Synthesis and pharmacological evaluation of 1,3,4-thiadiazole bearing pyrimidine derivatives as STAT3 inhibitor for treatment of breast cancer

Article abstract of DOI:10.1007/s13738-020-01932-z

Novel scaffold of 1,3,4-thiadiazolo-(3,2-α)-pyrimidine-6-carbonitrile derivatives was designed based on SAR of known inhibitors of STAT3 and synthesised using optimized reaction condition. These compounds were subjected to in silico studies using Discovery studio software and evaluated for their anticancer activity. A docking study of the newly synthesized compounds was performed; the results showed good binding mode in the active site of STAT3 enzyme. Among the synthesized compounds, compounds B4 and B5 show binding similar to standard drug static. The cellular evaluation indicates that the anticancer activity of compounds B4, B5 (IC₅₀ = 54.51 ± 2.02?μg/mL and 45.43 ± 1.05?μg/mL, respectively) is active against MDA-MB-232 cell line and compounds B5, B7 (IC₅₀ = 40.21 ± 3.01?μg/mL, 56.77 ± 2.96?μg/mL, respectively) are most active against MCF-7 cell lines. From in vitro result, compound B5 subjected to acute oral toxicity studies by OECD 423 guidelines and in vivo anti-cancer studies carried out using DMBA-induced model.

Full text of DOI:10.1007/s13738-020-01932-z

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-01932-z
ORIGINAL PAPER
Synthesis and pharmacological evaluation of 1,3,4‑thiadiazole bearing
pyrimidine derivatives as STAT3 inhibitor for treatment of breast
cancer
2
1
2
2
2
1
B. Sravanthi · L. Kaviarasan · T. K. Praveen · Pindiprolu S. S. Sai Kiran · C. Pavankumar · B. Gowramma
Received: 3 February 2020 / Accepted: 3 April 2020
©
Iranian Chemical Society 2020
Abstract
Novel scafold oꢀ 1,3,4-thiadiazolo-(3,2-α)-pyrimidine-6-carbonitrile derivatives was designed based on SAR oꢀ known
inhibitors oꢀ STAT3 and synthesised using optimized reaction condition. These compounds were subjected to in silico stud-
ies using Discovery studio soꢀtware and evaluated ꢀor their anticancer activity. A docking study oꢀ the newly synthesized
compounds was perꢀormed; the results showed good binding mode in the active site oꢀ STAT3 enzyme. Among the syn-
thesized compounds, compounds B4 and B5 show binding similar to standard drug static. The cellular evaluation indicates
that the anticancer activity oꢀ compounds B4, B5 (IC =54.51± 2.02 µg/mL and 45.43± 1.05 µg/mL, respectively) is active
5
0
against MDA-MB-232 cell line and compounds B5, B7 (IC =40.21± 3.01 µg/mL, 56.77± 2.96 µg/mL, respectively) are
5
0
most active against MCF-7 cell lines. From in vitro result, compound B5 subjected to acute oral toxicity studies by OECD
4
23 guidelines and in vivo anti-cancer studies carried out using DMBA-induced model.
Keywords STAT3 · Discovery studio · Anticancer activity · MDA-MB-231 and MCF-7 cell lines · DMBA-induced model ·
ADMET
Introduction
ꢀactor-2 (HER-2) receptors, breast cancer can be divided
+
+
−
into ꢀour main subtypes: (1) Luminal A (ER /PR /HER-2 ),
+
+
+
According to WHO, cancer is the second leading cause oꢀ
death and accounts ꢀor more than 9.6million death in 2018
globally. The global cancer burden is estimated to have risen
to 18.1 million cases and characterized by a large number
and variety oꢀ mutations in host genes. This results in the
destruction oꢀ the normal processes that afect cells to grow,
survival or diferentiation [1, 2]. Breast cancer is the most
prevalent cancer, which accounts ꢀor about 12% oꢀ all new
cancer cases and 25% oꢀ all types oꢀ cancer in women [3].
Breast cancer is the second leading cause oꢀ cancer deaths
worldwide. Based on the expression oꢀ progesterone receptor
(2) Luminal B (ER /PR /HER-2 ), HER-2 overexpressing
−
−
+
−
−
−
(ER /PR /HER-2 ) and triple negative (ER /PR /HER-2 )
breast cancer. Luminal A and B subtypes are derived ꢀrom
luminal epithelium oꢀ the breast ducts and express hormone
receptors, ER and PR. Luminal A and B cancers involve
70–80% breast cancers subtypes. Luminal A subtype is
penetrating to present hormonal therapy such as tamoxiꢀen,
ꢀulvestrant or aromatase inhibitors. Luminal B cancers have
a higher proliꢀeration rate than Luminal A and are resistant
to current hormonal therapy. HER-2 overexpressing breast
cancer comprises gene ampliꢁcation ꢀor the protooncogene,
ERBB2, and is currently treated with agents like trastu-
zumab or lapatinib. Triple negative breast cancer (TNBC)
which lacks expression oꢀ ER, PR and HER-2 is the aggres-
sive breast cancer subtype. TNBCs, thereꢀore, are insensi-
tive to hormonal therapy/receptor targeted therapy unlike
the other subtypes. TNBC is currently treated with DNA-
damaging agents (e.g., carboplatin) or tubulin-destabilizing
compounds (e.g., paclitaxel) [4].
(
PR), estrogen receptor (ER) and human epidermal growth
*
B. Gowramma
gowrammab@redifmail.com; gowrammab@jssuni.edu.in
1
Department oꢀ Pharmaceutical Chemistry, JSS College
oꢀ Pharmacy, JSS Academy oꢀ Higher Education
and Research, Ooty, Udhagamandalam, Tamilnadu, India
2
Department oꢀ Pharmacology, JSS College oꢀ Pharmacy,
JSS Academy oꢀ Higher Education and research,
Ooty, Udhagamandalam, Tamilnadu, India
Vol.:(0
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Journal of the Iranian Chemical Society
STAT3 signaling pathway
catalyst was reꢂuxed in a 25-mL round-bottom ꢂask. Aꢀter
culmination oꢀ reaction which was supervised by TLC, the
contents were decanted into a Petri plate, solvent was air-
dried and the resulting product was scrapped out. Careꢀully,
this product was washed with dilute HCl to remove the
excess base via neutralization, recrystallized by ethanol in
order to obtain puriꢁed product in high grade. The physico-
chemical character oꢀ synthesized compounds is shown in
Table 1. The purity oꢀ synthesized compounds was ensured
by melting point and TLC. The structures oꢀ synthesized
compounds were elucidated by various spectral analyses.
