Identification of a novel 1,2 oxazine that can induce apoptosis by targeting NF-κB in hepatocellular carcinoma cells

Article abstract of DOI:10.1016/j.btre.2020.e00438

Constitutive activation of NF-κB is associated with proinflammatory diseases and suppression of the NF-κB signaling pathway has been considered as an effective therapeutic strategy in the treatment of various cancers including hepatocellular carcinoma (HCC). Herein, we report the synthesis of 1,2 oxazines and their anticancer potential. The antiproliferative studies presented 3-((4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)methyl)-4-phenyl-4,4a,5,6,7,7a-hexahydrocyclopenta [e][1,2]oxazine(3i) as a lead cytotoxic agent against HCC cells. Flow cytometric analysis showed that 3i caused a substantial increase in the subG1 cell population. Annexin-V-FITC-PI staining showed a significant increase in the percentage of apoptotic cells on treatment with 3i. Transfection with p65 siRNA significantly reduced the 3i induced DNA fragmentation indicating that 3i may primarily mediate its proapoptotic effects by abrogating the NF-κB signaling. In addition, treatment of HCC cells with 3i decreased the DNA binding ability of NF-κB and NF-κB-dependent luciferase expression. Taken together, this report introduces 1,2-oxazine that potently targets the NF-κB signaling pathway in HCC cells.

Full text of DOI:10.1016/j.btre.2020.e00438

Journal Pre-proof
Identification of a Novel 1,2 Oxazine That Can Induce Apoptosis by
Targeting NF-␬B in Hepatocellular Carcinoma Cells
Chaithanya Somu, Chakrabhavi Dhananjaya Mohan, Sachin
Ambedkar, Dukanya, Shobith Rangappa, CP Baburajeev, Alexey
Sukhorukov, Srishti Mishra, Muthu K Shanmugam, Arunachalam
Chinnathambi, Tahani Awad Alahmadi, Sulaiman Ali Alharbi,
Basappa, Kanchugarakoppal S. Rangappa
PII:
S2215-017X(19)30729-5
https://doi.org/10.1016/j.btre.2020.e00438
BTRE 438
DOI:
Reference:
To appear in:
Biotechnology Reports
Received Date:
Revised Date:
Accepted Date:
13 December 2019
21 January 2020
17 February 2020
Please cite this article as: Somu C, Mohan CD, Ambedkar S, Dukanya, Rangappa S,
Baburajeev C, Sukhorukov A, Mishra S, Shanmugam MK, Chinnathambi A, Awad Alahmadi T,
Alharbi SA, Basappa, Rangappa KS, Identification of a Novel 1,2 Oxazine That Can Induce
Apoptosis by Targeting NF-␬B in Hepatocellular Carcinoma Cells, Biotechnology Reports
(2020), doi: https://doi.org/10.1016/j.btre.2020.e00438

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Identification of a Novel 1,2 Oxazine That Can Induce Apoptosis by
Targeting NF-κB in Hepatocellular Carcinoma Cells
Chaithanya Somua,#, Chakrabhavi Dhananjaya Mohanb,#, Sachin Ambedkarc,#, Dukanyad,
Shobith Rangappae, CP Baburajeevc, Alexey Sukhorukovf, Srishti Mishrag, Muthu K
Shanmugamg, Arunachalam Chinnathambih, Tahani Awad Alahmadii, Sulaiman Ali Alharbih,
Basappac,d,*, Kanchugarakoppal S. Rangappaa,*
aDepartment of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006,
India
bDepartment of Studies in Molecular Biology, University of Mysore, Manasagangotri, Mysore
570006, India
cLaboratory of Chemical Biology, Department of Chemistry, Bangalore University, Central
College Campus, Palace Road, Bangalore 560001, India
dLaboratory of Chemical Biology, Department of Studies in Organic Chemistry, University of
Mysore, Manasagangotri, Mysore 570006, India
eAdichunchanagiri Institute for Molecular Medicine, BG Nagara, Nagamangala Taluk,
Mandya district-571448, India
fN.D. Zelinsky Institute of Organic Chemistry, Leninsky Prospect, Moscow 119991, Russia
gDepartment of Pharmacology, Yong Loo Lin School of Medicine, National University of
Singapore 117600, Singapore
hDepartment of Botany and Microbiology, College of Science, King Saud University, Riyadh -
11451, Kingdom of Saudi Arabia
iDepartment of Pediatrics, College of Medicine and King Khalid University Hospital, King
Saud University Medical City, Riyadh 11461, Saudi Arabia
#These authors contributed equally to this work
*Corresponding authors
1

Highlights
 10 new 1,2-Oxazines were synthesized and evaluated for their anticancer
activity.
 3i is lead cytotoxic agent which increased SubG1 cell population of HCC
cells.
 p65 siRNA transfection significantly reduced the 3i induced DNA
fragmentation.
 3i decreased DNA binding and NF-κB-dependent luciferase reporter gene
expression.
Abstract
Constitutive activation of NF-κB is associated with proinflammatory diseases and suppression
of the NF-κB signaling pathway has been considered as an effective therapeutic strategy in the
treatment of various cancers including hepatocellular carcinoma (HCC). Herein, we report the
synthesis of 1,2 oxazines and their anticancer potential. The antiproliferative studies presented
3-((4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)methyl)-4-phenyl-4,4a,5,6,7,7a-
hexahydrocyclopenta [e][1,2]oxazine(3i) as a lead cytotoxic agent against HCC cells. Flow
cytometric analysis showed that 3i caused a substantial increase in the subG1 cell population.
Annexin-V-FITC-PI staining showed a significant increase in the percentage of apoptotic cells
on treatment with 3i. Transfection with p65 siRNA significantly reduced the 3i induced DNA
fragmentation indicating that 3i may primarily mediate its proapoptotic effects by abrogating
the NF-κB signaling. In addition, treatment of HCC cells with 3i decreased the DNA binding
ability of NF-κB and NF-κB-dependent luciferase expression. Taken together, this report
introduces 1,2-oxazine that potently targets the NF-κB signaling pathway in HCC cells.
Keywords: Oxazine; NF-κB; Anticancer; DNA fragmentation.
2

Introduction
The nuclear factor-κB (NF-κB) signaling pathway is a key player in the maintenance of
homeostasis by regulating growth, immune system, and inflammation [1-4]. The deregulation
of NF-κB signaling results in initiation and progression of several diseases such as cancer,
rheumatoid arthritis, colitis, atherosclerosis, asthma, diabetes, stroke, muscle wasting,
neurodegenerative diseases and viral infections [5-8]. NF-κB is a quiescent transcription factor
that ubiquitously resides in the cytoplasm of all the mammalian cells. It was initially discovered
by Ranjan Sen and David Baltimore in 1986 as a DNA binding protein that interacts with
enhancer elements of the immunoglobulin kappa light chain gene in B cells [9-12]. Since its
discovery, NF-κB has been recognized as a widely studied transcription factor and can regulate
the expression of about 500 genes [13-16]. NF-κB is associated with a family of inhibitory
proteins called IκB (IκBα/IκBβ/IκBε) in the cytoplasm of the unstimulated cell [17-20]. The
presence of ligand on the membrane receptor initiates the cascade of signaling events that
stimulate the phosphorylation, ubiquitylation, and degradation of IκB protein which leads to
the relocation of NF-κB into the nucleus. In the nucleus, NF-κB binds to specific DNA
elements and regulates the expression of genes that encodes apoptosis regulators (Bcl-2, Bcl-
xL, Bim, IAP, XIAP, TRAF-1/2), cell adhesion molecules (CD44, Fibronectin, ICAM-1,
VCAM-1, NCAM, P-Selectin), cytokines (BAFF, TNF-α/β, RANTES, CXCL5/6, Trefoil
factor 3), growth factors (VEGF-C, SCF, PIGF, NGF, M-CSF), and transcription regulators
(Bcl-3, c-rel, HIF-1, nfκb1, nfκb2, p53, Stat5a) [21-24]. NF-κB modulates the expression of
oncogenic genes involved in inflammation, cell growth, and survival, apoptosis and cancer
progression [25]. Mounting shreds of evidence suggest that NF-κB is often overactivated in
many tumors [26, 27]. NF-κB has been identified as a potent tumor promoter and several
chemical entities have been developed to target NF-κB in preclinical cancer models [28-30].
Notably, constitutive activation of NF-κB was found more frequently in hepatocellular
3

carcinoma tumor tissues than its normal counterpart suggesting its role in tumorigenesis [31-
33]. Therefore, NF-κB represents a novel and unique molecular target for therapeutic
intervention against several cancers including hepatocellular carcinoma.
Oxazine derivatives have been evaluated extensively and reported to possess good anticancer
potential in various preclinical tumor models [34-40]. Some of the oxazine derivatives have
been reported to interfere with the NF-κB signaling pathway to impart their anticancer activity.
γ- and -tocotrienol conjugated oxazines significantly decreased the levels of phosphorylated
NF-κB and IκB in the tumor tissues from mice [41]. In another study, 2-ethoxy-4,5-diphenyl-
1,3-oxazine-6-one (EDPOO) decreased the nuclear levels of NF-κB in LPS-treated rat
pheochromocytoma (PC12) cells [42]. In our previous study, we identified 2-((2-acetyl-6,6-
dimethyl-4-phenyl-5,6-dihydro-2H-1,2-oxazin-3-yl)methyl)isoindoline-1,3-dione (API) as an
inhibitor of NF-κB signaling pathway [37]. The structures of EDPOO and API are presented
in Figure 1. In our continued efforts to identify pharmacologically important small molecules
[43-48], and to identify potent NF-κB inhibitor than the previously reported oxazines, we
synthesized a panel of novel 1,2 oxazines and evaluated their anticancer and NF-κB inhibitory
activities in tumor cells. Several hepatocellular carcinoma cell lines including HepG2 and
HCCLM3 possess constitutive activation of NF-κB and we have demonstrated previously that,
NF-κB targeting compounds display good cytotoxicity towards HepG2 and HCCLM3 cells.
Therefore, we used HepG2 and HCCLM3 cells as representative cell lines to evaluate the
possible NF-κB inhibitory effect of new oxazines.
Material and methods
1
All chemicals used were purchased from Sigma-Aldrich. H and 13C NMR spectra were
recorded on a Bruker WH-200 (400MHz) spectrometer in CDCl3 or DMSO-d6 as a solvent,
using TMS as an internal standard and chemical shifts are expressed as ppm. Mass spectra were
determined on an Agilent ESI MS and the elemental analyses were carried out using an
4

