Synthesis, Molecular Docking and Preliminary Antileukemic Activity of 4-Methoxybenzyl Derivatives Bearing Imidazo[2,1-b][1,3,4]thiadiazole

Article abstract of DOI:10.1002/cbdv.202000800

In this study, we synthesized 22 compounds in a series with various substitution on imidazo[2,1-b][1,3,4]thiadiazole. The potential cytotoxic activity of these compounds investigated in leukemia cell lines by Differential Nuclear Staining (DNS). Our results identified two compounds, 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate and 6-(4-chlorophenyl)-2-(4-methoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde, exhibited the most cytotoxic effect against murine leukemia cells (L1210), human T-lymphocyte cells (CEM) and human cervix carcinoma cells (HeLa) with IC₅₀ values ranging between 0.79 and 1.6 μM. The results indicate that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate is inducing phosphatidylserine externalization and caspase-3 activation which are both a hallmark of apoptosis. Docking studies showed that 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate binds within the active sites of transforming growth factor beta (TGF-β) type I receptor kinase domain by strong hydrogen binding and hydrophobic interactions.

Full text of DOI:10.1002/cbdv.202000800

Title: Synthesis, molecular docking and preliminary antileukemic
activity of 4-methoxybenzyl derivatives bearing an imidazo[2,1-b]
[1,3,4]thiadiazoles
Authors: Subhas S. Karki, B Choodamani, Karla G Cano Hernandez,
Sujeet Kumar, Anna M Tony, Austre Y Schiaffino Bustamante,
Renato J Aguilera, Dominique Schols, and GopiMohan Gopi
C
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content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.202000800
Link to VoR: https://doi.org/10.1002/cbdv.202000800

10.1002/cbdv.202000800
Chemistry & Biodiversity
Synthesis, molecular docking and preliminary antileukemic
activity of 4-methoxybenzyl derivatives bearing an imidazo[2,1-
b][1,3,4]thiadiazoles
Chudamani B,a,b* Karla G. Cano Hernandez,c* Sujeet Kumar,a Ann Maria Tony,d
Austre Y. Schiaffino Bustamante,c Renato J. Aguilera,c Dominique Schols,e C
Gopi Mohan,d Subhas S. Karkia,b**
aDepartment of Pharmaceutical Chemistry, KLE College of Pharmacy, (A Constituent Unit of
KAHER-Belagavi) Bengaluru, 560010, Karnataka, India
bDr. Prabhakar B. Kore Basic Science Research Center, Off-Campus, KLE College of
Pharmacy, (A Constituent Unit of KAHER-Belagavi) Bengaluru, 560010, Karnataka, India
cThe Cellular Characterization and Biorepository Core Facility & Border Biomedical
Research Center& Department of Biological Sciences, The University of Texas at El Paso,
El Paso, 79968 TX, USA
dCentre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and
Research Centre, Amrita Vishwa Vidyapeetham, Kochi, 682041, Kerala, India
eRega Institute for Medical Research, Department of Microbiology, Immunology and
Transplantation, Laboratory of Virology and Chemotherapy, KU Leuven,
B-3000 Leuven, Belgium
*Equal first author
**Corresponding author: +91 8023325611 (Off); +91 8023425373 (fax);
subhasskarki@gmail.com (Subhas S Karki)
1
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10.1002/cbdv.202000800
Chemistry & Biodiversity
A B S T R A C T
In this study, we synthesized 22 compounds in a series with various substitution on
imidazo[2,1-b][1,3,4]thiadiazoles. The potential cytotoxic activity of these compounds
investigated in leukemia cell lines by Differential Nuclear Staining (DNS). Our results identify
two
compounds
2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-
thiocyanate and 6-(4-chlorophenyl)-2-(4’-
b][1,3,4]thiadiazol-5-yl
methoxybenzyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde exhibited as the most
cytotoxic effect against murine leukemia cells (L1210), human T-lymphocyte cells (CEM) and
human cervix carcinoma cells (HeLa) with IC50 values ranging between 0.79 to 1.6 μM. The
results indicate that compound 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-
b][1,3,4]thiadiazol-5-yl thiocyanate is inducing phosphatidylserine externalization and
caspase-3 activation which are both a hallmark of apoptosis. Docking studies showed that
compound 2-(4-methoxybenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-
5-yl thiocyanate binds within the active sites of transforming growth factor beta (TGF-β) type
I receptor kinase domain by strong hydrogen binding and hydrophobic interactions.
Keywords: Imidazothiadiazole, Cytotoxicity, TGF beta receptor kinase, Melphalan,
Levamisole
2
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Introduction
According to the American Cancer Society, in the United States, by 2020, more than 60,000 new
cases of leukemia are expected to be diagnosed. This year leukemia is expected to cause around
20,000 deaths.[1] Leukemia is a type of cancer characterized by uncontrolled proliferation of
hematopoietic stem cells in the bone marrow.[2] There are multiple types of leukemia often grouped
in acute leukemia, when cancer can progress rapidly and chronic leukemia that refers to slower
progression.[3] Furthermore, leukemia is divided on the type of bone marrow cell where cancer
starts. Lymphoblastic leukemia is the other type, which is characterized by starting in cells that
would regularly become lymphocytes.[4] Combining these grouping criteria, there are 4 main types
of leukemia that are; chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic
leukemia, and acute myeloid leukemia.[4] The development of treatments for leukemia has
advanced over the years. The main type of treatment given to patients is chemotherapy includes
drugs, such as, vincristine, daunorubicin, and cytarabine.[5] The improvements in leukemia
treatments has increased the 5-year survival rate to 63%.[6] However, the development of more
effective treatments is needed to decrease the number of fatalities caused by this disease.