The inꢀrared spectra oꢀ synthesized compounds revealed
stretching absorption bands within 3322.33 ꢀor N–H oꢀ pri-
mary amine, 2921.35 ꢀor CH oꢀ alkane, 2190.16 ꢀor C≡N oꢀ
carbonitrile, 1290.44 ꢀor C–O–C oꢀ ether and 1661.60 ꢀor
C=N oꢀ 1,3,4-thiadiazolo-[3,2-α]-pyrimidine moiety. The
Signal transducers and activators oꢀ transcription (STATs)
are a ꢀamily oꢀ transcription ꢀactors required ꢀor regulation
oꢀ growth, survival and diferentiation oꢀ cells [5, 6]. So
ꢀ
ar, seven STAT proteins have been accepted in mammalian
cells. Among all, STAT3 plays a key role in carcinogen-
esis by regulating the transcription oꢀ genes involved in cell
proliꢀeration, diferentiation, apoptosis, angiogenesis and
metastasis [7, 8]. IL-6/JAK is the canonical STAT3 activa-
tion signaling pathway, reported to be deregulated in cancer.
Since the receptor oꢀ IL-6 does not contain a kinase catalytic
domain, it induces STAT3 phosphorylation by stimulating
members oꢀ the JAK ꢀamily [9–11]. In various tumor types
including TNBC, STAT3 activation can promote tumor
growth, survival, stem cell enrichment, invasive/metastatic
properties and, ultimately, relapse.
1
H NMR spectra oꢀ synthesized compounds revealed sin-
glet signal at 8.26 ꢀor H oꢀ NH , singlet signal at 4.15 oꢀ H
2
Thiadiazoles
atom at chiral center oꢀ 1,3,4-thiadiazolo-[3,2-α]-pyrimidine
moiety and multiplet signal at 7–8 ꢀor H oꢀ aromatic ring.
The biological activity oꢀ the compounds depends upon the
molecular structure; several heterocyclic rings contain nitro-
gen and sulꢀur atoms. Thiadiazole has been oꢀ great inter-
est as core structures oꢀ antitumor agents ꢀor many years,
possibly due to the presence oꢀ toxophoric N–C–S moiety.
Diverse modiꢁcations oꢀ the thiadiazole rings at various
positions have led to a variety oꢀ novel compounds with
wide spectrum oꢀ pharmacological activities. From prelimi-
nary docking studies, it is evident that these compounds have
shown good in silico activity against STAT3 SH2 domain.
In the present study, therꢀore, an efort has been made to
design, synthesized and evaluate thiadiazole bearing pyrimi-
dine derivatives ꢀor there anti breast cancer activity.
Molecular docking
Molecular docking was carried out by using discovery studio
C docker. Among docked compounds, the compound B5
(−26.33) shows better aꢃnity towards active site oꢀ STAT3
protein when compared to standard drug static (− 21.22)
and ꢀollowed by compound B5, compound B8 (−25.16) has
better aꢃnity. The results ꢀor docking studies are shown in
Table 2. Among the docked molecules, most oꢀ them showed
good binding aꢃnity. The test molecules exhibited better
binding aꢃnity than standard molecule. The results, there-
ꢀore, indicate a potent SH2 domain (STAT3 protein: PDB
ID:4ZIA) binding potential oꢀ the test molecules. Figures 1,
2
, 3, 4 show the docking pose oꢀ compound B5, B7, B8 and
Results and discussion
B9.
Chemistry
In vitro cytotoxic evaluation
Herein, we report that the synthetic scheme which was ꢀol-
lowed ꢀor the preparation oꢀ a series oꢀ title compounds
B1–B9 is outlined in the Scheme 1. The intermediate
The targets compounds (B1–B9) were evaluated ꢀor their
anticancer activity against MDA-MB-231 and MCF-7 can-
cer cell lines. The IC values (concentration required to
5
0
5
-(1,3-benzodioxol-5-yl)-1,3,4-thiadiazol-2-amine was
growth inhibition oꢀ 50%) ꢀor the synthesized compounds
were determined using MTT assay. The anticancer evalu-
ation results and IC values are listed in Table 3. The
prepared by a mixture oꢀ thiosemicarbazide (50 mmol) and
piperonylic acid (50 mmol), and phosphoryl chloride (13 ml)
was heated in RBF at 65–75 °C ꢀor 35–45 min in a ꢀuming
cupboard. The contents were cooled to room temperature,
and to the above mixture distilled water (100 ml) was added
slowly. The pH was adjusted to pH 8 using 50% NaOH solu-
tion, to precipitate the product. The obtained compound
was reacted with propanedinitrile (0.001 mol) and corre-
sponding aromatic or heteroaromatic aldehyde (0.01 mol)
in absolute ethyl alcohol (10–12 mL) with NaOH (pellet) as
5
0
results indicated that the compounds B4 and B5 exhibited
signiꢁcant cell growth inhibition compared to reꢀerence
standard against MDA-MB-231 and MCF-7 cancer cell
lines, respectively. Compounds B4 and B5, which were
substituted with 4-chloro benzaldehyde and 2-chloro ben-
zaldehyde, were ꢀound to have IC values 54.51± 2.02 µg/
5
0
mL and 45.43 ± 1.05 µg/mL, respectively, against MDA-
MB-231 cell lines. The compounds B5 and B7, which were
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Journal of the Iranian Chemical Society
Step 1
S
COOH
O
H N
2
+
N
H
NH2
O
Thiosemicarbazide
benzo[d][1,3]dioxole-5-carboxylic acid
POCl3,
reflux for 65-75 C for 24hrs
0
Step 2
N
N
N
N
NH2
O
O
S
R-CHO
Substituted aldehyde
+
+
Propanedinitrile
A
5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-thiadiazol-2-amine
NaOH, Ethanol
reflux
N
H N
2
N
R
N
N
O
O
S
5-amino-2-(benzo[d][1,3]dioxol-5-yl)-7-substituted phenyl-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-6-carbonitrile
B (B1 - B9)
R =
O
Scheme 1 The synthetic scheme ꢀor preparation oꢀ designed compounds (B1–B9)
substituted with 2-chloro benzaldehyde and methoxy ben-
zaldehyde, were ꢀound to have IC values 45.43± 1.05 µg/
cell line. The detailed analysis oꢀ all in vitro result showed
that compound B5 acts as the most active compound among
other tested compounds against both the cell lines.