Elemental Vario Cube CHNS rapid analyzer. The progress of the reaction was monitored using
TLC pre-coated silica gel G plates. The small interfering RNA (siRNA) for NF-κB (sc-29410)
and scrambled control (sc-37007) was obtained from Santa Cruz Biotechnology. DNA
fragmentation assay kit was purchased from Roche Applied Science (CA, USA). HepG2 and
MDA-MB-231 cells were previously obtained from American Type Culture Collection
(ATCC) and the cells stock was maintained in our laboratory. DNA binding assay was
performed using TransAM NF-κB Kit (ActiveMotif, USA).
Chemistry
Typical procedure for the synthesis of bromomethyl 1,2 oxazine derivatives (1a-f)
The synthesis of bromomethyl 1,2 oxazine derivatives was carried out as described previously
[75]. In brief, the formation of 1,2-oxazine was achieved by inverse electron demand Diels–
Alder reaction of nitroalkenes derived from nitroethane to olefines. The appropriate 1,2-
oxazine-N-oxide (0.1 mol) was slowly added to a stirred solution of trimethylsilyl bromide
(39.6 mL, 0.3 mol) and Et3N (16.7 mL, 0.12 mol) in CH2Cl2 (0.5 L) at –80 °C. The resulting
mixture was stirred for 1 h at –80 °C, then MeCN (50 mL) was added and the temperature was
increased to –30 °C within 1 h. The mixture was kept at –30 °C for 3 h and then diluted with
EtOAc (0.5 L) and poured into a mixture of EtOAc (1.0 L) and a saturated aqueous solution of
NaHCO3 (0.5 L). The aqueous phase was back-extracted with EtOAc (2 × 100 mL), and the
combined organic layers were washed with H2O (2 × 100 mL) and brine (200 mL) and dried
(Na2SO4). The solvents were evaporated in vacuo and the residue was filtered through a thin
layer of silica gel. The obtained 3-bromomethyl-substituted 5,6-dihydro-4H-1,2-oxazines 1,
which serve as key precursors for the synthesis of new 1,2-oxazines (3a-j).
General procedure for the synthesis of N-alkylated 1,2 oxazine derivatives (3a-j)
To a solution of amines (250 mg) in 5 mL of 2-butanone, SCS-Bi2O3 (0.8 eq), and bromomethyl
1,2 oxazine (1a-f) (0.1 mmol) was added and the reaction mixture was stirred at room
5

temperature for 2 h. The progress of the reaction was monitored using thin-layer
chromatography. After completion of the reaction, contents were filtered to remove the base.
Subsequently, water (10 mL) was added and the product was extracted from the aqueous layer
using ethyl acetate (5 mL). The extraction with ethyl acetate was repeated thrice and dried with
magnesium sulfate, filtered, and concentrated in vacuum. The crude product was purified by
recrystallization using ethanol.
6-(tert-butyl)-8-fluoro-1-((4-(4-methoxyphenyl)-4a,5,6,7,8,8a-hexahydro-4H-
benzo[e][1,2]oxazin-3-yl)methyl)-2,3-dimethylquinolin-4(1H)-one (3a)
393.2mg (Yield 78%); Off-white solid; mp: 156-157 ºC; IR √max: 2928.94cm-1 √
(C-H)
,1601.74cm-1 √ (C=O) , 1510.77cm-1 √ (C-C) , 1246.71cm-1 √ (C-C), 1104.54cm-1 √ (C-F) , 946.45cm-
1
1
√
(C-O); H NMR (CDCl3 , 400 MHz) δ:7.52 (s , 1H), 7.36-7.33 (m, 1H), 7.11 (d ,J=8.4Hz,
2H), 6.88 (d, J=8.8Hz,2H), 4.54 (d, J=10.4Hz, 1H), 4.43 (d, J=10.8Hz, 1H), 3.79 (s, 3H),
3.77-3.76 (m,1H), 3.58 (s, 1H), 2.64 (s, 3H), 2.20 (s, 3H), 2.10 (d, J=13.6 Hz, 1H), 1.77 (d,
J=12Hz, 2H), 1.66 (d, J=12.8 Hz, 2H), 1.49-1.42 (m, 2H), 1.39-1.37 (m, 2H), 1.28 (s, 9H); 13C
NMR (CDCl3, 100 MHz): 160.3, 158.8, 158.6, 156.2, 152.3, 148.9, 148.9, 133.0, 129.2, 123.7,
122.3, 144.4, 114.1, 112.1, 111.7, 111.5, 76.6, 73.2, 69.2, 55.2, 43.1, 39.1, 35.4, 31.0, 29.2,
27.6, 24.9, 24.1, 19.9, 12.3; LCMS (ESI) [M+H]+: 505.33. Anal. Calcd for C31H37N2: C, 73.78;
H, 7.39; N, 5.55. Found: C, 73.81; H, 7.35; N, 5.58.
6-(tert-butyl)-1-((4-(4-chlorophenyl)-6,6-dimethyl-5,6-dihydro-4H-1,2-oxazin-3-yl)methyl)-
8-fluoro-2,3-dimethylquinolin-4(1H)-one (3b)
433.2mg (Yield 90%); Off-white solid; mp: 138-139 ºC; IR √max: 1650cm-1 √ (C=O) ,1491.93cm-
1
√
(C-C), 1011.25cm-1 √ (C-F), 694.78cm-1 √ (C-Cl) ; 1H NMR (CDCl3, 400 MHz) δ: 7.48 (s,1H),
7.38-7.34 (m, 3H), (d, J=8Hz, 2H), 4.53 ( d, J=12Hz, 1H), 4.29 (d, J=8Hz, 1H), 3.82-3.78 (m,
1H), 2.64 (s, 3H), 2.17-2.12 (m, 1H), 2.06 (s, 3H), 1.92-1.86 (m ,1H), 1.40-1.35 (m, 6H), 1.32
(s, 9H);13C NMR (CDCl3, 100 MHz): 160.3, 154.3, 149.1, 138.3, 133.4, 129.7, 129.4, 123.6,
6

122.2, 112.0, 111.7, 75.2, 73.5, 40.2, 37.0, 35.1, 31.0, 28.3, 24.0, 22.6, 12.2; LCMS (ESI)
[M+H]+: 483.27. Anal. Calcd for C28H32N2: C, 69.62; H, 6.68; N, 5.80. Found: C, 69.67; H,
6.73; N, 5.79.
6-(tert-butyl)-1-((6,6-dimethyl-4-phenyl-5,6-dihydro-4H-1,2-oxazin-3-yl)methyl)-8-fluoro-
2,3-dimethylquinolin-4(1H)-one (3c)
394.2mg (Yield 88%); Off-white solid; mp: 140-141 °C; IR √max: 1601.47cm-1
√
,
(C=O)
1
1394.94cm-1 √ (C-C), 1060.9cm-1 √ (C-F), 1009.51cm-1 √
; H NMR (CDCl3, 400 MHz) δ:
(C-O)
7.56 (s, 1H), 7.35-7.31 (m, 5H), 7.25-7.22 (m, 1H), 4.53 (s, 1H), 4.31 (d, J=12Hz, 1H), 3.80
(s,1H), 2.63 (s, 3H), 2.16 (s, 2H), 8 (s, 3H), 1.41-1.38 (m, 6H), 1.33 (s, 9H); 13C NMR (CDCl3
, 100 MHz): 160.3, 158.8, 154.9, 149.0, 139.8, 129.2, 128.3, 127.5, 123.7, 122.1, 112.2, 111.8,
111.6, 76.7, 75.2, 73.4, 40.4, 37.6, 35.1, 31.1, 28.3, 24.1, 22.7, 12.2; LCMS (ESI) [M+H]+:
449.29. Anal. Calcd for C28H33N2: C, 74.97; H, 7.42; N, 6.25. Found: C, 74.99; H, 7.45; N,
6.28.
4-(4-chlorophenyl)-3-((4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-6,6-
dimethyl-5,6-dihydro-4H-1,2-oxazine (3d)
428.6mg (Yield 79%); Off-white solid; mp: 132-133 °C; 1H NMR (CDCl3, 400 MHz) δ: 7.44-
7.39 (m, 4H), 7.28-7.21 (m, 9H), 5.60 (s, 1H), 4.17 (s, 2H), 2.78-2.61 (m, 9H), 1.98-1.81 (m,
2H), 1.34 (s, 3H), 1.23 (m, 3H); LCMS (ESI) [M+H]+: 523.52. Anal. Calcd for
C30H33Cl2N3OC, 68.96; H, 6.37; N, 8.04; Found: C, 68.98; H, 6.38; N, 8.06
3-((4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-6,6-dimethyl-4-phenyl-5,6-
dihydro-4H-1,2-oxazine (3e)
281.6mg (Yield 66%); White solid; mp: 141-142 °C; IR √max: 2971.8cm-1 √ (C-H) , 1489.64cm-
1
1
√
(C=C), 1294.03cm-1 √ (C-C), 1092.92cm-1 √ (C-O) , 720.15cm-1 √ (C-Cl); H NMR (CDCl3, 400
MHz) δ: 7.33-7.27 (m, 7H), 7.22-7.17 (m, 7H), 4.17 (s, 1H), 3.56 (t, J=9Hz, 1H), 2.79 (d,
7