Levamisole (I) was approved by the FDA in 1990 as a curative treatment for colon cancer.[7]
Prior to this, levamisole was used as an antirheumatic treatment in the 1970s and 1980s in
patients with rheumatoid arthritis.[8] Due to the protective effects of the immune system, the
drug has been studied in the treatment of various immune-mediated diseases, and some studies
have shown positive effects.[9] The drug has also been used in combination with other drugs to
treat various cancers.[8.9] In 1982, a clinical trial revealed that in acute myeloid leukemia
patients the combination of levamisole with 6-mercaptopurine and methotrexate resulted in
better remission of cancer, compared to those given only the chemotherapy.[10]
Transforming growth factor (TGF β) is a cytokine, which plays a key role in cell
proliferation, differentiation and apoptosis.[11] TGF β type 1 is activin receptor-like kinase 5
3
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10.1002/cbdv.202000800
Chemistry & Biodiversity
(ALK5) and plays a key role in TGF β signaling pathway. ALK5 receptor promotes
angiogenesis.[12] Various small molecules such as GW6604 (II),[13] galunisertib (LY-
2157299)[14] (III) imidazo[2,1-b],1,3,4]thiadiazole[15,16] (IV) as ALK5 inhibitors. Our group
reported cytotoxic activity of different imidazo[2,1-b][1,3,4]thiadiazole.[17-24] Considering the
importance of imidazothiadiazole fused ring, we prepared twenty two compounds of 2-(4-
methoxybenzyl)-imidazo[2,1-b][1,3,4]thiadiazole (V) as cytotoxic by bringing
phosphatidylserine externalization activity, which is a hallmark of apoptosis and binds within
the active sites of the TGF β receptor kinase (Figure 1).
Figure 1. Structures of various (fused) heterocyclic compounds (I-IV) as anticancer and
inhibitors of ALK 5 and synthesized cytotoxic compounds (V).
Results and Discussion
Chemistry
5-(4-methoxybenzyl)-1,3,4-thiadiazole-2-amine (3) was obtained by reacting 4-methoxyphenyl
acetic acid (1) and thiosemicarbazide (2) in sulfuric acid at 60-70 ⁰C for 8 h and made alkaline
with ammonia solution. Various 6-aryl substituted 2-(4-methoxybenzyl)-imidazo[2,1-
4
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10.1002/cbdv.202000800
Chemistry & Biodiversity
b][1,3,4]thiadiazoles (5a-h) obtained by reacting 3 with various 2-bromo ketones (4) in alcohol by
refluxing in ethyl alcohol for 12 h and neutralizing with aqueous sodium carbonate. The expected
5-substituted derivatives 6a-e, 7a-g and 8a, 8b was prepared by electrophilic substitution on
imidazo[2,1-b][1,3,4]thiadiazoles 5a-h (Scheme 1).
Scheme 1 Synthetic pathway of 2-(4-methoxybenzyl)-6-aryl-imidazo[2,1-b][1,3,4]-thiadiazole.
5
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10.1002/cbdv.202000800
Chemistry & Biodiversity
The structures of the newly prepared compounds have been confirmed on the bases
spectroscopic data (1H-NMR, 13C-NMR, HRMS and FTIR). Fourier transform infra-red
(FTIR) spectroscopy of the synthesized compounds (5a-8b) showed characteristic vibrations
between 3172-3006 cm-1 for C-H stretching, 2987-2750 cm-1 for C-H aliphatic stretching. In
addition, the compounds 5h and 7g showed vibrations between 1710-1668 cm-1 for carbonyl
stretching. While for the compounds (7a-g) containing -C≡N functional group exhibited
1
infrared vibrations between 2171-2163 cm-1. In H NMR, the imidazole proton (C5-H)
appeared between 8.91-8.48 δ ppm and that confirms the reaction of 2-amino-5-(4-
methoxybenzyl)-1,3,4-thiadiazole (3) with respective 2-bromoketone (4). The absence of
hydrogen at C5 position of imidazothiadiazole nucleus confirms the substitution at C5 position.
A singlet observed between 10.00 and 9.98 δ ppm for the compounds 8a and 8b confirmed for
the introduction of the formyl group by Vilsmeier-Haack reaction. Aromatic protons appeared
as multiplet signals between 8.02-6.94 δ ppm. Bridge headed -CH2- proton (at C2) exhibited as
a singlet between 4.50-4.34 δ ppm for all compounds. The presence of methoxy proton for all
the compounds observed between 3.84-3.74 δ ppm as a singlet. Compounds having CH3 (5g,
6e & 7f) proton appeared from 2.37-2.30 δ ppm as a singlet. The HR-MS spectrum of the
newly synthesized compounds showed their molecular ion peak with their respective molecular
weights and therefore confirms for the authenticity of the all compounds. Refer Scheme 1 for
the details of the synthesized compounds.
Cytotoxicity Evaluation
Cytotoxicity in murine & human cancer cell lines
Derivatives in the Scheme 1 have been screened towards L1210 cells, CEM cells and HeLa
cells to understand their cytotoxicity effect towards all cell lines tested. Table 1 shows a
comparison of the compounds with the anticancer drugs levamisole and melphalan.