5
0
mL and 56.77 ± 2.96 µg/mL, respectively, against MCF-7
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Table 1 Physical data oꢀ
Compound
B1
Substituents
Molecular ꢀormula
C H N O S
Molecular
weight
Melting point (°C)
202–204
5
5
-amino-2-(1,3-benzodioxol-
-yl)-7-(substituted phenyl/
ꢀ
uran)-1,3,4-thiadiazolo[3,2-α]
465
2
2
19
5
5
pyrimidine-6-carbonitrile (B1–
B9)
B2
C H N O S
391
194–196
1
9
13
5
3
B3
B4
C H N O S
375
409
185–186
184–186
1
9
13
5
2
C H N O SCl
1
9
12
5
2
B5
B6
C H N O SCl
409
435
214–218
199–201
1
9
12
5
2
C H N O S
2
1
17
5
4
B7
B8
C H N O S
405
395
221–224
311–313
2
0
15
5
3
C H N O S
1
9
17
5
3
B9
C H N O S
449
213–216
2
1
17
5
3
Table 2 The docking results oꢀ 1,3,4-thiadiazole bearing pyrimidine
Acute oral toxicity
derivatives against SH2 domain oꢀ STAT3 (PDB ID:4ZIA)
Acute toxicity study oꢀ pyrimidine base derivatives com-
pound B5 in mice was carried out. As per OECD guidelines
Name
C docker energy
Binding inter-
action energy
4
23, acute toxicity studies were started at a dose oꢀ 2000 mg/
B1
B2
B3
B4
B5
B6
B7
B8
B9
Static
−13.35
−17.91
−13.00
−11.36
−26.33
−10.98
−21.29
−25.16
−11.58
−21.22
28.44
26.58
27.46
29.52
30.69
31.85
26.51
26.83
31.11
23.83
kg and 300 mg/kg. The results are shown in Table 4.
In vivo anticancer efcacy
Body weight
The body weight results are given in Table 5. The animals
in Group 2 signiꢁcantly showed higher body weights com-
pared to animals in Group 1 (normal control). Group 3
animals showed (treatment with bendamustine) showed a
signiꢁcantly (p<0.05) prevented DMBA-induced changes
in bodyweight compared to Group 2 (disease control) ꢀrom
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Journal of the Iranian Chemical Society
Fig. 1 2D binding interaction
image oꢀ compound B5 with
STAT3 aꢀter docking using C
docker module
day 21. Treatment with compound B5 (Groups 4 &5) dose
dependently prevented the DMBA-induced bodyweight
changes ꢀrom day 28, compared to Group 2 (disease con-
trol) animals.
Discussion
In the present study based on the docking results, 9 com-
pounds oꢀ 1,3,4-thiadiazole bearing pyrimidine derivatives
are synthesized and characterized based on the FT-IR, 13C
Tumor volume (mm3)
1
and H-NMR and LC–MS both positive and negative ioniza-
tion was taken and m/z was calculated. The cellular evaluation
indicates that the anticancer activity oꢀ compounds B4, B5
(IC =54.51± 2.02 µg/mL and 45.43± 1.05 µg/mL, respec-
The tumor volumes oꢀ the DMBA-induced animals are
shown in Table 6 and Fig. 5. Treatment with bendamus-
tine signiꢁcantly (p < 0.05) reduced the tumor volume
compared to disease control. Compound B5 treatment at
doses 15 mg/kg., and 30 mg/kg, p.o signiꢁcantly (p<0.05)
reduced the tumor volume compared to diseases control.
Further, compound B5 dose dependently increased the
MST in DMBA-induced tumor bearing mice.
50
tively) are most active compounds against MDA-MB-232
cell line and compounds B5, B7 (IC =40.21± 3.01 µg/mL,
50
56.77± 2.96 µg/mL, respectively) are most active against
MCF-7 cell lines. From in vitro result, compound B5 sub-
jected to acute oral toxicity studies by OECD 423 guidelines.
The acute oral toxicity oꢀ compound B5 shows high mortal-
ity rate during the dose oꢀ 2000 mg/kg so we decreased the
dose 300 mg/kg. And the molecules are saꢀe with an oral LD
Mean survival time and percentage increase in mean sur‑
vival time in control and treatment groups
50
value≥300 mg/kg. DMBA has been used to induce tumors in
laboratory animals. The administration oꢀ DMBA is by oral
gavage that produces mammary tumors in rodents (18). The
breast cancer plays important role in various incidences. The
key role oꢀ environmental carcinogens is PAH that has been
studied in various mammary tumorigenesis in laboratory ani-
mals. Administration oꢀ DMBA is by oral gavage that produces
mammary tumors in rodents. The mechanism under which
mammary tumor ꢀormation includes, upregulation oꢀ cellular
cytosolic receptor ꢀor DMBA, the aryl hydrocarbon receptor
(Ahr). Upon ligand activation, the Ahr translocates into the
nucleus and associates with the coꢀactor ARNT. However,
The MST & % ILS results are shown in Table 7 and Fig. 6.
The mean survival time ꢀor disease control group was ꢀound
to be 42 days, and the animals are treated with bendamustine
(
70 mg/kg) in Group 3 compound B5 (15 & 30 mg/kg) in
group4 &5 are shown MST oꢀ 40, 41 and 42 days with a %
ILS oꢀ 31.57, 62.10, respectively, G-4&5 treated with low
dose and high dose oꢀ compound B5 shows a dose depend-
ent signiꢁcance activity in increase in MST dose dependent
efect.
