Synthesis, characterisation of new derivatives with mono ring system of 1,2,4-triazole scaffold and their anticancer activities

Article abstract of DOI:10.1016/j.molstruc.2019.06.066

In the present study, two important starting materials and 18 new 1,2,4-triazole compounds with mono ring system have been synthesized and characterized. The mono system showed 16 compounds of a Schiff base moiety attached to the triazole ring which was prepared from the corresponding starting material 5(A–B) or piperidinium salt system 6(A–B). All these compounds were characterized using Fourier Transform Infrared (FT-IR) and Nuclear Magnetic Resonance (NMR) spectroscopy and carbon hydrogen nitrogen (CHN) elemental analysis. The compounds were selected for in vitro anticancer study to test the therapeutic cytotoxic potential against cancer cells. The MTT test was conducted against human breast (MCF-7) and colorectal (HCT-116) cancer cells. Among all the compounds tested, 7A-i demonstrated more pronounced in vitro anticancer effect against MCF-7 and HCT-116 cells with IC₅₀ of 38 and 19.2 μM, respectively, comparable to that of the standard reference drugs, tamoxifen and 5-fluorouracil, respectively. Compound 7A-vi showed a considerable cytotoxic effect with IC₅₀ 53 and 41.2 μM against MCF-7 and HCT-116 cells, respectively. Compounds 7A-ii, 7A-iii and 7A-v exhibited moderate cytotoxicity with IC₅₀ 68, 91 and 85 μM, respectively against MCF-7 cells and also 59.3, 81.7 and 137.1 μM against HCT-116 cells, respectively. However, all other compounds tested in this study showed poor cytotoxicity against both the cell lines. Cellular morphological analysis revealed that the cytotoxicity induced by the compounds could probably due to autophagy. It can be concluded that 1,2,4-triazole derivatives can be promising therapeutic agents. Further studies will be done to investigate the antitumor efficacy of the 1,2,4-triazole derivatives using suitable preclinical models.

Full text of DOI:10.1016/j.molstruc.2019.06.066

Accepted Manuscript
Synthesis, characterisation of new derivatives with mono ring system of 1,2,4-
triazole scaffold and their anticancer activities
Mukhlif Mohsin Slaihim, Fouad Saleih R. Al-Suede, Melati Khairuddean,
Mohamed B. Khadeer Ahamed, Amin Malik Shah Abdul Majid
PII:
S0022-2860(19)30789-6
10.1016/j.molstruc.2019.06.066
MOLSTR 26708
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Accepted Date:
07 March 2019
18 June 2019
Please cite this article as: Mukhlif Mohsin Slaihim, Fouad Saleih R. Al-Suede, Melati Khairuddean,
Mohamed B. Khadeer Ahamed, Amin Malik Shah Abdul Majid, Synthesis, characterisation of new
derivatives with mono ring system of 1,2,4-triazole scaffold and their anticancer activities, Journal
of Molecular Structure (2019), doi: 10.1016/j.molstruc.2019.06.066
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ACCEPTED MANUSCRIPT
SYNTHESIS, CHARACTERISATION OF NEW DERIVATIVES WITH MONO
RING SYSTEM OF 1,2,4-TRIAZOLE SCAFFOLD AND THEIR ANTICANCER
ACTIVITIES
Mukhlif Mohsin Slaihima, Fouad Saleih R. Al-Suedeb, Melati Khairuddeanc,*, Mohamed B.
Khadeer Ahamedb and Amin Malik Shah Abdul Majidd,e
a Applied Science Faculty, Samarra University, Iraq
bEMAN Biodiscoveries Sdn. Bhd., A1-4, Lot 5, Persiaran 2/1, Kedah Halal Park, Kawasan
Perindustrian Sungai Petani, 08000 Sungai Petani, Kedah.
cSchool of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
d,School of Pharmaceutical Sciences , Universiti Sains Malaysia, 11800 Penang, Malaysia
e ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical
Research, Australian National Universityand Eman Research Ltd, Acton, Australia
Corresponding author:
*Melati Khairuddean
School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.
melati@usm.my

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ABSTRACT
In the present study, two important starting materials and 18 new 1,2,4-triazole
compounds with mono ring system have been synthesized and characterized. The mono
system showed 16 compounds of a Schiff base moiety attached to the triazole ring which was
prepared from the corresponding starting material 5(A-B) or piperidinium salt system 6(A-B).
All these compounds were characterized using Fourier Transform Infrared (FT-IR) and
Nuclear Magnetic Resonance (NMR) spectroscopy and carbon hydrogen nitrogen (CHN)
elemental analysis. The compounds were selected for in vitro anticancer study to test the
therapeutic cytotoxic potential against cancer cells. The MTT test was conducted against
human breast (MCF-7) and colorectal (HCT-116) cancer cells. Among all the compounds
tested, 7A-i demonstrated more pronounced in vitro anticancer effect against MCF-7 and
HCT-116 cells with IC50 of 38 and 19.2 µM, respectively, comparable to that of the standard
reference drugs, tamoxifen and 5-fluorouracil, respectively. Compound 7A-vi showed a
considerable cytotoxic effect with IC50 53 and 41.2 µM against MCF-7 and HCT-116 cells,
respectively. Compounds 7A-ii, 7A-iii and 7A-v exhibited moderate cytotoxicity with IC50
68, 91 and 85 µM, respectively against MCF-7 cells and also 59.3, 81.7 and 137.1 µM
against HCT-116 cells, respectively. However, all other compounds tested in this study
showed poor cytotoxicity against both the cell lines. Cellular morphological analysis revealed
that the cytotoxicity induced by the compounds could probably due to autophagy. It can be
concluded that 1,2,4-triazole derivatives can be promising therapeutic agents. Further studies
will be done to investigate the antitumor efficacy of the 1,2,4-triazole derivatives using
suitable preclinical models.
Keywords:1,2,4-triazole, pyridinium salt, piperidinium salt, anticancer activity, MTT assay,
anti-proliferation.