J=13.2Hz, 1H), 2.70 (d, J=12.8 Hz, 1H), 2.43-2.31 (m, 6H), 2.10 (s, 2H), 2.04-199 (m, 1H),
13
1.94-1.88 (m, 1H), 1.32 (s, 3H), 1.20 (m, 3H); C NMR (CDCl3 , 100 MHz):157.0, 142.2,
141.3, 140.7, 132.5, 129.1, 128.6, 128.6, 127.7, 127.1, 126.9, 75.6, 74.3, 59.5, 53.0, 52.1, 39.9,
37.9, 28.5, 22.8; LCMS (ESI) [M+H]+: 488.26. Anal. Calcd for C30H34N3: C, 73.83; H, 7.02;
N, 8.61. Found: C, 73.85; H, 7.04; N, 8.65.
3-((4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)-6,6-dimethyl-5,6-dihydro-
4H-1,2-oxazin-4-yl benzoate (3f)
259.7mg (Yield 70%); Off-white solid; mp: 146-147 °C; IR √max: 1612.09cm-1 √
,
(C=O)
1
1512.04cm-1 √
,1246.9cm-1
√
(C-C), 1034.19cm-1 √ (C-O),725.76cm-1 √
; H NMR
(C=C)
(C-Cl)
(CDCl3, 400 MHz) δ:7.99 (d, J=6Hz, 2H), 7.58 (s, 1H), 7.44-7.22 (m, 11H),
5.64 (s, 1H),
4.05 (s, 1H), 3.39 (d, J=12.8Hz, 1H), 3.08 (d, J=12.8Hz, 1H), 2.45-2.33 (m, 4H), 2.21 (s, 4H),
1.97-1.95 (m, 1H), 1.41-1.31 (m, 6H); 13CNMR: 165.33, 153.04, 142.22, 141.35, 133.32,
132.45, 129.62, 129.12, 128.53, 127.75, 127.09, 75.40, 74.32, 60.35, 58.86, 53.24, 51.63,
36.42, 26.02, 25.44; LCMS (ESI) [M+H]+: 532.26. Anal. Calcd for C31H34ClN3O3: C, 69.98;
H, 6.44; N, 7.90; Found: C,70; H, 6.46; N, 7.92.
3-((4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)methyl)-4-(4-methoxyphenyl)-4a,5,6,7,8,8a-
hexahydro-4H-benzo[e][1,2]oxazine (3g)
524.1mg (Yield 92%); White solid; mp: 141-142 °C; 1H NMR (CDCl3 , 400 MHz) δ: 7.51 (s,
2H), 7.20-7.08 (m, 2H), 6.98-6.90 (m, 2H), 6.81-6.9 (m,2H), 4.17 (s,2H), 3.77 (s, 3H), 3.58-
3.51(m,1H), 2.95-2.80 (m, 4H), 2.17-2.01 (m, 1H), 2.05-1.89 (m, 8H), 1.39 (s, 3H), 1.34 (s,
3H); 13CNMR (CDCl3 , 100 MHz): 158.5, 157.7, 157.6, 132.5, 129.6, 122.1, 114.6, 74.3, 59.8,
55.2, 54.3, 51.8, 39.9, 37.0, 36.4, 31.3, 31.1, 28.5. 22.8; LCMS (ESI) [M+H]+: 459.31. Anal.
Calcd or C28H34N4: C, 73.33; H, 7.47; N, 12.22. Found: C, 73.35; H, 7.49; N, 12.24.
8

3-((4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)methyl)-4-(4-methoxyphenyl)-6,6-dimethyl-
5,6-dihydro-4H-1,2-oxazine (3h)
419.3mg (Yield 78%); Off-white solid; mp: 135-136 °C ; IR √max: 1511.85cm-1
1246.29cm-1 √ (C-C),1032.8cm-1 (C-O) ; 1H NMR (CDCl3, 400 MHz) δ : 9.3 (s, 1H), 7.4-7.2
√
,
(NH)
√
(m, 2H), 7.1-7.0 (m, 2H), 6.9-6.8 (m, 4H) 5.0 (s, 2H), 4.1-4.0 (m,1H), 3.8 (, 3H), 3.1-3.0 (m,
1H), 2.8-2.7 (m, 2H), 2.2-1.6 (m, 8H), 1.4 (s, 3H), 1.2 (s, 3H); LCMS (ESI) [M+H]+: 433.27
Anal.Calcd for C26H32N4: C, 72.19; H, 7.46; N, 12.95. Found: C, 72.23; H, 7.42; N, 12.99.
3-((4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)methyl)-4-phenyl-4,4a,5,6,7,7a-
hexahydrocyclopenta[e][1,2]oxazine (3i)
318.9mg (Yield 77%) ; Off-white solid; mp: 137-138 °C; IR √max: 2928.89cm-1 √
,
(C-H)
1
1610.38cm-1 √ (C=O), 1247.58cm-1 √ (C-C), 1034.22cm-1 √ (C-O) ; H NMR (CDCl3, 400 MHz) δ:
7.59 (m, 2H), 7.48 (m, 2H), 7.29-7.13 (m, 5H), 4.11 (s, 2H), 3.41 (s, 1H), 2.43 (m, 2H), 2.17
(m, 2H), 2.02-1.88 (m, 4H), 1.71-1.41 (m, 10H); 13C NMR (CDCl3 , 100 MHz): 164.6, 156.1,
156.8, 140.7, 139.2, 132.5, 130.0, 129.6, 121.5, 117.6, 74.02, 56.7, 54.4, 51.8, 40.3, 39.6, 37.1,
36.2, 31.2, 30.9, 28.4, 22.7; HRMS (ESI) (M+Na)+: 437.2316.
3-((4-(1H-benzo[d]imidazol-2-yl)piperidin-1-yl)methyl)-6,6-dimethyl-5,6-dihydro-4H-1,2-
oxazin-4-yl benzoate (3j)
477.3mg (Yield 86%); Off-white solid; mp: 140-141 °C; IR √ max: 2934 cm-1 √ (C-H),1610cm-1
1
√
(C=O), 1510cm-1
√
(C=C), 1247cm-1 √ (C-C), 1034cm-1 √ (C-O); H NMR (CDCl3, 400 MHz) δ:
7.55 (s, 2H), 7.25-7.19 (m, 2H), 7.05-7.03 (m, 2H), 6.84-6.82 (m, 3H), 4.01 (s, 1H), 3.77 (s,
3H), 3.44 (m,1H), 3.08-2.87 (m, 4H),2.74 d, J=10Hz, 2H), 2.22-2.01 (m, 8H), 1.35 (s, 3H),
1.24 (s, 3H); 13C NMR (CDCl3 , 100 MHz): 158.4, 157.6, 154.2, 133.7, 129.3, 122.2, 114.3,
114.1, 60.1, 55.2, 54.4, 51.9, 42.9, 38.7, 36.2, 30.9, 30.7, 29.2, 27.5, 25.0, 19.9; LCMS (ESI)
[M+H]+: 447.24. Anal. Calcd for C26H30N4: C, 69.93; H, 6.77; N, 12.55. Found: C, 69.95; H,
6.79; N, 12.58.
9

Pharmacology
MTT assay
Cytotoxicity of the newly synthesized compounds was evaluated using MTT assay against
hepatocellular carcinoma cells as described previously [51]. Briefly, HepG2 and HCCLM3
liver cancer cells were cultured in DMEM containing 10% FBS. For cytotoxicity assay (5 X
103/well) were incubated in triplicate in a 96-well plate, in the presence of different
concentrations of compounds at a volume of 0.2 mL, for different time intervals at 37°C.
Thereafter, 20 μL MTT solution (5 mg/mL in PBS) was added to each well. After 2 h of
incubation at 37°C, the media was carefully removed, and the purple crystals were dissolved
in 0.1 mL of DMSO. Finally, the optical density (OD) at 570 nm was measured using the Tecan
plate reader.
Flow cytometric analysis
Flow cytometric analysis was performed to determine whether 3i can induce apoptosis of
HepG2 and HCCLM3 cells as reported earlier [76, 77]. Briefly, hepatocellular carcinoma cells
(5 X 105) were plated in a 60 mm petri dish and 24 h later the cells were exposed to compound
3i (50 μM) for 0, 24, 48 and 72 h. Thereafter cells were washed, fixed with 70% ethanol, and
incubated for 30 min at 37°C with 0.1% RNase A in PBS. Cells were washed again,
resuspended, and stained with PBS containing 25 μg/mL propidium iodide (PI) for 15 min at
room temperature. The cell cycle distribution across the various phases was analyzed using the
flow cytometer.
Annexin V/PI apoptosis assay
Phosphatidylserine exposure and cell death were assessed by FACS analysis using Annexin-
V-PI-stained cells as described previously [57, 58]. Briefly, 5×105 HepG2 cells were seeded in
a 60 mm petri dish and incubated with 3i (50 µM) for indicated time points and DMSO treated
samples were used as a control. Cells were then washed with Annexin V binding buffer (10mM
10

