NH), 6.79 (bs, 1H, NH), 7.26 (d, J = 8.5 Hz, 2H, Ar–H), 7.40 (d, J = 8.5 Hz, 2H, Ar–H), 7.65 (d,
J = 8.6 Hz, 2H, Ar–H), 7.91 (d, J = 8.6 Hz, 2H, Ar–H), 8.86 (s, 1H, CHO), 9.20 (bs, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6)
δ ppm 42.1 (CH2), 42.8 (CH2), 57.7, 60.1 (CH2), 119.0, 123.0 (Cq), 127.2, 127.3
(Cq), 127.8, 128.7, 131.9 (Cq), 135.9 (Cq), 140.4 (Cq), 143.2 (Cq), 156.0 (Cq), 159.4. MS (70 eV) m/z (%):
496:498 [M+]:[M + 2] (34/12), 468 (46), 440 (6), 391 (20), 115 (47), 43 (100). Anal. Calcd. C23H25ClN8O3:
C, 55.59; H, 5.07; N, 22.55; Found: C, 55.64; H, 4.98; N, 22.58.
3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-
◦
pyrazole-1-carbaldehyde (9f). White solid; 58% yield; m.p. 167–169 C. FT-IR (ATR) v (cm−1) Not
1
observed (N–H), 3273 (O–H), 3105 (=C–H), 1672 (C=O), 1649 and 1593 (C=N and C=C). H-NMR
(400 MHz, DMSO-d6)
δ ppm 3.16 (dd, J = 18.0, 4.8 Hz, 1H, H-4), 3.27–3.39 (m, 4H, CH2), 3.46–3.55
(m, 6H, CH2, OH), 3.86 (dd, J = 18.0, 11.7 Hz, 1H, H-4), 5.51 (dd, J = 11.7, 4.8 Hz, 1H, H-5), 6.66 (bs,
1H, NH), 6.78 (bs, 1H, NH), 7.19–7.12 (m, 2H, Ar–H), 7.30–7.23 (m, 2H, Ar–H), 7.64 (d, J = 8.7 Hz,
2H, Ar–H), 7.90 (d, J = 8.7 Hz, 2H, Ar–H), 8.85 (s, 1H, CHO), 9.19 (bs, 1H, NH). 13C-NMR (100 MHz,
DMSO-d6) δ
ppm 42.2 (CH2), 42.9 (CH2), 57.7, 60.1 (CH2), 115.5 (d, 2JCF = 21.5 Hz), 117.2 (Cq), 118.9,
123.1 (Cq), 127.2, 127.9 (d, 3JCF = 8.3 Hz), 137.7 (d, 4JCF = 3.0 Hz, Cq), 143.2 (Cq), 145.7 (Cq), 156.1
(Cq), 159.4 (CH=O), 161.4 (d, 1JCF = 243.5 Hz). MS (70 eV) m/z (%): 480 [M+] (45), 452 (75), 424 (100),
393 (43), 375 (23), 43 (14). Anal. Calcd. C23H25FN8O3: C, 57.49; H, 5.24; N, 23.32; Found: C, 57.48; H,
5.20; N, 23.39.
3-(4-((4,6-bis((2-Hydroxyethyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)-5-(4-(tri◦fluoromethyl)phenyl)-4,5-
Not observed (N–H), 3279 (O–H), 3099 (=C–H), 1649 (C=O), 1620 and 1593 (C=N and C=C). 1H-NMR
−1
dihydro-1H-pyrazole-1-carbaldehyde (9g). White solid; 35% yield; m.p. 160–164 C. FT-IR (ATR) v (cm
)
(400 MHz, DMSO-d6) ppm 3.20 (dd, J = 18.1, 5.0 Hz, 1H, H-4), 3.30–3.37 (m, 4H, CH2), 3.47–3.55
δ
(m, 6H, CH2, OH), 3.91 (dd, J = 18.1, 11.8 Hz, 1H, H-4), 5.60 (dd, J = 11.8, 5.0 Hz, 1H, H-5), 6.66
(bs, 1H, NH), 6.79 (bs, 1H, NH), 7.46 (d, J = 7.15 Hz, 2H, Ar–H), 7.64 (d, J = 7.2 Hz, 2H, Ar–H),
7.72 (d, J = 8.5 Hz, 2H, Ar–H), 7.90 (d, J = 8.5 Hz, 2H, Ar–H), 8.88 (s, 1H, CHO), 9.19 (bs, 1H, NH).