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1.
Introduction
The discovery and development of new effective and selective anticancer drugs are of
high importance in modern cancer researches. Cancer is a major worldwide problem which is
characterized by proliferation and spreading of abnormal cells uncontrollably and causes the
cells to eventually undergo structural changes to become malignant [1]. Treatment of cancer
by chemotherapy showed limitation due to the negative side effects. Since chemotherapeutic
drugs cannot differentiate between the cancer cells and normal cells, these drugs often
became toxic to human vital organs such as heart and kidney [2]. The emergence of
chemotherapeutic drug resistance in cancer cells also resulted in tumor recurrence [3,4].
Today, the discovery and development of effective anticancer agents are the key focus of
most researchers in medicinal chemistry. In spite of a large number of chemotherapeutic
drugs available for medical usage, the increasing resistance made it necessary to continue the
search for new potential anticancer drugs.
In the field of pharmaceutical organic chemistry, research is concentrated towards the
introduction of new and safe therapeutic agents of clinical importance. Recently, 5-membered
heterocyclic compounds have proved their importance as being the center of the biological
activities [5,6]. The nitrogen-containing heterocycles are found in abundance in most of the
medicinal compounds. Starting with imidazole as an important moiety of the medicinal agent,
research work has led to the introduction of triazole which is an isostere of imidazole, in
which one of the carbon atoms of imidazole is replaced by nitrogen [7-9].
H
H
H
N1
N1
N1
5
2
5
2
N
5
2
N
N3
imidazole
4 N
N3
1,2,3-triazole
3
4
4
1,2,4-triazole

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The discovery of 1,2,4-triazole ring represents an interesting class of compounds with
promising biological activities such as antibacterial [10,11], antimicrobial [12,13],
anticonvulsant [14], antifungal [15], antidepressant [16] and anti-tuberculosis [17,18].
However, these types of derivatives, targeting specifically on the anticancer activity have not
been widely explored [19-24]. This research work focused on the modification of 1,2,4-
triazole scaffold into various bioactive structures and their subsequent evaluation for
anticancer activities, focusing on the human breast (MCF-7) and colorectal (HCT-116) tumor
cells. New findings have established the fact that fused 1,2,4-triazole ring contributed great
significance in the field of medicinal chemistry due to their versatile biological properties
[14].
2.
Results and discussion
2.1
Spectral confirmation of the synthesized compounds
O
O
O
MeOH
NH2NH2.H2O
reflux, 14 h
Ar
Ar
Ar
reflux
7 h
OR
OH
NHNH2
2
1
R = -CH3 , -C2H5
KOH/EtOH
a
b
KOH/EtOH
CS2
CS
2, reflux
rt, 16-72 h
7-8 h
O
H
N N
N
SK
Ar
N
SH
Ar
O
4
H
S
3
NH2NH2.H2O
reflux, 10 h
N N
HO
,
N
.HCl
Ar =
N
,
SH
Ar
N
A
B
SA
NH2
5
Scheme 1: Synthesis pathways of 1,2,4-triazole derivatives

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In the series of esters, 1, all the spectral data for methyl and ethyl 4-substituted-
benzoate derivatives featured the main absorption peaks at 3078 cm-1 (Csp2-H aromatic
stretch), 2958 and 2855 (asymmetrical and symmetrical Csp3-H stretch), 1716 (C=O stretch),
1608 and 1520 cm-1 (C=C aromatic stretch). Compounds with hydroxyl substituent showed
the bands at 3369 cm-1 which corresponded to the O-H stretching. Since the hydroxyl groups
are powerful ortho- and para- activators, the accretions of electron density in these positions
led to the shielding effect which resulted in the upfield shifts of the NMR signal.
In the condensation reaction, carboxylic acid was transformed into the corresponding
acid hydrazide, 2. The absorption band in the IR spectrum showed the disappearance of the
absorption band (O-H stretch) of the carboxylic acid and the appearance of the absorption
band (N-H stretch) of the amide group. The 1H-NMR spectrum revealed the presence of two
doublets of the aromatic protons and two amino protons at  4.70 ppm (-NH2) and 10.14 ppm
(-NH). The reaction of acid hydrazide, 2 with CS2 at room temperature yielded potassium
dithiocarbazinate salts, 3, the intermediates used to prepare 1,2,4-triazole derivatives.
However, under reflux, the mixture gave 5-substituted-1,3,4-oxadiazole-2-thiol, 4, instead,
which were also used to prepare the corresponding 1,2,4-triazole derivatives (Scheme 1). In
the IR spectrum, the disappearance of the strong absorption of C=O in acid hydrazide and the
appearance of new bands of C=N in the range of 1604-1653 cm-1 confirmed the formation of
5-substituted-1,3,4-oxadiazole-2-thiol, 4. The 1H-NMR spectrum revealed the presence of the
aromatic protons as two doublets at  6.94 and 7.72 ppm and two broad singlets at  10.42
and 14.54 ppm, assigned for protons of -OH and -SH, respectively. In the 13C-NMR
spectrum, eight carbon signals were observed. Intermediates 3 and 4 were refluxed separately
with hydrazine hydrate to yield 1,2,4-triazole derivatives of salt 5SA and 4-hydroxyphenyl
derivatives of 5B, respectively. The IR spectrum of 5SA showed the appearance of a band at
1605 cm-1 (C=N stretch of the ring) and 2400 cm-1 (-+N-H, a triazole compound existed in the

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1
salt form). The H-NMR spectrum showed two singlets at  14.18 (-SH) and 5.87 (-NH2)
13
ppm and two doublets for two aromatic protons at  8.76 and 8.03 ppm while the C-NMR
spectrum showed seven carbon signals whereby five signals were assigned to the pyridyl
group and another two signals for carbons in the triazole ring [25].
A series of 1,2,4-triazole derivatives with Schiff base moiety were synthesized
according to two new procedures (Scheme 2). In the first procedure, piperidine was added
with intermediate 5A which underwent the dehydrohalogenation to produce the cyclic imine
[26]. In the second procedure, a mixture of 5A in absolute methanol with a few drops of
piperidine was refluxed before the addition of various aromatic aldehydes.
N
N
SH
Ar
N
NH2
5(A-B)
piperidine, MeOH,
reflux, 3-4 h
N
Ar =
HO
N N
B
A
H N
2
S
Ar
N
NH2
6(A-B)
piperidine, MeOH
1-methyl-pyrrolidin-2-one
reflux, 5-7 h
piperidine, MeOH
substituted benzaldehyde
reflux, 5-7 h
N
N
N N
H N
2
S
Ar
SH2N
H
Ar
N
N
N
N
N
7[A-B(i-vii)]
7(A-B)viii
H3C
X = OH, OCH3, CH3, Cl, Br, CN, NO2
ii iii iv v vi vii
X
i
Scheme 2: Synthesis pathway of 1,2,4-triazole derivatives with Schiff base moiety

