Synthesis of 1,2,4-triazole-linked urea/thiourea conjugates as cytotoxic and apoptosis inducing agents

Article abstract of DOI:10.1016/j.bmcl.2018.03.074

A new series of 1,2,4-triazole-linked urea and thiourea conjugates have been synthesized and evaluated for their in vitro cytotoxicity against selected human cancer cell lines namely, breast (MCF-7, MDA-MB-231), lung (A549) prostate (DU145) and one mouse melanoma (B16-F10) cell line and compared with reference drug. The compound 5t showed significant cytotoxicity on MCF-7 breast cancer cell line with a IC₅₀ value of 7.22 ± 0.47 μM among all the tested compounds. Notably, induction of apoptosis by compound 5t on MCF-7 cells was evaluated using different staining techniques such as acridine orange/ethidium bromide (AO/EB), annexin V-FITC/PI, and DAPI. Further, clonogenic assay indicates the inhibition of colony formation on MCF-7 cells by compound 5t. Moreover, the flow-cytometric analysis also revealed that compound 5t caused the arrest of cells at G0/G1 phase of cell cycle. In addition, the compounds when tested on normal human cells (L-132) were found to be safer with low cytotoxicity profile.

Full text of DOI:10.1016/j.bmcl.2018.03.074

Accepted Manuscript
Synthesis of 1,2,4-triazole-linked urea/thiourea conjugates as cytotoxic and
apoptosis inducing agents
Ramya Tokala, Swarna Bale, Ingle Pavan Janrao, Aluri Vennela, Niggula
Praveen Kumar, Kishna Ram Senwar, Chandraiah Godugu, Nagula Shankaraiah
PII:
S0960-894X(18)30279-8
https://doi.org/10.1016/j.bmcl.2018.03.074
BMCL 25728
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
13 December 2017
23 March 2018
27 March 2018
Please cite this article as: Tokala, R., Bale, S., Pavan Janrao, I., Vennela, A., Kumar, N.P., Ram Senwar, K., Godugu,
C., Shankaraiah, N., Synthesis of 1,2,4-triazole-linked urea/thiourea conjugates as cytotoxic and apoptosis inducing
agents, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.074
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Bioorganic & Medicinal Chemistry Letters
Synthesis of 1,2,4-triazole-linked urea/thiourea conjugates as cytotoxic and
apoptosis inducing agents
Ramya Tokala,a Swarna Bale,b Ingle Pavan Janrao,a Aluri Vennela,a Niggula Praveen Kumar,a Kishna Ram
Senwar,a Chandraiah Godugu,b* Nagula Shankaraiaha*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
bDepartment of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A new series of 1,2,4-triazole-linked urea and thiourea conjugates have been synthesized and
evaluated for their in vitro cytotoxicity against selected human cancer cell lines namely, breast
(MCF-7, MDA-MB-231), lung (A549) prostate (DU145) and one mouse melanoma (B16-F10)
cell line and compared with reference drug. The compound 5t showed significant cytotoxicity on
MCF-7 breast cancer cell line with a IC50 value of 7.22 ± 0.47 µM among all the tested
compounds. Notably, induction of apoptosis by compound 5t on MCF-7 cells was evaluated
using different staining techniques such as acridine orange/ethidium bromide (AO/EB), annexin
V-FITC/PI, and DAPI. Further, clonogenic assay indicates the inhibition of colony formation on
MCF-7 cells by compound 5t. Moreover, the flow-cytometric analysis also revealed that
compound 5t caused the arrest of cells at G0/G1 phase of cell cycle. In addition, the compounds
when tested on normal human cells (L-132) were found to be safer with low cytotoxicity profile.
Keywords:
1,2,4-Triazoles
Urea
Thiourea
Anticancer
Apoptosis
Flow-cytometry
Cancer is a major global health problem and one of the
leading causes of death when compared to other diseases. The
progression of cancer in a person is due to changes in DNA
(mutations)1 or a compromised immune system. Besides, the
preventive measures (vaccination) and avoiding behavioural risk
factors (smoking etc.,) the occurrence of the disease is still
accounted statistically.2 Evading apoptosis stands as the major
hallmark of cancer cellular pathways. The tolerance, along with
resistance and subsequent effect of existing anticancer agents,
demands the search for new chemical entities (NCEs) as
chemotherapeutic agents.
Examples of drugs with different medicinal importance
containing 1,2,4-triazole include8 (a) In breast cancer treatment-
letrozole (Figure 1) and anastrazole, (b) CNS acting agents like
etizolam, alprazolam, triazolam, (c) Antimicrobial agents like
ribavirin, (d) Antimycotics like propiconazole, fluconazole,
triadimefon, hexaconazole, myclobutanil, (e) In the treatment of
migraine headaches-rizatriptan. Further, 1,2,4-triazoles were
reported to have industrial applications as polymers, lubricants,
dyes and analytical reagents for heavy metal quantification.18
Moreover, the antiproliferative activity of the triazoles is
expressed in both individual, fused systems and highly effective
compounds with better selectivity can be obtained by the
chemical modulation of the triazole ring.
The chemistry involving triazoles has a significant role due to
their medicinal and industrial properties as drugs and
intermediates respectively in various areas.3,4 The aspects of
stability to metabolic degradation, association with drug targets,5
the formation of hydrogen-bonds makes triazole as
pharmacophore in many biologically active molecules.6,7 These
are well-known compounds for diverse biological activities like
anticancer,8,9 antimicrobial,10 antifungal,11 antitubercular,12
anthelmintic,
analgesic,13
anti-inflammatory,13,14
and
anticonvulsant.15,16 Also, 1,2,4-triazoles are evident for
possessing central nervous system (CNS) acting drug candidates
(stimulants, anxiolytics)8 and effective on enzymes like
cholinesterase inhibition,17 etc.