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Chemistry & Biodiversity
Table 1. Antiproliferative effects of the compounds on L1210, CEM and HeLa.
IC50* (µM)
Compound
CEM
> 250
> 250
> 250
> 250
> 250
NT
HeLa
> 250
> 250
> 250
> 250
> 250
NT
L1210
> 250
> 250
> 250
> 250
> 250
NT
5a
5b
5c
5d
5e
5f
> 250
5.0 ± 3.3
> 250
> 250
70 ± 4
> 250
> 250
90 ± 24
≥ 250
5g
5h
6a
> 250
46 ± 9
39 ± 5
> 250
> 250
46 ± 9
≥ 250
2020 ± 68
> 250
6b
6c
6d
13 ± 7
8.8 ± 2.4
28 ± 2
18 ± 0
17 ± 0
33 ± 10
42 ± 19
59 ± 29
87 ± 20
100 ± 15
65 ± 41
58 ± 40
1.6 ± 0.2
> 250
6e
7a
23 ± 0
7b
61 ± 6
21 ± 5
7c
59 ± 14
11 ± 7
20 ± 3
7d
16 ± 5
7e
84 ± 31
0.79 ± 0.35
> 250
63 ± 13
0.78 ± 0.13
96 ± 24
1.3 ± 0.2
NT
7f
7g
8a
0.94 ± 0.12
1.4±0.4
>250
1.1 ± 0.2
2.13±0.02
206
8b
Melphalan
Levamisole
>250
[*] The values are the mean ± SEM, (n = 3)
In order to evaluate the potential cytotoxic activity of the 22 derivatives of
imidazothiadiazoles, we performed a screening using L1210, HeLa and CEM cell lines. Our results
showed that compounds in the 5a-h series were not cytotoxic against all three cell lines tested,
except for compound 5h (IC50 5.0 μM) against human T-lymphocyte CEM cells. Substitution of
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10.1002/cbdv.202000800
Chemistry & Biodiversity
bromine atom at the 5th position of the imidazothiadiazole fused moiety gave 5-bromo derivative
6a-e. Among these bromo derivatives, namely 6d and 6e (IC50 13 to 46 μM) were moderate
cytotoxic to cell lines. The thiocyanate substitution at the C5 position of imidazo[2,1-
b][1,3,4]thiadiazole formed 5-thiocyanato derivatives like 7a-g, and there was much improvement
in cytotoxicity towards all three cells. Among them, compound 7g (IC50 0.78 – 1.6 μM) emerged
as the one with the most potent cytotoxic activity as it was twice more potent. Introduction of
formyl (-CHO) group was done at C5 position of imidazothiadiazole fused ring by Vilsmeier-
Haack reaction to get compounds 8a and 8b in good yield. Among these formyl series, compound
8b (IC50 0.94 – 1.3 micromoles) was twice more potent than the standard control compound against
all the cell lines tested. In general, 5-thiocyanate and 5-formyl derivatives were more potent in
comparison to the control levamisole.
To study the structure-activity relationship on 2-(4-methoxybenzyl)-imidazo[2,1-
b][1,3,4]thiadiazole ring, substitutions at the C5 by bromine, thiocyanate, formyl and at C6 by
phenyl/ substituted phenyl and coumarin-3-yl groups have been taken. Presence of bromine at C5
of imidazothiadiazole fused ring failed to produce any activity, exception with 5h. It has IC50 value
of 5 μM against CEM cells with the coumarin-3-yl moiety at C6 position. By introducing the
thiocyanate group at C5, most of these thiocyanate derivatives became moderate to good cytotoxic
derivatives 0.78-100 μM. Among these, compound 7g with coumarin-3-yl became most promising
with IC50 of 0.78 μM, 0.79 μM and 1.6 μM for HeLa, CEM and L1210 respectively. Introduction
of the -CHO substituent and coumarin-3-yl at 5th and 6th position of fused imidazothiadiazole ring
produced good activity for 8b with IC50 0.94 μM, 1.1 μM and 1.3 μM for CEM, L1210 and HeLa,
respectively.
The most potent compounds namely 7g and 8b were studied further against Jurkat
(Acute T Cell Leukemia), CCRF-CEM (Acute Lymphoblastic leukemia), and Hs27 (Non-
cancerous fibroblast) as described below.
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Compound 7g is selective towards leukemia cells compared to non-cancer cells
We examined the cytotoxic activity of compounds 7g and 8b in an additional leukemia cell line.
For this, we used the Jurkat human cell line, which is derived from a patient with Acute T Cell
Leukemia. The cytotoxic activity of compounds 7g and 8b was evaluated by using differential
nuclear staining (DNS) assay.[25] Different concentrations of the compounds were used to calculate
the concentration at which the compound is causing the death of 50% of the cell population.