HEPES/NaOH, pH7.4, 140mM NaCl, 2.5mM CaCl2), stained with Annexin V FITC for 30
min at room temperature in the dark, then washed again and resuspended in Annexin V binding
buffer containing PI. Samples were analyzed immediately using the flow cytometer.
DNA fragmentation assay
DNA fragmentation assay was performed using the Cell Death Detection ELISAPLUS assay kit
(Roche, CA, USA) according to the manufacturer’s instructions. Briefly, HepG2 cells were
plated in 6-well plates and allowed to adhere for 24 h. HepG2 cells were then transfected with
either control siRNA or NF-κB-siRNA for 48 h and then treated with compound 3i (25 or 50
µM) for an additional 72 h after which the media was aspirated, the cells were washed with
cold PBS and incubated on ice for 30 min in cell lysis buffer. The cells were then scraped and
the lysates were collected in a centrifuge tube and vortexed to break up the cell aggregates. The
lysates were cleared by centrifugation at 5,000 rpm for 10 min at 4C and the supernatant were
stored at -80C. The cell lysates were added to lysis buffer provided with the kit and pipetted
on streptavidin coated 96-well microtiter plate to which immunoreagent mix was added and
incubated for 2 h at room temperature with continuous shaking at 500 rpm. The wells were
then washed with washing buffer, and the color was developed by the addition of a substrate
solution, which was read at 405 nM against the blank, reference wavelength of 490 nm after
10–15 min. The enrichment factor (total amount of apoptosis) was calculated by dividing the
absorbance of the sample (A405 nm) by the absorbance of the controls without treatment (A490
nm).
Western blot assay
HepG2 and MDA-MB-231 cells were plated in 6-well plates and allowed to adhere for 24 h.
On the day of transfection, lipofectamine was added to control or NF-B siRNA (100 nM) in
a final volume of 1 mL of culture medium. After 48 hours of incubation following transfection,
the cells were lysed and 50 µg protein was taken for Western blot analysis. Briefly, 50 μg of
11

nuclear proteins extracted from treated or control cells were resolved in 10% SDS gel. After
electrophoresis, the proteins were electro-transferred to a nitrocellulose membrane (Bio-Rad,
USA), blocked with Blocking One (Nacalai Tesque, Inc, Japan), and probed with p65 and β-
actin primary antibody overnight at 4C. The blot was washed and probed with horseradish
peroxidase-conjugated secondary antibody for 1 h and finally examined by chemiluminescence
substrate (ECL, GE Healthcare, UK).
NF-κB DNA binding assay
Activation of NF-κB was evaluated by DNA binding assay using TransAM NF-κB Kit
according to the manufacturer’s instructions. Briefly, 50 μg of nuclear protein was extracted
from 0, 10, 25 and 50 µM 3i treated cells for 8 h and were added into 96-well plate coated with
an unlabelled oligonucleotide containing the discrete single-stranded consensus binding site
for NF-κB (5’-GGGACTTTCC-3’) and incubated for 1 h. The wells were washed and
incubated with antibodies against NF-κB p65 subunit. An HRP conjugated secondary antibody
was then applied to detect the bound primary antibody. HRP substrate was added and the color
produced provided the basis for colorimetric quantification as per kit manufacturer instructions.
NF-κB luciferase reporter assay
The effect of 3i on constitutive NF-κB-dependent reporter gene transcription in HepG2 and
HCCLM3 cells was determined. NF-κB responsive elements linked to a luciferase reporter
gene were transfected with wild-type or dominant-negative IκB. The transfected cells were
then treated with various doses of 3i for 6 h. Luciferase activity was measured with a Tecan
(Durham, NC, USA) plate reader and normalized to β-galactosidase activity. All luciferase
experiments were done in triplicate and repeated twice.
In silico interaction analysis
12

Discovery Studio 2.5 software from Accelrys was used for docking and visualization of the
results. Initially, we retrieved the crystal structure of the NF-κB p65 homodimer complex with
a DNA (PDB ID: 1RAM, 2.4 Å resolution) cleaned, minimized the energy, and identified the
spatial region of p65. All the energy calculations were performed using the CHARMM force
field. The three-dimensional structures of all 1,2-oxazines were prepared and docked toward
the p65 using the LigandFit protocol of Discovery Studio. The binding pose of ligands was
evaluated using the interaction score function in the Ligand Fit module of Discovery Studio.
Results
Chemistry
Preparation of 1,2 oxazines
The substituted bromomethyl 1,2-oxazine derivatives were prepared as described earlier [49].
Given the biological significance of oxazines in NF-κB inhibition, we synthesized the new
derivatives of (1) via a C-N bond formation reaction as presented in Figure 2. In the present
synthetic strategy, we utilized three different secondary amines namely, 6-(tert-butyl)-8-fluoro-
2,3-dimethylquinolin-4(1H)-one (2a), 1-((4-chlorophenyl)(phenyl) methyl)piperazine (2b),
and 2-(piperidin-4-yl)-1H-benzo[d]imidazole (2c) for the preparation of title compounds.
Based on our previous experience, the utilization of these secondary amine intermediates (2a-
c) resulted in products with good biological activity and therefore 2a-c were chosen for
coupling reactions in the present study. Based on our earlier report [38, 50], we utilized the
mild basic characteristic of combustion-derived Bi2O3 (SCS-Bi2O3) to prevent the
decomposition of base sensitive bromomethyl 1,2-oxazine (1) and for C-N bond formation
reaction. The synthesis of the final product (3a-j) was achieved in good yield. All the newly
1
13
synthesized oxazine derivatives were characterized by H NMR, C NMR, CHN, and mass
analysis and structures are shown in table 1 and spectra are provided as supporting information.
Pharmacology
13

1,2-Oxazines elicit growth inhibitory effect in HCC cells
Previous studies suggested that oxazine derived compounds possess good anticancer activity
towards various cancer models. Initially, we tested the anticancer potential of all the newly
synthesized compounds against HepG2 cells using MTT assay [51, 52]. Among the tested
compounds, 3e and 3i were found to be good anticancer agents with IC50 values of 47.6 and
46.4 µM. The IC50 values were determined using GraphPad Prism software[15]. The IC50 value
of other compounds towards HepG2 cells is provided in table 1. Since compound 3i showed
relatively better anticancer potential, we further investigated the effect of 3i on the proliferation
of HCCLM3 cells. Compound 3i exhibited good anticancer potential towards HCCLM3 with
an IC50 value of 40.9 µM. Furthermore, we treated HepG2 and HCCLM3 cells with 3i in
different time-points at different doses as indicated in Figure 3. Compound 3i significantly
reduced the proliferation of both the cell lines in a dose- and time-dependent manner (Figure
3). We next evaluated the effect of 3i against normal hepatocytes (LO2) and did not find
significant cytotoxicity of the compound towards normal cells (Data not shown). Doxorubicin
and paclitaxel are used as reference compounds and IC50 values are 10 and 8 nM, respectively
towards HCCLM3 cells. The results indicate that the presence of cyclopentane ring fused to
the 1,2-oxazine heterocycle enhances the cytotoxic effect towards HCC cells.
3i increases the subG1 population in HCC cells
The activation of executioner caspases and caspase-activated DNases during apoptosis leads to
the cleavage of genomic DNA into oligonucleotide fragments and the cells with reduced DNA
are detected as subG1 cell population [53, 54]. Therefore, we treated HepG2 and HCCLM3
cells with 3i for different time intervals and analyzed the distribution of cells in various phases
of the cell cycle using propidium iodide staining as described earlier [55, 56]. The results
indicated that 3i caused a substantial increase in the accumulation of the subG1 population to
14

20.2% in HepG2 and 48.8% in HCCLM3 cells in a time-dependent manner (Figure 4). The
results clearly demonstrated the apoptosis inducing effect of 3i in HCC cells.
3i induces apoptosis in HCC cells
The appearance of phosphatidylserine in the outer leaflet of the plasma membrane is one of the
biochemical events in the cell committed to apoptosis which can be detected using annexin V-
FITC-PI staining [57, 58]. We next examined the effect of 3i on the translocation of
phosphatidylserine in HepG2 cells. 3i significantly increased the percentage of apoptotic cells
(55%) compared with DMSO-treated cells (3.8%) (Figure 5A). These findings provide another
direct evidence for apoptosis inducing effect of 3i.
Transfection with p65 siRNA blocks 3i induced DNA fragmentation in HCC cells
DNA fragmentation is one of the key events in the cells undergoing apoptosis [59, 60]. Hence,
we investigated whether the knockdown of NF-κB using siRNA could inhibit the 3i induced
DNA fragmentation in HepG2 cells. In 3i-treated, control siRNA transfected cells, we observed
a significant increase in DNA fragmentation (Figure 5B). In the cells transfected with NF-κB
siRNA, we observed the significant decline in the 3i induced DNA fragmentation compared to
control siRNA treated group indicating that, 3i mediate its proapoptotic effects by abrogating
NF-κB signaling pathway in HepG2 cells. The efficiency of transfection was further confirmed
using western blotting analysis. The treatment of HepG2 and MDA-MB-231 cells with siRNA
efficiently downregulated the protein levels of NF- κB-p65 (Figure 5C).
3i inhibits the constitutive activation of NF-κB in HCC cells
Oh et al., identified 7-benzoyl-4-phenylcyclopenta[1,2]oxazine out of 7,243 diverse
compounds as a potent IKKβ inhibitor which leads to the abrogation of NF-κB signaling
pathway [61]. Hence, we evaluated whether 3i could inhibit the constitutive activation of NF-
κB in HepG2 and MDA-MB-231 cells. Initially, cancer cells were treated with 10, 25 and 50
15

