Design, synthesis and biological evaluation of some new 1,3,4-thiadiazine-thiourea derivatives as potential antitumor agents against non-small cell lung cancer cells

Article abstract of DOI:10.1016/j.bioorg.2019.103323

New 1,3,4-thiadiazine-thiourea derivatives have been synthesized. All the synthesized compounds were examined for in vitro cytotoxic activity against Non-Small Cell Lung Cancer (NSCLC) cell line A549, using MTT bioassay. Compounds 5d, 5i, 5j showed the highest cytotoxic activity with IC₅₀ values of 0.27 ± 0.01, 0.30 ± 0.02, and 0.32 ± 0.012 μM respectively with sorafenib as reference (IC₅₀ 3.85 ± 0.27 μM). These compounds were chosen for further investigations against various biological targets known to play roles in NSCLC specifically: vascular endothelial growth factor receptor 2 (VEGFR2), B-RAF and matrix metalloproteinase 9 (MMP9). Encouraging results were exhibited by the three compounds against the selected targets. Compound 5j was specially promising as it exhibited inhibitory activity of VEGFR2 close to sorafenib (IC₅₀ 0.11 ± 0.01 μM), most potent B-RAF activity inhibition (IC₅₀ 0.178 ± 0.004 μM) and MMP9 inhibition (IC₅₀ 0.08 ± 0.004 μM). Moreover, cell cycle analysis of A549 cells treated with 5j exhibited cell cycle arrest at G2-M phase and pro-apoptotic activity as indicated by Annexin V-FITC staining. Also, it reflected antinvasive and antimigration properties to A549 cells. Additionally, docking study of 5j on VEGFR2, B-RAF and MMP9 revealed that it binds to the target enzymes in a similar way as the co-crystallized ligand. The three compounds exhibited significantly high selectivity to A549 cancer cells against the normal human fetal lung fibroblast cell line WI-38 with higher selectivity index compared to sorafenib (5d IC₅₀ 136.76 ± 2.38 μM, SI = 506.52; 5i IC₅₀ 89.20 ± 2.11 μM, SI = 297.33; 5j IC₅₀ 79.60 ± 3.8 μM, SI = 248.75; sorafenib IC₅₀ 30.32 ± 2.41 μM, SI = 7.88). In conclusion, compounds 5d, 5i and 5j, specially 5j are promising anticancer agents targeting important pathways in NSCLC and warrant further preclinical and clinical trials.

Full text of DOI:10.1016/j.bioorg.2019.103323

BioorganicChemistry93(2019)103323
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of some new 1,3,4-thiadiazine-
thiourea derivatives as potential antitumor agents against non-small cell
lung cancer cells
Fatma A.F. Ragaba, Salah A. Abdel-Azizb, Marwa Kamelc, Abdelsalam Mohamed A. Oufb,
⁎
Heba Abdelrasheed Allama,
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo P.O. Box, 11562, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt
c Department of Cancer Biology, Unit of Pharmacology, National Cancer Institute, Cairo University, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
New 1,3,4-thiadiazine-thiourea derivatives have been synthesized. All the synthesized compounds were ex-
1,3,4-thiadiazine
Thiourea
amined for in vitro cytotoxic activity against Non-Small Cell Lung Cancer (NSCLC) cell line A549, using MTT
bioassay. Compounds 5d, 5i, 5j showed the highest cytotoxic activity with IC50 values of 0.27
0.01,
Non-small cell lung cancer
VEGFR2
0.30
0.02, and 0.32
0.012 μM respectively with sorafenib as reference (IC50 3.85
0.27 μM). These
compounds were chosen for further investigations against various biological targets known to play roles in
NSCLC specifically: vascular endothelial growth factor receptor 2 (VEGFR2), B-RAF and matrix metalloprotei-
nase 9 (MMP9). Encouraging results were exhibited by the three compounds against the selected targets.
Compound 5j was specially promising as it exhibited inhibitory activity of VEGFR2 close to sorafenib (IC50
B-RAF
MMP9
0.11
0.08
0.01 μM), most potent B-RAF activity inhibition (IC50 0.178
0.004 μM) and MMP9 inhibition (IC50
0.004 μM). Moreover, cell cycle analysis of A549 cells treated with 5j exhibited cell cycle arrest at G2-M
phase and pro-apoptotic activity as indicated by Annexin V-FITC staining. Also, it reflected antinvasive and
antimigration properties to A549 cells. Additionally, docking study of 5j on VEGFR2, B-RAF and MMP9 revealed
that it binds to the target enzymes in a similar way as the co-crystallized ligand. The three compounds exhibited
significantly high selectivity to A549 cancer cells against the normal human fetal lung fibroblast cell line WI-38
with higher selectivity index compared to sorafenib (5d IC50 136.76
2.38 μM, SI = 506.52; 5i IC50
89.20
2.11 μM, SI = 297.33; 5j IC50 79.60
3.8 μM, SI = 248.75; sorafenib IC50 30.32
2.41 μM,
SI = 7.88). In conclusion, compounds 5d, 5i and 5j, specially 5j are promising anticancer agents targeting
important pathways in NSCLC and warrant further preclinical and clinical trials.
1. Introduction
multi-targeted anticancer agents [3]. Vascular endothelial growth
factor (VEGF) represents a chief therapeutic target as it is a key an-
According to global cancer statistics 2018, lung cancer is a com-
monly diagnosed cancer (11.6% of the total cases) and the principal
cause of cancer death (18.4% of the total cancer deaths) [1]. It is ca-
tegorized into two main histological groups: Small cell Lung cancer
(SCLC) and Non-Small Cell Lung Cancer (NSCLC). NSCLC accounts for
about 85% of cell lung cancers and are generally subcategorized into
adenocarcinoma, squamous cell carcinoma, and large cell carcinoma
[2]. One of the main challenges of several clinical trials of therapy of
NSCLC is that blocking of one of the targeted pathways of signal
transduction allows others to act as escape mechanisms for cancer cells.
Therefore, a current strategy for cancer treatment involves the design of
giogenic factor involved in tumor blood vessels formation [4]. Over
expression of VEGF has been observed in a diversity of cancers in-
cluding lung cancers and VEGF is a validated target for NSCLC [5].
