Synthesis and evaluation of new 4(3H)-Quinazolinone derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.molstruc.2019.127097

A series of new 4(3H)-quinazolinones were synthesized and evaluated for their cytotoxic activity against a set of human cancer cell lines MDA-MB-231 and MCF-7 (breast), HCT-116 and HT-29 (colon) and A549 (lung). Among the tested compounds, 22a exhibited p

Full text of DOI:10.1016/j.molstruc.2019.127097

Journal of Molecular Structure 1200 (2020) 127097
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis and evaluation of new 4(3H)-Quinazolinone derivatives as
potential anticancer agents
Srikanth Gatadi a, Gauthami Pulivendala b, Jitendra Gour a, Satyaveni Malasala a,
Sushmitha Bujji a, Ramulu Parupalli a, Mujahid Shaikh a, Chandraiah Godugu b,
,
*
Srinivas Nanduri a
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, 500 037, India
b Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, 500 037, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 4(3H)-quinazolinones were synthesized and evaluated for their cytotoxic activity against
a set of human cancer cell lines MDA-MB-231 and MCF-7 (breast), HCT-116 and HT-29 (colon) and A549
(lung). Among the tested compounds, 22a exhibited promising cytotoxic activity against MDA-MB-231
Received 11 June 2019
Received in revised form
18 September 2019
Accepted 19 September 2019
Available online 19 September 2019
(IC50: 3.21 mM) and HT-29 (IC50: 7.23 mM) cell lines. The mechanism of action and the apoptosis
inducing effect of the compound 22a were studied using the breast cancer cell line MDA-MB-231.
Treatment of MDA-MB-231 cell line with compound 22a showed typical apoptotic morphology like
cell shrinkage, chromatin condensation and horseshoe shaped nuclei formation. Flow cytometric analysis
indicated that the compound induces G0/G1 phase of cell cycle arrest in a dose dependent manner. The
binding modes of the potent compounds with EGFR target protein were investigated by docking studies.
© 2019 Elsevier B.V. All rights reserved.
Keywords:
4(3H)-quinazolinone
Cytotoxicity
MDA-MB-231
Cell migration
Colony formation
1. Introduction
inhibition. Raffa et al. [14] reported the cytotoxic properties of new
2-styrylquinazolinone derivatives V. Suwaidan and co-workers
Cancer is a hyper proliferative disease with the potential to
invade to other parts of the body [1]. As the current chemothera-
peutic agents are not sufficient to tackle the spread of this dreadful
disease [2], search for new chemotherapeutic agents which can
arrest cell proliferation are being explored. In the quest for new
anticancer agents, efforts have been focused on the development of
compounds with 4(3H)-quinazolinone heterocycle. 4(3H)-Quina-
zolinone is a privileged structure possessing wide range of bio-
logical properties such as anticancer, antimicrobial, anti-
inflammatory and anticonvulsant activities. etc [3e8]. Idelalisib I,
a quinazolinone based drug used for the treatment of certain he-
matological malignancies acts as a phosphoinositide-3-kinase in-
hibitor [9,10]. Canertinib II is used as an experimental drug
candidate for the treatment of cancer [11]. Cao et al. [12] reported
the in vitro antitumor activity of 4(3H)-quinazolinones with
dithiocarbamate side chains III. Al-Rashood et al. [13] reported a
series of compounds IV with both antitumor potency and DHFR
reported a novel series of 3-benzyl-substituted-4(3H)-quinazoli-
nones VI as potential anti-tumor agents [15] (Fig. 1).
Additionally, several Quinazolinone based compounds have
been shown to exert their anti-cancer activity through EGFR kinase
inhibition. Erlotinib VII [16], used for the treatment of non-small
cell lung cancer and pancreatic cancer acts on the epidermal
growth factor receptor (EGFR). Lapatinib VIII [17], an orally active
drug for breast cancer and other solid tumours is found to be a dual
tyrosine kinase inhibitor interfering with the HER2/neu and
epidermal growth factor receptor (EGFR) pathways. Yin et al. [18]
reported the anticancer potential of new EGFR/HER2 dual in-
hibitors bearing an oxazolo[4,5-g]quinazolin-2(1H)-one moiety IX.