µM of 3i followed by preparation of nuclear extract and evaluation for NF-κB DNA activity.
We observed a significant decrease in DNA binding ability of NF-κB on 3i treatment in a dose
dependent fashion, thus indicating that 3i can also attenuate NF-κB activation in different
tumour cell lines (Figure 6). The binding characteristics were typical for NF-κB transcription
factors as noted by competition experiments with NF-κB-binding wild type (WT) DNA
oligonucleotides or mutated (MT) oligonucleotides in DNA binding assay (Figure 6). We next
examined the effect of 3i on the constitutive expression of the NF-κB-dependent reporter gene
in HCC cells. HCC cells were transfected with NF-κB responsive elements linked with
luciferase reporter gene with wild-type or dominant-negative IκB to study the effect of 3i on
constitutive activation of NF-κB. We observed a gradual decline in the expression of NF-κB-
dependent reporter gene in the presence of 3i with the maximum inhibition observed at 20 µM
(Figure 7A and B). These results indicate that 3i can potently affect the constitutive activation
of the NF-κB signaling pathway.
In silico interaction analysis of oxazines with p65
1,2-oxazines are reported as good anticancer agents and they induce anticancer activity via
inhibition of NF-κB at least partly in various cancer models. The oxygen-containing
heterocyclic compounds such as helenalin selectively alkylate the p65 subunit and inhibit the
activation of NF-κB [62]. Here we used the crystal structure of homodimer of p65 with DNA
for in silico interaction analysis targeting the key amino acids of p65 that interacts with DNA
using the Discovery Studio LigandFit protocol [63]. Initially, we retrieved the crystal structure
of the NF-κB p65 homodimer complex with a DNA (PDB ID: 1RAM, 2.4 Å resolution)
cleaned, minimized the energy, and identified the spatial region of p65. All the energy
calculations were performed using the CHARMM force field. The three-dimensional structures
of all 1,2-oxazines were prepared and docked toward the p65 using the LigandFit protocol of
Discovery Studio. The binding pose of ligands was evaluated using the interaction score
16

function in the LigandFit module of Discovery Studio. The docking results were summarised
in table 2. The molecular interaction analysis revealed that the oxazines showed favorable
interaction with the target protein with docking scores (DS) ranging from 48 to 62 kcal/mole.
Interestingly, compound 3i showed the DS of 54 kcal/mole indicating that it has a relatively
high affinity towards p65, Additionally, the 3i established hydrophobic interaction with Tyr36
and Arg187 of p65, which are present at the vicinity of the hydrophobic region near Cys38 of
p65 protein (Figure 8A-C). In addition, we compared the binding affinity of compound 3i with
a previously reported NF-κB inhibitor named CPP from our laboratory [21]. CPP presented the
docking score of 56 kcal/mole which is comparable to compound 3i and the interaction map of
CPP with p65. These results indicate that the compound 3i has a relatively better affinity for
p65.
Discussion
Oxazine is a six-membered heterocycle with oxygen and nitrogen as a heteroatom and this ring
system has been the part of various biologically active compounds. Oxazine derivatives were
demonstrated to exhibit good cytotoxic potential in different types of cancer cell lines and in
vivo models. In our previous study, an oxazine named CIMO inhibited the STAT3 signaling
in HCC cell lines and the orthotopic nude mice model [36]. Srinivas and colleagues showed
that 1,2-oxazines inhibit COX2 with high selectivity over COX1 [38]. In another study,
pyridine conjugated oxazinones abrogated NF-κB signaling and induced apoptosis of HCC
cells [14]. We also have shown that 1,2-oxazines inhibit the growth of colon cancer cells by
targeting the NF-κB signaling pathway and good anti-inflammatory activity was pronounced
in inflammatory bowel disease mice model [37]. Therefore, in continuation of our attempts to
explore oxazine derived potent NF-κB inhibitors, we prepared new 1,2-oxazine derivatives and
evaluated for cytotoxic potential towards HCC cells. We used HCC cells which possess
constitutive activation of NF-κB such as HepG2 and HCCLM3. Among the new chemical
17

reagents, 3i showed relatively good cytotoxicity than other structural analogs without
significantly affecting the viability of normal cells. Mansouri and colleagues also reported the
similar cytotoxicity profile of oxazine derivatives towards chronic lymphocytic leukemia,
colon and breast cancer cells and less toxic effects towards non-cancerous peripheral blood
mononuclear cells [64]. However, the cytotoxic effect of the lead compound (3i) was
significantly less compared to the standard therapeutic agents such as doxorubicin and
paclitaxel.
The genomic DNA is fragmented during apoptosis and these cells are recognized as subG1
population in flow cytometric analysis [65]. 3i presented the increase in subG1 cells indicating
its proapoptotic activity and this effect was further confirmed by annexin V-FITC-PI staining.
3i significantly increased the FITC-PI stained cells indicating phosphatidylserine
externalization and internalization of propidium iodide. Furthermore, a large number of human
cancers possess persistent activation of NF-κB due to oncogenic mutations and
proinflammatory microenvironment and NF-κB has been entangled with the initiation and
progression of cancer and metastasis [66-68]. We have previously demonstrated that NF-κB
modulators induce apoptosis in cancer cells [69-71]. Treatment of NF-κB-p65 siRNA
transfected cells with 3i did not show increased DNA fragmentation, whereas the treatment of
scrambled siRNA transfected cells with 3i showed elevated DNA fragmentation. These results
indicated that 3i mediates proapoptotic effect only via targeting NF-κB signaling and
knockdown of p65 nullified the proapoptotic effect of 3i. By this result, we predict that 3i may
not be an effective cytotoxic agent towards NF-κB-negative cancer cells. Persistent activation
of NF-κB is seen in a wide range of tumors and its overexpression can be a predictive marker
of negative prognosis [72-74]. Although all the assays were performed with HCC cell lines, we
were interested to understand the effect of 3i on breast cancer cell line which possesses
constitutively active NF-κB. Therefore, we selected two NF-κB expressing cancer cell lines
18

(HepG2 and MDA-MB-231) of different tissue origin (liver and breast respectively) and tested
the effect of 3i on the DNA binding ability of NF-κB. Compound 3i decreased the DNA binding
ability of NF-κB in both cell lines suggesting that 3i interferes with the transcriptional activity
of NF-κB. The transcription inhibitory effect of 3i was pronounced by the results of luciferase
reporter gene expression. In conclusion, we report a new oxazine derived NF-κB inhibitor in
HCC cells and validated in cell-based experiments and in silico analysis. Although the present
investigation establishes NF-κB as the major cellular target of oxazines, the further detailed
study is essential to learn about its off-targets, toxicity, and in vivo effects in suitable tumor
models.
Author Contributions
CS, SA, D, CPB, SM, and MKS performed the experiments. SR, AC, TAA, SAA, AS, CDM,
GS, B and KSR provided the resources. CDM, B, and KSR wrote the manuscript.
Conflict of interest
None declared
Acknowledgments
This research was supported by the Department of Biotechnology-NER
(BT/PR24978/NER/95/938/2017), Council of Scientific and Industrial Research
(No.02(0291)17/EMR-II), Vision group on Science and Technology, Government Of Karntaka
(CESEM/GRD637-2018) to Basappa. The authors extend their appreciation to the International
Scientific Partnership Program ISPP at King Saud University for funding this research work
through ISPP# 0091.
References
[1] S. Shishodia, B.B. Aggarwal, Nuclear factor-kappaB: a friend or a foe in cancer?, Biochem.
Pharmacol. 68 (2004) 1071-1080.
19