13C-NMR (100 MHz, DMSO-d6)
δ ppm 42.2 (CH2), 42.9 (CH2), 58.1, 60.2 (CH2), 119.0, 122.9 (Cq),
124.4 (q, 1JCF = 276.4 Hz, CF3), 125.6 (Cq), 125.8 (q, 3JCF = 3.9 Hz), 126.8, 127.3 (unresolved q), 128.2 (d,
2JCF = 31.7 Hz, Cq), 143.2 (Cq), 143. 3 (Cq), 146.0 (Cq), 156.2 (Cq), 159.6 (CH=O). MS (70 eV) m/z (%):
530 [M+] (93), 499 (35), 487 (19), 443 (24), 69 (54), 43 (100). Anal. Calcd. C24H25F3N8O3: C, 54.34; H,
4.75; N, 21.12; Found: C, 54.36; H, 4.70; N, 21.17.
3.2.6. General Procedure for the Synthesis of 2,20-((6-((4-(5-(aryl)-1-(3,5-Dichlorophenyl)-4,5-
dihydro-1H-pyrazol-3-yl)phenyll)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(ethanol)s 10a–g
A mixture of chalcone 7 (0.4 mmol) and 3,5-dichlorophenylhydrazine hydrochloride (2.6 mmol)
in ethanol (2.5 mL) was subjected to reflux for 2 h. The solid formed was filtered and washed with
ethanol. No further purification was required.
2,20-((6-((4-(1-(3,5-Dichlorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-triazine-2,4-
diyl)bis(azanediyl))bis(ethanol) (10a). Yellow solid; 65% yield; m.p. 207–208 ◦C. FT-IR (ATR) v (cm−1
)
Not observed (N–H), 3296 (O–H), 3130 (=C–H), 1647 and 1587 (C=N and C=C). 1H-NMR (400 MHz,
DMSO-d6) ppm 3.16 (dd, J = 17.7, 5.2 Hz, 1H, H-4), 3.37–3.49 (m, 4H, CH2), 3.52–3.63 (m, 6H, CH2,
OH), 3.94 (dd, J = 17.7, 12.1 Hz, 1H, H-4), 5.58 (dd, J = 12.1, 5.2 Hz, 1H, H-5), 6.80 (t, J = 1.7 Hz, 1H,
δ

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Ar–H), 6.93 (d, J = 1.7 Hz, 2H, Ar–H), 7.23–7.30 (m, 3H, Hp, Ar–H), 7.36 (t, J = 7.3 Hz, 2H, Ar–H),
7.81–7.71 (m, 4H, Ar–H), 8.23–8.84 (m, 2H, NH), 10.46 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ
ppm 43.2 (CH2), 59.0 (CH2), 59.2 (CH2), 62.4, 99.5 (Cq), 110.8, 116.9, 120.9, 125.8, 126.8, 127.8, 129.2,
134.4 (Cq), 141.4 (Cq), 145.7 (Cq), 149.8 (Cq). MS (70 eV) m/z (%): 578:580:582 [M+]:[M + 2]+:[M + 4]+
(100/67/11), 341 (13), 69 (32), 55 (39), 44 (57), 43 (66), 41 (28). Anal. Calcd. C28H28Cl2N8O2: C, 58.04; H,
4.87; N, 19.34; Found: C, 58.05; H, 4.89; N, 19.37.
2,20-((6-((4-(1-(3,5-Dichlorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-triazine-2,4-
diyl)bis(azanediyl))bis(ethanol) (10b). Yellow solid; 92% yield; m.p. 218–219 ◦C. FT-IR (ATR) v (cm−1
)
Not observed (N–H), 3309 (O–H), 3115 (=C–H), 1647 and 1585 (C=N and C=C). 1H-NMR (400 MHz,
DMSO-d6) ppm 2.25 (s, 3H, CH3), 3.12 (dd, J = 17.6, 5.1 Hz, 1H, H-4), 3.41–3.49 (m, 4H, CH2),
δ
3.53–3.61 (m, 6H, CH2, OH), 3.90 (dd, J = 17.6, 12.0 Hz, 1H, H-4), 5.53 (dd, J = 12.0, 5.1 Hz, 1H, H-5),
6.79 (t, J = 1.7 Hz, 1H, Ar–H), 6.92 (d, J = 1.7 Hz, 2H, Ar–H), 7.19–7.11 (m, 4H, Ar–H), 7.81–7.72 (m, 4H,
Ar–H), 8.20–8.88 (m, 2H, NH), 10.46 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 20.7 (CH3),
43.2 (CH2), 59.0 (CH2), 62.2, 99.5 (Cq), 110.8, 111.0 (Cq), 116.8, 120.9, 121.0 (Cq), 125.7, 126.7, 126.8 (Cq),
129.7, 134.4 (Cq), 137.0 (Cq), 138.4 (Cq), 145.7 (Cq), 149.8 (Cq). MS (70 eV) m/z (%): 592:594:596
[M+]:[M + 2]+:[M + 4]+ (100/78/15), 501 (13), 284 (11), 117 (14), 91 (15), 69 (20), 55 (22), 44 (27), 43 (36).