Inhibition of VEGF or VEGF receptors (VEGFR) has been achieved ei-
ther by using the monoclonal antibodies against VEGF such as bev-
acizumab [6] or by using tyrosine kinase inhibitors (TKIS) of VEGFR
[7]. Sorafenib I acts as a multitargeting kinase inhibitor. It inhibits
VEGFR2, VEGFR3, c-kit, PDGFRβ and RAF and is indicated for hepa-
tocellular carcinoma and advanced renal cell carcinoma. However, it
has demonstrated only a moderate antitumor efficacy with numerous
side effects in NSCLC, which greatly limits its clinical application [8].
⁎ Corresponding author.
E-mail address: heba.abdelkalek@pharma.cu.edu.eg (H.A. Allam).
https://doi.org/10.1016/j.bioorg.2019.103323
Received 30 April 2019; Received in revised form 19 September 2019; Accepted 26 September 2019
Availableonline26September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

F.A.F. Ragab, et al.
BioorganicChemistry93(2019)103323
Fig. 1. Examples of anticancer compounds containing urea or thiourea moieties.
Several sorafenib thiourea derivatives II have been synthesized by
substituting thiourea for urea moiety. Some of these derivatives de-
monstrated more potent inhibitory activities against cancer cells in
addition to potent inhibitory activities against the phosphorylation of
VEGFR [9–13].
The most active derivatives were further evaluated for their VEGFR2
kinase activity as well as other targets including B-RAF and MMP9.
Furthermore, cell cycle analysis, migration and invasion assays were
performed for the most promising compound in order to gain additional
insight about its mechanism of action. Also, molecular docking studies
were performed to examine the binding interaction of the most pro-
mising compounds with VEGFR, B-RAF and MMP9 active sites.
On the other hand, several heterocyclic thiourea derivatives play
important roles as anticancer agents against leukemia and solid tumors
such as benzothiazole III [14,15], pyrazole IV [16], benzimidazole V
[17], pyrimidine VI [18] and 1,3,4-thiadiazole VII [19]. 1,3,4-thia-
diazole thiourea derivatives were found to have potent anticancer ac-
tivity against A549 cell line [19]. However, the anticancer activity of
1,3,4-thiadiazines (ring expanded equivalent of 1,3,4-thiadiazole)
thiourea hybrids have not widely been investigated although some re-
ports mentioned that thiadiazine derivatives elicited inhibitory effect
against various carcinoma cell lines. Moreover, some thiadiazine deri-
vatives showed high inhibitory activities against different matrix me-
talloproteinases (MMPs) with high affinity to matrix metalloproteinase
9 (MMP9) VIII [20–23] (see Fig. 1). MMPs are enzymes which are
overexpressed in different kinds of cancer comprising lung cancer and
have critical roles in tumor invasion and metastasis [24].
2. Results and discussion
2.1. Chemistry
The general route for the synthesis of the target 1,3,4-thiadiazine-
thiourea hybrids 5a-p is represented in scheme I. Two general methods
were available to prepare the synthones 2-Amino-5-aryl-6H-1,3,4-
thiadiazine derivatives 3a-d, firstly by reacting of substituted α-halo
keto compounds with thiosemicarbazide in ethanol at 0 °C and the re-
sulting linear intermediates were isolated and then ring closed by
heating in ethanol/ H2O/HBr mixture to afford the 5-substituted-6H-
1,3,4-thiadiazine-2-Amine derivatives 3a-d as their hydrochloride salts
in good yields. In the second method the thiadiazine ring was con-
structed in one step from substituted α- halo keto compounds and
thiosemicarbazide hydrochloride or hydrobromide in methanol [20].
Consequently, the present investigation deals with the synthesis of a
series of 1-substituted phenyl-3-(1,3,4-thiadiazinyl) thioureas 5a-p to
be evaluated for their cytotoxic activity against NSCLC (A549 cell line).
2

F.A.F. Ragab, et al.
BioorganicChemistry93(2019)103323
Scheme 1. Synthesis of the target 1,3,4-thiadiazine thiourea derivatives. Reagents and conditions: (i) Conc HCl, EtOH, reflux; (ii) Aqueous ethanol, 7% solution of
NH3 (iii) Toluene, reflux.
to give the hydrochloride salt 2a-d. Neutralization of 2a-d by the ad-
dition of a 7% solution of NH4OH with cooling gave the thiadiazine
derivatives 3a-d [25]. The thiourea derivatives 5a-p were synthesized
by refluxing of 3a-d with various phenylisothiocyanates 4a-d in toluene
(Scheme 1).
Table 1
In vitro cytotoxic activity (IC50) of compounds 5a-p against A549 cancer cell
line using sorafenib as a reference drug
The structure of compounds 5a-p was confirmed by IR, 1H NMR, 13
C
.
NMR, mass spectra and elemental analysis. The IR spectra exhibited
two NeH stretching vibrations at 3286–3348, 3228–3273 cm−1 and
C]S stretching vibration at 1639–1720 cm−1
.
1H NMR spectra dis-
played the presence of a singlet signal at δ 3.60–3.85 (CH2 protons of
a
Compound
R
R1
IC50 μM
SE
the thiadiazine ring) and two singlet signals at
δ 12.30–12.60,
5a
H
H
1.48
10.32
1.39
0.27
2.05
4.47
2.50
3.23
0.30
0.32
1.70
0.97
2.77
3.55
13.86
21.99
3.85
0.07*
0.51*
0.07*
0.01*
0.10*
0.22
10.50–10.70 (NH protons, D2O exchangeable). Compounds 5c, 5g, 5k
and 5o revealed a characteristic singlet signal at δ 2.27–2.40 (CH3
protons of the p-tolyl part). Compounds 5i, 5j, 5k and 5l showed a
characteristic singlet signal at δ 2.37 (CH3 protons of the p-tolyl part
attached to thiadiazine ring). Compounds 5d, 5h, 5l and 5p revealed
the presence of a singlet signal at δ 3.74–3.80 (OCH3 protons).
5b
H
Cl
5c
H
CH3
OCH3
H
5d
H
5e
Cl
5f
Cl
Cl
5g
Cl
CH3
OCH3
H
0.12*
0.16
13C NMR spectra of compounds 5a, 5b, 5g, 5h, 5j, 5k, 5l, 5n, 5o
and 5p revealed the presence of CS carbon at δ 183.93–184.36. Also, C2
of the thiadiazine ring gave a signal at δ 165.45–167.43. In addition,
CH2 of the thiadiazine ring gave a signal at δ 23.99–24.03. Moreover,
compounds 5h, 5l, 5n, 5o and 5p showed a characteristic signal at δ
55.69–55.80 assigned for OCH3. Furthermore, compounds 5g, 5j, 5k
and 5l were characterized by a signal at δ 20.98–21.42 related to CH3.
