Journal of Molecular Structure 1274 (2023) 134549

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Journal of Molecular Structure

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Anticancer potency of N(4)-ring incorporated-5-methoxyisatin

thiosemicarbazones
Upendra Chaudhary a, Dawa Dawa b, Indranil Banerjee b, Shivani Sharma b, Kuldeep Mahiya c,
Abdur Rauf d, Yuba Raj Pokharel b,∗, Paras Nath Yadav a,∗
a
Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal
b
Faculty of Life Science and Biotechnology, South Asian University, Akbar Bhawan, Chanakyapuri, New Delhi-110021, India
c
Department of Chemistry, F G M Government College, Adampur, Mandi Adampur, Hisar-125052, Haryana, India
d
Department of Chemistry, University of Swabi, Anbar-23561, Khyber Pakhtunkhwa, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: (Z)-N’-(5-methoxy-2-oxoindolin-3-ylidene)thiomorpholine-4-carbothiohydrazide (MeOIstTmor), (Z)-N’-(5-

Received 24 August 2022 methoxy-2-oxoindolin-3-ylidene)-2,6-dimethylmorpholine-4-carbothiohydrazide (MeOIstDmMor), (Z)-N’-
Revised 6 November 2022
(5-methoxy-2-oxoindolin-3-ylidene)morpholine-4-carbothiohydrazide (MeOIstMor) and (Z)-2-(5-methoxy-
Accepted 12 November 2022
2-oxoindolin-3-ylidene)-N,N-dimethylhydrazine-1-carbothioamide (MeOIstDm) were synthesized and
Available online 17 November 2022
characterized by elemental analysis, FT-IR, 1 H NMR, 13 C NMR, UV–Vis, ESI-HRMS and single crystal X-
Keywords: ray analysis. Molecular docking studies showed that compound MeOIstDmMor interacted strongly with
Anticancer activities VEGFR2 via hydrogen bonding. The anticancer activities of the synthesized compounds were tested
Breast cancer against breast cancer (MCF-7), skin cancer (A431), and lung cancer (A549) for cell viability, cell cycle
Cell cycle arrest arrest and western blot analysis. The compounds exhibited significant anticancer potency in micromo-
Crystal structure lar concentration (IC50 , 2.52–7.41 μM). The compound MeOIstDmMor was found G0/G1 cell cycle arrest
Lung cancer
in A431 cells and inhibited C-Jun, β -catenin, Akt proteins involve in cell proliferation. Among the four
5-methoxyisatin, Skin cancer
Thiosemicarbazones
compounds, compound MeOIstDmMor exhibited strong anticancer potency in A549 (IC50 , 2.52 μM) than
rest of the compounds. Similarly, compound MeOIstMor exhibited high anticancer activity in MCF-7, IC50 ;
2.93 μM.
© 2022 Elsevier B.V. All rights reserved.

1. Introduction of 18.31->50 μg/mL [4]. Some cancer cell lines like MGC-803, PC-3
and SW620 were highly sensitive to moxifloxacin/gatifloxacin-
According to the recently released GLOBOCAN data estimated 1,2,3-triazoleisatin hybrid [5]. One of the derivatives of isatin,
2.3 million new cancer cases in 2020. Breast cancer is the most Sunitinib (SU011248) prevents many members of the split-kinase
common cancer in women in both developing and developed domain group of receptor tyrosine kinases (RTKs), such as the
countries [1]. Isatin and its derivatives such as tryptanthrin, vascular endothelial growth factor receptors (VEGFRs) types 1
2-oxindoles, indirubins, sunitinib, isatin thiosemicarbazone and and type 2 (FLT1 and FLK1/KDR), platelet-derived growth factor
others are pharmacologically active compounds with anticancer, receptors (PDGFR-α and PDGFR-β ), stem cell factor receptor c-KIT,
antiviral, antibacterial, anticonvulsant, antituberculosis and antidia- FLT3 and RET kinases [6]. For the treatment of solid tumors
betic activity, as well as other properties [2]. A potent microtubule including breast and lung cancer, GlaxoSmithKline (GSK) created
destabilizing agent 5,7-Dibromo-N-alkylisatins suppress initial the anti-cancer drug Lapatinib, which includes a sulphone moi-
tumor growth in vivo, induce apoptosis and depolymerize mi- ety. It has been shown to target the epidermal growth factor
crotubules [3]. Isatin derivative sparfloxacin showed anticancer pathway [7]. Isatin derivatives inhibit tyrosine kinases (TKIs)
potency against HepG2 (human hepatic carcinoma cells), SW480 and cyclin-dependent kinases (CDKs) through binding with ATP
(human colon adenocarcinoma cells), A549 (human lung carci- pocket as well as inhibiting caspases [8]. Thiosemicarbazones
noma cells) and HeLa (human cervical cancer cells) with an IC50 are effective against a variety of tumors, including leukemia,
pancreatic cancer, breast cancer, non-small cell lung cancer, cer-
vical cancer, prostate cancer, and bladder cancer [9]. According
∗
Corresponding authors.
to SAR investigations of the synthesized thiosemicarbazones, the
E-mail addresses: [email protected] (Y.R. Pokharel), [email protected] (P.N. Ya- existence of lipophilic/inductively electron withdrawing groups (Cl,
dav). F, F3 CO, NO2 ) at position-5 of the isatin moiety had a key role in

https://doi.org/10.1016/j.molstruc.2022.134549
0022-2860/© 2022 Elsevier B.V. All rights reserved.
U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