[2] Y.R. Puar, M.K. Shanmugam, L. Fan, F. Arfuso, G. Sethi, V. Tergaonkar, Evidence for the Involvement
of the Master Transcription Factor NF-kappaB in Cancer Initiation and Progression, Biomedicines. 6
(2018).
[3] R. Ningegowda, N.S. Shivananju, P. Rajendran, Basappa, K.S. Rangappa, A. Chinnathambi, F. Li, R.R.
Achar, M.K. Shanmugam, P. Bist, S.A. Alharbi, L.H.K. Lim, G. Sethi, B.S. Priya, A novel 4,6-disubstituted-
1,2,4-triazolo-1,3,4-thiadiazole derivative inhibits tumor cell invasion and potentiates the apoptotic
effect of TNFalpha by abrogating NF-kappaB activation cascade, Apoptosis. 22 (2017) 145-157.
[4] F. Li, J. Zhang, F. Arfuso, A. Chinnathambi, M.E. Zayed, S.A. Alharbi, A.P. Kumar, K.S. Ahn, G. Sethi,
NF-kappaB in cancer therapy, Arch. Toxicol. 89 (2015) 711-731.
[5] S.C. Gupta, C. Sundaram, S. Reuter, B.B. Aggarwal, Inhibiting NF-kappaB activation by small
molecules as a therapeutic strategy, Biochim. Biophys. Acta. 1799 (2010) 775-787.
[6] C. Kim, S.K. Cho, K.D. Kim, D. Nam, W.S. Chung, H.J. Jang, S.G. Lee, B.S. Shim, G. Sethi, K.S. Ahn,
beta-Caryophyllene oxide potentiates TNFalpha-induced apoptosis and inhibits invasion through
down-modulation of NF-kappaB-regulated gene products, Apoptosis. 19 (2014) 708-718.
[7] F. Li, M.K. Shanmugam, L. Chen, S. Chatterjee, J. Basha, A.P. Kumar, T.K. Kundu, G. Sethi, Garcinol,
a polyisoprenylated benzophenone modulates multiple proinflammatory signaling cascades leading
to the suppression of growth and survival of head and neck carcinoma, Cancer prev. Res. 6 (2013) 843-
854.
[8] G. Sethi, V. Tergaonkar, Potential pharmacological control of the NF-kappaB pathway, Trends
Pharmacol Sci. 30 (2009) 313-321.
[9] R. Sen, D. Baltimore, Multiple nuclear factors interact with the immunoglobulin enhancer
sequences, Cell. 46 (1986) 705-716.
[10] G. Sethi, M.K. Shanmugam, L. Ramachandran, A.P. Kumar, V. Tergaonkar, Multifaceted link
between cancer and inflammation, Biosci. Rep. 32 (2012) 1-15.
[11] K.S. Siveen, N. Mustafa, F. Li, R. Kannaiyan, K.S. Ahn, A.P. Kumar, W.J. Chng, G. Sethi,
Thymoquinone overcomes chemoresistance and enhances the anticancer effects of bortezomib
through abrogation of NF-kappaB regulated gene products in multiple myeloma xenograft mouse
model, Oncotarget. 5 (2014) 634-648.
[12] A.S. Nair, S. Shishodia, K.S. Ahn, A.B. Kunnumakkara, G. Sethi, B.B. Aggarwal, Deguelin, an Akt
inhibitor, suppresses IkappaBalpha kinase activation leading to suppression of NF-kappaB-regulated
gene expression, potentiation of apoptosis, and inhibition of cellular invasion, J. Immunol. 177 (2006)
5612-5622.
[13] C.D. Mohan, N.C. Anilkumar, S. Rangappa, M.K. Shanmugam, S. Mishra, A. Chinnathambi, S.A.
Alharbi, A. Bhattacharjee, G. Sethi, A.P. Kumar, Basappa, K.S. Rangappa, Novel 1,3,4-Oxadiazole
Induces Anticancer Activity by Targeting NF-kappaB in Hepatocellular Carcinoma Cells, Front. Oncol. 8
(2018) 42.
[14] C.D. Mohan, H. Bharathkumar, Dukanya, S. Rangappa, M.K. Shanmugam, A. Chinnathambi, S.A.
Alharbi, T.A. Alahmadi, A. Bhattacharjee, P.E. Lobie, A. Deivasigamani, K.M. Hui, G. Sethi, Basappa, K.S.
Rangappa, A.P. Kumar, N-Substituted Pyrido-1,4-Oxazin-3-Ones Induce Apoptosis of Hepatocellular
Carcinoma Cells by Targeting NF-κB Signaling Pathway, Front. Pharmacol. 9 (2018) 1125.
[15] A.W.L. Chua, H.S. Hay, P. Rajendran, M.K. Shanmugam, F. Li, P. Bist, E.S.C. Koay, L.H.K. Lim, A.P.
Kumar, G. Sethi, Butein downregulates chemokine receptor CXCR4 expression and function through
suppression of NF-κB activation in breast and pancreatic tumor cells, Biochem. Pharmacol. 80 (2010)
1553-1562.
[16] K.A. Manu, M.K. Shanmugam, L. Ramachandran, F. Li, C.W. Fong, A.P. Kumar, P. Tan, G. Sethi, First
evidence that gamma-tocotrienol inhibits the growth of human gastric cancer and chemosensitizes it
to capecitabine in a xenograft mouse model through the modulation of NF-kappaB pathway, Clin.
Cancer Res. 18 (2012) 2220-2229.
[17] H.K. Keerthy, C.D. Mohan, K. Sivaraman Siveen, J.E. Fuchs, S. Rangappa, M.S. Sundaram, F. Li, K.S.
Girish, G. Sethi, Basappa, A. Bender, K.S. Rangappa, Novel synthetic biscoumarins target tumor
20

necrosis factor-alpha in hepatocellular carcinoma in vitro and in vivo, J. Biol. Chem. 289 (2014) 31879-
31890.
[18] E.M. Shin, H.S. Hay, M.H. Lee, J.N. Goh, T.Z. Tan, Y.P. Sen, S.W. Lim, E.M. Yousef, H.T. Ong, A.A.
Thike, X. Kong, Z. Wu, E. Mendoz, W. Sun, M. Salto-Tellez, C.T. Lim, P.E. Lobie, Y.P. Lim, C.T. Yap, Q.
Zeng, G. Sethi, M.B. Lee, P. Tan, B.C. Goh, L.D. Miller, J.P. Thiery, T. Zhu, L. Gaboury, P.H. Tan, K.M. Hui,
G.W. Yip, S. Miyamoto, A.P. Kumar, V. Tergaonkar, DEAD-box helicase DP103 defines metastatic
potential of human breast cancers, J. Clin. Invest. 124 (2014) 3807-3824.
[19] K.A. Manu, M.K. Shanmugam, F. Li, L. Chen, K.S. Siveen, K.S. Ahn, A.P. Kumar, G. Sethi, Simvastatin
sensitizes human gastric cancer xenograft in nude mice to capecitabine by suppressing nuclear factor-
kappa B-regulated gene products, J. Mol. Med. (Berl). 92 (2014) 267-276.
[20] G. Sethi, K.S. Ahn, M.M. Chaturvedi, B.B. Aggarwal, Epidermal growth factor (EGF) activates
nuclear factor-kappaB through IkappaBalpha kinase-independent but EGF receptor-kinase dependent
tyrosine 42 phosphorylation of IkappaBalpha, Oncogene. 26 (2007) 7324-7332.
[21] M. Neelgundmath, K.R. Dinesh, C.D. Mohan, F. Li, X. Dai, K.S. Siveen, S. Paricharak, D.J. Mason,
J.E. Fuchs, G. Sethi, A. Bender, K.S. Rangappa, O. Kotresh, Basappa, Novel synthetic coumarins that
targets NF-kappaB in Hepatocellular carcinoma, Bioorg. Med. Chem. Lett. 25 (2015) 893-897.
[22] A. Dey, E. Wong, N. Kua, H.L. Teo, V. Tergaonkar, D. Lane, Hexamethylene bisacetamide (HMBA)
simultaneously targets AKT and MAPK pathway and represses NF kappaB activity: implications for
cancer therapy, Cell cycle, 7 (2008) 3759-3767.
[23] E.Z. Chai, K.S. Siveen, M.K. Shanmugam, F. Arfuso, G. Sethi, Analysis of the intricate relationship
between chronic inflammation and cancer, Biochem. J. 468 (2015) 1-15.
[24] C.D. Mohan, S. Hari, H.D. Preetham, S. Rangappa, U. Barash, N. Ilan, S.C. Nayak, V.K. Gupta,
Basappa, I. Vlodavsky, K.S. Rangappa, Targeting Heparanase in Cancer: Inhibition by Synthetic,
Chemically Modified, and Natural Compounds, iScience. 15 (2019) 360-390.
[25] M.H. Park, J.T. Hong, Roles of NF-κB in Cancer and Inflammatory Diseases and Their Therapeutic
Approaches, Cells. 5 (2016) 15.
[26] P.S. Son, S.A. Park, H.K. Na, D.M. Jue, S. Kim, Y.J. Surh, Piceatannol, a catechol-type polyphenol,
inhibits phorbol ester-induced NF-{kappa}B activation and cyclooxygenase-2 expression in human
breast epithelial cells: cysteine 179 of IKK{beta} as a potential target, Carcinogenesis. 31 (2010) 1442-
1449.
[27] F. Li, G. Sethi, Targeting transcription factor NF-kappaB to overcome chemoresistance and
radioresistance in cancer therapy, Biochim. Biophys. Acta. 1805 (2010) 167-180.
[28] X. Xiao, G. Yang, P. Bai, S. Gui, T.M.B. Nyuyen, I. Mercado-Uribe, M. Yang, J. Zou, Q. Li, J. Xiao, B.
Chang, G. Liu, H. Wang, J. Liu, Inhibition of nuclear factor-kappa B enhances the tumor growth of
ovarian cancer cell line derived from a low-grade papillary serous carcinoma in p53-independent
pathway, BMC Cancer. 16 (2016) 582.
[29] G. Sethi, B. Sung, B.B. Aggarwal, Nuclear factor-kappaB activation: from bench to bedside, Exp.
Biol. Med. (Maywood). 233 (2008) 21-31.
[30] A. Subramaniam, M.K. Shanmugam, T.H. Ong, F. Li, E. Perumal, L. Chen, S. Vali, T. Abbasi, S.
Kapoor, K.S. Ahn, A.P. Kumar, K.M. Hui, G. Sethi, Emodin inhibits growth and induces apoptosis in an
orthotopic hepatocellular carcinoma model by blocking activation of STAT3, Br. J. Pharmacol. 170
(2013) 807-821.
[31] D.I. Tai, S.L. Tsai, Y.H. Chang, S.N. Huang, T.C. Chen, K.S. Chang, Y.F. Liaw, Constitutive activation
of nuclear factor kappaB in hepatocellular carcinoma, Cancer. 89 (2000) 2274-2281.
[32] W. Li, D. Tan, M.J. Zenali, R.E. Brown, Constitutive activation of nuclear factor-kappa B (NF-kB)
signaling pathway in fibrolamellar hepatocellular carcinoma, Int. J. Clin. Exp. Pathol. 3 (2009) 238-243.
[33] L. Qiao, H. Zhang, J. Yu, R. Francisco, P. Dent, M.P.A. Ebert, C. Röcken, D.G. Farrell, Constitutive
Activation of NF-κB in Human Hepatocellular Carcinoma: Evidence of a Cytoprotective Role, Hum.
Gene. Ther. 17 (2006) 280-290.
21