Authors to whom correspondence should be addressed. Corresponding
Figure 1. The structures of sorafenib (A), letrozole (B), thiouracil (C),
and designed new derivatives (D).
author; Dr. Nagula Shankaraiah, E-mail: shankar@niperhyd.ac.in;
shankarnbs@gmail.com and Dr. Chandraiah Godugu, E-mail.:
chandra.niperhyd@gov.in

Small molecule kinase inhibitors including urea
derivatives (thioureas e.g., thiouracil (Figure 1) benzoyl ureas)19
are drawing substantial interest due to their inhibitory activity
against various kinases.20 Inhibition of protein and receptor
tyrosine kinases results in inhibition of tumor generation and
proliferation.21 Sorafenib (Figure 1) represents urea containing
kinase inhibitors with profound anticancer activity in specific to
primary kidney and in advanced liver cancer.22
that 5g, 5k, 5l, 5p, 5r, 5s, 5t, and 5y were active at less than 50
μM on all the tested cancer cell lines.
A broad spectrum of anticancer activity was observed in all
the cancer cell lines tested when thiourea congeners are
synthesized in conjunction with podophyllotoxin.23 In
continuation our earlier reports on 1,2,3-triazole24,25,26,27 as
significant anticancer agents, we pondered to explore antitumor
activity of these newly synthesized 1,2,4-triazole and
urea/thiourea congeners with various substitutions and explored
for their in vitro cytotoxicity profile against a panel of selected
human cancer cell lines.
The synthesis of designed triazolo-urea/thiourea conjugates
was outlined in Scheme 1. Schiff bases 3a–f were synthesized in
good yields by using commercially available aldehydes 1a–f and
4-amino-4H-1,2,4-triazole in presence of aluminium trichloride in
ethanol. Thus obtained Schiff bases were reduced to their
corresponding amines 4a–f employing sodium borohydride in
ethanol. Further, the reduced intermediates were condensed with
various isocyanates and isothiocyanates in tetrahydrofuran and
sodium hydride as the base to furnish triazolo-urea/thiourea 5a–y
congeners in quantitative yields respectively. The reactions were
monitored by thin layer chromatography (TLC) and all the
products were purified by column chromatography. The newly
1
synthesized compounds were characterized by HRMS, H, and
13C NMR spectroscopy. The HRMS (ESI) of all the compounds
showed a [M + H]+ peak equivalent to their molecular formulae.
The presence of broad singlet at a range of 6.5 to 7.0 ppm
accounts for NH of final compound confirms unambiguously.
The triazole protons which are deshielded appear as a singlet at a
range of 9.3 to 8.5 ppm. The 2 protons of methylene appeared at
4.8 ppm. Characteristic methoxy protons appeared as singlet at a
range of 3.7 ppm which accounts for 6 protons indicating the
presence of 2 methoxy groups and other aromatic protons lie in
range of 6.7 to 8.9 ppm. In 13C NMR spectrum of 5a, the amide