causing or boosting various activities, especially urease inhibition were synthesized by the procedure of Scovill [19]. (Z)-N’-(5-
[10]. 2-Pyridineformamide thiosemicarbazones exerted a cytotoxic methoxy-2-oxoindolin-3-ylidene)thiomorpholine-4-carbothiohy-
effect on PANC-1 cells in NDM, including apoptosis, in micromolar drazide (MeOIstTmor), (Z)-N’-(5-methoxy-2-oxoindolin-3-
concentration [11]. By using MTT assay, it was revealed to be more ylidene)−2,6-dimethylmorpholine-4-carbothiohydrazide (MeOIst-
powerful towards MCF-7, A431, A375, and Hela cell lines, with IC50 DmMor), (Z)-N’-(5-methoxy-2-oxoindolin-3-ylidene)morpholine-
of 0.9 μM for MCF-7 [12]. α -N-Heterocyclic thiosemicarbazones 4-carbothiohydrazide (MeOIstMor) and (Z)−2-(5-methoxy-2-
inhibit ribonucleotidereductase (RR) which repair DNA synthesis oxoindolin-3-ylidene)-N,N-dimethylhydrazine-1-carbothioamide
in cancer chemotherapeutics [13]. Thiosemicarbazones activity is (MeOIstDm) were synthesized by refluxing the equimolar mixture
believed to be the result of increased interactions between their of respective thiosemicarbazide and 5-methoxyisatin (2.82 mmol)
hydrophobic substituents and the hydrophobic residues in the TYR in absolute ethanol (20 mL) and glacial acetic acid (3-drops) at
cavity via van der Waal’s force, and even the potential of their 80 °C for the duration of 6 h (Scheme 1) [20]. The refluxed product
N–N–S tridentate scaffold to bind catalytic copper ions in the en- was allowed to cool at room temperature, washed with absolute
zyme [14]. Recently we have reported potent anticancer activity of alcohol and dried. The product was re-crystallized with EtOH and
5-nitrosatin-4-(1-(2-pyridyl)piperazinyl)−3-thiosemicarbazone, 5- again dried.
nitroisatin-4-thiomorpholinyl-3-thiosemicarbazone, 5-nitroisatin-
4,4-dimethyl-3-thiosemicarbazone and their copper complexes on 2.3.1. (Z)-N’-(5-methoxy-2-oxoindolin-3-ylidene)thiomorpholine-4-
MCF-7, MDA-MB-231, A431 and PNT2 cells [15,16,17]. Similarly, carbothiohydrazide
3-hydroxypyridine-2-carboxaldehyde N(4)-methyl and pyrrolidinyl (MeOIstTmor)
thiosemicarbazones and their Zn(II) complexes were investigated Yield: 56.70%; Color: Orange; MP: 198 °C; Anal. Calc (%) for:
from our laboratory for their ability to inhibit cell growth in PC3, C14 H16 N4 O2 S2 (336.43): C, 49.98; H, 4.79; N, 16.65; Found: C,
HeLa, DU145, A431 and A549 cells [18]. 49.42; H, 4.60; N, 16.26. FTIR (ν , cm−1 ): 3172–3037 (br, H–N; in-
In the present study, 5-methoxyisatin with N(4)-ring substi- dole&azomethine), 1685 (s, C=O), 1539 (s, C=N), 1174, 783 (s, C=S),
tuted thiosemicarbazones were synthesized with the anticipation 1155 (s, N-N), 1282 (s, O-CH3 ). 1 H NMR (δ , ppm): 13.23 (s, 1H, HN-
that such modification could result in significant anticancer activ- C=S), 11.10(s, 1H, Indole-NH), 7.01 (d, 1H, C7-H), 6.92 (d, 1H, C4-
ity. The molecular and isomeric structures of MeOIstDmMor and H), 6.86 (d, 1H,C6-H), 4.24 (s, 3H, -OCH3 ), 3.76 (t, 4H, aliphatic-
MeOIstDm were determined by X-ray single crystal diffraction anal- C11, C14-H), 2.81 (t, 4H, aliphatic-C12, C13-H). 13 C NMR (δ ,
ysis. Anticancer activities of the compounds were evaluated against ppm): 179.80(C10), 163.25(C2), 155.80(C5), 136.08(C3), 134.96(C9),
A549, MCF-7 and A431 cell lines. Additionally, molecular docking 121.20(C7), 117.63(C8), 112.43(C6), 105.95(C4), 56.07(OCH3 ), 53.36
study of MeOIstDmMor was completed on VEGFR2 enzyme. (C11, C14), 27.04 (C12, C13). ESI-HRMS [m/z, Found (Calc.)]:
337.0789 (337.4404) [M+H]+ , 359.0611 (359.4221) [M+Na]+ . UV–
2. Experimental Vis [λmax (nm) (CHCl3 )]:283, 356.

2.1. Materials and methods 2.3.2. (Z)-N’-(5-methoxy-2-oxoindolin-3-ylidene)−2,

6-dimethylmorpholine-4-carbothiohydrazide (MeOIstDmMor)
5-Methoxyisatin, thiomorpholine, 2, 6-dimethylmorpholine, Yield: 79.92%; Color: Red; MP: 206 °C; Anal. Calc (%) for:
morpholine, dimethylamine and N-methyl aniline were purchased C16 H20 N4 O3 S (348.42): C, 55.16; H, 5.79; N, 16.08; found: C,
from Alfa Aesar. Carbon disulphide (Qualigens fine chemicals), 55.11; H, 5.54; N, 16.04. FTIR (ν , cm−1 ): 3176–3064 (br, H–N;
sodium chloroacetate (Chemical center, India), hydrazine hydrate, indole&azomethine), 1681 (s, C=O), 1568 (s, C=N), 1179, 697(s,
98% (Fisher scientific), acetonitrile (Merck), methyl alcohol (Fisher C=S), 1151 (s, N-N), 1251 (s, O–CH3 ).1 H NMR (δ , ppm): 13.19
scientific), ethyl alcohol (Merk), diethyl ether (Merck), glacial acetic (s, 1H, HN-C=S), 11.04 (s, 1H, Indole-NH), 6.94 (d, 1H, C7-H),
acid (Fisher scientific), concentrated hydrochloric acid (Merck) and 6.94(d, 1H, C4-H), 6.88 (d, 1H,C6-H), 3.68 (t, 4H, aliphatic-C12,
sodium hydroxide (Fisher Scientific) were used as obtained. C13-H), 3.74 (s,3H,OCH3 ), 2.94 (t, 4H, aliphatic-C11, C14-H), 1.16
(d,6H,methyl-C15,C-16).13 C NMR (δ , ppm): 179.58 (C10), 163.18
2.2. Instruments (C2), 155.75 (C5), 136.02(C3), 134.80 (C9), 121.18 (C7), 117.53
(C8), 112.36 (C6), 105.65 (C4), 71.43 (C12,C13), 55.97 (OCH3 ),
FT-IR spectra were recorded using SHIMADZU, Tracer 100, melt- 55.32(C11,C14), 18.85 (C15,C16). ESI-HRMS [m/z, Found (Calc.)]:
ing points were determined on Philip Harris Melting Point Appara- 349.1332 (349.428) [M+H]+ , 371.1150 (371.40) [M+Na]+ .UV–Vis
tus, and UV–Visible spectra were recorded in the range of 600– [λmax (nm) (CHCl3 )]:284, 355.
200 nm using a SPECORD®200 PLUS UV-visible spectrophotometer
in MeOH and in CHCl3 solution at Central Department of Chem- 2.3.3. (Z)-N’-(5-methoxy-2-oxoindolin-3-ylidene)morpholine-4-
istry, Tribhuvan University (TU), Nepal. The elemental analysis was carbothiohydrazide (MeOIstMor)
performed using a LECO Truspec Micro analyzer, at IIT Madras, The compound (MeOIstMor) has been synthesized and charac-
India. NMR spectra were recorded in DMSO-d6 by using TMS as terized by K.N. Aneesrahman [21].
an internal standard on Bruker advance III HD NMR spectrometer,
400 MHz spectrometer and Mass spectra were recorded using ESI- 2.3.4. (Z)−2-(5-methoxy-2-oxoindolin-3-ylidene)-N,
HRMS on Bruker IMPACT HD liquid chromatography Mass Spec- N-dimethylhydrazine-1-carbothioamide (MeoIstDm)
trometer at the Department of Chemistry, Savitribai Phule Pune Yield: 65.95%; Color: Orange; MP: 122–124 °C; Anal. Calc (%)
University, India. Bruker D8 VENTURE diffractometer with PHOTON for: C12 H14 N4 O2 S (278.33): C, 51.78; H, 5.07; N, 20.13; found:
II detector was employed for single crystal data collection at IIT C, 51.87; H, 4.97; N, 20.02%. FTIR (ν , cm−1 ): 3267-3176 (m, H–
Madras, India. N; indole&azomethine), 1691 (s, C=O), 1537 (s, C=N), 1182, 775
(s, C = S), 1126 (s, N-N), 1300 (s, O-CH3 ). 1 H NMR (δ , ppm):
2.3. Preparation of N(4) substituted thiosemicarbazones 13.48 (s, 1H, HN-C = S), 11.07(s, 1H, Indole-NH), 7.06 (d, 1H,
C7-H), 6.89 (d, 1H, C4-H), 6.83 (d, 1H,C6-H), 3.73 (s, 3H, -
N(4)-Substituted thiosemicarbazides; Thiomorpholine-4-carbo- OCH3 –), 3.50 (t, 3H, aliphatic-C11), 3.36 (t, 3H, aliphatic-C12), 13 C
thiohydrazide, 2,6-dimethylmorpholine-4-carbothiohydrazide, Mor- NMR (δ , ppm): 180.37(C10), 163.39(C2), 156.64(C5), 133.86(C3,C9),
pholine-4-carbothiohydrazide, N,N-dimethylhydrazinecarbothioamide 121.59(C7), 118.28(C8), 112.68(C6), 106.56(C4), 56.26(-OCH3 –),