Vehicle (DMSO), H2O2 and the untreated control samples were also included. As expected, our
results indicate that compounds 7g and 8b exhibit cytotoxic effects against the Jurkat cell line and
the CC50 ranged from 1.65 μM to 4.73 μM. Compound 8b exerted more cytotoxic effect as shown
in Table 2. We then evaluated the selectivity of these compounds towards cancer cells by
analyzing their cytotoxic effect in the Hs27, a non-cancerous fibroblast cells. The CC50 obtained
from the leukemia cells (Jurkat and CEM) was compared to the noncancer cell line (Hs27) to
calculate the selectivity of the compounds towards cancer cells. The compound 7g exhibited
tumor-selective cytotoxicity showing high selectivity index (SCI) values on the Jurkat and CEM
cell lines. Whereas the compound 8b did not exhibit selectivity towards cancer cells. The Jurkat
cell line was selected for further experimentation due to its low CC50values with higher SCI.
Table 2. Concentration at which the compounds 7g and 8b causes the death of 50% of the
leukemia cells (Jurkat and CEM) as well as the human fibroblast cell line (Hs27). This table
depicts the selectivity index (SCI) of the compounds 7g and 8b towards cancer cells vs.
noncancer human fibroblast cells (Hs27).
Compound
7g
Cell line
CEM
Jurkat
Hs27
CC50[a]
4.73±0.22
2.36±0.10
31.45±0.74
SCI
6.65
13.33
3.62±0.26
1.65±0.19
2.36±0.03
0.65
1.43
CEM
Jurkat
Hs27
8b
[a] The CC50 was calculated by normalizing to our DMSO control average cell count
and using a linear interpolation equation to determine the CC50. Data are mean ± standard
deviation S.D. (n=3)
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Chemistry & Biodiversity
Compound 7g causes cell death by inducing apoptosis
We used Jurkat cells to further investigate the mechanism of action of compound 7g because
this cell line exhibits the lowest CC50 and higher selectivity index. Under normal conditions,
phosphatidylserine is localized in the inner side of the cell membrane. However, during
apoptosis, the cell membrane becomes compromised causing externalization of
phosphatidylserine. [26,27] We examined the levels of phosphatidylserine externalization in cells
treated with this compound. To measure the levels of phosphatidylserine externalization we
used the Annexin V-FITC assay. Annexin V binds phosphatidylserine with high affinity and
in this way, the levels of phosphatidylserine externalization can be measured. For this
experiment, two concentrations of compound 7g were used CC50 (2.36 μM) and the CC100 (4.72
μM). Our results indicate that this compound is inducing phosphatidylserine externalization in
a dose-response manner, as compared to the vehicle control Figure 2.A. This result suggests
that compound 7g is causing phosphatidylserine externalization, which is a hallmark of the
apoptotic pathway.
Compound 7g does not induce mitochondrial depolarization
Multiple apoptotic pathways can be initiated leading to cell death. One of them is intrinsic
apoptotic pathway that can be induced through the loss of mitochondrial transmembrane
potential.[28] We investigated the mitochondrial membrane potential of Jurkat cells treated with
compound 7g. For this experiment, we used the JC-1 polychromatic fluorescent reagent. JC-1
is a dye that in cells with normal mitochondrial potential produces red fluorescent J-aggregates
by its accumulation in the energized and negatively charged mitochondria. In unhealthy or
apoptotic cells this dye also enters the cells but to a lesser degree due to the reduction in
negative charge that is caused by the increased membrane permeability. Under these
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10.1002/cbdv.202000800
Chemistry & Biodiversity
conditions, there is a loss of electrochemical potential that does not allow JC-1 to concentrate
and form J aggregates, therefore, JC-1 remains in its original green color. Using flow cytometry
we were able to observe that compound 7g is not causing mitochondrial membrane
depolarization. Figure 2.B
Compound 7g induces Caspase-3 activation
Another apoptotic pathway that leads to cell death involves the activation of Caspase-3, which
the cleavage of multiple proteins that include, PARP-1, CAD, and others.[28] To extend the
findings that compound 7g induces apoptosis, the activation of Caspase-3 was measured in
Jurkat cells treated with the compound 7g CC50 (2.36 μM) and CC100 (4.72 μM) concentrations,
vehicle (DMSO) and untreated control for 7hrs. For this experiment, the NucView caspase-3
substrate was used and the cells were analyzed using flow cytometry. Our results indicate that
compound 7g induced statistically significant activation of caspase-3 when compared to the
controls (Figure 2.C). These findings provide further evidence to support that compound 7g
induces cell death via apoptotic events.
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10.1002/cbdv.202000800
Chemistry & Biodiversity
Figure 2 A. Annexin-V-FITC was used to detect phosphatidylserine externalization after
treatment with compound 7g. Results indicate that the compound induces phosphatidylserine
externalization which is a biochemical hallmark of apoptosis. B. Compound 7g mediated
cytotoxicity does not induce mitochondrial ΔΨm depolarization in Jurkat cells. C. Compound
7g treatment induces caspase-3 activation. Data are mean ± standard deviation S.D. (n=3) *P-
value<0.05
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Compound 7g prevents DNA synthesis and cytokinesis
In order to have a better understanding of the effect of compound 7g on cancer cells, we decided
to perform a cell cycle analysis on Jurkat cells treated with this compound. The cell cycle
profile of Jurkat cells was examined via flow cytometry utilizing the DNA intercalating agent,
DAPI (4’,6-diamidino-2-phenylindole). Jurkat cells were exposed to the compound 7g CC50
(2.36 μM) and CC100 (4.72 μM) concentrations and incubated for 48 hours and subsequently
treated with a nuclear isolation medium (NIM) containing DAPI as previously described.[29]
Cytometric analysis of cell cycle progression was performed to investigate if the DAPI
fluorescent signal of each treatment. It was measured to quantify the DNA content in the
different samples. A vehicle (DMSO) and an untreated control were added. An increase in
DNA fragmentation was found in cells treated with 7g which was indicated by an increase in
sub-G0 / G1 subpopulation Figure 3.C. Interestingly, we observed a decrease of cell
subpopulation in the G0-G1 phase and an increase of cell population in the S phase on the cells
treated with the CC50 Figure 3.C An additional increase of the cell population in the G2-M
phase can be observed as well.