Anal. Calcd. C29H30Cl2N8O2: C, 58.69; H, 5.10; N, 18.88; Found: C, 58.74; H, 5.11; N, 18.83.
2,20-((6-((4-(1-(3,5-Dichlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-
triazine-2,4-diyl)bis(azanediyl))bis(ethanol) (10c). Yellow solid; 85% yield; m.p. 226–228 ◦C. FT-IR (ATR)
v (cm−1) Not observed (N–H), 3307 (O–H), 3105 (=C–H), 1647 (C=N), 1585 (C=C) and 1251 (C–O).
1H-NMR (400 MHz, DMSO-d6)
δ ppm 3.12 (dd, J = 17.6, 5.1 Hz, 1H, H-4), 3.40–3.48 (m, 4H, CH2),
3.53–3.60 (m, 6H, CH2, OH), 3.70 (s, 3H, OCH3), 3.88 (dd, J = 17.6, 11.9 Hz, 1H, H-4), 5.51 (dd, J = 11.9,
5.1 Hz, 1H, H-5), 6.79 (t, J = 1.7 Hz, 1H, Ar–H), 6.90 (d, J = 8.7 Hz, 2H, Ar–H), 6.93 (d, J = 1.7 Hz, 2H,
Ar–H), 7.17 (d, J = 8.7 Hz, 2H, Ar–H), 7.81–7.72 (m, 4H, Ar–H), 8.23–8.80 (m, 2H, NH), 10.45 (s, 1H,
NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 43.2 (CH2), 55.1 (CH3), 59.2 (CH2), 61.9, 99.6 (Cq), 110.9,
114.6, 116.8, 120.9, 121.0 (Cq), 126.8, 126.9 (Cq), 127.1, 133.3 (Cq), 134.4 (Cq), 139.4 (Cq), 145.8 (Cq),
149.8 (Cq), 158.7 (Cq). MS (70 eV) m/z (%): 608:610:612 [M+]:[M + 2]+:[M + 4]+ (100/66/14), 501 (31),
83 (13), 69 (36), 55 (43), 44 (54), 43 (74). Anal. Calcd. C29H30Cl2N8O3: C, 57.15; H, 4.96; N, 18.38; Found:
C, 57.11; H, 5.03; N, 18.36.
2,20-((6-((4-(1-(3,5-Dichlorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)
1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(ethanol) (10d). Yellow solid; 92% yield; m.p. 202–204 ◦C. FT-IR
(ATR) v (cm−1) Not observed (N–H), 3273 (O–H), 3196 (=C–H), 1641 (C=N), 1585 (C=C) and 1228
1
(C–O). H-NMR (400 MHz, DMSO-d6)
δ ppm 3.22 (dd, J = 17.6, 6.2 Hz, 1H, H-4), 3.40–3.48 (m,
4H, CH2), 3.55–3.60 (m, 6H, CH2,OH), 3.63 (s, 3H, OCH3), 3.70 (s, 6H, OCH3), 3.92 (dd, J = 17.6,
12.0 Hz, 1H, H-4), 5.43 (dd, J = 12.0, 6.2 Hz, 1H, H-5), 6.60 (s, 2H, Ar–H), 6.83 (s, 1H, Ar–H), 6.97
(d, J = 1.6 Hz, 2H, Ar–H), 7.77 (s, 4H, Ar–H), 8.19–8.82 (m, 2H, NH), 10.48 (s, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6)
δ ppm 43.3 (CH2), 56.0 (CH3), 59.0 (CH2), 60.0 (CH3), 63.1, 103.1, 111.0, 117.1,
120.9, 126.8, 129.1 (Cq), 134.4 (Cq), 135.9 (Cq), 136.9 (Cq), 137.2 (Cq), 144.4 (Cq), 146.2 (Cq), 148.2 (Cq),
150.1 (Cq), 153.4 (Cq). MS (70 eV) m/z (%): 668:670:672 [M+]:[M + 2]+:[M + 4]+ (100/64/13), 501 (28),
84 (33), 69 (42), 55 (52), 44 (83), 43 (79). Anal. Calcd. C31H34Cl2N8O5: C, 55.61; H, 5.12; N, 16.74; Found:
C, 55.65; H, 5.18; N, 16.75.