5h
5i
Cl
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
0.02*
0.02*
0.09*
0.05*
0.13*
0.17
5j
Cl
5k
CH3
OCH3
H
5l
5m
5n
5o
Cl
CH3
OCH3
0.69*
1.09*
0.27
5p
Sorafenib
2.2. Biological evaluation
a
IC50 values are the means
SE of three separate experiments.
* Statistically significant from sorafenib at p ≤ 0.05.
2.2.1. MTT assay of the synthesized compounds
The newly synthesized compounds 5a-p were screened for their
cytotoxic activity against A549 cell line using MTT assay [26]. The
cytotoxic activity was measured as the concentration that reduces the
growth of the cancer cells by 50% represented as IC50 μM. The IC50 of
compounds 5a-p were presented in Table 1. Among the sixteen
In the present work, compounds 3a-d were prepared by a cyclo-
condesation reaction through stirring of phenacylbromide derivatives
1a-d with thiosemicarbazide in ethanol at 0 °C for 3 h. Catalytic amount
of hydrochloric acid was added to the stirred mixture then reflux for 1 h
3

F.A.F. Ragab, et al.
BioorganicChemistry93(2019)103323
Table 2
metalloproteinases [35]. Overexpression of MMP9 has been reported in
NSCLC and is thus an interesting target for adjuvant anticancer
therapy in NSCLC using specific inhibitors of MMP9 [36,37]. Thereby,
in this study compounds 5d, 5i and 5j were evaluated for their
MMP9 inhibition response where they displayed promising inhibitory
effect for the enzyme activities as represented in Table 3.
Interestingly, compound 5j exerted the most favorable response
VEGFR2, B-RAF and MMP9 activity assay results of compounds 5d, 5i and 5j
using sorafenib as reference drug.
Compound
VEGFR2 IC50
B-RAF IC50
MMP9 IC50
a
a
a
μM
SE
μM
SE
μM
SE
5d
0.10
0.18
0.11
0.11
0.01
0.66
2.50
0.18
0.04
0.03*
0.07*
0.004*
0.002
3.015
2.067
0.075
0.02
0.08*
0.04*
0.01*
5i
0.01*
0.004
0.004
(IC50 = 0.08
0.01 μM).
5j
Sorafenib
0.004
2.2.5. Cell cycle analysis of compound 5j
a
IC50 values are the means
SE of three separate experiments.
Based on the favorable effect of 5j in inhibiting the activity of key
enzymes involved in NSCLC, we were interested in the behavior of this
compound on the cell cycle. Thus, influence of 5j on A549 cells has
been determined using flow cytometric analysis. Significant shifts in
phases of the cell cycle resulted upon treatment of A549 cells with IC50
concentration of 5j (0.32 µM) for 24 h. The percentage of apoptotic cells
at the pre-G phase was increased (12.64%) on exposure to 5j compared
to control (2.61%). Moreover, as shown in Fig. 2, there was a reduction
in cell number at G0-G1 and S phases while there was an increased cell
accumulation at G2-M phases which makes us speculate that compound
5j inhibits the cell proliferation by means of cell cycle arrest at G2-M
phase, thus prompting apoptotic cell death
* Statistically significant from sorafenib at p ≤ 0.05.
compounds tested, twelve compounds showed more potent cytotoxic
activity than sorafenib with IC50 range 0.27
0.013 to
3.55
0.17 µM (sorafenib IC50 = 3.85
0.02, 0.32
0.27 µM). Compounds 5d,
0.01,
5i and 5j were the most potent derivatives with IC50 0.27
0.30
0.02 μM respectively. Variation of the aryl sub-
stituents attached to both the 5-position of thiadiazine nucleus and
thiourea scaffold affected the cytotoxic activity. In general, it was ob-
served that compounds 5i-l substituted with a p-tolyl group at 5-posi-
tion of thiadiazine nucleus exhibited more potent effect
(IC50 = 0.30
0.02–1.70
0.09 µM) than their analogues with
0.10–4.47 0.22 µM)
5m-p (IC50 = 2.77
2.2.6. Annexin V-FITC apoptosis assay
4-chlorophenyl group 5e-h (IC50 = 2.05
or with 4-methoxyphenyl group
0.13–21.99
Additional assessment of the pro-apoptotic activity of compound 5j
was performed by Annexin V-FITC and propidium iodide double
staining. Flow cytometric analysis of the differential binding of the cells
to Annexin V-FITC and propidium iodide displayed a notable influence
on the fraction of apoptotic cells. A549 cells treated with 0.32 µM of 5j
(IC50) presented a rise in the percentage of Annexin V-FITC positive
apoptotic cells (UR + LR) by 10.53% (6 folds) for 5j (Fig. 3).
1.09 µM) which gives an indication that the p-tolyl
group at the thiadiazine nucleus is more beneficial for activity. In ad-
dition, derivatives with unsubstituted phenyl attached to thiadiazine
nucleus showed promising activity except compound 5b, while the
most active compound was 5d (IC50 = 0.27
0.01 µM).
2.2.2. VEGFR2 kinase activity assay of compounds 5d, 5i and 5j
The most potent cytotoxic derivatives 5d, 5i, 5j were further eval-
uated for their effects on VEGFR2 kinase activity. As indicated in
Table 2, all the three compounds reduced the activity of the enzyme
where compounds 5d and 5j exhibited VEGFR2 Kinase activity in-
2.2.7. Cell invasion and migration assays
Since compound 5j exhibited a promising effect on MMP9 activity,
it was further assessed for its cell invasion and migration potentials. As
shown in Fig. 4(A) 5j decreased the invasion and migration of A549 by
almost 2.5 and 2.4 folds respectively relative to control untreated cells.
Similar effect was observed in Fig. 4(B). Collectively, these results de-
monstrate the promising effect of 5j as an antimetastatic agent although
it wasn’t superior to sorafenib in this respect.
hibition very close to sorafenib (5d IC50 = 0.10
0.01 μM; 5j
IC50 = 0.11
0.004 μM; sorafenib IC50 = 0.11
0.004 μM).