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The IR spectrum of 6A showed the absorption bands at 3418 (N-H stretch of
piperidine); 3271 and 3160 (N-H stretch of triazole); 3058 (Csp2-H stretch); 1605 (C=N
stretch); 1571 and 1478 (C=C stretch); 1413 (C–N); 1217, 1085 and 1003 (C–N–C); 937, 823
1
and 687 cm-1 (1,2,4-triazole ring). The H-NMR spectrum of 6A revealed three different
protons. The methylene protons showed three different signals. A signal at  2.99 ppm was
integrated to four methylene protons of the piperidinium ring while two multiplets at  1.64
and 1.56 ppm were assigned for six methylene protons. The second type was the aromatic
protons which featured two doublets at  8.64 and 8.03 ppm, integrated to four protons of the
pyridyl group while the third type were the protons of the amine and ammonium groups
13
which showed a singlet at  5.71 and 5.31 ppm, respectively. The C-NMR spectrum of 6A
showed eight signals which represented all the 12 carbons. Signals at  149.8, 120.2 and
134.8 ppm were assigned to five carbons of the pyridyl group while signals at  168.4 and
146.2 ppm were assigned to two carbons of the triazole ring and signals at  22.1 to 44.3 ppm
were for carbons of the piperidinium moiety of 6A. The DEPT-135 NMR spectrum
confirmed the methine and quaternary carbons in compound 6A. The 2D-NMR correlation
(1H-1H COSY and 1H-13C HMQC spectra) confirmed the assignment of the aromatic signals,
1
especially the triazole protons. Further confirmation of 6A provided by H-13C HMBC
showed the short-range correlations observed between the carbon atoms of the aryl/phenyl.
For 1,2,4-triazole Schiff base derivatives (7Ai-viii), compound 7Ai was used as a
representative. The IR spectrum of 7Ai (Figure 1) indicated the absence of the -NH2 group
but the appearance of the bands at 3331 (O-H stretch) and 1651 cm-1 (C=N stretch) which
confirmed the formation of the Schiff base in 7Ai. Other absorption bands were observed at
3001 (Csp2-H stretch), 2955 (Csp3-H stretch), 1592 and 1514 (C=C stretch) and 710 cm-1 (C-
S).

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Figure 1: IR spectrum of 7Ai
1
The H NMR spectrum (Figure 2) of 7Ai revealed signals of nine aromatic protons and five
piperidine protons. The 13C-NMR spectrum (Figure 3) of 7Ai displayed signals of 14
aromatic carbons and five piperidine carbons.
Figure 2: 1H-NMR spectrum of 7Ai (500 MHz, DMSO-d6)

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Figure 3: 13C-NMR spectrum of 7Ai (125 MHz, DMSO-d6)
The methine and quaternary carbons in compound 7Ai were confirmed using DEPT-
1
135 NMR spectrum (Figure 4). The 2D-NMR correlation using H-1H COSY (Figure 5) and
1H-13C HMQC (Figure 6) spectra confirmed the assignment of the aromatic protons. The
COSY spectra of all the Schiff base derivatives, 7A(i-viii) showed clear correlations of the
neighboring protons between H-2’/H-6’ and H-3’/H-5’ in Ring 1 (aryl group) and H-2”/H-6”
1
and H-3”/H-5” ring 2 (phenyl group). Further confirmation of 7Ai using H-13C HMBC
(Figure 7) showed the short-range correlations between the carbon atoms of the aryl/phenyl.
All the synthesized Schiff base compounds have two aromatic rings and one piperidinium
ring. Two substituents attached to the benzene ring were the aryl group (pyridin-4-yl and 4-
hydroxyphenyl) of the triazole ring (Ring 1) and a phenyl group of the aromatic aldehyde
(Ring 2). In conclusion, the spectroscopic data led to the clear identification of all the
compounds.

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Figure 4: DEPT-135 NMR spectrum of 7Ai (125 MHz, DMSO-d6)
Figure 5: 1H-1H COSY NMR spectrum of 7Ai (500 MHz, DMSO-d6)

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Figure 6: 1H-13C HMQC NMR spectrum of 7Ai (500 MHz, DMSO-d6)
Figure 7: 1H-13C HMBC NMR spectrum of 7Ai (500 MHz, DMSO-d6)
2.2
Cytotoxicity study
Compounds of the mono system with different moieties attached to the triazole ring
were tested for cytotoxicity activity against two different cell lines of human breast cancer
(MCF-7) cell and human colorectal cancer (HCT-116) cell. The IC50 values of these

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compounds were compared with the standard references used, Tamoxifen as a positive
control for MCF-7 and 5-fluorouracil as a positive control for HCT-116. Cytotoxic activity is
an ability of a test sample to cause toxic effects in the cancer cells which can be measured by
assessing the proliferation of the cells [27-30]. The dose-dependent antiproliferative effect of
the compounds tested against human breast tumor cells (MCF-7) is presented in Figure 8.
Figure 8: Graphical illustration of the dose-pendent response of the compounds tested on
human breast cancer cells (MCF-7). The data is presented as mean percentage inhibition ±
SD (n = 3). Tam = tamoxifen.
Figure 9 shows the human breast cancer (MCF-7) cell images observed under an
inverted phase-contrast microscope attached to a digital camera, after 48 hours being treated
with compounds 7A(i-vi). Tamoxifen was used as the reference standard and treated cell
group with 0.1% DMSO used as the negative control. Cells from the negative control group
exhibited full confluent compact monolayer of the proliferating MCF-7 cells. However, cells
treated with the standard drug, tamoxifen (IC50 = 8.2 µM) demonstrated significant (p < 0.01)
inhibitory effect on the proliferation of the cells. Images of the standard-treated cells showed
clear toxic signs such as reduced cellular population, round shaped cells and loss of the
pseudopodial cellular projections. Pseudopodial projections are the cytoplasm-filled

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projection of the cell membrane. These projections help the cells to get attached to another
cell or a surface of the tissue or flask [31]. Basically, these cellular projections originate with
the help of cytoskeleton of the cells to support and maintain the structural features of the cells
[32]. Among the compounds tested, compound 7Ai demonstrated significantly potent
inhibitory effect (IC50 38 µM) on cellular proliferation, as the cellular population was reduced
drastically. Compounds 7Aii, 7Av and 7Avi demonstrated the considerable cytotoxic activity
with IC50 of 68, 85 and 53 µM, respectively. On the other hand, compounds 7Aiii and 7Aiv
exhibited a moderate inhibitory effect on the proliferation of human breast cancer cells with
IC50 91 and 101 µM, respectively.

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Figure 9: Images of human breast cancer (MCF-7) cell under an inverted phase-contrast
microscope with a digital camera at 48ꢀhours after treatment with compounds 7A(i-vi).