2
U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

Scheme 1. Synthesis of N(4) Thiomorpholinyl/ (2,6-dimethyl)morpholinyl/ Morpholinyl/ Dimethyl amine 5-methoxyisatin Thiosemicarbazones.

29.92 (C11,C12). ESI-HRMS [m/z, Found (Calc.)]: 279.091 (279.338) clumping of cells. After that samples were centrifuged and given a
[M+H]+ , 301.0734 (301.31) [M+Na]+ . UV–Vis [λmax (nm)(CHCl3 )]: DPBS wash twice. After adding PI, cells were incubated at 40 °C for
284, 350. 30 min. The cells were then analyzed with Flow Cytometry.

2.4. Anticancer screening 2.4.5. Cell cycle analysis

For cell cycle analysis, around 1.5 × 105 cells/well were seeded
2.4.1. Cell lines in a six-well plate and were harvested after 48 h of treatment. Af-
A549, MCF-7 and A431 cell lines were cultured in complete ter three PBS washes, cells were fixed with 80% ice-cold ethanol.
DMEM media. After incubation with RNase and PI stain for 30 min, cells were
analyzed by FACS machine and cells in different phase were quan-
2.4.2. Cell viability assay tified. The percentage of cell with G0/G1 DNA content was used as
Cell viability of A431, MCF-7, and A549 cells were assessed by a measure of apoptosis.
crystal violet assay. Approximately 5 × 103 cells were seeded in
each well of 96 wells plate. Cells were treated with different con- 2.4.5. Immunoblotting
centration of compounds and incubated for 72 h. After 72 h, the 2.4.5.1. Cell lysate preparation. After 48 h incubation, cells were
media was discarded. Cells were stained with 80 mL (0.4%) crys- washed with DPBS and 50–80 μL of 2X SDS lysis buffer was added
tal violet that was prepared in 50% methanol and incubated for (depends upon confluence of cells). The cells were collected with
30 min on a bench rocker with 20 oscillations per minute. Af- the help of cell scrapper and collected into the MCT. After that cells
ter that, cells were washed by dipping in a beaker filled with were sonicated at 30% of amplification for 10 s with 10 s interval
tap water; this prevents the washout of cells. Culture plates were for three cycles. Sonication was done by keeping MCT in a 50 mL
kept overnight for air dry at room temperature. Next day, 150 μL beaker filled with ice to avoid protein degradation. Samples were
of methanol was added in each well and kept on a rocker for centrifuged at 16,0 0 0 g for 20 min at 40 °C. Supernatant was col-
30 min. Finally, optical density was measured in micro-plate reader lected in fresh MCT and kept at −80 °C.
at 570 nm.
2.4.5.2. Protein estimation by bca kit. Pierce TM BCA protein assay
2.4.3. Colony formation assay kit was used to know the total protein concentration in the sam-
A431 cells were analyzed to form colonies. 10 0 0 cells were ples. BCA reagent A and reagent B were mixed in 50:1 ratio respec-
seeded in each well of six-well plates and incubated for 24 hrs. tively. Then, 200 μL mixtures were added in each well containing
The cells were treated with the respective compounds in differ- 25 μL of protein samples (Protein samples can be diluted by adding
ent concentration (0.1% DMSO as a control, 0.3 μM, 1 μM, 3 μM, PBS). After that, plate was incubated for 30 min at 37 °C. The ab-
and 10 μM). Fresh media was added by replacing the used one in sorbance was taken at 562 nm. After plotting the absorbance (Y-
every 72 h, followed by the treatment with respective concentra- axis) vs concentration graph (X-axis) we got an equation. From that
tion of compounds. The cell culture was maintained for 10 days equation the concentration of protein samples was calculated. To
with change of media after every two days. After 10 days colonies see the expression of particular protein, in each well of SDS gel,
were washed with DPBS, fixed with methanol and stained with we should add equal amount (25 μg) of total protein. Thus, while
0.4% crystal violet which was prepared in 50% methanol. The cul- dividing 25 μg with concentration of protein each MCT gives the
ture plate was air dried and colonies were counted using Image J volume of protein should be withdrawn for one well. Protein con-
software. centration and loading dye should be in 1:0.25.