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Chemistry & Biodiversity
Figure 3. Cell cycle analysis of Jurkat cells treated with compound 7g. A. Increase in the sub-
G0-G1 phase subpopulation on the cells treated with the compound’s CC50. B. Decrease of the
cell population in the G0-G1 phase on the cells treated with the compound’s CC50. C. The
increase of cell population can be identified in the S phase on the cells treated with the
compound’s CC50. D. The increase of the cell population in the G2-M phase can be observed
on the cells treated with the compound’s CC50. Data are mean ± standard deviation S.D. (n=3)
*P-value<0.05
In silico analysis
Molecular docking studies
Increased expression of TGF β receptor kinase I is an important hallmark of malignant
cancer.[30,31] In the present study, molecular docking of two potent synthesized compounds 7g
and 8b were docked against the active site of TGF β receptor kinase I using Glide module. Co-
crystallized ligand i.e. heteroaryl substituted 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole in this
protein (PDB: 1RW8) was used as a reference molecule in our docking simulations. The root
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10.1002/cbdv.202000800
Chemistry & Biodiversity
mean square deviation (RMSD) between the docked and co-crystallized ligand was 0.273 Å,
suggesting that our docking protocol was accurate to test the newly synthesized molecules.
Docking of the well-known compound Melphalan was also performed to reveal its interaction
with this kinase target. Details of the Glide docking score and interacting residues are depicted
in Table 3. Figure 4 shows the molecular mechanism of interaction between (a) 7g (b) 3-(4-
fluorophenyl)-2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b] pyrazole and (c)
melphalan with the TGF β type I receptor kinase domain. The most potent in vitro molecule
7g showed best docking score (binding affinity) in comparison to 5,6-dihydro-4H-pyrrolo[1,2-
b] pyrazole derivative as depicted in Table 3. The potent molecule 7g made strong hydrogen
bonding with S280 and Y282, hydrophobic interaction with A230, K232, L260, V219, A350,
L340 and I211 residues, which is in good agreement with the 3-(4-fluorophenyl)-2-(6-
methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b] pyrazole binding site residues of kinase I
receptor domain (Table 3)[32] Melphalan and molecule 8b showed moderate binding affinities
and hydrogen bonding interactions with the kinase active site residues (Table 3, Figure 4).
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10.1002/cbdv.202000800
Chemistry & Biodiversity
Table 3. Interaction details between TGF β type I receptor kinase domain and potent molecules
using Glide docking technique.
Molecular interactions towards TGF β type I receptor
Molecule
XPGlide
score
kinase domain
(kcal/mol)
Hydrogen
bond
Hydrophobic
interactions
Other
-7.012
-4.864
-8.784
S280
Y282
A230
V219
I211
V219
A230
K213
L260
V219
A230
K232 L260
A350 L340
--
7g
8b
K232
S280
G212
H283
Y282
D351
K232 L278
L260 L340
D290 (Electrostatic)
H283 (Halogen)
[a]3-(4-
fluorophenyl)-
2-(6-
L278 Y249
K232 A350
L340
methylpyridin
-2-yl)-5,6-
dihydro-4H-
pyrrolo[1,2-
b]pyrazole
Melphalan
-5.291
I211
E284
S280
A230
L340
K232 V219
L278 (Halogen)
[a]Reference molecule used in Glide docking.
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Chemistry & Biodiversity
Figure 4. Molecular interactions between TGF beta kinase I receptor with the (a) most potent
synthesized molecule 7g (b) co-crystallized ligand 3-(4-fluorophenyl)-2-(6-methylpyridin-2-
yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole and (c) Melphalan using Glide docking technique.
The molecular interaction figures are reproduced using PyMol software.