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Tamoxifen is the reference standard and treated cell group with 0.1 DMSO as a negative
control (Magnification × 200).
The dose-dependent antiproliferative effect of the compounds tested against human
colorectal tumor cells (HCT-116) is graphically presented in Figure 10.
Figure 10: Graphical illustration of the dose-pendent response of the compounds tested on
human colorectal tumor cells (HCT-116). The data is presented as mean percentage inhibition
± SD (n = 3). 5-FU = 5-fluorouracil.
Photomicrographic images (Figure 11) of human colorectal tumor (HCT-116) cell were
captured using a digital camera observed under an inverted phase-contrast microscope at
magnification power of × 200. The images were taken at 48ꢀhours after the treatment. The
cells from the negative control group (treated with 0.1% DMSO) displayed a complete
confluent monolayer of aggressively growing HCT-116 cells while the cells from the positive
control group (treated with the standard drug 5-fluorouracil) demonstrated significant
inhibitory effect (p<0.01) on the proliferation of the cells with IC50 6.7 µM. The standard
reference, 5-fluorouracil caused the manifestation of obvious toxic signs in the treated cells,
such as round shaped morphology and affected the normal pseudopodial like cellular
projections in the cells. Among the synthetic compounds tested in this study, 7Ai revealed

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more pronounced antiproliferative effects with IC50: 19.2 µM. The cellular morphological
analysis revealed that compound 7Ai might have caused autophagy in the treated cells, as it is
clearly evident from plenty of autophagic vacuoles in the cytoplasm of the treated cells.
Compounds 7Aii and 7Avi showed moderate cytotoxicity with IC50: 59.3 and 41.2 µM,
respectively. Similar to compounds 7Ai, 7Aii and 7Aiii, autophagic vacuoles were also
observed in the treated cells. On the other hand, compounds 7Aiii, 7Aiv and 7Av
demonstrated a moderate inhibitory effect on the proliferation with IC50: 81.7, > 300 and
137.1 µM, respectively, as the population of cells was reduced considerably, however cellular
morphology remained more or less similar to that of the negative control.

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Figure 11: Photomicrographic images of human colorectal tumor (HCT-116) cells under an
inverted phase-contrast microscope with a digital camera at 48ꢀhours after treatment with

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compounds 7A(i-vi). The reference standard is 5-fluorouracil (5-FU) and treated cell group
with 0.1% DMSO as a negative control (Magnification × 200).
All the IC50 values of compounds 5(A-B), 6(A-B), 7A(i-viii) and 7B(i-viii) on MCF-7
and HCT-116 cancer cell lines are summarized in Table 1.
Table 1: IC50 values of the synthesized compounds against MCF-7 and HCT-116 cancer cell
lines. Each value is the average of triplicate experiments with standard deviation
IC50 values (µM) on the cancer cells
Compounds
MCF-7
HCT-116
5A
5B
92
74.1
231
87
366.2
112.4
184.6
19.2
6A
6B
>300
38
7Ai
7Aii
68
59.3
7Aiii
7Aiv
7Av
91
81.7
101
85
>300
137.1
41.2
7Avi
7Avii
7Aviii
7Bi
53
>300
178.9
122.3
106
172
>300
>300
>300
>300
>300
8.8
>300
>300
224
7Bii
132
7Biii
7Biv
7Bv
152.1
97.7
106.2
155.6
107.7
211.43
7Bvi
7Bvii
7Bviii
Tamoxifen
5-FU
--
12.8

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2.3 Structure Activity Relationship (SAR)
The analysis of SARs correlates the effects of a chemical structure and its biological activity
to develop the most effective drug. Through the analysis of the results obtained on human
breast cancer cell MCF-7 and colon cancer cell HCT-116 (Table 1), it is possible to deduce
some relationships between the structures of these newly synthesized compounds and their
measured cytotoxicity in order to optimize and identify a potent anti-tumor drug.
For drug candidates, the molecular configuration of chemical structure plays a highly crucial
role in the therapeutic efficacy, as a relatively minor change in the structure can lead to major
changes in medicinal efficacy [33]. So, in this study an attempt is made to recognize the
functional groups which are important for pronounce activity. At first stage, 2 derivatives of
1,2,4-triazole were synthesized with pyridine (5A) and phenolic (5B) rings. The results of the
study revealed an interesting phenomenon with respect to presence of the adjacent pyridine or
phenolic ring at the 5th carbon of 1,2,4-triazole nucleus. Pyridine ring with 1,2,4-triazole (5A)
showed to have cytotoxic activity against both the cell lines (MCF-7 and HCT 116), whereas,
phenolic ring with 1,2,4-triazole (5B) showed poor cytotoxic effect against the cell lines.
Further structural modifications was done by attaching a piperidine moiety to the thiol group
of 1,2,4-triazole of 5A and 5B. However, the biological activity of the compounds was not
much affected, as the cytotoxicity of the compounds (6A and 6B) remains same as that of
their parental compounds (5A and 5B). In the next stage, Schiff base complexes were derived
from 6A and 6B by adding either benzaldehyde or 1-methyl-pyrrolidin-2-one to the amino
group. Consistently, the results for the B-series compounds (with phenolic ring at 5th carbon
of 1,2,4-triazole) showed no improvement in the cytotoxic effect, as all the compounds of the
B-series remain poorly cytotoxic. Note worthily, addition of methyl pyrrolidin functional
group to 1,2,4-triazole-pyridin complex, which yielded the Schiff base 7Aviii, resulted in loss
of cytotoxic activity of the compound. Addition of benzaldehyde to amino group of 1,2,4-

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triazole – pyridine complex and attaching different functional groups at the 4th carbon of the
benzaldehyde resulted in a series of compounds (7Ai to 7Avii) with differential biological
activity. Among the compounds, 7Ai found to be the most potent cytotoxic compound.
Findings from the MTT assay against human breast tumor cells (MCF-7) showed that
compounds 5A, 6A and 7A(ii-vi) exhibited moderate anti-proliferation activity while
compounds 5B, 6B, and 7B(iv-viii) showed much weaker activity. The other remaining
compounds were considered inactive for MTT assay against MCF-7 cells, hence the IC50
values for such compounds could not be determined. The MTT assay against human
colorectal tumor (HCT-116) cell showed that compounds 7Ai and 7Avi demonstrated
significant cytotoxic effect with IC50 19.2 and 41.2, respectively, while compounds 6A, 6B,
7Aiii, 7Av and 7B(ii-viii) showed moderate to weaker activity while other compounds
showed poor activity with the IC50 of > 200, as shown in Table 1.
The results suggested that the hydroxyphenyl ring of piperidinium-4H-1,2,4-triazole-
3-thiolate strongly affected the activity. Furthermore, changing the hydroxyphenyl ring with a
pyridyl ring led to the loss of antiproliferative activity against HCT 116 cells. These
modifications and structure-activity relationship studies revealed that the hydroxyphenyl ring
of piperidinium-4H-1,2,4-triazole-3-thiolate played a critical role in their anti-proliferation
activity[34].
Furthermore, the results suggested that the hydroxyphenyl group at position-4 of 1,2,4-
triazole ring attached to 5-hydroxyphenyl and 3-piperidinium-4H-thiolate affected the
activity. Changing the p-hydroxyphenyl ring (7Ai) with p-cyanide phenyl ring (7Avi) led to
the loss of antiproliferative activity against MCF-7 cells. An overview of the structure-
cytotoxicity activity relationship study can be highlighted as follows: (i) The 1,2,4-triazole
ring framework is an important pharmacophore for the design and synthesis of novel
anticancer agents with significant pharmacological activity and low toxicity. (ii) The