2,20-((6-((4-(5-(4-Chlorophenyl)-1-(3,5-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-
triazine-2,4-diyl)bis(azanediyl))bis(ethanol) (10e). Yellow solid; 36% yield; m.p. 223–225 ◦C. FT-IR (ATR)
v (cm−1) Not observed (N–H), 3269 (O–H), 3086 (=C–H), 1637 and 1585 (C=N and C=C). 1H-NMR
(400 MHz, DMSO-d6)
δ ppm 3.17 (dd, J = 17.6, 4.8 Hz, 1H, H-4), 3.36–3.49 (m, 4H, CH2), 3.54–3.61 (m,
6H, CH2), 3.93 (dd, J = 17.6, 12.0 Hz, 1H, H-4), 5.63 (dd, J = 12.0, 4.8 Hz, 1H, H-5), 6.81 (s, 1H, Ar–H),
6.93 (s, 2H, Ar–H), 7.28 (d, J = 8.3 Hz, 2H, Ar–H), 7.42 (d, J = 8.3 Hz, 2H, Ar–H), 7.67–7.83 (m, 4H,
Ar–H), 8.24–8.83 (m, 2H, NH), 10.46 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 42.9 (CH2),

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43.3 (CH2), 59.1 (CH2), 63.1, 110.9, 111.8 (Cq), 117.1, 117.2 (Cq), 120.0, 126.7 (Cq), 126.8, 127.8, 127.8,
129.2, 132.3 (Cq), 134.5 (Cq), 140.3 (Cq), 145.6 (Cq), 149.9 (Cq). MS (70 eV) m/z (%): 612:614:616:618
[M+]:[M + 2]+:[M + 4]+:[M + 6]+ (100/81/32/5), 284 (16), 84 (30), 69 (50), 55 (60), 43 (82). Anal. Calcd.
C28H27Cl3N8O2: C, 54.78; H, 4.43; N, 18.25; Found: C, 54.73; H, 4.47; N, 18.22.
2,20-((6-((4-(1-(3,5-Dichlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-
triazine-2,4-diyl)bis(azanediyl))bis(ethanol) (10f). Yellow solid; 68% yield; m.p. 193–195 ◦C. FT-IR (ATR)
v (cm−1) Not observed (N–H), 3282 (O–H), 3116 (=C–H), 1641 and 1583 (C=N and C=C). 1H-NMR
(400 MHz, DMSO-d6)
δ ppm 3.17 (dd, J = 17.7, 5.0 Hz, 1H, H-4), 3.36–3.49 (m, 4H, CH2), 3.53–3.62
(m, 6H, CH2, OH), 3.93 (dd, J = 17.7, 12.0 Hz, 1H, H-4), 5.62 (dd, J = 12.0, 5.0 Hz, 1H, H-5), 6.81 (t,
J = 1.7 Hz, 1H, Ar–H), 6.93 (d, J = 1.7 Hz, 2H, Ar–H), 7.19 (t, J = 8.8 Hz, 2H, Ar–H), 7.31 (dd,
J = 8.2 and JHF = 5.6 Hz, 2H, Ar–H), 7.82-7.72 (m, 4H, Ar–H), 8.21-8.80 (m, 2H, NH), 10.46 (s, 1H,
NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 43.1 (CH2), 43.3 (CH2), 59.1 (CH2), 61.7, 110.9, 116.0 (d,
2JCF = 21.5 Hz), 117.0, 120.9, 126.9, 126.9 (Cq), 128.0 (d, 3JCF = 7.2 Hz), 134.5 (Cq), 135.9 (Cq), 137.6 (d,
1
4JCF = 2.7 Hz, Cq), 145.6 (Cq), 145.8 (Cq), 149.6 (Cq), 149.9 (Cq), 161.5 (d, JCF = 243.6 Hz, C-F).
MS (70 eV) m/z (%): 596:598:600 [M+]:[M + 2]+:[M + 4]+ (100/67/11), 124 (21), 84 (14), 69 (22), 55 (24),
43 (40). Anal. Calcd. C28H27Cl2FN8O2: C, 56.29; H, 4.56; N, 18.75; Found: C, 56.33; H, 4.53; N, 18.70.
0
2,2 -((6-((4-(1-(3,5-Dichlorophenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-amino)
-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(ethanol) (10g). Yellow solid; 68% yield; m.p. 222–223 ◦C. FT-IR
(ATR) v (cm−1) Not observed (N–H), 3307 (O–H), 3126 (=C–H), 1647 and 1587 (C=N and C=C).