carbonyl carbon appeared at δ 154.1 ppm and the aromatic
methoxy-substituted carbons resonated around δ 149.1 ppm. The
equivalent carbons of the triazole appear at δ 144.2 ppm. The sp
hybridized carbon of the nitrile group resonates at 119.1 ppm.
The methoxy carbons appeared at 55.8 ppm whereas the signal
attributed to the methylene carbon appeared at 54.8 ppm. The
aromatic carbons resonate around δ 111.7-144.2 ppm. A similar
Scheme 1. Synthesis of 1,2,4-triazole-linked urea and thiourea
conjugates (5a–y).
From the cytotoxicity results, it was evident that compound 5t
was exhibiting significant cytotoxic activity in all the cell lines
screened compared to other compounds. 5s is showing selective
toxicity towards B16-F10 at a concentration <10 μM. From the
IC50 values, it is evident that the thiourea congeners 5o-5y are
potent when compared to urea derivatives 5a-5n. In urea
congeners, heterocyclic aldehydes were found to be potent than
simple aldehydes and halogen containing derivatives 5f, 5g
showed better activity than alkyl derivatives 5k, 5l. Though
activity of 5t is potent in B16-F10 cell line compared to MCF-7
cell line and molecular level studies were performed on MCF-7
cell line due to human derived cancer nature.
1
pattern was observed in the H NMR and 13C NMR of all the
other derivatives 5b–y. The HRMS (ESI) of these compounds
showed a [M + H]+ peak equivalent to their respective molecular
formula.
The newly synthesized triazolo-urea and thiourea conjugates
5a–y were subjected to in vitro cytotoxicity studies on MCF-7
and MDA-MB-231 (breast), A549 (non small cell-lung), DU 145
(Prostate), B16-F10 (mouse melanoma), cancer cell lines by
DAPI (4', 6-diamidino-2-phenylindole) is a fluorescent dye
used to detect the nuclear damage, it binds strongly to A-T rich
regions in DNA.28 Dose dependent nuclear changes were
remarkably observed with the compound 5t compared to control.
As concentration of the treatment is increased (4 µM, 8 µM),
normal oblong shaped cells got shrunk and obtained round shape,
whereas at higher concentration of 12 µM, horse shoe shaped
(pyknotic) nucleus along with DNA fragmentation that appears
as bright color was observed, a typical hallmark of apoptosis was
observed as shown in Figure 2. DAPI staining demonstrated that
compound 5t is inducing significant nuclear morphological
changes.
performing
the
3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide (MTT) assay. The IC50 (μM) values
(concentration required to inhibit 50% of cancer cells growth) of
tested compounds 5a–y and reference standard (5-fluorouracil)
has been listed in Table 1. Results from the Table 1 indicated
that some of the synthesized compounds exhibited potential
cytotoxicity against B16-F10 and MCF-7 cancer cell lines and
were found to be active in the range of 4.51± 0.05 to 7.22 ± 0.47
μM. From the close examination of IC50 values, it is observed