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Journal of the Iranian Chemical Society
Fig. 2 2D binding interaction
image oꢀ compound B7 with
STAT3 aꢀter docking using C
docker module
Fig. 3 2D binding interaction
image oꢀ compound B8 with
STAT3 aꢀter docking using C
docker module
there is an evidence that regulates cell growth and survival,
suggesting that there is additional mechanism through which
Ahr may contribute mammary tumorigenesis. DMBA admin-
istration ꢁnally leads to mammary carcinogenesis. In the pre-
sent study, in vivo anti-breast cancer activity oꢀ compound B5
was evaluated by DMBA-induced mammary tumor model and
compound B5 shows good activity in vitro assays due to the
presence oꢀ chlorine group at ortho position. Compound B5
thereꢀore possess potentially in the therapy oꢀ breast cancer.
Materials and methods
ꢀ
ound that compound B5 inhibited tumors dose dependently.
In silico docking study
Thereꢀore, compound B5 can be a potential anticancer activity.
Transcription ꢀactor STAT3/DNA complex-4ZIA was
downloaded ꢀrom the protein data bank (RCSB PDB-4ZIA)
Docking was carried out by Discovery studio 4.1(Accelrys)
soꢀtware.
Conclusion
In the present study, 1, 3, 4-thiadiazole bearing pyrimidine
derivatives were synthesized and evaluated ꢀor there in
silico, in vitro, in vivo anticancer activity. Among these,
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Journal of the Iranian Chemical Society
Fig. 4 2D binding interaction
image oꢀ compound B9 with
STAT3 aꢀter docking using C
docker module
Table 3 In vitro cytotoxic evaluation oꢀ synthesized compounds
loops, optimize side chains oꢀ missing residues.
against MDA-MB-231 and MCF-7 cell line
Preparation of ligand molecule
Sample description MDA-MB-231 IC μg/ MCF 7 (IC μg/mL)
5
0
50
mL
Ligand molecules are prepared by 2D to 3D conversion
by enumerating ionization states tautomers and isomers.
Multiple rapid and exhaustive generation methods are also
employed. Prior to molecular docking, active site oꢀ the
prepared protein was generated. The docking scores oꢀ the
designed molecules were associated with static a known
STAT3 inhibitor.
B1
B2
B3
B4
B5
B6
B7
B8
B9
331.30± 3.05
335.42± 2.02
99.74± 1.05
54.51± 2.02
45.43± 1.05
213.48± 1.05
155.94± 2.25
99.24± 2.01
215.10± 2.08
187.93± 1.05
107.12± 2.21
153.21± 1.94
> 150
40.21± 3.01
145.31± 2.56
56.77± 2.96
189.92± 2.43
82.43± 1.06
Docking was prepared by both LibDock and C docker
modes
The Dock Ligands (LibDock) protocol is an interꢀace
to the LibDock program developed by Diller and Merz
Preparation of the protein
[
Diller and Merz, 2001. A polar Hotspot is preꢀerred by a
In this step, protein is generated to identiꢀy potential prob-
lems. It involves automatically ꢁxing and building missing
polar ligand atom ꢀor example a hydrogen bond donor or
acceptor and is preꢀerred by a carbon atom. The receptor
Table 4 Acute oral toxicity oꢀ pyrimidine base derivatives B5 in mice at the dose 2000 mg/kg and 300 mg/kg
Animal marking Body weight Drug Dose (mg) Volume (mL) Body weight change (g)
(g)
Day 0 Day 1
Day 7 Weight change Day 14 Weight change Mortality
Head
Tail
Body
Head
Body
Tail
28.5
30
31
28.9
28
29
28
2000 mg/kg 56
0.28
0.29
0.3
0.28
0.27
0.28
28.9
30
31
28.4
28
28.8
0.9
0.5
0.5
0.4
0.5
0.3
28.9
30
31
28.5
29
29.4
0.9
0.5
0.5
0.5
1.5
1.9
2
29.5
30.5
28
27.5
28.5
59
61
8.4
300 mg/kg
0
8.25
8.5
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Journal of the Iranian Chemical Society
Table 5 Efect oꢀ compound B5 1,3,4-thiadiazole bearing pyrimidine derivatives on body weight (g) oꢀ albino mice ꢀollowing oral treatment
Days
Normal control
Disease control
Bendamustine
70 mg/kg)
Compound B5 (15 mg/kg)
Compound B5 (30 mg/kg)
(
Day 0
Day 7
Day 14
Day 21
Day 28
Day 35
Day 42
26.84± 2.85
24.06± 3.06
26.34± 4.50
25.16± 3.26
26.13± 3.50
26.84± 2.85
25.06± 3.06
25.51± 4.80
26.16± 2.87
27.12± 2.38
29.40± 2.17
31.51± 4.80
33.16± 2.87
34.56± 3.40
26.14± 3.36
28.4± 2.93
25.56± 3.40
28.68± 3.16
31.67± 3.66
30.16± 3.26*
29.13± 3.50*
25.80± 4.10
25.00± 2.12
25.81± 3.17
24.23± 2.85
28.21± 4.12*
28.78± 3.90*
28.21± 2.63*
25.18± 3.20
24.90± 4.00
26.73± 2.71
23.30± 3.52*
23.11± 3.62*
24.65± 2.81*
†
†
$
†
#
#$
†
#
#$
23.00± 2.13*
†
#
The values are expressed as mean± S.D. n=6 by; p<0.05 versus normal control; *p<0.05 versus disease control; p<0.05 versus; bendamus-
$