Tsou et al. [19] reported a series of new 6-substituted-4-(3-
bromophenylamino)quinazoline derivatives X that might function
as irreversible inhibitors of EGFR and human epidermal growth
factor receptor (HER-2) tyrosine kinases. Soliman et al. [20]
explored a series of new N-alkyl-2-[(4-oxo-3-(4-sulfamoylphenyl)-
3,4-dihydroquinazolin-2-yl)thio]acetamide derivatives XI as anti-
cancer agents. Soliman et al. [21] also reported the series of new
sulphonamide benzoquinazolinones XII as dual EGFR/HER2 in-
hibitors (Fig. 2).
* Corresponding author.
E-mail address: nandurisrini92@gmail.com (S. Nanduri).
https://doi.org/10.1016/j.molstruc.2019.127097
0022-2860/© 2019 Elsevier B.V. All rights reserved.

2
S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
F
On the other hand, 1,2,3-triazole (Fig. 3) could also be consid-
F
O
N
ered as an important scaffold due to its high stability, hydrogen
bonding ability and broad applicability in medicinal chemistry [22].
In recent years, researchers have increasingly focused on its anti-
cancer potential. Duan et al. [23] reported the 1,2,3-triazole-
dithiocarbamate hybrids XIII as potential anticancer agents. Kamal
et al. [24] reported the anticancer properties of new 1,2,3-triazole-
linked pyrrolobenzodiazepine conjugates XIV. Recently, Sumit and
co-workers reported a series of new 1H-1,2,3-triazole tethered
nitroimidazoleꢀisatin conjugates XV as potent anti-cancer agents
[25].
HN
N
Cl
H
N
S
O
N
N
N
N
R1
N
S
NH
CH3
O
O
R2
NH
N
H
N
N
N
III
II
I
O
O
N
O
O
O
O
N
N
Cl
R
N
H2N
N
S
N
O
S
Hybridization of the two dissimilar bioactive pharmacophores
in a single molecule is a frequently used approach for the explo-
ration of novel compounds which may have synergistic effect [26].
In the current work, we have designed and synthesized new 1,2,3-
CH3
HN
O
O
VI
V
IV
O
Fig. 1. Structure of some reported bioactive quinazolinones as anticancer agents.
HN
N
O
O
O
O
N
VII
O
N
F
R1
R2
R3
N
O
O
S
O
R4
NH2
O
N
HN
Cl
N
O
S
HN
N
O
S
N
H
N
O
O
R
XII
VIII
N
O
Br
N
N
N
N
22a
Compound
IC50 on
MDA-MB-231 cell line: 3.21 µM
O
S
O
NH2
Cl
R2
R1
O
O
O
O
N
HN
N
N
S
N
O
H
N
N
N
R
XI
HN
H
Br
O
IX
N
N
O
N
X
Fig. 2. Design of 4(3H)-Quinazolinone derivatives as new EGFR inhibitors.
S
N
N
O
N
H3CO2C
N
O
n
O
N
S
N
N
N
N
N
O
N
H3CO2C
N
N
N
O
N
N
N
O
F
N
O
O2N
R
N
XIII
H3CO2C
XV
XIV
CO2CH3
Fig. 3. Structure of some reported bioactive 1,2,3-triazoles as anticancer agents.

S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
3
O
O
R1
HO
COOH
NH2
HO
Ac2O
O
O
Cl
R2
H2N
Reflux
2hrs
N
7
O
O2N
2
1
6
acetonitrile,
0
pyridine
reflux, 4 h
oC-rt, 4 h
H2N
COOMe
O
O
R1
O
O
N
N
H
R2
HO
N
O
O2N
3
O
8
1. SnCl2 2H2O, ethanol, reflux, 5 h
2. HCl, NaNO2, NaN3, 0 oC-rt, 3 h
propargyl bromide
K2CO3, DMF, 0 oC-rt,
6 hrs
O
R1
OH
N
O
N
H
R2
O
R1
Cl
R1 N3
O
O
O
N
4
N3
Sodium ascorbate (10 mol%)
CuSO4.5H2O (2 mol%)
Sodium ascorbate (10 mol%)
CuSO4.5H2O (2 mol%)
tert-butanol:H2O 1:1
10-12 h
O
N
Sodium ascorbate (5 mol%)
CuSO4.5H2O (2 mol%)
9
tert-butanol:H2O 1:1
tert-BuOH/H2O (1:1)
12-14 h
8 h
O
N
O
O
R1
O
R1
O
O
R1
N
O
O
N
N
O
N
N
H
R2
N
N
O
O
O
N
N
10
N
11
N
5
10a: R1
10b: R1
=
=
4-chloro phenyl
4-methyl phenyl
11a: R1
11b: R1
11c: R1
11d: R1
=
3-chloro phenyl
=
=
=
4-methoxy phenyl
3-methyl phenyl
5a: R
5b: R
1 = benzyl; R2 = CH3
1 = isopropyl; R2 = CH2CH3
3,4,5- trimethyl phenyl
Scheme 1. Synthesis of 4-(4-phenyl-1H-1,2,3-triazol-1-yl)benzamide 5a-5b and methyl 2-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetate 10a-10b, 11a-11d derivatives.