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introduction of the substituted aromatic moiety at position-5, piperidinium-4H-thiolate at
position -3 and a series of para-substituted phenyl ring at position-4 played a crucial role in
modulating the anticancer activities of 1,2,4-triazole derivatives. (iii) Different substituents at
position-4 (hydroxy and cyanide) are suitable candidates as powerful cytotoxic agents.
The most probable reason for the enhanced cytotoxicity of 7Ai can be attributed to its
polarity. Comparatively, the compound 7Ai has a polar functional group (hydroxyl group),
whereas the compounds of the series have non-poplar groups, such as methoxy (OCH3),
methyl (CH3), chloro (Cl), bromo (Br), nitrile/cyano (CN) and nitro (NO2) groups. Unlike the
other compounds of the series, 7Ai has both hydrophobic side groups (pyridine and
piperidine) as well as a hydrophilic group (hydroxyl). Because of this, the compound 7Ai
may exhibit amphipathic property in biological system which enhanced the penetrability of
the compound through the cell membranes and allows the compound to effectively interact
with biomolecules such as proteins, RNA and DNA.
3.
Experimental
3.1
Chemicals
The chemicals and reagents used in the syntheses, characterisation and application
work of all the synthesised compounds include acetic acid glacial, acetone, chloroform,
dichloromethane, diethyl ether, ethyl acetate, n-hexane, toluene, tetrahydrofuran,methanol,
(99.5%), ethanol (99.7%) dimethyl sulfoxide-d6, chloroform-d, sulphuric acid (95-97%),
hydrochloric acid (37%), pyridine (99.5%), piperidine (99%), carbon disulphide (99%)
hydrazine monohydrate (80%) 4-pyridinecarboxylic acid hydrazide (99%), 4-
hydroxybenzhydrazide (98%), p-tolualdehyde (98%), anisaldehyde (98%), 3-
hydroxybenzaldehyde (97%), 4-cyanobenzaldehyde (97%), p-nitrobenzaldehyde (99%),
magnesium sulphate anhydrous, methanol-d4, potassium bromide (FT-IR grade), sodium

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hydrogen carbonate, calcium chloride anhydrous, potassium hydroxide, sodium hydroxide,
methyl Paraben, TLC silica gel 60 F254 (aluminium sheet, 20 cm × 20 cm). These chemicals
and solvent were purchased from Merck (Germany), BDH (England), Sigma-Aldrich (USA),
Aldrich Chemicals Co Ltd (England), Fluka (Germany), System, UNILAB (Sydney,
Australia), Fisher Scientific (UK), ARMAR Chemicals (Switzerland), R & M Chemicals
(UK) and QRëC. All chemical were used directly without further purifications.
3.2
Instruments
All the instruments used for the characterization work are located at the School of
Chemical Sciences, Universiti Sains Malaysia, Penang, Malaysia. Infrared Spectroscopy (IR):
Samples were prepared as potassium bromide (KBr) discs and were recorded on a Perkin-
Elmer System 2000 FT-IR spectrometer. All the 1D and 2D NMR spectra were recorded on a
Bruker Avance 500 MHz. The elemental analyses were executed on Perkin Elmer II, 2400
CHN analyzer. The instrument for the cytotoxic assay used includes NuAire Biosafety
cabinet class II, from NuAire (USA). Galaxy® CO2 incubator from RS Biotech (UK).
Axiovert 25 inverted phase-contrast microscope from Carl Zeiss (Germany). FM-2034 High-
Quality Elisa Reader machine (USA). AMG EVOS fI inverted microscope from Electron
Microscopy Sciences (USA).
3.3
Cytotoxicity assay
The reagents and chemicals used include DMEM and RPMI-1640 medium with L-
glutamine, heat-inactivated foetal bovine serum (HIFBS), penicillin (10000 U/mL) –
streptomycin (10 mg/mL) and trypsin-EDTA (10 ) from GIBCO (USA). Phosphate buffered
saline (PBS) tablets and sodium hydrogen carbonate from Sigma-Aldrich (USA).
Dimethylsulfoxide from Fisher Scientific (UK), 3-[4,5-dimethylthiazol-2-yl]-2,5-

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diphenyltetrazolium bromide (MTT) from PhytoTechnology Laboratories (USA). The tissue
culture materials include tissue culture flasks (25 cm2 and 75 cm2) with 0.2 µm polysulphone
filter cap, 96-well flat bottom tissue culture plates, 10 mL serological pipettes, vacuum
filtration system with PES membrane, syringe filters (0.22 µm) and cryotubes from Techno
Plastic Products (Switzerland) and 15 mL plastic centrifuge tubes from Corning Life Sciences
(USA). The cell lines include estrogen-dependent human breast cancer (MCF-7) and human
colorectal cancer (HCT-116) cell lines from the American Type Culture Collection (USA).
For cytotoxicity assay, stock solutions (10 mg/mL) of all the test samples were prepared in
DMSO. Using serial dilutions of the stock solutions, various concentrations (12.5 to 200
µg/mL) of the test samples were prepared with cell culture media.
3.4
Synthesis method
3.4.1 General procedure for the synthesis of methyl 4-hydroxybenzoate, 1
A mixture of 4-hydroxybenzoic acid (1.38 g, 0.01 mol) in 50 mL methanol and 5 mL
concentrated sulphuric acid was refluxed and the reaction progress was monitored by TLC.
After 12 hours, the mixture was poured into ice water and treated with 1N sodium
bicarbonate solution to give a white precipitate. The product was filtered, dried and
recrystallized from methanol.
Yield: (95%) as white crystalline; mp: 126-128°C (Lit: 125-128°C). Molecular weight:
152.15. IR (cm-1): 3286 (O-H stretch); 3034 (Csp2-H stretch); 2963 and 2855 (Csp3-H stretch);
1
1677 (C=O); 1605 and 1512 (C=C aromatic); 1105 (C-O). H-NMR (500 MHz, CDCl3) ,
ppm: 7.98 (2H, d, J=8.60 Hz, H-2, H-6); 6.91 (2H, d, J=8.60 Hz, H-3, H-5); 3.93 (3H, s, H-
13
8). C NMR (125 MHz, CDCl3) , ppm: 167.5 (C-7); 160.3 (C-4); 133.0 (C-2, C-6); 122.3
(C-1); 115.3 (C-3, C-5); 52.1 (C-8).