tine; p<0.05 versus compound B5 (15 mg/kg). (ANOVA ꢀollowed by Dunnett’s test)
Table 6 Efect oꢀ compound B5 1,3,4-thiadiazole bearing pyrimi-
Table 7 Mean survival time and percentage increase in mean survival
3
dine derivatives on tumor volume (mm ) DMBA-induced mammary
time in control and treatment groups
tumors in albino mice
Groups
MST (days)
Increase in MST (%)
Groups
Tumor volume (mm3)
Day 0 Day 7
Disease control
Bendamustine
Compound B5 (15 mg/kg)
Compound B5 (30 mg/kg)
15.83± 5.08
25.83± 8.89*
35.66± 9.18*
46.50± 9.20*
–
Day 14
31.57
62.10*
193.68*
#
#
#
Disease control
Bendamustine (70 mg/kg)
2.43± 0.33 2.72± 0.38 3.47± 0.27
2.20± 0.23 1.92± 0.18* 1.57± 0.16*
2.58± 0.34 2.26± 0.29* 1.95± 0.25*
$
#
Compound B5 (15 mg/
The values are mean± S.D. n=6 *p<0.05 versus disease control;
kg), p.o
#
$
p<0.05 versuss; bendamustine; p<0.05 versus compound B5
Compound B5 (30 mg/
kg), p.o
2.62± 0.39 2.06± 0.34* 1.84± 0.27*
(
15 mg/kg). (ANOVA ꢀollowed by Dunnett’s test)
4
3
2
1
0
Day 0
Day 7
Day 14
l
)
)
)
l
)
)
)
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)
)
g
)
o
/K /Kg /Kg
g
o
/K /Kg /K
g
g
o
g
r
r
r
t
t
t
/K /K
n
g
(15
g
g
n
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g
g
n
o
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o
o
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a
(
Fig. 6 Efect oꢀ compound B5 on mean survival time and percentage
increase in mean survival time oꢀ DMBA-induced mammary tumor
bearing albino mice
e
e
n
5
5 (30me
e
n
5
5
e
e
n
5
Dis in
B
B
is in
B
B
is in
B
st
d
d
D
st
d
d
D
st
d
n
n
nd B5 (30mg/Kg
u
u
u
u
u
u
u
u
u
m
o
o
m
o
o
m
o
p
o
a
p
p
a
p
p
a
d
m
C
m
d
m
m
d
m
mp
o
n
o
o
n
o
o
n
e
o
e
e
C
C
C
C
C
B
B
B
Group
The investigated compounds were docked into the crystal
structure oꢀ STAT3 homodimer bound to DNA (PDB ID:
3
Fig. 5 Efect oꢀ compound B5 on tumor volume (mm ) oꢀ DMBA-
induced mammary tumor bearing albino mice
4
ZIA) using static as a standard. The observed compounds
were saved in mol2 ꢀormat. In discovery studio, the hydro-
gen bonds were added and molecules were imported. PDB
ꢀormat which is obtained was saved. The C docker and inter-
action energy were calculated ꢀor each pose [12].
Hotspot ꢁle is calculated prior to the docking procedure.
The 10 compounds having diferent aromatic aldehydes were
designed and geometrically optimized by docking study.
1
3

Journal of the Iranian Chemical Society
Chemistry
5‑amino‑2‑[(1,3‑benzodioxol‑5‑yl) methyl]‑7‑(3,4,5‑trimeth
oxyphenyl)‑1,3,4‑thiadiazolo [3,2‑α] pyrimidine‑6‑carbon‑
itrile (B1)
Synthesis
of 5‑(1,3‑benzodioxol‑5‑yl)‑1,3,4‑thiadiazol‑2‑amine
Yield: 55.4%. Solvent oꢀ crystallization: ethanol and pet
−
1
A mixture oꢀ thiosemicarbazide (50 mmol) and piperonylic
acid (50 mmol) and phosphoryl chloride (13 ml) was heated
in a RBF at 65–75 °C ꢀor 35–45 min in a ꢀuming cupboard.
The contents were cooled to room temperature, and to the
above mixture distilled water (100 ml) was added slowly
and pH was adjusted to pH 8 using50% NaOH solution to
precipitate the product [13, 14]. The product was ꢁltered and
recrystallized by using absolute ethanol. (Scheme 1).
ether. M.P:110–120 °C; IR ν
cm : 3326.7 (C–NH ),
max 2
3071.2 (aromatic C–H stretching), 2885 (aliphatic C–H
stretching), 2198.3 (C≡N), 1687.3 (C=N), 1590 (aromatic
1
C=C), 1235,1000.7 (C–O–C), 713.3 (C–S–C); H NMR
(DMSO): δ 7–8 (9H, ArH), 6.7 (2H, NH ), 4.5 (2H, CH ),
2
2
3.5–4 (9H, OCH ), 3.25 (1H, 1,3,4-thiadiazole-[3,2-a]
3
1
3
pyrimidine). C NMR (CDCl , 400 MHz): 40.18, 76.69,
3
77.01, 77.33, 111.75, 129.87; Anal. Calcd ꢀor C H N O S:
26
23
5
4
C, 62.26; H, 4.62; N, 13.96; O, 12.76; S, 6.39. Found: C,
6
0.23, H, 5.12, N, 41.42, S, 5.98. Molecular weight: 501.
N
N
5
‑amino‑2‑[(1,3‑benzodioxol‑5‑yl)methyl]‑7‑(4‑hydroxyphe
NH2
nyl)‑1,3,4‑thiadiazolo [3,2‑α] pyrimidine‑6‑carbonitrile (B2)
O
S
Yield: 67.2%. Solvent oꢀ crystallization: ethanol and pet
−
1
ether. M.P:115–120 °C, IR ν cm : 3497 (C–OH), 3385.2
max
O
(
C–NH ), 3050 (aromatic C–H stretching), 2885 (aliphatic
2
C–H stretching), 2214.3 (C≡N), 1628.8 (C=N), 1585 (aro-
matic C=C), 1256.2 (C–O–H), 1213.6,1043.3 (C–O–C),
1
7
50.56 (C–S–C). H NMR (CDCl , 400 MHz): 1.675 (2H
3
Synthesis of 5‑amino‑2‑(1,3‑benzodioxol‑5‑yl)‑7‑sub‑
stitiuted aldehyde‑7H‑ [1, 3, 4] ‑thiadiazole[3,2‑a]pyrimi‑
dine‑6‑carbonitrile
methyl), 3.209 (chiral proton in pyrimidine ring), 6.984 (1H.