[34] S. Ananthula, P. Parajuli, F.A. Behery, A.Y. Alayoubi, S. Nazzal, K. El Sayed, P.W. Sylvester, -
Tocotrienol Oxazine Derivative Antagonizes Mammary Tumor Cell Compensatory Response to CoCl2-
Induced Hypoxia, Biomed. Res. Int. 2014 (2014) 13.
[35] H. Bharathkumar, C.D. Mohan, S. Rangappa, T. Kang, H.K. Keerthy, J.E. Fuchs, N.H. Kwon, A.
Bender, S. Kim, Basappa, K.S. Rangappa, Screening of quinoline, 1,3-benzoxazine, and 1,3-oxazine-
based small molecules against isolated methionyl-tRNA synthetase and A549 and HCT116 cancer cells
including an in silico binding mode analysis, Org. Biomol. Chem. 13 (2015) 9381-9387.
[36] C.D. Mohan, H. Bharathkumar, K.C. Bulusu, V. Pandey, S. Rangappa, J.E. Fuchs, M.K. Shanmugam,
X. Dai, F. Li, A. Deivasigamani, K.M. Hui, A.P. Kumar, P.E. Lobie, A. Bender, Basappa, G. Sethi, K.S.
Rangappa, Development of a novel azaspirane that targets the Janus kinase-signal transducer and
activator of transcription (STAT) pathway in hepatocellular carcinoma in vitro and in vivo, J. Biol. Chem.
289 (2014) 34296-34307.
[37] A.C. Nirvanappa, C.D. Mohan, S. Rangappa, H. Ananda, A.Y. Sukhorukov, M.K. Shanmugam, M.S.
Sundaram, S.C. Nayaka, K.S. Girish, A. Chinnathambi, M.E. Zayed, S.A. Alharbi, G. Sethi, Basappa, K.S.
Rangappa, Novel Synthetic Oxazines Target NF-kappaB in Colon Cancer In Vitro and Inflammatory
Bowel Disease In Vivo, PloS one. 11 (2016) e0163209.
[38] V. Srinivas, C.D. Mohan, C.P. Baburajeev, S. Rangappa, S. Jagadish, J.E. Fuchs, A.Y. Sukhorukov,
Chandra, D.J. Mason, K.S. Sharath Kumar, M. Madegowda, A. Bender, Basappa, K.S. Rangappa,
Synthesis and characterization of novel oxazines and demonstration that they specifically target
cyclooxygenase 2, Bioorg. Med. Chem. Lett. 25 (2015) 2931-2936.
[39] N.B. Sulaiman, C.D. Mohan, S. Basappa, V. Pandey, S. Rangappa, H. Bharathkumar, A.P. Kumar,
P.E. Lobie, K.S. Rangappa, An azaspirane derivative suppresses growth and induces apoptosis of ER-
positive and ER-negative breast cancer cells through the modulation of JAK2/STAT3 signaling pathway,
Int. J. Oncol. 49 (2016) 1221-1229.
[40] F. Uzma, C.D. Mohan, A. Hashem, N.M. Konappa, S. Rangappa, P.V. Kamath, B.P. Singh, V. Mudili,
V.K. Gupta, C.N. Siddaiah, S. Chowdappa, A.A. Alqarawi, E.F. Abd Allah, Endophytic Fungi-Alternative
Sources of Cytotoxic Compounds: A Review, Front. Pharmacol. 9 (2018) 309.
[41] S. Ananthula, P. Parajuli, F.A. Behery, A.Y. Alayoubi, K.A. El Sayed, S. Nazzal, P.W. Sylvester,
Oxazine derivatives of gamma- and delta-tocotrienol display enhanced anticancer activity in vivo,
Anticancer Res. 34 (2014) 2715-2726.
[42] N. Ansari, F. Khodagholi, M. Amini, F. Shaerzadeh, Attenuation of LPS-induced apoptosis in NGF-
differentiated PC12 cells via NF-kappaB pathway and regulation of cellular redox status by an oxazine
derivative, Biochimie. 93 (2011) 899-908.
[43] K.S. Rakesh, S. Jagadish, T.R. Swaroop, C.D. Mohan, N. Ashwini, K.B. Harsha, F. Zameer, K.S. Girish,
K.S. Rangappa, Anti-Cancer Activity of 2,4-Disubstituted Thiophene Derivatives: Dual Inhibitors of
Lipoxygenase and Cyclooxygenase, Med. Chem. 11 (2015) 462-472.
[44] N.C. Anilkumar, M.S. Sundaram, C.D. Mohan, S. Rangappa, K.C. Bulusu, J.E. Fuchs, K.S. Girish, A.
Bender, Basappa, K.S. Rangappa, A One Pot Synthesis of Novel Bioactive Tri-Substitute-Condensed-
Imidazopyridines that Targets Snake Venom Phospholipase A2, PloS one. 10 (2015) e0131896.
[45] N. Ashwini, M. Garg, C.D. Mohan, J.E. Fuchs, S. Rangappa, S. Anusha, T.R. Swaroop, K.S. Rakesh,
D. Kanojia, V. Madan, A. Bender, H.P. Koeffler, Basappa, K.S. Rangappa, Synthesis of 1,2-benzisoxazole
tethered 1,2,3-triazoles that exhibit anticancer activity in acute myeloid leukemia cell lines by
inhibiting histone deacetylases, and inducing p21 and tubulin acetylation, Bioorg. Med. Chem. 23
(2015) 6157-6165.
[46] C.D. Mohan, V. Srinivasa, S. Rangappa, L. Mervin, S. Mohan, S. Paricharak, S. Baday, F. Li, M.K.
Shanmugam, A. Chinnathambi, M.E. Zayed, S.A. Alharbi, A. Bender, G. Sethi, Basappa, K.S. Rangappa,
Trisubstituted-Imidazoles Induce Apoptosis in Human Breast Cancer Cells by Targeting the Oncogenic
PI3K/Akt/mTOR Signaling Pathway, PloS one, 11 (2016) e0153155.
[47] M. Gilandoust, K.B. Harsha, C.D. Mohan, A.R. Raquib, S. Rangappa, V. Pandey, P.E. Lobie, Basappa,
K.S. Rangappa, Synthesis, characterization and cytotoxicity studies of 1,2,3-triazoles and 1,2,4-triazolo
[1,5-a] pyrimidines in human breast cancer cells, Bioorg. Med. Chem. Lett. 28 (2018) 2314-2319.
22

[48] A. Sebastian, V. Pandey, C.D. Mohan, Y.T. Chia, S. Rangappa, J. Mathai, C.P. Baburajeev, S.
Paricharak, L.H. Mervin, K.C. Bulusu, J.E. Fuchs, A. Bender, S. Yamada, Basappa, P.E. Lobie, K.S.
Rangappa, Novel Adamantanyl-Based Thiadiazolyl Pyrazoles Targeting EGFR in Triple-Negative Breast
Cancer, ACS Omega. 1 (2016) 1412-1424.
[49] A.Y. Sukhorukov, A.C. Nirvanappa, J. Swamy, S.L. Ioffe, S. Nanjunda Swamy, Basappa, K.S.
Rangappa, Synthesis and characterization of novel 1,2-oxazine-based small molecules that targets
acetylcholinesterase, Bioorg. Med. Chem. Lett. 24 (2014) 3618-3621.
[50] S. Anusha, B.S. Anandakumar, C.D. Mohan, G.P. Nagabhushana, B.S. Priya, K.S. Rangappa,
Basappa, C.G. T, Preparation and use of combustion-derived Bi2O3 for the synthesis of heterocycles
with anti-cancer properties by Suzuki-coupling reactions, RSC Adv. 4 (2014) 52181-52188.
[51] S. Anusha, C.D. Mohan, H. Ananda, C.P. Baburajeev, S. Rangappa, J. Mathai, J.E. Fuchs, F. Li, M.K.
Shanmugam, A. Bender, G. Sethi, Basappa, K.S. Rangappa, Adamantyl-tethered-biphenylic compounds
induce apoptosis in cancer cells by targeting Bcl homologs, Bioorg. Med. Chem. Lett. 26 (2016) 1056-
1060.
[52] C.C. Woo, A. Hsu, A.P. Kumar, G. Sethi, K.H.B. Tan, Thymoquinone Inhibits Tumor Growth and
Induces Apoptosis in a Breast Cancer Xenograft Mouse Model: The Role of p38 MAPK and ROS, PloS
one. 8 (2013) e75356.
[53] R. Roopashree, C.D. Mohan, T.R. Swaroop, S. Jagadish, B. Raghava, K.S. Balaji, S. Jayarama,
Basappa, K.S. Rangappa, Novel synthetic bisbenzimidazole that targets angiogenesis in Ehrlich ascites
carcinoma bearing mice, Bioorg. Med. Chem. Lett. 25 (2015) 2589-2593.
[54] C.P. Baburajeev, C. Dhananjaya Mohan, H. Ananda, S. Rangappa, J.E. Fuchs, S. Jagadish, K.
Sivaraman Siveen, A. Chinnathambi, S. Ali Alharbi, M.E. Zayed, J. Zhang, F. Li, G. Sethi, K.S. Girish, A.
Bender, Basappa, K.S. Rangappa, Development of Novel Triazolo-Thiadiazoles from Heterogeneous
"Green" Catalysis as Protein Tyrosine Phosphatase 1B Inhibitors, Sci. Rep. 5 (2015) 14195.
[55] K.N. Nandeesh, H.A. Swarup, N.C. Sandhya, C.D. Mohan, C.S. Pavan Kumar, M.N. Kumara, K.
Mantelingu, S. Ananda, K.S. Rangappa, Synthesis and antiproliferative efficiency of novel bis(imidazol-
1-yl)vinyl-1,2,4-oxadiazoles, New J. Chem. 40 (2016) 2823-2828.
[56] S.M. Tan, F. Li, P. Rajendran, A.P. Kumar, K.M. Hui, G. Sethi, Identification of beta-escin as a novel
inhibitor of signal transducer and activator of transcription 3/Janus-activated kinase 2 signaling
pathway that suppresses proliferation and induces apoptosis in human hepatocellular carcinoma cells,
J. Pharmacol. Exp. Ther. 334 (2010) 285-293.
[57] H.K. Keerthy, M. Garg, C.D. Mohan, V. Madan, D. Kanojia, R. Shobith, S. Nanjundaswamy, D.J.
Mason, A. Bender, Basappa, K.S. Rangappa, H.P. Koeffler, Synthesis and characterization of novel 2-
amino-chromene-nitriles that target Bcl-2 in acute myeloid leukemia cell lines, PloS one, 9 (2014)
e107118.
[58] V. Pandey, B. Wang, C.D. Mohan, A.R. Raquib, S. Rangappa, V. Srinivasa, J.E. Fuchs, K.S. Girish, T.
Zhu, A. Bender, L. Ma, Z. Yin, Basappa, K.S. Rangappa, P.E. Lobie, Discovery of a small-molecule
inhibitor of specific serine residue BAD phosphorylation, Proc. Natl. Acad. Sci. U S A. 115 (2018)
E10505-e10514.
[59] K.S. Rakesh, S. Jagadish, K.S. Balaji, F. Zameer, T.R. Swaroop, C.D. Mohan, S. Jayarama, K.S.
Rangappa, 3,5-Disubstituted Isoxazole Derivatives: Potential Inhibitors of Inflammation and Cancer,
Inflammation. 39 (2016) 269-280.
[60] S.S. Singh, W.N. Yap, F. Arfuso, S. Kar, C. Wang, W. Cai, A.M. Dharmarajan, G. Sethi, A.P. Kumar,
Targeting the PI3K/Akt signaling pathway in gastric carcinoma: A reality for personalized medicine?,
World. J. Gastroenterol. 21 (2015) 12261-12273.
[61] K.S. Oh, S. Lee, J.K. Choi, B.H. Lee, Identification of novel scaffolds for IkappaB kinase beta inhibitor
via a high-throughput screening TR-FRET assay, Comb. Chem. High T. Scr. 13 (2010) 790-797.
[62] G. Lyss, A. Knorre, T.J. Schmidt, H.L. Pahl, I. Merfort, The anti-inflammatory sesquiterpene lactone
helenalin inhibits the transcription factor NF-kappaB by directly targeting p65, J. Biol. Chem. 273
(1998) 33508-33516.
23