1H-NMR (400 MHz, DMSO-d6)
δ ppm 3.22 (dd, J = 17.7, 4.8 Hz, 1H, H-4), 3.37–3.48 (m, 4H, CH2),
3.53–3.60 (m, 6H, CH2,OH), 3.97 (dd, J = 17.7, 12.1 Hz, 1H, H-4), 5.75 (dd, J = 12.1, 4.8 Hz, 1H, H-5),
6.83 (d, J = 1.6 Hz, 1H, Ar–H), 6.94 (d, J = 1.6 Hz, 2H, Ar–H), 7.48 (d, J = 8.0 Hz, 2H, Ar–H), 7.82–7.71
(m, 6H, Ar–H), 8.08–8.76 (m, 2H, NH), 10.44 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm
1
42.9 (CH2), 43.2 (CH2), 59.0 (CH2), 61.7, 110.8, 117.2, 120.8, 122.7 (Cq), 124.7 (q, JCF = 243.5 Hz,
CF3), 125.4 (Cq), 126.0 (Cq), 126.1 (q, 4JCF = 3.5 Hz), 126.6 (Cq), 126.7, 126.9 (unresolved q), 128.3 (q,
2JCF = 32.1 Hz), 134.5 (Cq), 145.5 (Cq), 146.0 (Cq), 150.0 (Cq). MS (70 eV) m/z (%): 646:648:650 [M+]:[M
+ 2]+:[M + 4]+ (100/66/14), 615 (14), 124 (20), 69 (26), 55 (26), 43 (48). Anal. Calcd. C29H27Cl2F3N8O2:
C, 53.80; H, 4.20; N, 17.31; Found: C, 53.89; H, 4.21; N, 17.35.
3.2.7. General Procedure for the Synthesis of 2,20-((6-((4-(1-(4-Chlorophenyl)-5-(4-(aryl)-4,5-
dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(ethanols) 11a–g
A mixture of chalcone 7 (0.4 mmol) and 4-chlorophenylhydrazine hydrochloride (2.6 mmol) in
methanol (2.5 mL) was subjected to reflux for 2 h. The solid formed was filtered and washed with
methanol. No further purification was required.
2,20-((6-((4-(1-(4-Chlorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-triazine-2,4-diyl)
bis(azanediyl))bis(ethanol) (11a). Yellow solid; 92% yield; m.p. 225–226 ◦C. FT-IR (ATR) v (cm−1) Not
1
observed (N–H), 3290 (O–H), 3099 (=C–H), 1645 and 1595 (C=N and C=C). H-NMR (400 MHz,
DMSO-d6) δ ppm 3.10 (dd, J = 17.5, 5.9 Hz, 1H, H-4), 3.39–3.50 (m, 4H, CH2), 3.53–3.62 (m, 6H, CH2,
OH), 3.91 (dd, J = 17.5, 12.2 Hz, 1H, H-4), 5.48 (dd, J = 12.2, 5.9 Hz, 1H, H-5), 6.97 (d, J = 9.0 Hz, 2H,
Ar–H), 7.17 (d, J = 9.0 Hz, 2H, Ar–H), 7.22–7.29 (m, 3H, Ar–H), 7.37–7.30 (m, 2H, Ar–H), 7.69–7.79
(m, 4H, Ar–H), 8.11–8.76 (m, 2H, NH), 10.44 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 43.2
(CH2), 43.3 (CH2), 59.1 (CH2), 63.0, 99.5 (Cq), 114.3, 120.9, 121.4 (Cq), 122.0 (Cq), 125.8, 126.3, 127.5,
127.6 (Cq), 128.8, 129.1, 135.9 (Cq), 142.1 (Cq), 143.1 (Cq), 147.8 (Cq). MS (70 eV) m/z (%): 544:546
[M+]:[M + 2]+ (100/36), 467 (10), 284 (7), 125 (22), 90 (11), 43 (20). Anal. Calcd. C28H29ClN8O2: C,
61.70; H, 5.36; N, 20.56; Found: C, 61.68; H, 5.40; N, 20.53.
0
2,2 -((6-((4-(1-(4-Chlorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-triazine-2,4-diyl)
bis(azanediyl))bis(ethanol) (11b). Yellow solid; 55% yield; m.p. 184–186 ◦C. FT-IR (ATR) v (cm−1) Not
1
observed (N–H), 3290 (O–H), 3184 (=C–H), 1651 and 1597 (C=N and C=C). H-NMR (400 MHz,

Molecules 2018, 23, 1956
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DMSO-d6)
δ ppm 2.24 (s, 3H, CH3), 3.07 (dd, J = 17.4, 5.9 Hz, 1H, H-4), 3.34–3.48 (m, 4H, CH2),
3.54–3.62 (m, 6H, CH2, OH), 3.88 (dd, J = 17.4, 12.2 Hz, 1H, H-4), 5.43 (dd, J = 12.2, 5.9 Hz, 1H, H-5),
6.97 (d, J = 8.9 Hz, 2H, Ar–H), 7.21–7.09 (m, 6H, Ar–H), 7.67–3.79 (m, 4H, Ar–H), 8.17–8.79 (m, 2H,
NH), 10.43 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 20.7 (CH3), 43.1 (CH2), 43.2 (CH2),
59.0 (CH2), 62.8, 114.3, 120.9, 121.0 (Cq), 121.9 (Cq), 125.8, 126.3, 126.5 (Cq), 127.4 (Cq), 128.6, 129.6,
136.7 (Cq), 139.1 (Cq), 143.1 (Cq), 147.8 (Cq), 151.0 (Cq). MS (70 eV) m/z (%): 558:560 [M+]+:[M + 2]+
(100/35), 467 (16), 284 (8), 125 (21), 91 (13), 44(23), 43 (35). Anal. Calcd. C29H31ClN8O2: C, 62.30; H,
5.59; N, 20.04; Found: C, 62.36; H, 5.60; N, 20.08.