2.4.4. Propidium iodide staining 2.4.5.3. SDS gel running. According to the molecular weight of pro-
Around 1.5 × 105 cells/well were seeded in a six-well plate tein that is of interest, the percentage of gel is determined. For
and after 24 h, treated with different concentration of compound higher molecular weight protein gel with lower percentage of gel
(0.1% DMSO as a control, 0.3 μM, 1 μM, 3 μM, 10 μM). Cells were is prepared and vice-versa. After transferring the gel into running
incubated for 48 h. The cell along with media from the same buffer and then samples were loaded into the separate well.
tube was harvested after 48 h in 1.5 ml micro-centrifuge tubes.
Micro-centrifuge tubes were centrifuged at 30 0 0 rpm for 5 min 2.4.5.4. Transferring proteins from gel to PVDF membrane. 1X trans-
and washed with DPBS twice. Immediately after that, 80% ice- fer buffer was prepared and kept in ice to chill down. Foam pads
cold ethanol was added while vortexing the tube to minimize the and filter paper was soaked in 1X transfer buffer. PVDF membrane

3
U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

was taken the same size of the gel and activated by dipping it in at the range of 180.37–179.58 ppm [37]. The characteristic –
Methanol for 2 min. On a transfer cassette, the gel sandwich was C = N (C3) and –C = O (C2) peaks were observed at 136.08–
prepared in the order: Sponge, filter paper, gel, PVDF membrane, 133.86 ppm and 163.39–163.18 ppm respectively [38,39]. The aro-
filter paper, and sponge. While putting PVDF membrane above gel, matic carbons (C4–C9) of the isatin ring were observed at 106.56–
1X transfer buffer was flooded to avoid the bubbles in between. 105.65(C4), 156.64–155.75 (C5), 112.43–111.68 (C6), 121.59–121.18
The transfer cassette was then arranged in a transfer apparatus, (C7), 118.28–117.53 (C8) and 134.96–133.86 ppm (C9) [40,41]. The
which was kept on the gel tank filled with 1X transfer buffer. Ice- C(5) (156.64–155.75) carbons atom shifted downfield due to the
pack is kept inside the tank and tank is kept on the ice box. Trans- presence of methoxy group (OCH3 –) and its carbon peaks were
fer was done at 90 Vs for 2 h. observed at 56.26–55.97 ppm [22]. The signals of N(4) thiomor-
pholinyl group (MeOIstTmor) carbon atoms (C11-C14) were seen at
2.4.5.5. Blocking the membrane. After the transfer is over, the PVDF 53.36–27.04 ppm [21] whereas the signals in N(4) 2,6-dimethyl
membrane is kept in 5% skimmed milk for one hour for the block- morpholinyl group (MeOIstDmMor) carbons atoms (C11–C14) of
ing of unspecific proteins. 5% skimmed milk was prepared in TBST. cyclic ring were observed at 71.43–55.32 ppm [14]. The signals of
3% BSA is also used as a blocking agent. methyl carbon atoms (C15-C16) in the compound (MeOIstDmMor)
were observed at 18.85 ppm whereas the signal of methyl car-
bon atoms (C11-C12) in the compound (MeOIstDm) were seen at
2.4.6. Data and statistical analysis
29.92 ppm [42] (Supplementary data S8–S10).
The results are expressed as the mean ± standard deviation
(SD). A Paired t-test was used to determine the significant differ-
3.3. ESI-HRMS spectrometry
ence between groups. A p-value< 0.05 was considered as signifi-
cant.
All the obtained mass spectral data of the synthesized thiosemi-
carbazones were in agreement with the calculated data of the pro-
3. Results and discussion posed molecular structures.
The protonated and alkali adduct analyte molecules were seen
3.1. FTIR spectroscopy using the positive mode of the ESI-HRMS investigations for the
mass spectral peaks of the compounds. The protonated molec-
The broad symmetrical stretching band in the range of 3267– ular ion [M+H]+ peaks of thiosemicarbazones were observed at
3037 cm−1 was assigned to v(N-H) of indole and azomethine v(N- m/z = 337.0789 (calc., 337.4404) (MeOIstTmor), m/z = 349.1332
H) moieties of thiosemicarbazones [22]. The absence of a stretch- (calc., 349.4280) (MeOIstDmMor) and m/z = 279.0910 (279.338)
ing band at about 250 0–260 0 cm−1 , specifies to v(S-H) [23] and (MeOIstDm) [21,43]. Beside the protonated peaks, the thiosemicar-
the presence of two strong bands specific to v(C = S) at 1182–1174 bazones showed the molecular ion [M+Na]+ peaks: m/z = 371.1150
cm−1 and 783–697 cm−1 indicated the existence of thiosemicar- (calc., 371.40 0 0) (MeOIstDmMor), m/z = 343.0838 (calc., 343.3565)
bazone in the thione tautomer [24]. Strong stretching bands ap- (MeOIstMor) and m/z = 301.0734 (calc., 301.31) (MeOIstDm).
peared in the thiosemicarbazone MeOIstTmor at 1685 cm−1 and In the compound MeOIstDmMor, the fragment N(4)−2,6-
1539 cm−1 ,respectively, assigned to v(C = O) and (C = N) of dimethylmorpholinyl [C16 H20 N4 O3 + H]+ cation caused the
thiosemicarbazones [25,26]. Similarly, the synthesized thiosemicar- prominent peak at m/z 317.1606 (calc., 317.3630) [44]. The pres-
bazones had two significant stretching bands at the range 1691– ence of fragment N(4) dimethylamine [C12 H14 N4 O2 + H]+ cation
1681 cm−1 and 1568–1537 cm−1 , respectively, that assigned to in the compound MeOIstDm accounts for the prominent peak at
v(C = O) and (C = N) [27]. The strong bands due to v(N-N) m/z 247.1185 (calc., 247.2731). Fragmentation is characterized by
of thiosemicarbazones appeared at 1155–1126 cm−1 [28]. Strong loss of one sulfur atom, abbreviated SH (m/z; 33 amu) [45]. Mass
bands of isatin moieties of thiosmecarbazones appeared at 1300– spectral data of the thiosemicarbazones agree well with those
1251 cm−1 , which were assigned to v(-OCH3 –) [29,30] (Supple- estimated for their putative structures [46] (Supplementary data
mentary data S5–S7). S11–S13).

3.2. NMR spectroscopy 3.4. UV–Vis spectroscopy

In the 1 H NMR (DMSO-d6 ) spectra of N(4) substituted thiosemi- UV-visible spectral data of the thiosemicarbazones were
carbazones the signals of highly polar H-N-C = S and indol-NH recorded in CHCl3 in the 60 0–20 0 nm range. The compounds ex-
protons were observed downfield as a singlet at 13.48–13.19 ppm hibited two broad absorption bands with varying intensities and
[21] and 11.10–11.04 ppm respectively [31]. A singlet peak at 11.10– shoulder like appearance in the region 284 nm and 356–350
11.04 ppm related to the NH adjacent to C = S, but absence of nm[37] attributed to n→π ∗ intraligand electronic transition viz.
peak at 4 ppm attributed to the S–H proton in the thiosemicar- the bands due to the electronic transition of azomethine (-C = N)
bazones indicate the existence of thione tautomer [32]. All the aro- and that due to thioamide (-HN-C = S) group [44,47,48]. The π →
matic protons of isatin moiety were seen as singlet or doublet sig- π ∗ transitions of the imine C = N double bond were seen at
nals at 7.06–6.83 ppm [33]. In the case of N(4) thiomorpholinyl 284 nm for all the synthesized thiosemicarbazones, the bands at
group (MeOIstTmor), the signals of ring -CH2 protons were found 356 nm are assigned to the electronic transition n → π ∗ of the
as a quartet at 3.76 ppm whereas in N(4) 2,6-dimethyl morpholinyl thiosemicarbazone moiety (C = S) [42,49]. Similar observations
group (MeOIstDmMor), the signals of ring -CH2 protons were ob- about the peak position and nature has been observed with isatin
served as a quartet at 3.68–2.94 ppm [34] and that of methyl pro- thiosemicarbazones [50] (Supplementary data S14–S15).
tons were observed as a triplet at 3.50 ppm [35] in the compound
MeoIstDm and triplet at 1.16 ppm in the compound MeoIstDmMor 3.5. X-ray crystallography
[21,27]. The signals of methoxy (-OCH3 –) protons were observed as
a singlet at 4.24–3.73 ppm in the thiosemicarbazones [36] (Supple- Single crystals of MeOIstDm and MeOIstDmMor suitable for X-
mentary data S5–S7). ray diffraction studies were grown by slow evaporation in 1:4 mix-
In the 13 C NMR (DMSO-d6 ) spectra of the compounds, the tures of CHCl3 and EtOH. The single crystal diffraction data were
HN–C = S (C10) signal of the thioamide carbon was observed collected at 298 K on Bruker D8 VENTURE diffractometer with