Discussion
In this study, we synthesized 22 variants of 2-(4-methoxybenzyl)-6-substituted-
imidazo[2,1-b][1,3,4]-thiadiazole that were expected to have cytotoxic activity against
leukemia. Our results indicate that compound 7g exhibits potent cytotoxicity on one murine
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10.1002/cbdv.202000800
Chemistry & Biodiversity
leukemia cells and two human leukemia cancer cells. In addition, compound 7g exhibited
favorable selective cytotoxicity towards leukemia cells when compared to the Hs27 non-
cancerous fibroblast cells. We investigated the mechanism of action of compound 7g by
performing different assays. First, we evaluated if the compound was causing cell death by
inducing the apoptotic pathway by using the Annexin V-FITC-PI assay. Our results indicate
that the compound induces high levels of phosphatidylserine externalization. Furthermore, we
investigated the effect of compound 7g in Jurkat cell membrane potential. We found that
compound 7g does not induce mitochondrial depolarization by the absence of JC-1
dimerization compared to the controls. Caspase-3 activation was analyzed, and our findings
show strong caspase-3 activation upon compound 7g treatment, suggesting that compound 7g
activates the intrinsic apoptotic pathway. To further understand the effect of compound 7g in
leukemia cells, we performed cell cycle analysis. We observed an increase of the sub-G0-G1
phase that represents the subpopulation with DNA fragmentation. Moreover, results show a
decrease in the G0-G1 phase with an increase in the S and G2/M phases, where DNA is
synthesized, and cytokinesis occurs respectively. Together, our cycle analysis assay suggests
that the compound is preventing the cells from proceeding from the S phase by affecting DNA
synthesis. The high population of cells in the G2-M phase suggests that the compound could
be blocking cytokinesis that is the last step during cell replication. Molecular docking analysis
showed that compound 7g has better binding affinity in comparison to 8b and Melphalan. 7g,
the most potent synthesized compound also made hydrogen bonding and other week
interactions at the active sites of TGF β receptor kinase I domain as explained above (Figure
4a). Anti-cancer activity and selectivity of compounds 7g and 8b clearly shown that 7g
exhibited high SCI in different cancer cell lines by preventing DNA synthesis and cytokinesis,
that could be further correlated with the higher binding affinity of this molecule (-7.012
kcal/mol) obtained from our docking simulations, as shown in Table 3. Further, SAR studies
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presumes that the good cytotoxicity of 7g molecule might be due to the presence of SCN
substituent at 5th position of fused imidazothiadiazole nucleus and that contributed in better
interaction at the active sites of the TGF β receptor kinase I resulting in high binding affinity
(Table 3 and Figure 4a). Thus, the present integrated SAR study discovered a novel molecule
7g showing potent anti-cancer activity for the treatment of leukemia.
Conclusions
22 compounds with different substitution on imidazothiadiazoles fused ring were prepared,
1
characterized by H/13C-NMR, HR-MS and FTIR. All these derivatives tested for cytotoxic
activity against a murine leukemia cell line and two human leukemia cell lines. SAR study
displayed that the presence of -SCN and -CHO on the 5th position of imidazothiadiazole
displayed moderate to good cytotoxicity. Overall, 7g and 8b resulted to be more toxic to the
cell lines tested. Therefore, these two compounds were further screened using the leukemia cell
line Jurkat, and the non-cancer human fibroblast cell line Hs27. Compound 7g but not 8b
showed favorable selective cytotoxic activity against leukemia cells compared to the human
non-cancer fibroblast cells (Hs27). Compound 7g induces high levels of phosphatidylserine
externalization and caspase-3 activation in the Jurkat cell line. These results confirm that the
compound is inducing cell death in the Jurkat cell line by initiating the apoptotic pathway. Our
cell cycle analysis suggests that the compound interferes with DNA synthesis denoted by the
increase of cell population in the S-Phase. The cell population increase in the G2-M phase
suggests that the compound is inhibits cytokinesis. Furthermore, molecular docking showed
that 7g binds within the active site of TGF β type I receptor kinase domain by strong hydrogen
bonding and hydrophobic interactions. Our results identify compound 7g as a novel potential
anti-leukemia compound. Further studies should be performed to evaluate the effect of this
compound in xenograft mouse models.
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Experimental Section
Chemicals and reagents
Jasco 430+ instrument was used for recording FTIR spectra in KBr by using diffuse reflectance
technique; the 1H/13C NMR spectrum was taken in CDCl3/ DMSO-d6 on a Jeol JNM-ECZ400S/
Bruker (400 MHz). XEVO G2-XS QTOF Mass Spectrometer was used to report HR-MS. 2-
bromoacetophenone,[33] 2-bromo-4’-chloroacetophenone,[34] 2,4’-dibromoacetophenone,[35] 2-
bromo-4’-methoxyacetophenone,[36]
2-bromo-4’-methylacetophenone,[37]
2-bromo-4’-
fluoroacetophenone,[38] 2-bromo-4’-nitroacetophenone,[39] 3-(bromoacetyl)coumarin,[40] 2-
amino-5-(4’-methoxybenzyl)-1,3,4-thiadiazole (3), compounds 5a, 5c, 5d, 5e, 5f, 5g and 8b
prepared as per literature.[41]
Cell lines and culture conditions
The cell lines used in this study consists of Jurkat (Acute T Cell Leukemia), CCRF-CEM
(Acute Lymphoblastic leukemia), and Hs-27 (Non-cancerous fibroblast). The Jurkat and
CCRF-CEM cell lines were supplemented with RPMI-1640 medium and an antibiotic mixture
of 25μg/ml amphotericin B, 1000 U/ml penicillin, and 1000 μg/ ml streptomycin, and 10%
heat-inactivated fetal bovine serum. The Hs-27 cell line was grown in DMEM media with the
same additives added to the RPMI media.
Cytotoxicity in human and murine tumor cell lines
The synthesized derivatives have been screened for their cytotoxicity study as per the published
procedure.[21] Experiments have been performed in triplicate and the IC50 values were
calculated and expressed in micromoles.
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Cytotoxic activity in human tumor cell lines
Cytotoxic activity was based on inhibition of HEL cell growth. HEL cells were incorporated
into 5 X 103 cells / well into 96 microtiter plates and allowed to replicate for 24 h. After that,
the medium containing a different concentration of the test compounds was added. After 3 days
of incubation at 37 ° C, a cell number was found with the Coulter counter. The cytotoxic
concentration was calculated as CC50 (the compound concentration required to reduce cell
growth by 50% relative to the number of cells in the untreated controls). CC50 values were
estimated from graphic plots of the number of cells (percentage of control) as a function of the
concentration of the test compounds. Cytotoxicity was expressed as a minimum cytotoxic
concentration (MCC) or the compound concentration that causes a microscopically detectable
alteration of cell morphology. All tests were performed and repeated three times.