2.2.3. B-RAF kinase activity assay of compounds 5d, 5i and 5j
RAF kinases (A-RAF, B-RAF and C-RAF (known also as RAF1)
comprise important components of the RAS–RAF–MEK–ERK signaling
cascade (ERK signaling) [27–29]. B-RAF mutations are present in many
human tumors [30] such as melanomas (50%) [31], papillary thyroid
cancers (45%) [32] and NSCLC (10%) [33]. Compounds 5d, 5i, 5j were
evaluated for their B-RAF inhibitory activities (Table 3). The most po-
2.2.8. Cytotoxic activity against WI-38 human fetal lung fibroblast cell line
In order to assess the safety profile of compounds 5d, 5i and 5j, MTT
cytotoxicity assay was performed against normal human fetal lung fi-
broblast cell line WI-38 using sorafenib as a reference drug. The results
infer that our selected compounds have significantly higher selectivity to
A549 cancer cells compared to sorafenib (5d IC50 136.76
2.38 μM,
tent compound was 5j (IC50 = 0.18
0.004 μM).
SI = 506.52; 5i IC50 89.20
2.11 μM, SI = 297.33; 5j IC50
79.6
3.8 μM, SI = 248.75; sorafenib IC50 30.32
2.41 μM,
SI = 7.88).
2.2.4. MMP9 inhibition screening assay of compounds 5d, 5i and 5j
MMPs are family of zinc metalloproteinases which degrade the ex-
tracellular matrix and basement membranes. They play key roles in
angiogenesis, inflammation, tumor development, cell proliferation and
apoptosis [34]. Their activity is regulated by tissue inhibitors of
2.3. Docking study
The docking studies were performed to discover the possible
binding mode of the most promising compound 5j and its interaction
with the main amino acids in the VEGFR2, B-RAF and MMP9 active
sites. The 5j was docked in the active sites of crystal structure of
VEGFR2, B-RAF co-crystallized with sorafenib and MMP9 co-crystalized
with reverse hydroximate inhibitor. To ensure the accuracy of the
docking protocol, validation was performed by redocking the co-crys-
tallized ligand (sorafenib) into VEGFR2, B-RAF binding sites and re-
verse hydroximate inhibitor into MMP9 active site. The docking poses
were compared with the initial pose based on the binding mode and
root mean square deviation (RMSD). The docking validation results
displayed that, sorafenib docked nearly at the same position
(RMSD = 0.691 Å) with energy score of −15.35 kcal/mol for VEGFR2
Table 3
In vitro cytotoxic activity (IC50) of compounds 5d, 5i and 5j against WI-38 cell
line using sorafenib as a reference drug.
a
Compound
WI-38 IC50 μM
SE
Selectivity index (SI)
5d
136.76
89.20
79.6
2.38*
2.11*
3.8*
2.41
506.52
297.33
248.75
7.88
5i
5j
Sorafenib
30.32
a
IC50 values are the means
SE of three separate experiments.
* Statistically significant from sorafenib at p ≤ 0.05.
4

F.A.F. Ragab, et al.
BioorganicChemistry93(2019)103323
Fig. 2. Effect of 5j on DNA-ploidy flow cytometric analysis of A549 cells.
and B-RAF (RMSD = 0.6497 Å) with energy score of −14.75 kcal/mol
while in the case of MMP9, RMSD was 0.9292 Å with energy score of
−11.08 kcal/mol.
with the side chain of His574.
2.3.3. Docking results of compound 5j in MMP9
Compound 5j docked at the same position as reverse hydroximate
inhibitor and displayed good interaction as indicated by hydrogen
bonding with essential glutamic acid residue (402) as well as the co-
ordination bond of the catalytic zinc ion by His401, His405 and His411
is completed by both nitrogen atoms of the thiadiazine ring (Fig. 7).
2.3.1. Docking results of compound 5j in VEGFR2
Compound 5j docked at the same position as sorafenib and dis-
played good interaction as indicated by hydrogen bonding with two of
the key amino acids in the active site (Fig. 5). As a H-bond donor, the
thiourea NeH attached to the thiadiazine ring interacts with Asp1046
(2.7 Å), while the anilino NeH interacts with the carboxylate side chain
of Glu885 (2.6 Å) of the αC helix where the chlorophenyl moiety is
fitted into the hydrophobic back pocket which is lined with the hy-
drophobic side chains of Ile888, Leu889, Val899, Cys1024 and Ile1025.
3. Conclusion
In summary, new hybrids containing thiadiazine and thiourea
fragments have been synthesized. All the synthesized compounds were
investigated for their cytotoxic activity against A549 cell line.
Compounds 5d, 5i and 5j showed the highest cytotoxic activities with
2.3.2. Docking results of compound 5j in B-RAF
IC50 range 0.27
0.01 to 0.32
0.02 μM. These compounds have
Compound 5j docked at the same position as sorafenib and dis-
played good interaction as indicated by hydrogen bonding with two of
the key amino acids in the active site (Fig. 6). As a H-bond donor, the
thiourea NeH attached to the thiadiazine ring interacts with Asp594
(2.7 Å), while the anilino NeH interacts with the carboxylate side chain
of Glu501 (2.5 Å) of the αC helix where the chlorophenyl moiety is
fitted into the hydrophobic back pocket lined with the hydrophobic side
chains of Leu514, Ile527, Val471 and Val482. Moreover, the phenyl
group connected to the thiadiazine ring was included in π-π interaction
been selected to examine their effects on vital enzymes known to play
roles in the pathogenesis of NSCLC namely VEGFR2, B-RAF and MMP9
using sorafenib as a reference drug. The three compounds exerted
promising reduction to the enzyme activities and compound 5j was of
special interest in this regards. 5j also exhibited cell cycle arrest at G2-
M phase and pro-apoptotic, antinvasive and antimigration properties to
A549 cells. Also, docking study of 5j revealed that it bound to the
VEGFR2, B-RAF and MMP9 in a similar pattern as the co-crystallized
5

F.A.F. Ragab, et al.
BioorganicChemistry93(2019)103323
Fig. 3. Effect of 5j on the percentage of Annexin V-FITC-positive staining A549 cells.
ligand. The three compounds displayed safety against the normal lung
fibroblast cell line WI-38 which indicates high selectivity against A549
cancer cells and potential safety profile to normal cells. Thus, based on
this study we suggest that compounds 5d, 5i and 5j are promising
anticancer agents targeting key pathways implicated in NSCLC which
qualifies them as suitable candidates for further preclinical and clinical
trials.