OH proton), 7.059, 7.7076, 7.093, 7.321, 7.338, 7.600 (aro-
1
3
matic protons, 11H); C NMR (CDCl , 400 MHz): 76.69,
3
7
7.01, 77.33, 120.29, 128.78; Anal. Calcd ꢀor C H N O S:
23 17 5 2
Mixture consisting oꢀ 5-(1,3-benzodioxol-5-yl)-1,3,4-thi-
adiazol-2-amine (0.01 mol), propanedinitrile (0.001 mol)
and corresponding aromatic or heteroaromatic aldehyde
C, 64.62; H, 4.01; N, 16.38; O, 7.49; S, 7.50. Found:
C,64.71; H,4.37; N, 16.13; O, 8.02; S, 6.97. Molecular
+
weight: 397, LC–MS mass: M+2H+=452(Na peak).
(
0.01 mol) in absolute ethyl alcohol (10–12 mL) with NaOH
pellet) as catalyst was reꢂuxed in a 25-mL round-bottom
(
5
‑amino‑2‑(1,3‑benzodioxol‑5‑yl)‑7‑phenyl‑1,3,4‑thiadia‑
ꢂask. Aꢀter culmination oꢀ reaction which was supervised
by TLC, the contents were decanted into a Petri plate, sol-
vent was air-dried, and the resulting product was scrapped
out. Careꢀully, this product was washed with dilute HCl to
remove the excess base via neutralization, recrystallized by
ethanol in order to obtain puriꢁed product in high grade. The
synthesis oꢀ designed compounds (B1–B9) was outlined in
Scheme 1.
zolo [3,2‑α] pyrimidine‑6‑carbonitrile (B3)
Yield: 78.6%. Solvent oꢀ crystallization: ethanol and pet
−
1
ether. M.P: 90–95 °C, IR ν
cm : 3332 (C–NH ), 3034
2
max
(
aromatic C–H stretching), 2885 (aliphatic C–H stretch-
ing), 2198.3 (C≡N), 1628.8 (C=N), 1580 (aromatic C=C),
1
1
261.5,1043.3 (C–O–C), 702.6 (C–S–C); H NMR (DMSO,
4
00 MHz): δ 6.15–7.9 (12H, ArH), 5.4 (2H, NH ), 4.45
2
(
2H, CH ), 4.25 (1H, 1,3,4-thiadiazole-[3,2-a]pyrimidine).
2
1
3
C NMR (CDCl , 400 MHz): 76.69, 77.01, 77.33, 125.29,
3
1
30.78; Anal. Calcd ꢀor C H N OS: C, 67.13; H, 4.16;
2
3
17
5
N, 17.02; O, 3.89; S, 7.79. Found: C, 67.45; H, 4.24, N,
7,45; O, 4.01; S, 8.34. Molecular weight: 375, LC–MS
mass: M+=375.
1
1
3

Journal of the Iranian Chemical Society
5‑amino‑2‑(1,3‑benzodioxol‑5‑yl)‑7‑(4‑chlorophenyl)‑1,3,4
5‑amino‑2‑(1,3‑benzo‑
‑thiadiazolo [3,2‑α] pyrimidine‑6‑carbonitrile (B4)
dioxol‑5‑yl)‑7‑(4‑methoxyphenyl)‑1,3,4‑thiadiazolo [3,2‑α]
pyrimidine‑6‑carbonitrile (B7)
Yield: 70.8%. Solvent oꢀ crystallization: ethanol and pet
−
1
ether; M.P: 90–92 °C, IR ν
cm : 3348 (C–NH ), 3060
Yield: 57.6%. Solvent oꢀ crystallization: ethanol and pet
max
2
−
1
(
aromatic C–H stretching), 2885 (aliphatic C–H stretch-
ether. M.P:105–110 °C, IR ν
cm : 3326.7 (C–NH ),
max 2
ing), 2198.3 (C≡N), 1628.8 (C=N), 1585 (aromatic C=C),
3039.3 (aromatic C–H stretching), 2885 (aliphatic C–H
1
1
219,1096.5 (C–O–C), 819.76 (C–Cl), 745.24 (C–S–C); H
stretching), 2214.3 (C≡N), 1628.8 (C=N), 1511.7 (aro-
1
NMR (CDCl , 400 MHz):8.059 (2H, NH proton), 7.944,
matic C=C), 1256.2,1027 (C–O–C), 710 (C–S–C). H
3
2
7
.935, 7.926, 7.566, 7.547, 7.527, 7.520, 7.515, 7.506 (aro-
NMR (CDCl , 400 MHz): 8.56 (NH protons, 2H), 7.97,
3
2
1
3
matic protons, 11H), 7.428 (CH proton, 2H); C NMR
7.93, 7.59, 7.48, 7.39, 7.23, 7.12, 6.87 (aromatic protons,
2
1
3
(
CDCl , 400 MHz): 76.69, 77.01, 77.21, 77.33, 124.32,
11H), 3.83 (chiral proton and methoxy protons); C NMR
3
1
1
24.54, 126.32, 128.48, 128.90, 129.32, 131.40, 137.63,
(CDCl , 400 MHz): 76.71, 77.02, 77.34, 128.50, 129.35,
3
49.66. 165.38; Anal. Calcd ꢀor C H ClN OS: C, 61.95;
130.22, 133.79, 171.93; Anal. Calcd ꢀor C H N O S:
2
3
16
5
24 19
5
2
H, 3.62; Cl, 7.95; N, 15.71; O, 3.59; S, 7.19. Found: C,
C, 65.29; H, 4.34; N, 15.86; O, 7.25; S, 7.26. Found: C,
65.45; H, 4.14; N, 14.86; O, 7.62; S, 7.12. Molecular
weight: 405.
6
2.45; H, 4.26; Cl, 8.54; N, 15.23; O, 3.51; S, 7.22. Molecu-
+
lar weight: 409, M+2=434(Na ).