Differential Nuclear Staining Assay
In order to analyze the potential cytotoxicity of the compounds that were synthesized, the
Differential Nuclear Staining Assay (DNS) was used which has been validated for high
throughput compound screening.[25] The DNS assay involves the use of two dyes, propidium
iodide (PI) and Hoechst. While Hoechst permeates and stains the nuclei of healthy and dead
cells, PI can only penetrate and stain nuclei of cells with compromised membranes. The cells
with both Hoeschst (blue) and PI staining (red) are recognized and defined as the dead cell
population.[25] Before each assay, cell viability was analyzed to ensure that the cells were at
least 95% viable using PI staining and flow cytometry. For the initial cytoxicity screening, cells
were seeded in a 96-well microplate at a density of 10,000 cells per well in 100μL of media
and incubated for 2 h for non-adherent cells and overnight for adherent cells. A concentration
gradient of the compounds from 0.1 to 10 μM was tested. The following controls were added
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in each experiment: untreated cells, 1% v/v DMSO solvent, and a positive control for cell death
of 1mM H202. Each experimental point, as well as the controls, were assessed in triplicate.
Furthermore, two incubation times were performed, 48 h for non-adherent cells, and 72 h for
adherent cells. Two hours before the end of the incubation period, a mixture of Hoescht and PI
(5 μg/mL final concentration) was added to each well, and the plates were incubated for the
remaining 2 hours. After incubation, four images were obtained to create a 2x2 montage using
a 10X target and two fluorescent channels (Hoechst and PI signals) using the IN Cell Analyzer
2000 system. The images were separated and analyzed in both live and dead cells using
software IN Cell Analyzer Workstation 3.2, to determine the percentage of each cell and each
source.
Mitochondrial depolarization assay
Cells are grown in a clear 24-plate plate at a density of 200,000 cells / well. After 5 h of
compounds treatment, the cells were subjected to cationic polychromatic JC-1 (5 ', 6,6'-
tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide) reagent at 2 μM
(MitoProbe; Life Technologies; M34152). When mitochondria are degraded, JC-1 cannot form
a compound and remains as a monomer emitting a green fluorescence signal.[42] After
incubation with JC-1, the samples were analyzed using flow cytometry (Gallios; Beckman
Coulter). Similar controls have been used as in other apoptosis experiments (see above).
Cytotoxic concentration 50% and selective cytotoxicity index values
CC50 has been described as a combined concentration needed to kill 50% of the cell
population. To remove the base of the dead cells, the percentage of dead cells found in the
DMSO vehicle control was removed from each test sample, and the applied values were used
to calculate the CC50. After this, a linear interpolation equation was used to obtain CC50
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values.[43,44] Due to the short duration of compound exposure and dose response, cells are
exposed to the CC50 and double that compound concentration. The selected cytotoxicity index
describes the potential combination of experimental compounds that kill cancer cells that cause
minor or insignificant damage to non-cancer cells. SCI values were calculated by separating
the CC50 of the non-cancer control cell line by the CC50 of the cancer cell line as described
earlier. [29,43,44]
Annexin V-FITC/PI assay
Jurkat cells are propagated in a mass of 100,000 cells / well with 1 ml of cultural media on a
24-well plate in 1 ml medium. After this, the cells were treated with CC50 concentration of 7g
for 24 h. Next, cells are collected and marked with PI and annexin V-FITC according to
manufacturer's instructions (Beckman Coulter; IM3546). After incubation, the samples were
analyzed using flow cytometry (Cytomics FC 500; Beckman Coulter). The same positive and
negative controls have been used as in previous sections. About 10,000 events (cells) were
detected per sample and analyzed using the CXP (Beckman Coulter) software tool as described
earlier. [42]
Mitochondrial membrane potential polychromatic assay
Jurkat cells are grown in a mass of 100,000 cells / well in a clear 24-plate plate. Jurkat cells
were treated with CC50 concentrations per compound for 5 h. Thereafter, the cells were
subjected to cationic polychromatic JC-1 (5 ′, 6,6'-tetrachloro-1,1 ′, 3,3'-tetraethyl
benzimidazolylcarbocyanineiodide) reagent in the final 1 μM (MitoProbe; Life Technologies;
(M34152). In cells with a stable mitochondrial membrane, JC-1 dye binds inside the inner
mitochondrial membrane causing a transition from green (~ 529 nm) to red (~ 590 nm). As
soon as the mitochondria are broken down, JC-1 cannot form a compound and remains as a
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[42]
monomer emitting green fluorescence signal.
After JC-1 contamination, samples were
analyzed using flow cytometry (Cytomics FC 500; Beckman Coulter). Similar controls were
used as in other experiments for apoptosis (see above).
Caspase- activation assay
Jurkat cells (100,000 cells / well) were seeded in a 24-source plate per 1 ml complete RPMI-
1640 medium. Next, the cells were treated for 7 h with CC50 of the composite compound
which subsequently found caspase-3 activation using a fluorogenic NucView 488 caspase-3
substrate designed to identify active caspase-3 within live cells (Biotium; 30,029 ); as described
earlier. [42] After cytometry (Cytomics FC 500; Beckman Coulter) green fluorescence signal
cells were classified as apoptotic cells with activated caspase 3. The same positive and negative
controls as in other apoptosis trials were also used in this series of tests.