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U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

Table 1
Crystal data and structure refinement parameters.

Identification code MeOIstDm MeOIstDmMor

Empirical formula C12 H14 N4 O2 S C16 H22 N4 O4 S

Formula weight 278.33 366.43
Temperature/K 296.15 296.15
Crystal system monoclinic orthorhombic
Space group P21 /c Pna21
a/Å 9.357(2) 16.091(3)
b/Å 9.9370(19) 8.6078(14)
c/Å 14.207(3) 27.098(5)
α /° 90 90
β /° 104.929(9) 90
γ /° 90 90
Volume/Å3 1276.4(5) 3753.3(11)
Z 4 8
ρ calc g/cm3 1.448 1.297
μ/mm-1 0.258 0.200
F(000) 584.0 1552.0
Crystal size/mm3 0.35 × 0.25 × 0.2 0.15 × 0.12 × 0.1
Radiation Mo/Kα (λ = 0.71073) Mo/Kα (λ = 0.71073)
2θ range for data collection/° 5.06 to 64 6.932 to 49.998
Index ranges −13 ≤ h ≤ 13, -14 ≤ k ≤ 14, -21 ≤ l ≤ 21 −19 ≤ h ≤ 19, -10 ≤ k ≤ 10, -32 ≤ l ≤ 32
Reflections collected 47,022 74,084
Independent reflections 4418 [Rint = 0.0439, Rsigma = 0.0250] 6574 [Rint = 0.1318, Rsigma = 0.0648]
Data/restraints/parameters 4418/0/175 6574/25/467
Goodness-of-fit on F2 1.096 1.202
Final R indexes [I ≥ 2σ (I)] R1 = 0.0646, wR2 = 0.1546 R1 = 0.0997, wR2 = 0.1943
Final R indexes [all data] R1 = 0.1035, wR2 = 0.1768 R1 = 0.1280, wR2 = 0.2063
Largest diff. peak/hole / e Å−3 0.64/−0.42 0.34/−0.30
Flack parameter – 0.10(9)

PHOTON II detector with Mo/Kα radiation (λ = 0.71073 Å). The Table 2

Bond Lengths for MeOIstDm.
data were corrected for Lorentz and polarization effects. Multi-scan
absorption correction was applied. The structure was solved by di- Atom Atom Length/Å Atom Atom Length/Å
rect methods using ShelXT [51] and refined by full-matrix least- S1 C10 1.6735(19) N4 C5 1.416(3)
squares refinement techniques on F2 , using ShelXL-2018/3 [52]. All O1 C1 1.430(3) N4 C9 1.358(3)
calculations were done with the help of OLEX2 version 1.5 crys- O1 C2 1.377(2) C2 C3 1.390(3)
O2 C9 1.228(3) C2 C7 1.394(3)
tallographic software [53]. For the molecular graphics, the pro-
N1 N2 1.354(2) C3 C4 1.398(3)
gramme OLEX2 [53] and Mercury [54] were used. All non-hydrogen N1 C8 1.293(2) C4 C5 1.371(3)
atoms were refined anisotropically. All hydrogen atoms were fixed N2 C10 1.378(2) C5 C6 1.399(3)
geometrically with Uiso values of 1.2 times the Uiso values of their N3 C10 1.335(2) C6 C7 1.380(3)
respective carrier atoms. The hydrogen on lattice water were lo- N3 C11 1.467(3) C6 C8 1.455(3)
N3 C12 1.448(3) C8 C9 1.518(3)
cated from difference fourier map and refined using the riding
model. For MeOIstDm, a total of 47,022 reflections were measured
of which 4418 were unique and 3148 were considered observed (I
> 2σ (I)]. The final residual index are; R1 = 0.0646, wR2 = 0.1546 Rint is 13.2% and the Poor Data/Parameter Ratio of 7.21. The crystal
for the observed and R1 = 0.1035, wR2 = 0.1768 for all reflections is a weak diffractor and diffract very weakly beyond 2θ = 420 .
using 175 parameters. For MeOIstDmMor, a total of 74,084 reflec- Figs. 1 and 3
tions were measured of which 6574 were unique and 5048 were In compound MeOIstDmMor, a comparison of C-N and N-N
considered observed (I > 2σ (I)]. The final residual index are; R bond distances with typical single and double bond lengths
0.0997, Rw 0.1943 for the observed and R 0.1280, Rw 0.2063 for [C(9)-N(1) 1.322, C(5)-N(1) 1.405, C(10-N(3) 1.358, C(10)-N(4)
all reflections using 467 parameters 25 restraints. Details of the 1.334, C(16)-N(4) 1.473, C(11)-N(4) 1.428, C(8)=N(2) 1.279, N(2)-
crystallographic data and structure refinement for MeOIstDm, and N(3)1.354 Å] indicates that charge delocalization is widespread
MeOIstDmMor are given in Table 1. Selected bond lengths are given throughout the thiosemicarbazone skeleton. Similarly in com-
in Tables 2 and 3. CCDC reference numbers 2,194,144 (MeOIstDm) pound (MeOIstDm), the comparison of C-N and N-N bond dis-
and 2,194,145 (MeOIstDmMor) contains the supplementary crystal- tances with typical single and double bond lengths [C(9)-N(4)
lographic data for this paper. 1.358, C(10)-N(2) 1.378, C(10)-N(3) 1.335, C(11)-N(3) 1.467, C(12)-
N(3) 1.448, C(8)-N(1) 1.293, N(1)-N(2)1.354 Å] also indicates that
charge delocalization is widespread throughout the thiosemicar-
3.5.1. Crystal structure description bazone skeleton [55]. All bond distances are normal in the 5-
The structure of compound MeOIstDm and MeOIstDmMor to- methoxyisatin moiety in the compound (MeOIstDmMor), except
gether with the atom labeling scheme is shown in Fig. 2 and for the elongated C(9)-C(8) single bond is 1.525 Å whereas in
Fig. 3 respectively. The compound MeOIstDm crystallizes in mon- compound (MeOIstDm) C(9)-C(8) single bond is 1.518 Å. In 5-
oclinic space group P21 /c with one molecule in the asymmetric methoxyisatin moiety C(9)-O(2) distance for studied MeOIstDmMor
unit (Fig. 2) and the compound MeOIstDmMor crystallizes in or- crystal structure is 1.242 Å whereas in (MeOIstDm) C(9)-O(2) bond
thorhombic Pna21 space group with two molecule of compound distance is 1.228 Å [49]. In comparison to the 5-methoxyisatin
and two lattice water molecules in the asymmetric unit (Fig. 3). derivative (Z)-N-(5-methoxy-2-oxoindolin-3-ylidene)pyrrolidine-1-
The quality of data for MeOIstDmMor is not very good. The value of carbothiohydrazide, the bond lengths C-N, N-N, C(9)-C(8), and