0
2,2 -((6-((4-(1-(4-Chlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-triazine
◦
−1
)
-2,4-diyl)bis(azanediyl))bis(ethanol) (11c). Yellow solid; 83% yield; m.p. 160–163 C. FT-IR (ATR) v (cm
Not observed (N–H), 3286 (O–H), 3093(=C–H), 1622 (C=N), 1595 (C=C) and 1244 (C–O). 1H-NMR
(400 MHz, DMSO-d6) ppm 3.07 (dd, J = 17.4, 5.9 Hz, 1H, H-4), 3.37–3.48 (m, 4H, CH2), 3.54–3.61 (m,
δ
6H, CH2, OH), 3.70 (s, 3H, OCH3), 3.86 (dd, J = 17.4, 12.1 Hz, 1H, H-4), 5.42 (dd, J = 12.1, 5.9 Hz, 1H,
H-5), 6.88 (d, J = 8.8 Hz, 2H, Ar–H), 6.98 (d, J = 8.8 Hz, 2H, Ar–H), 7.13–7.20 (m, 4H, Ar–H), 7.67–7.79
(m, 4H, Ar–H), 8.19–8.75 (m, 2H, NH), 10.43 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 43.2
(CH2), 43.3 (CH2), 55.1 (CH3), 59.1 (CH2), 62.6, 114.4, 114.4, 121.0, 121.1 (Cq), 122.0 (Cq), 126.3, 126.7
(Cq), 127.1, 127.6 (Cq), 128.6, 133.9 (Cq), 136.0 (Cq), 143.1 (Cq), 147.8 (Cq), 158.6 (Cq). MS (70 eV)
m/z (%): 574:576 [M+]:[M + 2]+ (100/34), 467 (26), 377 (40), 125 (42), 55 (58), 43 (98). Anal. Calcd.
C29H31ClN8O3: C, 60.57; H, 5.43; N, 19.49; Found: C, 60.60; H, 5.40; N, 19.55.
0
2,2 -((6-((4-(1-(4-Chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-
triazine-2,4-diyl)bis(azanediyl))bis(ethanol) (11d). Yellow solid; 64% yield; m.p. 176–178 ◦C. FT-IR (ATR)
v (cm−1) Not observed (N–H), 3275 (O–H), 3086 (=C–H), 1637 (C=N), 1591 (C=C) and 1228 (C–O).
1H-NMR (400 MHz, DMSO-d6)
δ ppm 3.14 (dd, J = 17.5, 7.0 Hz, 1H, H-4), 3.35–3.50 (m, 4H, CH2),
3.52–3.61 (m, 6H, CH2, OH), 3.62 (s, 3H, OCH3), 3.69 (s, 6H, OCH3), 3.88 (dd, J = 17.5, 12.1 Hz, 1H,
H-4), 5.33 (dd, J = 12.1, 7.0 Hz, 1H, H-5), 6.58 (s, 2H, Ar–H), 7.01 (d, J = 9.0 Hz, 2H, Ar–H), 7.20 (d,
J = 9.0 Hz, 2H, Ar–H), 7.68–7.79 (m, 4H, Ar–H), 8.09–8.75 (m, 2H, NH), 10.41 (s, 1H, NH). 13C-NMR
(100 MHz, DMSO-d6)
δ ppm 43.3 (CH2), 55.9 (CH3), 59.1 (CH2), 59.9 (CH3), 99.6 (Cq), 102.9, 114.5,
118.6 (Cq), 120.9, 122.8 (Cq), 125.9 (Cq), 126.4, 127.3 (Cq), 128.7, 128.8 (Cq), 135.9 (Cq), 136.6 (Cq), 137.9
(Cq), 143.6 (Cq), 148.2 (Cq), 153.3 (Cq). MS (70 eV) m/z (%): 634:636 [M+]:[M + 2]+ (60/22), 439 (33),
437 (86), 270 (24), 125 (56), 43 (100). Anal. Calcd. C31H35ClN8O5: C, 58.63; H, 5.55; N, 17.64; Found: C,
58.68; H, 5.48; N, 17.55.