triazole linked 4(3H)-quinazolinone and also some benzamido ac-
etate derivatives and evaluated them for their cytotoxic potential
against a number of human cancer cell lines. We have also studied
their binding affinity to EGFR protein kinase through molecular
docking studies.
Treatment of 9 with various substituted azides under click condi-
tions gave the corresponding triazole derivatives 11a-11d. When
the intermediate 9 was reacted with oxime gave isoxazole de-
rivatives 10a and 10b (Scheme 1).
Similarly, when a mixture of benzoxazinone intermediate 7 and
substituted benzylamine in glacial acetic acid were refluxed, it
yielded 3-benzyl-6-hydroxy-2-methylquinazolin-4(3H)-one deriv-
ative 14 in moderate to good yields (Scheme 2). The intermediate
14 was further subjected to O-propargylation by using propargyl-
bromide to afford corresponding 5-O-propargylated quinazolinone
derivative 15. Treatment of 15 with various substituted azides un-
der click conditions gave the corresponding triazole derivatives
16a-16j. Further, intermediates 16a & 16b were reacted with 4-
cyano benzaldehyde in glacial acetic acid under reflux conditions
for overnight to afford the new 2-styrylquinazolin-4(3H)-one de-
rivatives 17a & 17b as final compounds (Scheme 2) in moderate to
excellent yields.
2. Results and discussion
2.1. Chemistry
A series of new 1,2,3-triazolylbenzamido acetate derivatives
were synthesized as shown in Scheme 1. 4-nitro benzoyl chloride 1
and amino acid ester 2 were reacted in acetonitrile at room tem-
perature to afford the intermediate 3. The nitro group in interme-
diate 3, was reduced in the presence of tin(II)chloride under reflux
conditions in ethanol. Further, the obtained aniline derivative was
allowed to undergo azidation reaction using sodium nitrite and
sodium azide in 2N HCl to afford the azide intermediate 4. Finally,
alkyl 2-(4-(4-phenyl-1H-1,2,3-triazol-1-yl)benzamido)acetate de-
rivatives 5a-5b were synthesized by reacting the azide intermedi-
ate 4 with phenylacetylene by employing Cu(I)-catalyzed azide-
alkyne cycloaddition (CuAAC) reaction.
Besides, a series of new quinazolin-4(3H)-one derivatives were
synthesized as shown in Schemes 1, 2 & 3. 5-hydroxy anthranilic
acid 6 were converted into benzoxazinone intermediate 7 by
treating with acetic anhydride under reflux for 2 h. The benzox-
azinone intermediate 7 was dissolved in pyridine and glycine ester
was added and the reaction mixture was refluxed for 4e5 h to give
In Scheme 3, the 4 or 5-nitro anthranilic acid 18 was reacted
with formamide at room temperature to afford
6 or 7-
nitroquinazolin-4(3H)-one intermediates 19. Further, the in-
termediates were treated with substituted benzyl bromide in
presence of sodium hydride as base in dry DMF at room tempera-
ture to afford 6 or 7-nitro 3-benzylquinazolin-4(3H)-one de-
rivatives 20. The intermediate 20 was further converted into 21 by
reduction of the nitro group in 20 with tin(II) chloride under reflux
conditions in ethanol and subsequent azidation reaction using so-
dium nitrite and sodium azide in 2N HCl. Synthesis of the designed
compounds 22a-22b, 22’a and 23a-23d were accomplished by
reacting the azide intermediate 6 or 7-azido-3-benzylquinazolin-
4(3H)-one 21 with various substituted phenylacetylenes or phe-
noxymethylacetylene by employing Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction [27e29]. Structures of all the newly
the
corresponding
methyl
2-(6-hydroxy-2-methyl-4-
oxoquinazolin-3(4H)-yl)acetate 8. The intermediate 8 was sub-
jected to O-propargylation by using propargyl bromide to afford
corresponding 5-O-propargylated quinazolinone derivative 9.