5
U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

Fig. 1. Structure and numbering of 5-MethoxyisatinThiosemicarbazones.

shows H-bonding with hydrogen atom H8A of lattice water

molecule (O8–H8A……S2 = 2.535(4) Å), this water molecule
further involved in H-bonding with second water molecule
(O7–H7C……O8 = 2.097(10) Å), and oxygen atom (O7) of this
water molecule involved in H-bonding with hydrogen atom
attached to ring nitrogen (N1) of neighboring molecule (N1–
H1……O7 = 2.080(8) Å). The oxygen atom on this molecule
involved in intra as well as intermolecular H-bonding, it shows in-
tramolecular H-bonding with hydrogen (H3A) attached to nitrogen
(N3–H3A……O2 = 1.989(6) Å) and intermolecular H-bonding with
hydrogen H5 attached to the ring nitrogen (N5) of neighboring
molecule (N5–H5……O2 = 2.028(6) Å) forming a two dimensional
H-bonding network (Fig. 5).
Fig. 2. ORTEP diagram of compound MeOIstDm drawn in 30% thermal probability
ellipsoids showing atomic numbering scheme.
3.6. Molecular docking studies of compound MeOIstDmMor

Molecular docking studies were carried out by using Molec-

C(9)-O(2) of the compounds MeOIstDmMor and MeOIstDm are sim- ular Operating Environment (MOE) software package. Three-
ilar [21]. Indole-3-thiosemicarbazone, {1.696 (3) Å}, and 5-bromo dimensional (3D) crystal structure of VEGFR2 was obtained from
indole-3-thiosemicarbazone, {1.699(2) Å} have identical C = S Protein Data Bank (PDB). The accession code for the downloaded
bond lengths of 1.679(9) Å in MeOIstDmMor and 1.6735(19) Å in enzyme was 4ASD. Before docking studies, the docking protocol
MeOIstDm [56]. The bond angle of N2-N1-C8-C6 is 179.62(19)°, in- was validated by using re-dock method. Native co-crystallized lig-
dicating that MeOIstDm of the imine N2 atom undergone sp2 hy- and sorafenib was re-docked into the binding site of prepared en-
bridization whereas the torsion angle C5-C4-C8-N2 is 179.11(9)°, zyme. Comparison of the binding orientation was carried out be-
indicating that the compound MeOIstDmMor has an E (trans) tween the re-docked ligand and experimental ligand. The validated
configuration, resulting in the double bond character of C = N protocol with root-mean square deviation less than 2 Å was used
[47] (Supplementary data S16–S31). for further docking simulations. The 3-D / 2-D interaction plots of
The two molecules in the asymmetric unit of MeOIstDm- native ligand sorafenib are shown in Fig. 6: (a-b). Native ligand
Mor are involved in intra as well as intermolecular H-bonding sorafenib forms hydrogen bond interactions with Asp1044, Cys919
via two lattice water molecules (Fig. 4). The sulfur atom S2 and Cys919. While a π -π stacking interaction was also observed

Fig. 3. ORTEP diagram of MeOIstDmMor drawn in 20% thermal probability ellipsoids showing atomic numbering scheme. The asymmetric unit contains two crystallographi-
cally independents units and two water molecule in the crystal lattice.

6
U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

Fig. 4. Hydrogen bonding interactions in the crystal lattice of MeOIstDmMor viewed along the crystallographic c-axis (Hydrogen bonding interactions with max D–A distance
2.9 Å and minimum angle 120°).

Fig. 5. Two dimensional hydrogen bonding network in the crystal lattice of MeOIstDmMor viewed along crystallographic a-axis. (Hydrogen bonding interactions with max
D–A distance 2.9 Å and minimum angle 120°).

7
U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

Fig. 6. (a-b) 3D and 2D interaction plots of native ligand sorafenibinto the binding site of VEGFR2 enzyme (PDB ID = 4ASD).