Cell cycle analysis
Jurkat cells were placed in a 24-well plate where 100,000 cells combine containing 1 mL of
culture media per well. Cells were treated with CC25 and CC50 compound concentration for a
period of 72 h. The controls included in this series of tests were described in previous sections.
The cells are then collected in cytometry flow tubes, separated by centrifugation at 262g for 5
minutes and gently regenerated into 100 μL of new media. Thereafter, 200 μL of nuclear
isolation medium (NIM) -DAPI solution was added to the cell configuration and immediately
analyzed by flow cytometry.[29,45] The NIM-DAPI reagent can locate nuclei of cells that allow
the synthesis of DNA content using a FL-9 detector and a 405-nm laser for excitation purposes.
This FL-9 detector picks up a fluorescence signal released when DAPI is attached to DNA and
leaves a signal when it is attached to RNA. To obtain a well-defined cell cycle distribution
profile, 100,000 cells were detected per sample and analyzed with Kaluza Flow Cytometry
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Software. Each test point, and their corresponding controls were analyzed simultaneously and
tested three times.
Statistical analysis
All values were expressed as mean ± SEM. The intergroup variance between different groups
was measured by one-way ANOVA using Graph Pad Prism, version 6.01. Here, the results
were considered statistically significant when * p <0.05.
Protein Preparation
The X-ray coordinates of TGF-beta receptor 1 kinase in complex with heteroaryl substituted
5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole ligand at a resolution of 2.4 Å was retrieved from
protein databank (PDB) with PDB ID 1RW8.[32] The complex was prepared using the Protein
Preparation Wizard module of Schrödinger software (Schrödinger, LLC, New York, 2017).[46]
Initially, the 3D structure of the protein was pre-processed by assigning bond orders and adding
hydrogen atoms. The protein complex was viewed in PyMol software to check for interactions
with water. Water molecules that mediate hydrogen bonding interactions between ligand and
the receptor were retained after removing the remaining water molecules. Geometry was
optimized using the Optimized Potential for Liquid Simulations 2005 (OPLS-2005) force field
in order to avoid any structural errors.
Ligand Preparation
7g and 8b molecular structures were constructed using software ChemBioDraw Ultra version
11.0 and minimized. 3D structure of Melphalan was downloaded from DrugBank database.[47]
All three ligands were further prepared using the LigPrep module in Schrödinger software.[48]
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3D structure of the ligand was optimized using the OPLS 2005 force field while retaining its
specified chirality.
Receptor grid generation
Glide (Grid-based Ligand Docking with Energetics) program of Schrödinger suite was used to
generate a receptor grid for docking.[49] The ligand bound to the protein in the complex was
picked and a receptor grid was automatically generated by the software around the binding site
of the protein. Receptor grids were calculated for prepared proteins such that various ligand
poses bind within the active site during docking. Grids were generated keeping the default
parameters of van der Waals scaling factor 1.00 and charge cut-off 0.25 subjected to OPLS
2005 force field. A cubic box of specific dimensions 20Å × 20Å × 20Å was generated in the
TGF-beta receptor 1 kinase active site for docking simulations.
Molecular Docking
Molecular docking was performed in two stages: The first stage includes SP (standard-
precision) docking followed by XP (extra-precision) docking with higher accuracy and less
false positives. The Glide XP binding free energy is calculated using the following equation:
XP Glide score = Ecoul + EvdW + Ebind + Epenalty
Where,Ecoul, EvdW, and Ebind represent the electrostatic potential energy, van der Waals energy,
and binding interaction energy respectively. Epenalty is the strain energy from protein, ligand,
or both, loss of entropy of the ligand and protein and desolvation of the ligand or protein.46The
binding interaction of these ligands to the active site of TGF beta receptor kinase was ranked
based on the Glide XP scoring function. Hydrogen bonding (H-bonding) and hydrophobic
interactions between these complex systems were analyzed by Discovery Studio v19 and
PyMol software.
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Acknowledgement
Many thanks to NMR Research Centre-Indian Institute of Science, Bengaluru (Karnataka)
India for 1H-NMR, Sophisticated Analytical Instrument Facility (SAIF)-Dharwad, (Karnataka)
India for 13C-NMR spectroscopy. We sincerely thank facility for HR-MS, Dept. of Chemistry,
Indian Institute of Technology, Ropar (Punjab) India. CGM acknowledges Centre for
Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham, Kochi for
computational infrastructure support. This research was funded by NIH-MARC Program under
Grant No. T34GM008048-35 and was performed in facilities funded by the National Institute
on Minority Health and Health Disparities (NIMHD) Grant no. 5U54MD007592 to the Border
Biomedical Research Center UTEP.
Author contribution statement
C. B. and S. K. Performed the synthetic parts of experiment. D. S. Carried out cytotoxicity
study. K. G. C. H. and A. Y. S. B. performed biochemical study. A. M. T. designed and
performed the docking study. C. G. M. analyzed and compiled the docking study. S. S. K. and
R. J. A. designed the experiment, analyzed spectral and biochemical data, compiled and wrote
the paper.
27
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