Table 1. In vitro cytotoxic activity (IC50 in µM)a of triazole-linked urea/thiourea conjugates 5a–y
Compound
MCF-7b
A-549c
MDA-MB 231d
DU-145e
24.12±1.03
20.59±1.00
20.41±0.36
>50
B16-F10f
>50
5a
5b
5c
5d
5e
5f
>50
>50
>50
>50
20.69±0.10
44.21±0.41
29.27±5.81
25.96±3.56
21.42±2.32
26.24±0.52
24.85±4.56
21.98±4.21
>50
>50
21.03±1.56
42.16±5.34
>50
>50
>50
>50
>50
>50
>50
31.00±0.66
20.41±0.36
24.13±0.88
20.15±3.19
21.23±0.59
>50
28.85±1.61
>50
31.16±0.25
30.06±0.38
>50
40.27±0.51
29.60±0.66
>50
5g
5h
5i
22.07±3.24
23.65±5.23
31.00±0.09
35.80±1.46
39.67±0.27
35.19±0.66
31.08±0.01
>50
>50
5j
46.94±2.35
34.02±0.70
38.89±0.53
>50
44.06±4.07
33.17±1.21
39.54±0.92
>50
5k
5l
43.66±1.25
21.90±1.18
22.10±1.42
20.14±1.81
21.52±1.50
>50
5m
5n
>50
>50
>50
>50
34.72±3.20
5o
5p
>50
24.80±0.02
>50
28.41±0.11
17.19±0.29
22.22±1.82
16.18 ± 0.76
21.02±2.83
9.39 ± 1.61
21.52±0.69
>50
>50
24.77±0.04
>50
>50
22.90±0.60
24.71±1.39
29.88±8.05
8.54 ± 1.94
5.62 ± 0.32
4.51 ± 0.05
39.48±3.48
29.38±2.51
20.71±2.32
46.78±6.70
20.67±3.36
-
21.37±1.22
26.29±0.50
26.39±1.74
23.14±0.68
11.75 ± 1.29
29.81±2.53
>50
5q
5r
15.47 ± 1.13
23.79±0.93
7.22 ± 0.47
>50
25.28±0.35
24.92±6.28
9.28 ± 0.12
>50
5s
5t
5u
5v
>50
>50
5w
5x
>50
17.38±0.37
22.78±1.62
21.02±0.69
39.86
>50
20.91±0.25
20.83±0.11
23.32±0.35
39.17±1.25
>50
>50
5y
5-FUg
30.07±1.76
16.7±1.5
27.91±3.05
35.91±4.82
a50% Inhibitory concentration after 48 h of compound treatment. b,d Breast cancer cells, cLung cancer cells, eProstate cancer cells, f Mouse melanoma cells, g5-
Fluorouracil: Reference standard.
the concentration of 4 μM induced morphological changes like
membrane blebbing. At the concentration of 8 μM, DNA
fragmentation and chromatin condensation are also observed
along with membrane blebbing, resulting in loss of membrane
permeability. Further, at a concentration of 12 μM, the number of
green cells was reduced and orange cells were increased
indicating cells underwent late apoptotic stage. These results
explore that the compound may induce dose dependent apoptosis
as evident by observed hall marks of apoptosis as shown in
Figure 3.
Figure 2. Morphological nuclear changes by 5t in MCF-7 cells on 48 h
exposure as indicated by DAPI staining. Control cells indicating normal
oblong shape, 4 μM, 8 μM, and 12 μM, treated cells indicating rounding,
shrinkage, pyknotic nucleus of the cells, respectively. Arrows indicate the
altered nuclear morphological (horse shoe nucleus and fragmentation)
changes in a dose dependant manner.
Acridine Orange and Ethidium Bromide (AO/EB) fluorescent
staining assay was carried out for differentiation of live,
apoptotic and necrotic cells.29 This staining solution is a mixture
of AO and EB, in which AO is used for live cell identification
and EB for identification of dead cells. The AO permeates into
the intact cell membrane and thus stains live cells to appear green
whereas, EB stained dead cells to appear red when visualized by
fluorescent microscopy due to disruption of membrane integrity.
It is observed from the results of AO/EB, that control cells
showed normal cell morphological features while compound 5t at
Figure 3. Apoptosis induction by 5t in MCF-7cells using AO/EB
staining. Control cells exhibiting normal green structure. Treatment with 4
μM, 8 μM, and 12 μM. Treated cells indicating rounding, shrinkage, pyknotic
nucleus of the cells, respectively. Arrows indicate the altered cell
morphological (membrane blebbing and apoptotic bodies) changes at
different concentrations of 5t.