4
S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
O
O
H2N
O
12
O
N
propargyl bromide
K2CO3, 0 oC-rt, 8 h
O
O
N
O
13
HO
HO
O
HO
O
OH
N
N
reflux, 2 h
AcOH, reflux,
4 h
NH2
N
14
7
6
15
Sod.ascorbate (10 mol%)
CuSO4.5H2O (2 mol%)
tert-butanol:H2O (1:1)
N3
R1
rt, 12-14 h
N
N
N
N
N
N
O
O
N
CHO
R1
R1
O
O
N
N
NC
N
16
AcOH, reflux, 16-18 h
17
CN
16a: R
16b: R
1 = 3-chloro phenyl
17a: R
17b: R
1 = 3-chloro phenyl
1 = 4-chloro phenyl
1 = 3-chloro-4-fluoro phenyl
16c: R
1 = 3-methyl phenyl
16d: R
16e: R
1 = 3-chloro-4-fluoro phenyl
1 =2-methyl-4-nitro phenyl
1 = 3,4,5-trimethyl phenyl
16f: R
16g: R
16h: R
1 = 3,4,5-trimethoxy phenyl
1 = benzyl
16i: R
16j: R
1 = 4-bromo benzyl
1 = 4-nitro benzyl
Scheme 2. Synthesis of 6-((1H-1,2,3-triazol-4-yl)methoxy)-3-benzylquinazolin-4(3H)-one derivatives 16ae16j & 17a-17b.
Br
O
O
O
N
COOH
NH2
H
NH2
R1
O2N
N
NH
O2N
O2N
dry DMF, NaH,
rt, overnight
N2, rt, 5 h
N
R1
18
19
20
1. SnCl2 2H2O, ethanol, reflux, 5 h
2. HCl, NaNO2, NaN3, 0 oC-rt, 3 h
O
N
O
N
N
N
R2
O
N
O
R2
N
N
N
R1
N
N
R1
N
N
N3
Sod. ascorbate (10 mol%)
CuSO4.5H2O (2 mol%)
tert-BuOH/H2O 1:1
Sod. ascorbate (10 mol%)
CuSO4.5H2O (2 mol%)
tert-BuOH/H2O 1:1
R1
22
O
R2
R2
23
R1 = H; R2 = phenyl
R1 = Br; R2 = 2,5-dimethyl phenyl
R1 = Br; R2 = 2-methoxy-5-ethyl phenyl
R1 = Br; R2 = 4-tert-pentyl phenyl
21
12-14 h
12-14 h
23a:
23b:
23c:
23d:
22a:
22b:
R
1 = Br; R2 = phenyl
Sod. ascorbate (10 mol%)
CuSO4.5H2O (2 mol%)
R
1 = Br; R2 = 4-methyl phenyl
R2
tert-BuOH/H2O 1:1
12-14 h
N
O
N
N
N
R2
N
R1
22'a:
R1 = H; R2 = phenyl
Scheme 3. Synthesis of 3-benzyl-7-(1H-1,2,3-triazol-1-yl)quinazolin-4(3H)-one derivatives 22a-22b, 22’a, and 23a-23d.

S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
5
synthesized compounds were confirmed by 1H NMR, 13C NMR and
HRMS (ESI) spectroscopic techniques.
metastatic cascade [31]. Given the importance of cell migration in
pathobiology of cancer, we have investigated the effect of com-
pound 22a on MDA-MB-231 cells by performing wound scratch
assay. In order the evaluate the effect of 22a on cell motility, an
artificial wound was created in culture well plate with confluent
monolayer of cells using sterile tip as described in section 6.3. As
depicted in Fig. 4, it was observed that artificially created wound
was closed in control cells reflecting the rapid proliferation and
migratory property of cancer cells. Furthermore, 22a treated cells
showed significant inhibition of cell migration into the wound area.