5‑amino‑2‑(1,3‑benzodioxol‑5‑yl)‑7‑(2‑chlorophenyl)‑1,3,4
‑thiadiazolo [3,2‑α] pyrimidine‑6‑carbonitrile (B5)
5‑amino‑2‑(1,3‑benzodioxol‑5‑yl)–7‑(furan‑2‑yl)‑1,3,4‑thia‑
diazolo [3,2‑α] pyrimidine‑6‑carbonitrile (B8)
Yield: 60.5%. Solvent oꢀ crystallization: ethanol and pet ether.
−
1
M.P:155–160 °C, IR ν
cm : 3278.8 (C–NH ), 3065.9
Yield: 73.7%. Solvent oꢀ crystallization: ethanol and pet
max
2
−
1
(
aromatic C–H stretching), 2879.6 (aliphatic C–H stretching),
ether. M.P:155–160 °C, IR ν
cm : 3342.7 (C–NH ),
max 2
2
219.6 (C≡N), 1628.8 (C=N), 1505 (aromatic C=C), 1256.2,
3060.6 (aromatic C–H stretching), 2885 (aliphatic C–H
1
1
027.3 (C–O–C), 841.05 (C–Cl), 745.24 (C–S–C). H NMR
stretching), 2198.3 (C≡N), 1634.1 (C=N), 1590 (aro-
1
(
CDCl , 400 MHz):8.059 (2H, NH2 proton), 7.944, 7.935,
matic C=C), 1219, 1075.2 (C–O–C), 707.98 (C–S–C). H
3
7
.926, 7.566, 7.547, 7.527, 7.520, 7.515, 7.506 (aromatic
NMR (CDCl , 400 MHz): 8.26 (NH proton), 9.97, 7.93,
3
2
1
3
protons, 11H), 7.428 (CH proton, 2H); C NMR (CDCl ,
7.59,7.58, 7.48, 7.39, 7.23, 6.08, 6.04 (aromatic protons,
2
3
1
3
4
1
1
1
00 MHz): 39.32, 39.53, 39.73, 40.15, 40.36, 40.56, 120.06,
22.47, 123.13, 127.18, 127.32, 127.37, 128.20, 128.27,
28.20, 128.27, 128.20, 128.27, 128.46, 128.62, 128.70,
31.56, 131.75, 132.59, 134.32, 136.60, 165.65; Anal. Calcd
9H), 4.15 (chiral proton and CH protons); C NMR
2
(CDCl , 400 MHz): 76.70, 77.02, 77.33, 119.04, 127.24,
3
128.28, 128.34, 128.50, 128.63, 128.76, 129.23, 130.20,
132.64, 133.64, 133.64, 136.58, 147.02, 157.17, 159.61;
Anal. Calcd ꢀor C H N O S: C, 62.83; H, 3.77; N, 17.45;
ꢀ
or C H ClN OS: C, 61.95; H, 3.62; Cl, 7.95; N, 15.71; O,
23
16
5
21 15
5
2
3
.59; S, 7.19. Found: C, 62.45; H, 4.26; Cl, 8.54; N, 15.23;
O, 7.97; S, 7.99. Found: C, 62.55; H, 3.53; N, 17.17; O,
+
O, 3.51; S, 7.22. Molecular weight: 409, M+2=434(Na ).
7.64; S, 8.19. Molecular weight: 395.
5‑amino‑2‑(1,3‑benzodioxol‑5‑yl)‑7‑(3,4‑dimethoxyphenyl)
‑
1,3,4‑thiadiazolo [3,2‑α] pyrimidine‑6‑carbonitrile (B6)
5‑amino‑2‑(1,3‑benzodioxol‑5‑yl)‑7‑(3‑hydroxy‑4‑methoxy
phenyl)‑1,3,4‑thiadiazolo [3,2‑α] pyrimidine‑6‑carbonitrile
(B9)
Yield: 65.7%. Solvent oꢀ crystallization: ethanol and pet
−
1
ether. M.P:190–195 °C, IR ν
cm : 3230.9 (C–NH ),
2
max
3
097.8 (aromatic C–H stretching), 2885 (aliphatic C–H
Yield: 88.7%. Solvent oꢀ crystallization: ethanol and pet
−
1
stretching), 2214 (C≡N), 1628.8 (C=N), 1596.9 (aromatic
ether. M.P:145–150 °C, IR ν
cm : 3284.1 (C–NH ),
max 2
1
C=C), 1213.6,1043.3 (C–O–C), 745.24 (C–S–C); H NMR
3023.3 (aromatic C–H stretching), 2885(aliphatic C–H
stretching), 2219.6 (C≡N), 1628.8 (C=N), 1510 (aro-
matic C=C), 1256.2 (C–O–H), 1219,1032.7 (C–O–C), 710
(
CDCl , 400 MHz): 8.915 (NH proton 2H), 7.858, 7.725,
3
2
7
.705, 7.624, 7.607, 7.599, 7.590, 7.581, 7.572 (aromatic
1
proton, 10H), 7.111 (CH proton), 3.003 (methoxy pro-
(C–S–C); H NMR (DMSO): δ 9.65 (1H, OH), 7–8 (10H,
2
1
3
tons and chiral proton); C NMR (CDCl , 400 MHz):
ArH), 6.8 (2H, NH ), 5.45 (2H, CH ), 3.80 (3H, OCH )
3
2
2
3
1
3
4
0.18, 76.69, 77.01, 77.33, 111.75, 129.87; Anal. Calcd
1.6 (1H, 1,3,4-thiadiazole-[3,2-a]pyrimidine): C NMR
ꢀ
or C H N O S: C, 63.68; H, 4.49; N, 14.85; O, 10.18; S,
(CDCl , 400 MHz): 76.70, 77.02, 77.33, 119.04, 127.24,
2
5
21
5
3
3
6
.80. Found: C, 63.76; H, 4.21; N, 15.05; O, 9.68; S, 6.71.
128.28, 128.34, 128.84, 128.54, 128.76, 129.23, 130.64,
Molecular weight: 435.
135.64, 137.43, 143.54, 146.81, 147.02, 159.77, 160.87;
1
3

Journal of the Iranian Chemical Society
Anal. Calcd ꢀor C H N O S: C, 63.01; H, 4.19; N, 15.31;
ꢀed with standard pellet diet (M/s Amrit ꢀeeds Ltd, Banga-
lore, India) and water ad libitum. The experiment was con-
ducted with prior approval oꢀ institutional animal ethical
committee oꢀ JSS College oꢀ Pharmacy, Ooty, (Approval
number: JSSCP/OT/M.PHARM/06/2018–2019).