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3.4.2 General procedure for the synthesis of 4-hydroxybenzohydrazide, 2
A mixture of methyl 4-hydroxybenzoate, 1 (15.0 g, 0.10 mol) and hydrazine hydrate 80%
(9.0 g, 0.18 mol) in 15 ml methanol was refluxed for 8 h. The reaction mixture was then
poured into ice water. Hydrochloric acid (10%) was added to neutralize the mixture. The
precipitate formed was vacuum filtered and dried overnight in the oven at 55oC. The product
was recrystallized from 90% ethanol. Yield: 11.46 g (76.4%), light brown crystal; mp: 262-
264°C (Lit.: 266°C). IR (cm-1): 3318 (O-H stretch); 3280 and 3197 (-NH2); 3009 (Csp2-H
1
aromatic); 1619 (C=O); 1537 and 1467 (C=C aromatic); 1326 (C-N); 1255 (C-O). H-NMR
(500 MHz, pyridine-d5) , ppm: 10.67 (1H, s, -OH); 8.28 (2H, d, J=10.0 Hz, H-2, H-6); 7.24
(2H, d, J=10.0 Hz, H-3, H-5); 5.23 (2H, s, -NH2). 13C-NMR (125 MHz, pyridine-d5) , ppm:
168.8 (C-7); 162.4 (C-4); 130.4 (C-2, C-6); 125.8 (C-1); 116.5 (C-3, C-5).
3.4.3 General procedure for the synthesis of potassium 2-(4-hydroxybenzoyl)
hydrazinecarbo-dithioate, 3
4-Hydroxybenzohydrazide, 2 (0.10 mol) and potassium hydroxide (0.26 mol) were
dissolved in 100 mL ethanol. The mixture was cooled in an ice bath before the dropwise
addition of carbon disulphide (0.23 mol). The mixture was stirred for a day. The precipitate
formed was collected by vacuum filtration, washed with anhydrous diethyl ether (100 mL)
and dried in vacuum oven. Yield: 80.8%, white powder; mp: 280-282°C. Molecular weight:
266.38. IR (cm-1): 3380 (O-H stretch); 3148 (N-H); 3016 (Csp2-H aromatic); 1646 (C=O);
1605 and 1491 (C=C aromatic); 1330 (NCS); 1077 (C-S). 1H-NMR (500 MHz, DMSO-d6) ,
13
ppm: 7.68 (2H, d, J=8.70 Hz, H-2, H-6); 6.85 (2H, d, J=8.70 Hz, H-3, H-5). C-NMR (125
MHz, DMSO -d6) , ppm: 168.2 (C-7); 160.5 (C-4); 129.8 (C-2, C-6); 128.7 (C-10); 122.6
(C-1); 115.7 (C-3, C-5). CHN Elemental analysis, Calculated for C8H7KN2O2S2: C, 36.07; H,
2.65; N, 10.52 Found: C, 33.38; H, 2.45; N, 9.49.

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3.4.4 General procedure for the synthesis of 4-Amino-5-(4-hydroxyphenyl)-4H-[1,2,4]
triazole-3-thiol, 5
A suspension of potassium 2-(4-hydroxybenzoyl)hydrazinecarbodithioate, 3 (0.10
mol), hydrazine hydrate, 80% (15 mL, 0.30 mol) and water (5 mL) was refluxed at 110-
120oC for 8 hours. H2S evolved after a clear solution resulted. The mixture was diluted with
cold water (25 mL) and subsequent acidification with dilute HCl formed a precipitate which
was then filtered, washed with cold water and dried in the oven at 55oC. Yield: 78.7%, white
powder; mp: 263-265°C. Molecular weight: 208.24. IR (cm-1): 3305 (O-H stretch); 3270 and
3100 (-NH2); 3030 (Csp2-H aromatic); 2591 (S-H); 1604 (C=N); 1512 and 1477 (C=C
aromatic); 1435 (C-N); 1222, 1066 and 1003 (C-N-C); 944, 825 and 693 (1,2,4-triazole ring).
1H-NMR (500 MHz, DMSO-d6) , ppm: 13.77 (1H, s, -SH); 10.04 (1H, s, OH); 7.87 (2H, d,
13
J=8.60 Hz, H-2’, H-6’); 6.88 (2H, d, J=8.60 Hz, H-3’, H-5’); 5.75 (2H, s, -NH2). C-NMR
(125 MHz, DMSO-d6) , ppm: 166.3 (C-3); 159.3 (C-4’); 149.5 (C-5); 129.6 (C-2’, C-6’);
116.4 (C-1’); 115.2 (C-3’, C-5’). CHN Elemental analysis: Calculated for C8H8N4OS: C,
46.14; H, 3.87; N, 26.90. Found: C, 44.87; H, 3.59; N, 25.68.
3.4.5 General procedure for synthesis of piperidinium 4-amino-5-(4-sub-aryl)-4H-1,2,4-
triazole-3-thiolate, 6(A-B)
A mixture of compound 5A or 5B (0.01 mol) and piperidine (0.01 mol) in absolute
methanol (50 mL) was refluxed for 3-5 hours. The reaction progress was monitored by TLC.
A precipitate formed was filtered and dried. Purification using column chromatography gave
a pure product.
Compound 6A. Yield: (82.7%), khaki powder; mp:178-180°C. IR (cm-1): 3418 (-+NH2 at
piperidine ring); 3271 and 3160 (-NH2 at triazole ring); 3058 (Csp2-H aromatic); 2938 and
2852 (Csp3-H aliphatic); 2435 (-+NH at pyridine ring); 1605 (C=N); 1571 and 1478 (C=C