Table 3 Table 4
Bond Lengths for MeOIstDmMor. Cell Viability assay (μM) of the compounds in different cell lines with
their IC50 Values for 72 h.
Atom Atom Length/Å Atom Atom Length/Å
Compd↓ Cell lines→ MCF-7 A549 A431
C1 O1 1.411(12) C18 C19 1.389(14)
C2 C3 1.374(13) C18 C23 1.415(14) MeOIstTmor 2.93 6.81 5.29
C2 C7 1.396(14) C18 O4 1.382(12) MeOIstDmMor 4.41 2.52 4.80
C2 O1 1.378(12) C19 C20 1.407(12) MeOIstMor 5.41 5.64 6.84
C3 C4 1.425(12) C20 C21 1.391(12) MeOIstDm 7.41 3.55 7.25
C4 C5 1.405(12) C20 C24 1.440(14)
C4 C8 1.424(13) C21 C22 1.368(14)
C5 C6 1.361(13) C21 N5 1.396(12)
C5 N1 1.405(12) C22 C23 1.399(15) Arg1027.The computed binding energy value for MeOIstDmMor, it
C6 C7 1.389(14) C24 C25 1.504(11) was −6.3185 Kcal mol−1 .
C8 C9 1.525(11) C24 N6 1.319(11)
C8 N2 1.279(11) C25 N5 1.316(12)
C9 N1 1.322(13) C25 O5 1.235(12)
3.7. Anticancer activity
C9 O2 1.242(12) C26 N7 1.356(13)
C10 N3 1.358(12) C26 N8 1.333(14) Breast cancer cells (MCF-7), Lung cancer cells (A549), and Skin
C10 N4 1.334(13) C26 S2 1.688(10) cancer cells (A431) were cultured in 96 well plates in DMEM
C10 S1 1.679(9) C27 C28 1.487(19)
medium for 12 h and treated cells with different concentration
C11 C12 1.435(19) C27 N8 1.484(14)
C11 N4 1.428(15) C28 C29 1.524(17) (i.e., 1, 3, 10, and 30 μM of MeOIstTmor, MeOIstDmMor, MeOIstMor
C12 C13 1.50(2) C28 O6 1.437(16) and MeOIstDm respectively and incubated for 72 h as shown in
C12 O3 1.449(18) C30 C31 1.50(2) Fig. 9: A, B, C and D respectively. All the synthesized compounds
C14 C15 1.59(2) C30 C32 1.458(18) showed high inhibitory effects toward cell viability. Table 4, shows
C14 C16 1.439(19) C30 O6 1.414(16)
C14 O3 1.377(18) C32 N8 1.482(15)
the IC50 value of different synthetic compounds against different
C16 N4 1.473(14) N2 N3 1.354(10) cell lines, the compound MeOIstDmMor has exhibited broad spec-
C17 O4 1.399(14) N6 N7 1.351(12) trum activity towards A549 with IC50 value 2.52 μM than rest
of compounds whereas the compound MeOIstTmor has exhibited
high anticancer activity against MCF-7 with IC50 ; 2.93 μM. All the
compounds have shown moderate activity towards A431 with IC50
ranging from 4.80 to 7.25 μM.
Cells were seeded at a density of 30 0 0–40 0 0 cells/well in a 96
well plate and incubated at 37 °C for 24 h. Then, the medium was
removed, and freshly prepared solutions of the compounds were
added (Control, 0.3 μM, 1 μM, 3 μM, and 10 μM). After a 72 h
treatment, crystal violet assay was performed. A, B, C, D shows
the effect of MeOIstTmor, MeOIstDmMor, MeOIstMor and MeOIstDm
on different cell lines respectively. Data are represented as mean
± SD of three independent experiments. With an IC50 of 2.93 μM
Fig. 7. (a-b) 3D and 2D interaction plots of MeOIstDmMor into the binding site of
VEGFR2 enzyme (PDB ID = 4ASD). compared to other compounds, the compound MeOIstTmor demon-
strated strong anticancer activity against MCF-7.
Out of screened compounds for their effects in cell viability in
between Phe1047 and Phenyl ring of sorafenib. Trifluoromethyl different cancer cell lines MeOIstDmMor showed a better antipro-
(CF3 ) group forms halogen interactions with Ile1044. The synthe- liferative effects against the A431 cell line. It was selected for fur-
sized 5-methoxyisatin derivative was also docked into the bind- ther studies to determine the ability of the cells to from colonies
ing site of VEGFR2 enzyme. Its 3-D / 2-D dimensional interaction in presence of MeOIstDmMor in a dose dependent manner. A431
plots are shown in Fig. 7: (a-b). The synthesized compound in- cells were seeded in six well plates with low numbers of 30 0 0
teracts with Asp1046 and Lys868 via hydrogen bond interactions. cells/well and treated compound in different concentrations (C, 0.3,
The strength of ligand-enzyme complex was computed in terms 1, 3, and 10 μM), every after 72 h fresh media was added with
of binding energy of the docked poses. The computed binding en- compound. After two weeks of incubation, it was observed that
ergy value for native sorafenib was −9.0855 Kcal mol−1 . While for MeOIstDmMor inhibited the size and number of colonies in dose
isatin derivative, it was −6.4015 Kcal mol−1 . The interaction plots dependent manner (Fig. 10: A and B). We found colonization of cell
of 5-methoxyisatin derivative are shown in Fig. 8: (a-b). The com- was inhibited (100%) above 3 μM as compared to control after two
pound forms two hydrogen bond interactions with Ile1025 and weeks. The number of colonies drastically decreased as compare

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U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

Fig. 8. (a-b) 3D and 2D interaction plots of MeOIstDmMor into the binding site of VEGFR2 enzyme (PDB ID = 4ASD).

Fig. 9. Cell Viability assay synthetic compounds with different concentration.

to that of control with compound treatment, however the ability pared to control, significant percentage of cells were arrested in
to form colony was completely inhibited in 3 μM and 10 μM of the G0/G1 phase treated with 0.3 μM, and 1 μM (Fig. 11: A, B, C,
concentration. D). It was found that MeOIstDmMor changed the profile of the cell
To analyze the effect on cell death of A431 cells by flow cy- cycle with an increase in G0/G1 cell population associated with de-
tometry assay using propidium iodide staining, cells were treated cline G2/M and S cell population. This increase in percentage of
in different concentrations, i.e., control, 0.3 μM, 1 μM, 3 μM, and cells in S phase arrest might be attributed to irreparable DNA dam-
10 μM of MeOIstDmMor. As shown in Fig. 10, in comparison with age [57].
the control, treatment with MeOIstDmMor led to an increase in A431 cells were seeded in six well plates with low numbers of
apoptotic cell population significantly in a concentration depen- 30 0 0 cells/well and treated compound in different concentrations
dent manner, which might be due to DNA damage or other survival (C, 0.3, 1, 3, and 10 μM), every after 72 h fresh media was added
stresses induced by MeOIstDmMor similar to many other thiosemi- with compound for 14 days and crystal violet staining was per-
carbazone derivatives [12]. Furthermore, the effect of MeOIstDm- formed. B. A431 cells were seeded at a density of 30,0 0 0 cells/well
Mor in cell cycle was evaluated, A431cells were treated with a in a 12-well plate and incubated at 37 °C for 24 h and treated com-
lower dose of MeOIstDmMor i.e., control, 0.3 μM, and 1 μM. Com- pound in different concentrations of MeOIstDmMor (C, 0.3, 1, 3, and

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U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

Fig. 10. Colony Formation assay and Flow Cytometric analysis of apoptosis by Propidium Iodide (PI) Staining A.

Fig. 11. MeOIstDmMor causes G0/G1 cell cycle arrest in A431 cell line (A) Control (DMSO). Hectograph representing PI area vs number of cells when treated with 0.3 (B)
and 1 μM (C) of MeOIstDmMor were fixed and permeabilized with 80% chilled ethanol and stored for overnight at 4 °C. Then cells were washed and subjected to RNase
treatment and PI staining for 30 min at room temperature for flow cytometry to analyze DNA content.

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U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

Fig. 12. A; Western blotting analysis of compound MeOIstDmMor in A431 cell line.