2
4
19
5
3
O, 10.49; S, 7.01. Found: C, 62.71; H, 4.31; N, 14.91; O,
9
.95; S, 7.21. Molecular weight: 449.
In vitro anti‑cancer activity evaluation
Acute toxicity studies (OECD) (ATC method 423) Acute oral
toxicity studies oꢀ novel 1,3,4-thiadiazole bearing pyrimi-
dine moiety molecules will be studied as per OECD 423
guideline Female Swiss Albino mice oꢀ weight (20–30 g)
will be taken ꢀor the study; limit test at a dose oꢀ 2000 mg/
kg, p.o, will be carried out using 3 ꢀemales per step (6 ani-
mals ꢀor acute toxicity). Aꢀter drug administration, all the
animals will be practical ꢀor clinical signs and mortality at
0.0.5, 1.0, 2.0 and 4 h and thereaꢀter daily ꢀor a period oꢀ
14 days. During the study period, the weekly body weight
oꢀ all the animals will be recorded. At the end oꢀ the study,
all the survived animals will be culled through deep ether
anesthesia and subjected to gross necropsy analysis.
Cell culture and conditions
MDA-MB231 (TNBC) cell lines (ATCC) were maintained
in DMEM, supplemented with 10% FBS, penicillin (100 U/
mL) and streptomycin (100 μg/mL). The cells were grown
at 37 °C with 5% CO2.
MTT (3(4,5 dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium
bromide) assay
Principle The principle elaborates in the cleavage oꢀ tetra-
zolium salt MTT (3-(4,5 dimethyl thiazole-2yl)-2,5-diphe-
nyl tetrazolium bromide). The number oꢀ cells was ꢀound
to be proportional to the extent oꢀ ꢀormazan production oꢀ
cells used.
In vivo anticancer eꢀcacy
Preparation of DMBA (40 mg/kg) 4 mg/mL suspension oꢀ
DMBA was prepared in corn oil and administered as a dose
volume oꢀ 10 ml/kg p.o, body weight.
5
Procedure The cell culture was adjusted to 1.0 × 10 cells/
mL using DMEM medium containing 10%FBS. To each
well oꢀ a 96-well ꢂat bottom micro titer plate, 100 µL
oꢀ distilled cell suspension (approximately 10,000cells/
well) was added. Aꢀter 24 h, the cell population was ꢀound
adequate, the cell was centriꢀuged, and the pellets were
suspended with 100 µL oꢀ diferent test samples concen-
tration prepared in maintenance media. The plates were
Preparation of bendamustine(70 mg/kg) 7 mg/mL suspen-
sion oꢀ Bendamustine was prepared in 0.5% CMC adminis-
tered at a dose volume oꢀ 10 ml/kg p.o, body weight.
Preparation of compound (15 mg/kg and 30 mg/kg) 1.5 mg/
mL, 3.0 mg/mL suspensions oꢀ compound were prepared in
0.5%CMC administered at a dose volume oꢀ 10 ml/kg p.o,
body weight.
incubated at 37 °C ꢀor 48 h in 5% CO atmosphere, and
2
examination was carried the observations recorded every
2
4 h. Aꢀter 48 h, 20 µL oꢀ MTT (2 mg/mL) in MEM-PR
(
MEM without phenol red) was added. The plates were
incubated ꢀor 2 h at 37 °C (in 5% CO atmosphere). The
Induction of breast cancer in mice 7,12 Dimethylbenz(α)
anthracene (DMBA) dissolved in corn oil (4 mg/mL stock
concentration) was administered to mice at a dose oꢀ 40 mg/
kg, p.o., weekly ꢀor 4 consecutive weeks [15, 16].
2
1
00 µL oꢀ DMSO was added plates were shaken to solubi-
lize the ꢀormed ꢀormazan. The absorbance was measured
using a microplate reader at a wavelength oꢀ 540 nm. The
percentage cell viability was calculated using the ꢀormula
Treatment Aꢀter attaining measurable tumor volume, the
animals are divided into 5 groups oꢀ 6 each. The animals
in Group 1 and Group 2 received vehicle (CMC 0.5%w/v,
Mean OD of individual sample
%
cell viability =
× 100
Mean OD of control
1
0 ml/kg, p.o) and served as normal control and disease con-
trol, respectively. Group 3 received Bendamustine (70 mg/kg
p.o,); Group 4 and 5 received test compound (15 mg/kg p.o,)
and (30 mg/kg p.o,). The animals were observed weekly ꢀor
clinical signs and tumors volume was measured by using
Vernier caliper at 7 days’ intervals. And the volumes were
calculated by using the ellipsoid ꢀormula [17–19].
Acute oral toxicity study
Healthy, ꢀemale Swiss albino mice (25–40 g) were pro-
duced ꢀrom in animal house ꢀacility ꢀrom J.S.S College oꢀ
pharmacy, Ooty. The animals were housed under standard
conditions oꢀ temperature (22± 3 °C) and relative humidity
Length (cm) × width (mm) × Height (cm)
3
Tumor volume (mm ) =
(
30–70%) with a 12:12 light/dark cycle. The animals were
2
1
3

Journal of the Iranian Chemical Society
H. Yu, R. Jove, Nat. Rev. Cancer 4(2), 97–105 (2004)
8
9
.
.
Statistical analysis
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1. T.N. Dechow, L. Pedranzini, A. Leitch, K. Leslie, W.L. Gerald,
Proc. Natl. Acad. Sci. 101, 10602–10607 (2004)
Statistical signiꢁcance was determined by one-way analy-
sis oꢀ variance (ANOVA), ꢀollowed by Dunnett’s multiple
comparison test (Graph Pad Prism, Version 6, Graph Pad
Soꢀtware Inc., La Jolla, USA). The values with p<0.05 were
considered signiꢁcant [20].
1
1
1
1
1
1
1
1
1
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