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1
aromatic); 1413(C-N); 1217, 1085, 1003 (C-N-C); 937, 823, 687 (1,2,4-triazole ring). H-
NMR (500 MHz, DMSO-d6) , ppm: 8.64 (2H, d, J=6.15 Hz, H-2’, H-6’); 8.03 (2H, d,
J=6.15 Hz, H-3’, H-5’); 5.71 (2H, s, -NH2); 5.31 (2H, br. s, -+NH2pip); 2.99 (4H, t, J=5.53 Hz,
H-6, H-10); 1.62-1.64 (4H, m, H-7, H-9); 1.54-1.56 (2H, m, H-8). 13C-NMR (125 MHz,
DMSO-d6) , ppm: 168.4 (C-3); 149.8 (C-2’, C-6’); 146.2 (C-5); 134.8 (C-4’); 120.2 (C-3’,
C-5’); 44.3 (C-6, C-10); 23.2 (C-7, C-9); 22.1 (C-8).
Compound 6B. Yield: (88.7%), orange powder; mp: 162-164°C. IR (cm-1): 3418 (-+NH2 at
piperidine ring); 3304 and 3208 (-NH2 at triazole ring); 3304 (-OH, superimposed with the N-
H bands); 3030 (Csp2-H aromatic); 2927 and 2853 (Csp3-H aliphatic); 1607 (C=N stretch);
1530 and 1464 (C=C aromatic); 1394 (C–N); 1253, 1057, 1015 (C-N-C); 945, 815, 690
1
(1,2,4-triazole ring). H-NMR (500 MHz, DMSO-d6) , ppm: 7.86 (2H, d, J=8.25 Hz, H-2’,
H-6’); 6.88 (2H, d, J=8.25 Hz, H-3’, H-5’); 6.37 (2H, br. s, -+NH2 at piperidine ring); 5.67
13
(2H, s, -NH2); 2.84 (4H, t, J=4.68 Hz, H-6, H-10); 1.51-1.53 (6H, m, H-7, H-8, H-9). C-
NMR (125 MHz, DMSO-d6) , ppm: 166.2 (C-3); 159.2 (C-4’); 149.1 (C-5); 129.1 (C-2’, C-
6’); 117.2 (C-1’); 115.3 (C-3’, C-5’); 45.2 (C-6, C-10); 24.5 (C-7, C-9); 23.4(C-8).
3.4.6 General procedure piperidinium (E)-4-(4-sub-benzylideneamino)-5-(pyridin-4-yl)-4H-
1,2,4-triazole-3-thiolate, 7A(i-viii)
Schiff base compounds of piperidinium (E)-4-(4-sub-benzylideneamino)-5-(pyridin-4-
yl)-4H-1,2,4-triazole-3-thiolate, 7A(i-vii) and piperidinium (E)-4-(1-methylpyrrolidin-2-
ylideneamino)-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiolate, 7Aviii were prepared according
to two new procedures. In the first procedure, piperidine was added in the system which
undergoes dehydrohalogenation to produce cyclic imine. In the second procedure, 4-amino-5-
(pyridin-4-yl)-4H-1,2,4-triazole-3-thiole, 5A (0.0026 mol) in absolute methanol (50 mL) with

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a few drops of piperidine (0.5 mL) were refluxed for 30 mins before the addition of various
aromatic aldehydes (0.0026 mol) and the mixture was continued to reflux for 7 hours. A solid
precipitate formed was filtered off, dried under vacuum overnight and purified to give (E)-4-
(4-sub-benzylideneamino)-5-(pyridin-4yl)-4H-1,2,4-triazole-3-thiolate, 7A(i-vii) and 7Aviii.
In Scheme 2, Schiff base compounds, 7A(i-viii) can be synthesized from starting material 6A
using the first procedure or from starting material 5A by using the second procedure.
Compound 7Ai. Yield: 82.9%, khaki powder; mp:150-152°C. IR (cm-1): 3331 (O-H stretch);
3001 (Csp2-H aromatic); 2955 (Csp3-H aliphatic); 1651 (C=N);1592 and 1514 (C=C aromatic);
1
710 (C-S). H-NMR (500 MHz, DMSO-d6) , ppm: 9.80 (1H, s, H-12); 8.63 (2H, d, J=6.15
Hz, H-2’, H-6’); 7.90 (2H, d, J=6.15 Hz, H-3’, H-5’); 7.73 (2H, d, J=8.65 Hz, H-2”, H-6”);
6.95 (2H, d, J=8.65 Hz, H-3”, H-5”); 3.02 (4H, t, J=5.60 Hz, H-6, H-10); 1.63-1.65 (4H, m,
H-7, H-9); 1.54-1.56 (2H, m, H-8). 13C-NMR (125 MHz, DMSO-d6) , ppm: 164.4 (C-3);
162.3 (C-12); 161.3 (C-4”); 149.9 (C-2’, C-6’); 146.2 (C-5); 135.0 (C-4’); 130.3 (C-2”, C-
6”); 123.7 (C-1”); 120.5 (C-3’, C-5’); 116.0 (C-3”, C-5”); 44.0 (C-6, C-10); 22.8 (C-7, C-9);
22.1 (C-8). CHN Elemental analysis: Calculated for C19H22N6OS: C, 59.66; H, 5.80; N,
21.97. Found: C, 58.76; H, 5.82; N, 21.55.
Compound 7Aii. Yield: 92.6%, yellow-green powder; mp:182-184°C. IR (cm-1): 3007 (Csp2-
H aromatic); 2934 (Csp3-H aliphatic); 1599 (C=N);1568 and 1511 (C=C aromatic); 729 (C-S).
1H-NMR (500 MHz, DMSO-d6) , ppm: 9.75 (1H, s, H-12); 8.69 (2H, d, J=6.15 Hz, H-2’, H-
6’); 7.88 (2H, d, J=6.15 Hz, H-3’, H-5’); 7.87 (2H, d, J=8.82 Hz, H-2”, H-6”); 7.12 (2H, d,
J=8.82 Hz, H-3”, H-5”); 3.86 (3H, s, -OCH3); 3.05 (4H, t, J=5.65 Hz, H-6, H-10); 1.65-1.67
(4H, m, H-7, H-9); 1.54-1.56 (2H, m, H-8). 13C-NMR (125 MHz, DMSO-d6) , ppm: 164.6
(C-3); 163.6 (C-12); 162.7 (C-1”); 150.1 (C-2’, C-6’); 146.3 (C-5); 133.8 (C-4’); 130.5 (C-