10 μM). After a 72hPI staining was performed. Data represented as

mean ± SD of three independent experiments p∗∗ < 0.01, ∗ p<0.05.
Bar graph showing the percentage of cell death compared to vehi-
cle control (DMSO).
To identify the mechanism of action affected by treatment of
MeOIstDmMor in skin cancer cell line, we examined downstream
molecules associated with MAPK signaling path ways that regulate
major cellular events including cell proliferation, survival and mi-
gration. Improper functioning of MAPK pathways leads to the de-
velopment and progression of cancer [18]. We found that MeOIstD-
mMor treatment in A431 skin cancer cell line, downregulate the
expression of MAPK pathway molecules c-jun. Where in cancer
condition, the highly express c-Jun are associated with cell pro-
liferation and angiogenesis [12]. MeOIstDmMor induced cell death
and apoptosis in A431 cells significantly, to understand its effects
in the expression of proteins which are responsible for cancer cell
proliferation, we have performed western blot analysis. As shown
in Fig. 11:A, the expression level of β -catenin, C-Jun and Akt signif- Fig. 13. Graphical abstract of MeoIstDmMor mediated anticancer activity in A431
icantly inhibited after the treatment of cells with MeOIstDmMor in cells lines.
concentration dependent manner. It shows that compound MeOIst-
DmMor induce cytotoxic activity with the regulation of β -catenin,
C-Jun and Akt which are responsible for cell proliferation, migra-
tion and apoptosis in cancer. 4. Conclusions
Cleavage of chromosomal DNA into oligonucleosomal size frag-
ment is an integral part of apoptosis. So, to evaluate the role The single crystal X-ray diffraction study has proved that
of compound MeOIstDmMor in A431cell, DNA fragmentation assay the thiosemicarbazones exist as the thione tautomer. Molecular
was conducted. As shown in Fig. 12: B that uncut DNA that is con- docking studies showed that compound MeOIstDmMor interacted
trol as well as drug control shows distinct band of DNA. However, strongly with VEGFR2 via two hydrogen bonding with Il1025 and
typical ladder DNA fragments of 180–200 base pairs and multiples Arg1027. Our investigation displayed promising anticancer activity
thereof on an agarose gel should be present as the concentration of MeOIstTmor, MeOIstDmMor, MeOIstMor and MeOIstDm with IC50
of the drugs increases but, in our experiment, only the formation of 2.52 to 7.41 μM respectively, which may serve as anticancer
of DNA smear has been observed as concentration of drugs has in- drugs for skin cancer in the future. MeOIstDmMor significantly in-
creases. hibited cancer cell proliferation in low dose and inhibited A431
A431 cells were treated with different concentrations of the cancer cell colony formation, PI staining was conducted and found
MeOIstDmMor (Control, 0.3 μM, 1 μM, 3 μM and 10 μM) and β - to undergo apoptosis in a dosage dependent, with G0G1 phase
actin was used as loading control. MeOIstDmMor inhibit β -Catenin, cell cycle arrest. Western blotting analysis of MeOIstDmMor treated
C-Jun and AKT protein expression in dose dependent manner. B; skin cancer cell lines showed downstream molecules of MAPK (C-
Agarose gel showing fragmentation of DNA, Lane 1 contains control Jun), β -catenin and Akt. Experimental data obtained suggest that
DNA sample while lane 2 contain drug control i.e., DMSO. Lane 3 MeOIstDmMor has a significant cytotoxic effect on skin cancer cell
and 4 contain samples for MeOIstDmMor 3 μM and 10 μM respec- viability, and we conclude that this compound can be used as a
tively. therapeutic agent for the treatment of cancer in future.

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U. Chaudhary, D. Dawa, I. Banerjee et al. Journal of Molecular Structure 1274 (2023) 134549

Declaration of Competing Interest [16] N.K. Singh, S. Shrestha, N. Shahi, R.K. Choudhary, A.A. Kumbhar, Y.R. Pokharel,
P.N. Yadav, Study on Enhancement of Anticancer Activity of N(4)1-(2-
Pyridyl)piperazinyl 5-Nitroisatin Thiosemicarbazone on Chelation with Cop-
Submission of this manuscript complies with no conflict of in- per(II), Asian J. Chem. 33 (2022) 557–564, doi:10.9734/bpi/cacs/v9/1762A.
terest that exists among the authors to declare. [17] N.K. Singh, S. Shrestha, N. Shahi, R.K. Choudhary, A.A. Kumbhar, Y.R. Pokharel,
P.N. Yadav, Anticancer potential of N(4)substituted 5-nitroisatin thiosemicar-
Data Availability bazones and their copper(II) complexes, Rasayan J. Chem. 14 (2021) 1600–
1610, doi:10.31788/RJC.2021.1436341.
[18] N. Shahi, V. Pandey, A. Pathak, R.S. Thapa, P. Pokhrel, Y.R. Pokharel, P. N.Yadav,
No data was used for the research described in the article. Anticancer potential of 3-hydroxypyridine-2-carboxaldehyde N(4)-methyl and
pyrrolidinylthiosemicarbazones and their Zn(II) complexes in different cancers
Acknowlegements via targeting MAPK superfamily signaling pathway, Results Chem 3 (2021) 1–9,
doi:10.1016/j.rechem.2021.100104.
[19] J.P. Scovill, A facile synthesis of thiosemicarbazides and thiosemicarbazones
The authors sincerely acknowledge Nepal Academy of Science by the transamination of 4-methyl-4-phenyl-3-thiosemicarbazide, Phosphorus.
and Technology (NAST), Khumaltar, Lalitpur, Nepal for Ph.D. fellow- Sulfur. Silicon Relat. Elem. 60 (1991) 15–19, doi:10.1080/10426509108233920.
ship grant (2018/2019 AD) to UC. The authors gratefully acknowl- [20] A. Qasem, S. Guan, A. Salhin, N. Eltaher, Synthesis of isatin thiosemicarbazones
derivatives : in vitro anti-cancer, DNA binding and cleavage . activities Spec-
edge IIT Madras, India for CHN analysis and single crystal data col- trochimica Acta Part A : molecular and Biomolecular Spectroscopy Synthesis of
lection and Dr. Anupa A Kumbhar, Department of Chemistry at Sav- isatin thiosemicarbazones derivatives : in vitro anti-cance, Mol. Biomol. Spec-
itribai Phule Pune University, India for providing NMR and mass trosc. 125 (2014) 440–448, doi:10.1016/j.saa.2014.01.086.
[21] K.N. Aneesrahman, K. Ramaiah, G. Rohini, G.P. Stefy, N.S.P. Bhuvanesh,
spectral data. A. Sreekanth, Synthesis and characterisations of copper(II) complexes of
5-methoxyisatin thiosemicarbazones: effect of N-terminal substitution on
Supplementary materials DNA/protein binding and biological activities, Inorganica Chim. Acta 492 (2019)
131–141, doi:10.1016/j.ica.2019.04.019.
[22] H. Muğlu, Synthesis, characterization, and antioxidant activity of some
Supplementary material associated with this article can be new N 4-arylsubstituted-5-methoxyisatin-β -thiosemicarbazone derivatives,
found, in the online version, at doi:10.1016/j.molstruc.2022.134549. Res. Chem. Intermed. 46 (2020) 2083–2098, doi:10.1007/s11164- 020- 04079- x.
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