2,20-((6-((4-(1,5-bis(4-Chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-triazine-2,4-diyl)-bis
(azanediyl))bis(ethanol) (11e). Yellow solid; 63% yield; m.p. 229–230◦C. FT-IR (ATR) v (cm−1) Not
1
observed (N–H), 3292 (O–H), 3099 (=C–H), 1643 and 1593 (C=N and C=C). H-NMR (400 MHz,
DMSO-d6) δ ppm 3.12 (dd, J = 17.4, 5.7 Hz, 1H, H-4), 3.36–3.50 (m, 4H, CH2), 3.52–3.65 (m, 6H, CH2,
OH), 3.91 (dd, J = 17.4, 12.1 Hz, 1H, H-4), 5.52 (dd, J = 12.1, 5.7 Hz, 1H, H-5), 6.97 (d, J = 8.9 Hz, 2H,
Ar–H), 7.19 (d, J = 8.9 Hz, 2H, Ar–H), 7.28 (d, J = 8.3 Hz, 2H, Ar–H), 7.40 (d, J = 8.3 Hz, 2H, Ar–H),
3.66–3.81 (m, 4H, Ar–H), 8.23–8.80 (m, 2H, NH), 10.44 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ
ppm 42.9 (CH2), 43.3 (CH2), 59.0, 62.3 (CH2), 114.3, 120.9, 121.0 (Cq), 122.2, 123.3 (Cq), 126.4, 127.8,
127.9 (Cq), 128.7, 129.0, 132.0 (Cq), 139.8 (Cq), 141.0 (Cq), 142.9 (Cq), 147.9 (Cq). MS (70 eV) m/z (%):
578:580 [M+]:[M + 2]+:[M + 4]+ (100/63/12), 467 (12), 284 (10), 125 (38), 90 (17), 43 (36). Anal. Calcd.
C28H28Cl2N8O2: C, 58.04; H, 4.87; N, 19.34; Found: C, 58.07; H, 4.81; N, 19.32.
0
2,2 -((6-((4-(1-(4-Chlorophenyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,5-triazine-2,
4-diyl)bis(azanediyl))bis(ethanol) (11f). Yellow solid; 90% yield; m.p.129–230 ◦C. FT-IR (ATR) v (cm−1
)
Not observed (N–H), 3292 (O–H), 3101 (=C–H), 1645 and 1595 (C=N and C=C). 1H-NMR (400 MHz,
DMSO-d6) ppm 3.10 (dd, J = 17.5, 5.8 Hz, 1H, H-4), 3.39–3.49 (m, 4H, CH2), 3.55–3.61 (m, 6H, CH2,
δ
OH), 3.90 (dd, J = 17.5, 12.1 Hz, 1H, H-4), 5.51 (dd, J = 12.1, 5.8 Hz, 1H, H-5), 6.97 (d, J = 9.0 Hz,
2H, Ar–H), 7.22–7.12 (m, 4H, Ar–H), 7.29 (dd, J = 8.0 and JHF = 5.7 Hz, 2H, Ar–H), 7.69–7.79 (m, 4H,

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Ar–H), 8.11–8.78 (m, 2H, NH), 10.43 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ
ppm 43.1 (CH2),
43.3 (CH2), 59.1 (CH2), 62.3, 114.4, 115.9 (d, 2JCF = 21.8 Hz), 120.9, 121.0 (Cq), 122.2 (Cq), 126.4, 127.4
(Cq), 128.0 (d, 3JCF = 8.5 Hz), 128.7, 136.0 (Cq), 138.2 (d, 4JCF = 2.48 Hz, Cq), 143.0 (Cq), 144.2 (Cq),
147.9 (Cq), 161.4 (d, 1JCF = 243.1 Hz, C-F). MS (70 eV) m/z (%): 562:564 [M+]:[M + 2]+ (100/37), 467
(11), 284 (8), 125 (26), 69 (14), 44 (20), 43 (24). Anal. Calcd. C28H28ClFN8O2: C, 59.73; H, 5.01; N, 19.90;
Found: C, 59.76; H, 4.97; N, 20.06.
0
2,2 -((6-((4-(1-(4-Chlorophenyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenyl)amino)-1,3,
◦
5-triazine-2,4-diyl)bis(azanediyl))bis(ethanol) (11g). Yellow solid; 24% yield; m.p. 197–199 C. FT-IR (ATR)
v (cm−1) Not observed (N–H), 3288 (O–H), 3095 (=C–H), 1645 and 1599 (C=N and C=C). 1H-NMR
(400 MHz, DMSO-d6)
δ ppm 3.16 (dd, J = 17.7, 5.8 Hz, 1H, H-4), 3.36–3.49 (m, 4H, CH2), 3.53–3.61
(m, 6H, CH2, OH), 3.95 (dd, J = 17.7, 12.2 Hz, 1H, H-4), 5.63 (dd, J = 12.2, 5.8 Hz, 1H, H-5), 6.97 (d,
J = 9.0 Hz, 2H, Ar–H), 7.20 (d, J = 9.0 Hz, 2H, Ar–H), 7.48 (d, J = 8.0 Hz, 2H, Ar–H), 7.67–7.83 (m, 6H,
Ar–H), 8.07–8.80 (m, 2H, NH), 10.42 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-d6)
δ ppm 42.6 (CH2),
42.9 (CH2), 58.8 (CH2), 62.1, 114.3, 121.0, 124.2 (q, 1JCF = 286.1 Hz, CF3), 126.1 (d, 4JCF = 3.14 Hz),
2
126.5, 126.8, 128.0 (Cq), 128.5 (d, JCF = 26.9 Hz, Cq), 128.8 (unresolved q), 133.9 (Cq), 134.4 (Cq),
135.9 (Cq), 136.0 (Cq), 142.8 (Cq), 146.7 (Cq), 148.0 (Cq). MS (70 eV) m/z (%): 612:614 [M+]:[M + 2]+
(100/35), 581 (11), 284 (9), 125 (24), 111 (14), 69 (16), 43 (26). Anal. Calc. C29H28ClF3N8O2: C, 56.82; H,
4.60; N, 18.28; Found: C, 56.86; H, 4.63; N, 18.20.