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3”, C-5”); 124.8 (C-4”); 121.1 (C-3’, C-5’); 114.7 (C-2”, C-6”); 55.5 (OCH3); 43.7 (C-6, C-
10); 22.3 (C-7, C-9); 21.7 (C-8).
Compound 7Aiii. Yield: 91.4%, lemon powder; mp:155-157°C. IR (cm-1): 3012 (Csp2-H
aromatic); 2947 (Csp3-H aliphatic); 1599 (C=N); 1567 and 1511 (C=C aromatic); 718 (C-S).
1H-NMR (500 MHz, DMSO-d6) , ppm: 10.05 (1H, s, H-12); 8.65 (2H, d, J=6.50 Hz, H-2’,
H-6’); 7.89 (2H, d, J=6.50 Hz, H-3’, H-5’); 7.79 (2H, d, J=7.85 Hz, H-2”, H-6”); 7.38 (2H, d,
J=7.85 Hz, H-3”, H-5”); 1.69 (3H, s, -CH3); 3.05 (4H, t, J=5.48 Hz, H-6, H-10); 1.53-1.55
13
(4H, m, H-7, H-9); 1.06-1.08 (2H, m, H-8). C-NMR (125 MHz, DMSO-d6) , ppm: 164.1
(C-3); 162.1 (C-12); 149.9 (C-2’, C-6’); 146.4 (C-5); 142.4 (C-1”); 134.6 (C-4’); 130.3 (C-
4”); 129.7 (C-3”, C-5”); 128.3 (C-2”, C-6”); 120.8 (C-3’, C-5’); 43.9 (C-6, C-10); 22.5 (C-7,
C-9); 21.9 (C-8); 21.2 (-CH3).
Compound 7Aiv. Yield: 23.1%, lemon powder; mp: 208-211°C. IR (cm-1): 3026 (Csp2-H
aromatic); 2936 (Csp3-H aliphatic); 1603 (C=N); 1559 and 1475 (C=C aromatic); 725 (C-S).
1H-NMR (500 MHz, DMSO-d6) , ppm:10.23 (1H, s, H-12); 8.65 (2H, d, J=6.20 Hz, H-2’,
H-6’); 7.87 (2H, d, J=6.30 Hz, H-3’, H-5’); 7.90 (2H, d, J=8.50 Hz, H-2”, H-6”); 7.63 (2H, d,
J=8.50 Hz, H-3”, H-5”); 3.04 (4H, t, J=5.70 Hz, H-6, H-10); 1.63-1.65 (4H, m, H-7, H-9);
1.54-1.56 (2H, m, H-8). 13C-NMR (125 MHz, DMSO-d6) , ppm: 163.6 (C-3); 162.0 (C-12);
150.0 (C-2’, C-6’); 146.6 (C-5); 131.5 (C-4’); 129.3 (C-3”, C-5”); 136.9 (C-4”); 130.1 (C-2”,
C-6”); 134.0 (C-1”); 121.2 (C-3’, C-5’); 43.7 (C-6, C-10); 22.2 (C-7, C-9); 21.7 (C-8).CHN
Elemental analysis: Calculated for C19H21ClN6S: C, 56.92; H, 5.28; N, 20.96. Found: C,
57.14; H, 5.24; N, 20.99.

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Compound 7Av.Yield: 26.9%, banana powder; mp: 224-226°C. IR (cm-1): 3025 (Csp2-H
aromatic); 2926 (Csp3-H aliphatic); 1604 (C=N stretch); 1561 and 1477 (C=C aromatic); 724
(C-S). 1H-NMR (500 MHz, DMSO-d6) , ppm: 10.16 (1H, s, H-12); 8.67 (2H, d, J=6.20 Hz,
H-2’, H-6’); 7.87 (2H, d, J=6.30 Hz, H-3’, H-5’); 7.84 (2H, d, J=8.50 Hz, H-2”, H-6”); 7.77
(2H, d, J=8.50 Hz, H-3”, H-5”); 3.05 (4H, t, J=5.68 Hz, H-6, H-10); 1.65-1.67 (4H, m, H-7,
H-9); 1.55-1.57 (2H, m, H-8). 13C-NMR (125 MHz, DMSO-d6) , ppm: 163.9 (C-3); 161.0
(C-12); 150.0 (C-2’, C-6’); 146.5 (C-5); 134.2 (C-4’); 132.2 (C-3”, C-5”); 132.1 (C-4”);
130.1 (C-2”, C-6”); 125.6 (C-1”); 121.0 (C-3’, C-5’); 43.7 (C-6, C-10); 22.3 (C-7, C-9); 21.7
(C-8).
Compound 7Avi. Yield: 55.3%, peach powder; mp: 260C. IR (cm-1): 3033 (Csp2-H
aromatic); 2947 (Csp3-H aliphatic); 2232 (CN stretch); 1601 (C=N stretch); 1547 and 1468
(C=C aromatic);713 (C-S). 1H-NMR (500 MHz, DMSO-d6) , ppm: 10.70 (1H, s, H-12); 8.62
(2H, d, J=6.15 Hz, H-2’, H-6’); 7.87 (2H, d, J=6.05 Hz, H-3’, H-5’); 8.04 (2H, d, J=8.30 Hz,
H-2”, H-6”); 7.98 (2H, d, J=8.30 Hz, H-3”, H-5”); 3.00 (4H, t, J=5.58 Hz, H-6, H-10); 1.58-
1.60 (4H, m, H-7, H-9); 1.50-1.52 (2H, m, H-8). 13C-NMR (125 MHz, DMSO-d6) , ppm:
164.5 (C-3); 156.1 (C-12); 149.6 (C-2’, C-6’); 14.7 (C-5); 135.3 (C-4’); 132.9 (C-3”, C-5”);
113.1 (C-4”); 128.5 (C-2”, C-6”); 138.2 (C-1”); 120.5 (C-3’, C-5’); 119.4 (C-N); 47.4 (C-6,
C-10); 25.2 (C-7, C-9); 19.2 (C-8).
Compound 7Avii. Yield: 31.9%, dark-orange powder; mp: 242-244°C. IR (cm-1): 3036 (Csp2-
H aromatic); 2928 (Csp3-H aliphatic); 1606 (C=N stretch); 1581 and 1519 (C=C aromatic);
1
1429 and 1340 (-NO2 asymmetrical and symmetrical stretching); 749 (C-S). H-NMR (500
MHz, DMSO-d6) , ppm: 10.20 (1H, s, H-12); 8.40 (2H, d, J=8.50 Hz, H-3”, H-5”); 8.20
(2H, d, J=8.50 Hz, H-2”, H-6”); 7.88 (2H, d, J=5.50 Hz, H-3’, H-5’); 8.76 (2H, d, J=5.50 Hz,