Cell cycle analysis by quantification of DNA content is a
reliable method to investigate at which phase cell cycle has been
arrested, wherein Propidium Iodide dye is used which binds in
proportion to the amount of DNA present in the cell.31 Results of
cell cycle analysis using 5t against MCF-7 cell lines by flow
cytometry showed a transient G0/G1 arrest of cells peaking at 24
h. The results revealed that 5t caused an increase in % of G0/G1
arrest (61.18% and 69.97% at 4 and 8 µM respectively) than
control cells (53.01%).
Figure 4. Effect of 5t on cell cycle progression of MCF-7 using flow
cytometry. Cells treated with 4 and 8 μM concentrations of 5t exhibiting
concentration dependent arrest of G0/G1 (61.18% and 69.97%) respectively
compared to control cells.
Figure 7. Estimation of apoptotic population by Annexin
V and
Propidium Iodide (PI) staining by compound 5t on MCF-7 cells after 48 h
using BD C6 Accuri flowcytometer. 10,000 cells from each sample were
analysed using flow cytometry. The percentage of cells positive for Annexin
V-FITC and/or Propidium Iodide is reported inside the quadrants. Cells in the
lower left quadrant (L: AV-/PI-): live cells; lower right quadrant (EA:
AV+/PI-): early apoptotic cells; upper right quadrant (LA: AV+/PI+): late
apoptotic cells and upper left quadrant (D: AV-/PI+): necrotic cells.
The clonogenic cell reproduction assay determines the ability
of a cell to proliferate indefinitely, thereby retaining its
proliferative ability to form large colonies.30 This assay also
serves to determine long term viability of cells and growth arrest
potential of the compound. Figure 5 clearly shows inhibition of
colony formation by compound 5t in concentration- dependent
manner. At the highest concentration (12 µM) maximum
inhibition of colony was observed in comparison to control
(Figure 6). Total colony count was made possible using
molecular imaging system Vilber Fusion Fx software and
represented as a total number of colonies verses concentration.
The colony count was found to be significantly decreased in 5t
treated cells concentration dependently compared to untreated
control cells.
To confirm the induction of apoptosis, Annexin V binding
studies by flow cytometer was performed. To detect early
apoptosis and late apoptosis or necrosis, FITC conjugated human
phospholipid phosphatidyl serine binding protein, Annexin V,
conjugated with FITC is used to stain cells in combination with
Propidium Iodide (PI). As phosphatidyl serine externalization is
the hallmark of apoptotic cells, Annexin V binds to the
membrane and gives fluorescence. Thus, Annexin V stained cells
represent cells with intact membranes and externalized
phosphatidyl serine. Cells that stained positive for Annexin V/PI
represents the cells in the late apoptotic stage that have lost
membrane integrity. MCF-7 cells on treatment with 4, 8 and 12
µM for 48 h have shown that increase in the concentration of 5t
resulted in increased Annexin V–FITC binding population when
compared to vehicle-treated control cells. It indicates the
Control
4 μM
8 μM
12 μM
compound 5t induces apoptosis in MCF-7 cells in
a
concentration dependent manner. The percentage of total
apoptotic cells (late apoptotic cells) was increased to 38.35%
after treatment with 12 µM concentration of compound 5t for 48
h, in comparison to the control (7.4%) cells (Figure 7).
Figure 5. Effect of 5t on clonogenic assay. Control cells having multiple
colonies, while 5t at concentrations of 4, 8 and 12 µM showing inhibition of
number of colonies respectively when compared to control.
In cancer disease condition, specificity of the compounds
towards cancer cell lines, while unaffecting the normal cell
functioning gains prime significance. Therefore, performing cell
viability assay in the normal cell line of any anticancer agent
demonstrates its specific activity towards cancer cell line, and
thus can be used further until it doesn’t affect normal cell
physiology. The L-132 lung epithelial cell was used as a normal
cell line to investigate the effect of compound 5t and found a
two-fold difference of cytotoxic activity in L-132 cells compared
to MCF-7 cell line. IC50 value of 5t in normal cell line was found
to be 15.18 ± 0.84 µM, whereas, on MCF-7 cell line, the IC50
value was 7.22 ± 0.47 µM indicating the cancer specificity of
compound 5t while the same concentration is not significantly
toxic to normal cell line (Figure 8).
Figure 6. Inhibition of colony formation of 5t on MCF-7 cell line.
Significant decrease in cell reproduction is observed with increase in
concentration. All the values are expressed as Mean ± SEM of three
experiments in which each treatment was performed in triplicate wells.
****p<0.0001 versus control cells.