University, Egypt. Infrared Spectra were performed on Shimadzu-FTIR
spectrophotometer, Faculty of Pharmacy, Cairo University, Egypt and
expressed in wave number (cm−1), using potassium bromide discs.
Compounds 1a-d and 3a-d have been synthesized as reported
[23,38–40].
4.1.1. Synthesis of compounds 5a-p
A mixture of 2-amino-5-aryl-6H-1,3,4-thiadiazine derivatives 3a-d
(2 mmol), and phenylisothiocyanate derivatives 4a-d (2 mmol) was
refluxed for 1 h in toluene. The reaction mixture was cooled to room
temperature and the acquired yellow precipitate was filtered, washed
with toluene then hexane, dried and crystalized using absolute ethanol.
4. Experimental
4.1. Chemistry
Unless otherwise mentioned, all chemicals were picked up from
commercial suppliers and used without any further purification.
Reactions were observed by TLC (Kieselgel 60 F254 precoated plates, E.
Merck, Germany) and the spots were visualized by exposure to UV lamp
at λ 254 nm. Determination of melting points was obtained by using of
an electro thermal melting point apparatus (Stuart Scientific Co.). 1H
NMR and 13C NMR spectra were determined in DMSO‑d6 and recorded
on 400 MHz spectrophotometer for 1H NMR and 100 MHz spectro-
photometer for 13C NMR (Bruker AG, Switzerland) at the Faculty of
Pharmacy, Cairo University; Chemical shift (δ) values are expressed in
parts per million (ppm) and coupling constants (J) in Hertz (Hz).
Elemental microanalyses and mass spectrometry were carried out at the
regional center for microbiology and biotechnology, Al-Azhar
4.1.1.1. 1-phenyl-3-(5-phenyl-6H-1,3,4-thiadiazin-2-yl) thiourea (5a).
Yield, 89%; mp: 180–182 °C; IR (KBr) νmax/cm−1 3305, 3263, 2924,
1519. 1H NMR δ 12.52 (s, 1H, NH, D2O exchangeable), 10.69 (s, 1H,
NH, D2O exchangeable), 7.90–7.88 (m, 2H, Ar-H), 7.56–7.50 (m, 5H,
Ar-H), 7.31 (t, J = 7.5 Hz, 2H, Ar-H), 7.08 (s, 1H, Ar-H), 3.87 (s, 2H,
CH2). 13C NMR δ 184.36, 166.90, 149.39, 140.41, 134.55, 130.80,
129.32, 128.84, 127.03, 123.83, 122.09, 23.99. MS (m/z): 326 (M+),
327 (M++1). Anal. Calcd for C16H14N4S2 (326.44): C, 58.87; H, 4.32;
N, 17.16. Found: C, 59.12; H, 4.39; N, 17.34.
4.1.1.2. 1-(4-chlorophenyl)-3-(5-phenyl-6H-1,3,4-thiadiazin-2-yl)thiourea
(5b). Yield, 90%; mp: 196–198 °C; IR (KBr) νmax/cm−1 3292, 3257,
6

F.A.F. Ragab, et al.
BioorganicChemistry93(2019)103323
Fig. 4. Effect of 5j on invasion and migration of A549 cells as indicated by (A) Transwell membrane assay and (B) Wound healing assay.
2902, 1519. 1H NMR δ 12.60 (s, 1H, NH, D2O exchangeable), 10.76 (s,
1H, NH, D2O exchangeable), 7.89–7.89 (m, 2H, Ar-H), 7.66–7.65 (m,
2H, Ar-H), 7.51–7.50 (m, 3H, Ar-H), 7.35 (d, J = 7.9 Hz, 2H, Ar-H),
3.88 (s, 2H, CH2). 13C NMR δ 184.26, 167.43, 149.47, 139.38, 134.47,
130.87, 129.33, 128.69, 127.49, 127.09, 123.51, 24.03. MS (m/z): 360
(M+), 361 (M++1), 362 (M++2). Anal. Calcd. for C16H13ClN4S2
(360.88): C, 53.25; H, 3.63; N, 15.53. Found: C, 53.48; H, 3.72; N,
15.61.
1H, NH, D2O exchangeable), 7.89–7.87 (m, 2H, Ar-H), 7.51–7.40 (m,
5H, Ar-H), 7.11–7.10 (m, 2H, Ar-H), 3.87 (s, 2H, CH2), 2.27 (s, 3H,
CH3). MS (m/z): 340 (M+), 341 (M++1). Anal. Calcd. for C17H16N4S2
(340.46): C, 59.97; H, 4.74; N, 16.46. Found: C, 59.62; H, 4.39; N,
16.64.
4.1.1.4. 1-(4-methoxyphenyl)-3-(5-phenyl-6H-1,3,4-thiadiazin-2-yl)
thiourea (5d). Yield, 90%; mp: 176–178 °C; IR (KBr) νmax/cm−1 3309,
3248, 2924, 1506. 1H NMR δ 12.40 (s, 1H, NH, D2O exchangeable),
10.59 (s, 1H, NH, D2O exchangeable), 7.91–7.87 (m, 3H, Ar-H),
7.60–7.49 (m, 3H, Ar-H), 7.41 (d, J = 7.8 Hz, 1H, Ar-H), 6.88–6.86
(m, 2H, Ar-H), 3.87 (s, 2H, CH2), 3.74 (s, 3H, OCH3). MS (m/z): 356
4.1.1.3. 1-(5-phenyl-6H-1,3,4-thiadiazin-2-yl)-3-(p-tolyl)thiourea
(5c). Yield, 87%; mp: 182–184 °C; IR (KBr) νmax/cm−1 3300, 3267,
2916, 1508. 1H NMR δ 12.46 (s, 1H, NH, D2O exchangeable), 10.62 (s,
Fig. 5. The best docking pose of 5j in the active site of VEGFR-2 in 2D and 3D style.
7

F.A.F. Ragab, et al.
BioorganicChemistry93(2019)103323
Fig. 6. The best docking pose of 5j in the active site of B-RAF in 2D and 3D style.
(M+), 357 (M++1). Anal. Calcd. for C17H16N4OS2 (356.46): C, 57.28;
H, 4.52; N, 15.72. Found: C, 57.49; H, 4.58; N, 15.68.
(M+), 375 (M++1), 376 (M++2). Anal. Calcd. for C17H15ClN4S2
(374.91): C, 54.46; H, 4.03; N, 14.94. Found: C, 54.67; H, 4.07; N,
15.08.