3.3. 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 experiment, cells were
inoculated into 96-well microtiter plates. After cell inoculation, the microtiter plates were incubated
◦
at 37 C, 5% CO2, 95% air, and 100% relative humidity for 24 h prior to the addition of the tested
compounds. After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a
measurement of the cell population for each cell line at the time of sample addition (Tz). The samples
were solubilized in dimethyl sulfoxide (DMSO) at 400-fold the desired final maximum test concentration
and stored frozen prior to use. At the time of compound addition, an aliquot of frozen concentrate
was thawed and diluted to twice the desired final maximum test concentration with complete medium
containing 50
to provide a total of five drug concentrations plus the control. Aliquots of 100
sample dilutions were added to the appropriate microtiter wells already containing 100
µ
g/mL gentamicin. An additional four 10-fold or 1/2 log serial dilutions were made
L of these different
L of medium,
39]. After the tested compounds were added,
µ
µ
resulting in the required final sample concentrations [37
–
the plates were incubated for an 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 fixed in situ by
the gentle addition of 50
60 min at 4 ◦C. The supernatant was discarded, and plates were washed five times with tap water and
air dried. Sulforhodamine B (SRB) solution (100 L) at 0.4% (w/v) in 1% acetic acid was added to each
µL of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for
µ
well, and plates were incubated for 10 min at room temperature. After staining, unbound dye was
removed by washing five times with 1% acetic acid and the plates were air dried. Bound stain was
subsequently solubilized with 10 mM trizma base, and the absorbance 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 five concentration levels
(Ti)], the percentage growth was calculated at each of the drug concentrations levels. Percentage growth
inhibition was calculated as: [(Ti
[(Ti Tz)/Tz] 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)]
100 = 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated
−
Tz)/(C
−
Tz)]
×
100 for concentrations for which Ti > Tz, and
−
×
−
−
×
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

Molecules 2018, 23, 1956
18 of 20
drug treatment as compared to that at the beginning), indicating a net loss of cells; calculated from
[(Ti Tz)/Tz] 100 = 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 [37–39,41].
4. Conclusions
In summary, the trisubstituted [
microwave irradiation after a short reaction time. Then, starting from
intermediates , four new series of 1,3,5-triazine-2-pyrazoline hybrids (8– )a–g
1,
3,
5]-triazine precursor
5
was obtained in good yield under
and mediated by chalcone
11 were successfully
5
7
prepared in acceptable to excellent yields through a multistep method. Seventeen of the obtained
compounds were subjected under evaluation of their in vitro growth inhibition properties at one- and
five-dose levels against a panel of 58 human cancer cell lines by the U.S. NCI. The screening results
showed that chalcone 7g and 3,5-dichlorophenylpyrazolines 10d
activities against several cancer cell lines (i.e., renal, cns and breast), with GI50 values in the range of
0.569–16.6 M and low toxicity. Therefore, compounds 7g and 10d are promising building blocks
,e,g exhibited the more remarkable
µ
,e,g
for further structural modifications in order to improve their activities and hence develop possible
new antitumor agent leaders.
Author Contributions: B.I. designed the experiments; L.M.M. and J.R.P. performed the experiments; L.M.M., J.Q.,
R.A., J.R.P. and B.I. analyzed and discussed the experimental and biological results, all spectral data and wrote the
manuscript. All authors read and approved the final manuscript.
Funding: This work was financially supported by Colciencias, Universidad del Valle and the Science, Technology
and Innovation Fund-General Royalties System (FCTeI-SGR) under contract No. BPIN 2013000100007.
Acknowledgments: The authors thank The Developmental Therapeutics Program (DTP) of the National Cancer
Institute of the United States for performing the anticancer screening of the obtained compounds.
Conflicts of Interest: The authors declare no conflicts of interest.
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