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Figure 8. Cytospecificity of compound 5t towards breast cancer cell line.
The cytotoxic effect of compound 5t was examined on both cancer (MCF-7)
and normal cell line (L-132) using MTT assay. The values were expressed as
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In conclusion, a new series of 1,2,4-triazole-urea/thiourea
conjugates have been synthesized and evaluated for their in vitro
cytotoxicity against different human cancer cell lines including
melanoma mouse cancer cell lines. From the preliminary
screening, 1,2,4-triazole-thiourea congeners were found to be
potential on MCF-7 cancer cell line. Interestingly, compound 5t
displayed a broad spectrum of activity against all tested cancer
cell lines. Specificity in cytotoxicity towards cancer cell lines
was studied by performing cell viability assay on healthy lung
epithelial cells (L-132) and two-fold lesser cytotoxicity was
observed in comparison to MCF-7 cell line. Additionally, the
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staining, Annexin V/Propidium Iodide assay suggested that the
compound 5t induces apoptosis in a dose-dependent manner.
Colony formation in MCF-7 was effectively inhibited by
compound 5t. Moreover, the compound 5t caused cell cycle
arrest at G0/G1 phase by disruption of cytoskeleton in MCF-7
cancer cells. Overall, the current studies demonstrate that the
triazole-linked thiourea hybrids have the potential to be
developed as lead and their further structural modifications may
generate promising new anticancer agents in breast cancer
therapy.
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Acknowledgments
The authors are thankful to DoP, Ministry of Chemicals &
Fertilizers, Govt. of India, for the award of NIPER fellowship.
Dr. N. Shankaraiah gratefully acknowledges SERB, DST, Govt.
of India for research grant (YSS-2015-001709).
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Graphical Abstract
Synthesis
of
1,2,4-triazole-linked
urea/thiourea conjugates as cytotoxic and
apoptosis inducing agents
Ramya Tokala,a Swarna Bale,b Ingle Pavan Janrao,a
Aluri Vennela,a Niggula Praveen Kumar,a Kishna
Ram Senwar,a Chandraiah Godugu,b* Nagula
Shankaraiaha*
aDepartment of Medicinal Chemistry, National
Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bDepartment of Regulatory Toxicology, National
Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India

Research Highlights

A new series of 1,2,4-triazole-urea/thiourea
conjugates were synthesized.
Compound 5t induced apoptosis and cell
cycle arrest in G0/G1 phase in MCF-7 cell
line.



Compound 5t inhibited colony formation in
MCF-7 cell line.
Compound 5t was almost 2 times more
selective on MCF-7 cells compared to L-132
cells.