4.1.1.5. 1-(5-(4-chlorophenyl)-6H-1,3,4-thiadiazin-2-yl)-3-phenylthiourea
(5e). Yield, 85%; mp: 179–180 °C; IR (KBr) νmax/cm−1 3321, 3244,
2924, 1525. 1H NMR δ 12.54 (s, 1H, NH, D2O exchangeable), 10.72 (s,
1H, NH, D2O exchangeable), 7.90 (d, J = 8.5 Hz, 2H, Ar-H), 7.57 (d,
J = 8.6 Hz, 4H, Ar-H), 7.31 (t, J = 7.3 Hz, 2H, Ar-H), 7.08 (s, 1H, Ar-
H), 3.87 (s, 2H, CH2). MS (m/z): 360 (M+), 361 (M++1), 362
(M++2). Anal. Calcd. for C16H13ClN4S2 (360.88): C, 53.25; H, 3.63;
N, 15.53. Found: C, 53.44; H, 3.60; N, 15.69.
4.1.1.8. 1-(5-(4-chlorophenyl)-6H-1,3,4-thiadiazin-2-yl)-3-(4-
methoxyphenyl) thiourea (5h). Yield, 80%; mp: 178–180 °C; IR (KBr)
νmax/cm−1 3317, 3251, 2927, 1504. 1H NMR δ 12.44 (s, 1H, NH, D2O
exchangeable), 10.64 (s, 1H, NH, D2O exchangeable), 7.89 (d,
J = 8.3 Hz, 2H, Ar-H), 7.60–7.54 (m, 3H, Ar-H), 7.41 (d, J = 7.2 Hz,
1H, Ar-H), 6.96–6.87 (m, 2H, Ar-H), 3.94 (s, 2H, CH2), 3.74 (s, 3H,
OCH3). 13C NMR δ 183.93, 166.24, 156.85, 156.09, 148.13, 147.82,
135.48, 133.65, 133.43, 129.35, 128.74, 125.10, 123.70, 114.07,
113.85, 55.69, 23.77. MS (m/z): 390 (M+). Anal. Calcd. for
C17H15ClN4OS2 (390.90): C, 52.23; H, 3.87; N, 14.33. Found: C,
52.19; H, 3.90; N, 14.58.
4.1.1.6. 1-(4-chlorophenyl)-3-(5-(4-chlorophenyl)-6H-1,3,4-thiadiazin-2-
yl) thiourea (5f). Yield, 78%; mp: 178–180 °C; IR (KBr) νmax/cm−1
3348, 3228, 2900, 1512. 1H NMR
δ 12.62 (s, 1H, NH, D2O
exchangeable), 10.78 (s, 1H, NH, D2O exchangeable), 7.91 (d,
J = 8.6 Hz, 2H, Ar-H), 7.71–7.51 (m, 4H, Ar-H), 7.35 (d, J = 8.0 Hz,
2H, Ar-H), 3.87 (s, 2H, CH2). MS (m/z): 394 (M+) 395 (M++1) 396
(M++2). Anal. Calcd. for C16H12Cl2N4S2 (395.33): C, 48.61; H, 3.06; N,
14.17. Found: C, 48.50; H, 3.12; N, 14.05.
4.1.1.9. 1-phenyl-3-(5-(p-tolyl)-6H-1,3,4-thiadiazin-2-yl)thiourea
(5i). Yield, 89%; mp: 186–188 °C; IR (KBr) νmax/cm−1 3304, 3253,
2916, 1519. 1H NMR δ 12.48 (s, 1H, NH, D2O exchangeable), 10.67 (s,
1H, NH, D2O exchangeable), 7.79 (d, J = 8.0 Hz, 2H, Ar-H), 7.72–7.57
(m, 2H, Ar-H), 7.31 (d, J = 7.8 Hz, 4H, Ar-H), 7.08 (s, 1H, Ar-H), 3.84
(s, 2H, CH2), 2.37 (s, 3H, CH3). MS (m/z): 340 (M+), 341 (M++1).
Anal. Calcd. for C17H16N4S2 (340.46): C, 59.97; H, 4.74; N, 16.46.
Found: C, 60.17; H, 4.76; N, 16.80.
4.1.1.7. 1-(5-(4-chlorophenyl)-6H-1,3,4-thiadiazin-2-yl)-3-(p-tolyl)
thiourea (5 g). Yield, 83%; mp: 184–186 °C; IR (KBr) νmax/cm−1 3286,
3267, 2920, 1516. 1H NMR δ 12.48 (s, 1H, NH, D2O exchangeable),
10.65 (s, 1H, NH, D2O exchangeable), 7.90 (d, J = 8.5 Hz, 2H, Ar-H),
7.57 (d, J = 8.5 Hz, 3H, Ar-H), 7.39 (s, 1H, Ar-H), 7.16–7.11 (m, 2H,
Ar-H), 3.86 (s, 2H, CH2), 2.27 (s, 3H, CH3). 13C NMR δ 184.20, 166.44,
148.15, 1380.34, 137.91, 135.48, 134.39, 133.44, 130.81, 129.37,
129.06, 128.75, 128.51, 126.22, 122.13, 23.78, 20.98. MS (m/z): 374
4.1.1.10. 1-(4-chlorophenyl)-3-(5-(p-tolyl)-6H-1,3,4-thiadiazin-2-yl)
thiourea (5j). Yield, 90%; mp: 190–192 °C; IR (KBr) νmax/cm−1 3288,
3267, 2920, 1517. 1H NMR δ 12.54 (s, 1H, NH, D2O exchangeable),
10.73 (s, 1H, NH, D2O exchangeable), 7.79 (d, J = 7.9 Hz, 2H, Ar-H),
Fig. 7. The best docking pose of 5j in the active site of MMP9 in 2D and 3D style.
8

F.A.F. Ragab, et al.