[63] R. Chandramohanadas, Basappa, B. Russell, K. Liew, Y.H. Yau, A. Chong, M. Liu, K. Gunalan, R.
Raman, L. Renia, F. Nosten, S.G. Shochat, M. Dao, R. Sasisekharan, S. Suresh, P. Preiser, Small molecule
targeting malaria merozoite surface protein-1 (MSP-1) prevents host invasion of divergent plasmodial
species, J. Infect. Dis. 210 (2014) 1616-1626.
[64] S.G. Mansouri, H. Zali-Boeini, K. Zomorodian, B. Khalvati, R.H. Pargali, A. Dehshahri, H.A. Rudbari,
M. Sahihi, Z. Chavoshpour, Synthesis of novel naphtho[1,2-e][1,3]oxazines bearing an arylsulfonamide
moiety and their anticancer and antifungal activity evaluations, Arab. J. Chem. 13 (2020) 1271-1282.
[65] D. Plesca, S. Mazumder, A. Almasan, DNA damage response and apoptosis, Method. Enzymol. 446
(2008) 107-122.
[66] Y. Xia, S. Shen, I.M. Verma, NF-κB, an Active Player in Human Cancers, Cancer Immunol. Res. 2
(2014) 823-830.
[67] S.K. Manna, R.S. Aggarwal, G. Sethi, B.B. Aggarwal, G.T. Ramesh, Morin (3,5,7,2',4'-
Pentahydroxyflavone) abolishes nuclear factor-kappaB activation induced by various carcinogens and
inflammatory stimuli, leading to suppression of nuclear factor-kappaB-regulated gene expression and
up-regulation of apoptosis, Clin. Cancer Res. 13 (2007) 2290-2297.
[68] M. Sawhney, N. Rohatgi, J. Kaur, S. Shishodia, G. Sethi, S.D. Gupta, S.V. Deo, N.K. Shukla, B.B.
Aggarwal, R. Ralhan, Expression of NF-kappaB parallels COX-2 expression in oral precancer and cancer:
association with smokeless tobacco, Int. J. Cancer. 120 (2007) 2545-2556.
[69] K.S. Ahn, G. Sethi, M.M. Chaturvedi, B.B. Aggarwal, Simvastatin, 3-hydroxy-3-methylglutaryl
coenzyme A reductase inhibitor, suppresses osteoclastogenesis induced by receptor activator of
nuclear factor-kappaB ligand through modulation of NF-kappaB pathway, Int. J. Cancer. 123 (2008)
1733-1740.
[70] K.S. Ahn, G. Sethi, B.B. Aggarwal, Reversal of chemoresistance and enhancement of apoptosis by
statins through down-regulation of the NF-kappaB pathway, Biochem. Pharmacol. 75 (2008) 907-913.
[71] G. Sethi, K.S. Ahn, B. Sung, B.B. Aggarwal, Pinitol targets nuclear factor-kappaB activation pathway
leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and
angiogenesis, Mol. Cancer Ther. 7 (2008) 1604-1614.
[72] D.K. Sarkar, D. Jana, P.S. Patil, K.S. Chaudhari, B.K. Chattopadhyay, B.R. Chikkala, S. Mandal, P.
Chowdhary, Role of NF-κB as a Prognostic Marker in Breast Cancer : A Pilot Study in Indian Patients,
Indian J. Surg. Oncol. 4 (2013) 242-247.
[73] L. Gu, Z. Wang, J. Zuo, H. Li, L. Zha, Prognostic significance of NF-κB expression in non-small cell
lung cancer: A meta-analysis, PloS one, 13 (2018) e0198223.
[74] K.S. Ahn, G. Sethi, A.K. Jain, A.K. Jaiswal, B.B. Aggarwal, Genetic deletion of NAD(P)H:quinone
oxidoreductase 1 abrogates activation of nuclear factor-kappaB, IkappaBalpha kinase, c-Jun N-
terminal kinase, Akt, p38, and p44/42 mitogen-activated protein kinases and potentiates apoptosis, J.
Biol. Chem. 281 (2006) 19798-19808.
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Figure legends
Figure 1: Structures of the previously reported biologically active oxazines that target NF-κB.
Figure 2: The synthetic route for the preparation of title compounds (3a-j). 1a-f represents the
bromides that are used in the preparation of 1,2-oxazines (3a-j) and structures of bromides are
provided in Table 1.
Figure 3: 3i elicits growth inhibitory effects in HCC cells. HepG2 and HCCLM3 cells were
treated with different concentrations (25, 50 and 100 µM) of 3i at indicated time intervals (24,
48, and 72h) and then subjected to MTT assay as described in materials and methods (*p <
0.05).
Figure 4: 3i induced apoptosis in HCC cells. The distribution of cell cycle in 3i treated HepG2
and HCCLM3 cells were examined using flow cytometry. Cells were exposed to 3i (50 μM) at
indicated times (0, 24, 48 and 72 h), after which cells were harvested and stained with
propidium iodide.
Figure 5: (A) 3i induced apoptosis in HepG2 cells. Cells were exposed to 3i (50 μM) for 48 h,
after which cells were harvested and stained with Annexin V and propidium iodide. The
percentage of early and late apoptotic cells were detected using flow cytometry. Results show
early apoptosis, defined as annexin V-positive and PI-negative cells, and late apoptosis, defined
as annexin V-positive and PI-positive cells. (B) The knockdown of p65 by small interfering
RNA (siRNA) reduces the 3i-induced DNA fragmentation. HepG2 cells were transfected with
either control or p65 specific siRNA (50 nM). After 48 h, the cells were treated with 3i (25 or
50 μM) for 72 h, and the DNA fragmentation was analyzed by the ELISA assay kit. The data
is expressed as mean ± SD, compared with the untreated control, (*p < 0.05). (C) Western blot
analysis revealed that siRNA efficiently knocked down NF-B-p65 in both the cell lines tested.
25

Figure 6: (A) The effect of 3i on the constitutive DNA-binding activity of NF-κB. 3i
downregulated NF-κB DNA binding ability in HepG2 and MDA-MB-231 cells. The cells were
treated with 0, 10, 25 and 50 µM 3i for 8 hr and nuclear extracts were prepared and 50 µg of
nuclear extract protein was used for DNA binding assay. The NF-κB DNA binding was
inhibited in a dose-dependent manner. WT (wild type oligonucleotide); MT (mutant
oligonucleotide). 3i was found to reduce the DNA-binding activity of constitutively activated
NF-κB.
Figure 7: (A and B) 3i suppresses constitutive activation of reporter gene expression. HepG2
(5 × 105/mL) and HCCLM3 (5 × 105/mL) cells were transfected with NF-κB luciferase and β-
galactosidase reporter plasmid using lipofectamine, incubated for 24 h, and then treated with
3i for 5, 10, and 20 h. Cells were lysed in reporter lysis buffer and analyzed for luciferase
activity and normalized with β-galactosidase activity. Results are expressed as fold activity
over the activity of vector control. *p < 0.05.
Figure 8: In silico molecular interactions between p65 of NF-κB complex and the oxazines:
(A) DNA bound p65 subunit is shown, where the stick form of the amino acid Cys38 region
was used for docking studies (B & C) The surface view and interaction map for the lead
compound 3i interaction with p65 subunit in the hydrophobic region is shown. 3i established
hydrophobic interaction with Tyr 36 and Arg 187 of p65, which are present at the vicinity of
the hydrophobic region near Cys38 of p65 protein.
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