BioorganicChemistry93(2019)103323
7.67–7.64 (m, 2H, Ar-H), 7.35–7.30 (m, 4H, Ar-H), 3.84 (s, 2H, CH2),
2.37 (s, 3H, CH3). 13C NMR δ 184.20, 167.72, 149.54, 140.81, 139.41,
131.67, 129.92, 128.67, 127.05, 123.57, 123.46, 23.97, 21.42. MS (m/
z): 374 (M+), 375 (M++1), 376 (M++2). Anal. Calcd. for
νmax/cm−1 3312, 3265, 2921, 1506. 1H NMR δ 12.34 (s, 1H, NH,
D2O exchangeable), 10.54 (s, 1H, NH, D2O exchangeable), 7.84 (d,
J = 8.6 Hz, 2H, Ar-H), 7.60–7.41 (m, 2H, Ar-H), 7.05 (d, J = 8.6 Hz,
2H, Ar-H), 6.96–6.87 (m, 2H, Ar-H), 3.83 (s, 5H, CH2, OCH3), 3.74 (s,
3H, OCH3). 13C NMR δ 183.70, 166.73, 155.95, 149.22, 133.79,
129.09, 128.70, 126.83, 125.08, 123.57, 114.84, 114.71, 114.04,
55.85, 55.69, 23.92. MS (m/z): 386 (M+), 387 (M++1). Anal. Calcd.
for C18H18N4O2S2 (386.49): C, 55.94; H, 4.69; N, 14.50. Found: C,
55.74; H, 4.74; N, 14.43.
C
17H15ClN4S2 (374.91): C, 54.46; H, 4.03; N, 14.94. Found: C, 54.62;
H, 4.06; N, 15.12.
4.1.1.11. 1-(p-tolyl)-3-(5-(p-tolyl)-6H-1,3,4-thiadiazin-2-yl)thiourea
(5k). Yield, 86%; mp: 185–187 °C; IR (KBr) νmax/cm−1 3315, 3273,
2926, 1506. 1H NMR δ 12.42 (s, 1H, NH, D2O exchangeable), 10.59 (s,
1H, NH, D2O exchangeable), 7.78 (d, J = 8.0 Hz, 2H, Ar-H), 7.62–7.40
(m, 2H, Ar-H), 7.31 (d, J = 8.0 Hz, 2H, Ar-H), 7.11–7.09 (m, 2H, Ar-H),
3.83 (s, 2H, CH2), 2.37 (s, 3H, CH3), 2.27 (s, 3H, CH3). 13C NMR δ
184.10, 166.77, 149.39, 140.67, 137.98, 131.79, 130.81, 129.90,
129.64, 129.26, 126.97, 126.79, 126.21, 122.06, 23.93, 21.42, 20.98.
MS (m/z): 354 (M+), 355 (M++1). Anal. Calcd. for C18H18N4S2
(354.49): C, 60.99; H, 5.12; N, 15.81. Found: C, 61.23; H, 5.21; N,
15.97.
4.2. Biological evaluation
Biological evaluation was performed in the laboratory of the
Egyptian company for the production of vaccines, sera and drugs
(VACSERA, Giza, Egypt).
4.2.1. MTT cell viability assay
A549 or WI-38 cells were originally provided by American Type
Culture Collection (ATCC, Manassas, VA, USA). The cells were rinsed
with 0.25% (w/v) trypsin, 0.53 mM EDTA solution and Ca2+/Mg2+
free PBS (pH 7.2). Dulbecco’s Modified Eagle’s Medium (DMEM) sup-
plemented with 10% FBS, 10 µg/ml of insulin and 1% penicillin/
streptomycin was used for the cell culture. After that they were plated
(100 µl/well) in 96-well cell culture plate overnight at 37 °C with 5%
CO2 and 95% humidity. 100 µl of serial 10-fold diluted sterile tested
compounds were added to final concentrations of 0.01–100 µM and
cultures were incubated for 24 h. The cell viability was identified using
MTT assay kit (Sigma-aldrich, Saint Louis, Missouri, USA). Supernatants
were wasted, 50 µl/well of (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide) (MTT) solution (5 mg/ml) was added and in-
cubated at 37 °C with 5% CO2 for 4 h. To dissolve the dark blue crystals,
100 µl of 0.04 N HCI in isopropanol was added to every well and mixed
thoroughly. The absorbance was measured on Robonik P2000 spec-
trophotometer at a wave length of 490 nm using DMEM as a blank
control. Results from three separate experiments were recorded and the
viable cells percentage was calculated [26].
4.1.1.12. 1-(4-methoxyphenyl)-3-(5-(p-tolyl)-6H-1,3,4-thiadiazin-2-yl)
thiourea (5l). Yield, 84%; mp: 187–190 °C; IR (KBr) νmax/cm−1 3319,
3248, 2924, 1525. 1H NMR δ 12.36 (s, 1H, NH, D2O exchangeable),
10.57 (s, 1H, NH, D2O exchangeable), 7.77 (d, J = 8.0 Hz, 2H, Ar-H),
7.61 (s, 1H, Ar-H), 7.42 (d, J = 7.3 Hz, 1H, Ar-H), 7.31 (d, J = 8.0 Hz,
2H, Ar-H), 6.87 (d, J = 7.6 Hz, 2H, Ar-H), 3.83 (s, 2H, CH2), 3.74 (s,
3H, OCH3), 2.36 (s, 3H, CH3). 13C NMR δ 183.79, 166.58, 149.36,
140.67, 133.75, 131.83, 129.89, 126.96, 125.09, 123.60, 114.06,
113.84, 55.69, 23.89, 21.41. MS (m/z): 370 (M+). Anal. Calcd. for
C
18H18N4OS2 (370.49): C, 58.35; H, 4.90; N, 15.12. Found: C, 58.46; H,
4.98; N, 15.40.
4.1.1.13. 1-(5-(4-methoxyphenyl)-6H-1,3,4-thiadiazin-2-yl)-3-
phenylthiourea (5m). Yield, 85%; mp: 183–185 °C; IR (KBr) νmax/cm−1
3293, 3246, 2907, 1522. 1H NMR
δ 12.45 (s, 1H, NH, D2O
exchangeable), 10.62 (s, 1H, NH, D2O exchangeable), 7.85 (d,
J = 8.8 Hz, 2H, Ar-H), 7.59 (s, 2H, Ar-H), 7.30 (t, J = 7.5 Hz, 2H, Ar-
H), 7.06 (d, J = 8.9 Hz, 3H, Ar-H), 3.83 (s, 5H, CH2, OCH3). MS (m/z):
356 (M+), 357 (M++1), 358 (M++2). Anal. Calcd. for C17H16N4OS2
(356.46): C, 57.28; H, 4.52; N, 15.72. Found: C, 57.21; H, 4.50; N,
15.66.
4.2.2. VEGFR2 kinase activity assay
VEGFR2 kinase activity was evaluated by means of VEGFR2 (KDR)
Kinase Assay Kit (San Diego, CA, USA) according to manufacturer's
instructions. In short, master mix (5X kinase buffer, 500 µM ATP, 50X
PTK substrate and water) and compounds 5d, 5i and 5j were added to
corresponding wells. The reaction was initiated by adding VEGFR2
(positive control) to their designated wells. The plates were incubated
at 30 °C for 45 min. After that, Kinase-Glo® MAX (Promega) was added
to the reaction mixture as a detection reagent and the reaction was
incubated again for 15 min at room temperature after which lumines-
cence was measured using TECAN spark microplate reader. The con-
centration of the test solutions which inhibited the activity of the en-
zyme by 50% was determined.
4.1.1.14. 1-(4-chlorophenyl)-3-(5-(4-methoxyphenyl)-6H-1,3,4-
thiadiazin-2-yl)thiourea (5n). Yield, 80%; mp: 182–184 °C; IR (KBr)
νmax/cm−1 3298, 3251, 2931, 1515. 1H NMR δ 12.49 (s, 1H, NH,
D2O exchangeable), 10.69 (s, 1H, NH, D2O exchangeable), 7.86 (d,
J = 8.7 Hz, 2H, Ar-H), 7.67–7.48 (m, 2H, Ar-H), 7.34 (d, J = 7.9 Hz,
2H, Ar-H), 7.06 (d, J = 8.7 Hz, 2H, Ar-H), 3.83 (s, 5H, CH2, OCH3). 13
C
NMR δ 183.93, 166.24, 156.85, 156.09, 148.13, 147.82, 135.48,
133.65, 133.43, 129.35, 128.74, 125.10, 123.70, 114.07, 113.85,
55.69, 23.77. MS (m/z): 390 (M+). Anal. Calcd. for C17H15ClN4OS2
(390.90): C, 52.23; H, 3.87; N, 14.33. Found: C, 52.47; H, 3.76; N,
14.58.
4.2.3. B-RAF kinase activity assay
4.1.1.15. 1-(5-(4-methoxyphenyl)-6H-1,3,4-thiadiazin-2-yl)-3-(p-tolyl)
thiourea (5o). Yield, 83%; mp: 176–178 °C; IR (KBr) νmax/cm−1 3300,
3253, 2914, 1506. 1H NMR δ 12.38 (s, 1H, NH, D2O exchangeable),
10.57 (s, 1H, NH, D2O exchangeable), 7.85 (d, J = 8.5 Hz, 2H, Ar-H),
7.61–7.31(m, 2H Ar-H), 7.26–7.04 (m, 4H Ar-H), 3.83 (s, 5H, CH2,
OCH3), 2.27 (s, 3H, CH3). 13C NMR δ 183.96, 166.91, 161.48, 149.25,
137.99, 129.26, 128.71, 126.83, 123.19, 122.05, 114.72, 55.84, 23.97,
20.97. MS (m/z): 370 (M+), 371 (M++1), 372 (M++2). Anal. Calcd.
for C18H18N4OS2 (370.49): C, 58.35; H, 4.90; N, 15.12. Found: C, 58.72;
H, 4.82; N, 14.93.
The inhibitory effects of 5d, 5i and 5j on B-RAF (V600E) kinase
activity were evaluated by means of B-RAF V600E Kinase Assay Kit
(San Diego, CA, USA) according to manufacturer's instructions. In short,
master mix (5X kinase buffer, 500 µM ATP, 5X B-RAF substrate, and
water) and the compounds were added to corresponding wells. The
reaction was initiated by adding B-RAF (V600E) or B-RAF WT (positive
control) to their designated wells. The plates were incubated at 30 °C
for 45 min. After that, Kinase-Glo® MAX (Promega) was added to the
reaction mixture as a detection reagent and the reaction was incubated
again for 15 min at room temperature after which luminescence was
measured using TECAN spark microplate reader. The concentration of
the test solutions which inhibited the activity of the enzyme by 50%
was determined.
4.1.1.16. 1-(4-methoxyphenyl)-3-(5-(4-methoxyphenyl)-6H-1,3,4-
thiadiazin-2-yl)thiourea (5p). Yield, 78%; mp: 171–173 °C; IR (KBr)
9

F.A.F. Ragab, et al.
BioorganicChemistry93(2019)103323
4.2.4. MMP9 inhibition screening assay
compounds. (4) Placement method was adjusted to triangle matcher
in case of VEGFR2 and MMP9 while in case of B-RAF, the placement
method was adjusted to pharmacophore generated based on the in-
teraction of sorafenib with Glu501 and Asp594.5). The obtained poses
were ranked using London dG as the scoring function and were exposed
to ForceField refinement using the same scoring function. (6) Finally,
the best scoring poses of the docked compounds were recognized and
examined for protein-ligand interactions of the complexes.
The inhibitory effects of 5d, 5i and 5j on MMP9 were assayed using
MMP9 Inhibitor Screening Assay Kit ab139448 (Abcam, UK) according
to manufacturer's instructions. Briefly, MMP chromogenic substrate
(25 mM Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5), in-
hibitor (1.3 mM NNGH in DMSO), and human recombinant enzyme
(2.68 U/μL) were diluted and brought to reaction temperature (37 °C).
Then, colorimetric assay buffer was added to appropriate wells fol-
lowed by MMP enzyme, MMP inhibitor and compounds 5d, 5i and 5j
were incubated for 60 min at 37 °C. The reaction was initiated by
adding MMP substrate to each well and the colour intensity was mea-
sured at 412 nm using Robonik p2000 EIA reader. The concentration of
the compounds which inhibited the activity of the enzyme by 50% was
determined and compared to that of sorafenib.
Declaration of Competing Interest
The authors have declared no conflict of interest
Appendix A. Supplementary material
4.2.5. DNA-flow cytometry analysis
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103323.
A549 cells at a density of 1 × 106 cells were subjected to 0.32 µM of
compound 5j or to 0.002% DMSO as a control for 24 h. The cells were
separated by trypsinization, rinsed in ice-cold PBS and fixed in ice cold
70% ethanol. They were preserved in ethanol at 4 °C for at least 2 h then
harvested by centrifugation. Subsequently, cells were washed in PBS
and stained through Cycle TEST™ plus DNA Reagent Kit (ab139418
Propidium Iodide Flow Cytometry Kit) (Becton Dickinson Biosciences,
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