A concentration dependent effect was observed where the low dose
3. Pharmacology
3.1. In vitro cytotoxic activity
The newly synthesized quinazolin-4(3H)-one derivatives were
screened for their cytotoxic activity against human cancer cell lines
MDA-MB-231 and MCF-7 (breast), HCT-116 and HT-29 (colon) and
A549
(lung),
using
3-(4,5-dimethylthiazol-2-yl)-2,5-
(2
treatment showed wound mild closure where as the highest dose
of 6 M displayed conspicuous suppression of cell migration as
mM) showed partial closure of the denuded area, mid dose (4 mM)
diphenyltetrazolium bromide (MTT) assay [30]. IC50 value is
defined as the concentration required to cause 50% inhibition of
cancer cell growth. The IC50 (mM) values of the test compounds 5a-
5b, 10a-10b, 11a-11d, 16a-16j, 17a-17b, 22a-22b, 22’a, 23a-23d are
given in Table 1. Doxorubicin is used as the positive control. The
results indicated that some of the synthesized compounds
possessed moderate to potent growth inhibition against the tested
cancer cells. Among the tested compounds, compound 22a
exhibited potent cytotoxic activity against HT-29 & MDA-MB-
m
evident from the scantly present cells in the denuded area. These
results suggest the potential of 22a inhibition migration and
motility of cancer cells.
3.3. Acridine orangeeethidium bromide (AOeEB) staining
AO/EB staining is dual fluorescent staining assay commonly
used to detect tumor cell apoptosis. AO/EB staining identifies
apoptosis and necrotic-associated changes occurring in cell mem-
branes. This assay distinguishes viable cells from the non-viable
ones based upon the cell membrane integrity [32]. Changes per-
taining to apoptotic cell death upon treatment with 22a were thus
studied by AO/EB staining. AO is up-taken by cells with intact cell
membrane and is visualized as green fluorescence. Viable cells are
impermeable to EB and can be taken up by cells with loss of
membrane integrity Therefore, early apoptotic cells have bright
green nucleus with condensed chromatin and fragmented nuclei;
and late apoptotic cells show orange colored condensed and frag-
mented chromatin. Cell death due to necrosis is visualized as a
structurally normal orange nucleus. From Fig. 5, untreated cells did
not show any morphological changes pertaining to apoptosis/ne-
crosis. Cells treated with 22a showed significant apoptosis changes
characterized by bright green dots representing chromatic
condensation. Concentration dependent increase in apoptosis was
apparent upon treatment with 22a. Also, both doses showed
chromatin condensation and blebbing in cells treated with 22a.
231 cell lines with IC50 of 7.29
It is significant to note that the IC50 of Doxorubicin (3.77
comparable to the IC50 value of 22a against MDA-MB-231 cell line.
The promising cytotoxic activity of compound 22a on MDA-MB-231
cancer cells encouraged us to study its effects at cellular level.
m
M and 3.21
m
M values respectively.
mM) is
3.2. In vitro cell migration assay/wound healing assay
Cell migration is a central and integral process of various
important biological mechanisms like tissue development, tissue
wound healing and pathological conditions like cancer. Uncon-
trolled cell migration is the first step and hallmark of cancer cell
metastasis, which is composed of sequential events called
Table 1
IC50 values (mM) of the tested compounds against selected human cancer cell lines.
Compound name
HCT-116
HT-29
A549
MDA-MB-231
MCF-7
5a
5b
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
7.53
>30
>30
>30
>30
>30
>30
>30
10.82
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
7.23
>30
>30
>30
>30
>30
>30
9.56
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
9.01
>30
>30
3.95
>30
>30
>30
>30
>30
29.34
>30
16.57
>30
>30
>30
>30
>30
>30
>30
5.74
>30
>30
>30
12.57
3.21
>30
>30
>30
14.49
>30
>30
3.77
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
13.50
>30
>30
>30
>30
0.32
10a
10b
11a
11b
11c
11d
16a
16b
16c
16d
16e
16f
3.4. DAPI staining
4,6-Diamidino-2-phenylindole (DAPI) is a fluorescent dye which
specifically binds to adenine-thymine clusters of double stranded
DNA and stains it blue. Healthy cells display intact round nuclei
with distinct margins upon staining with DAPI, where as apoptotic
cells show intense blue fluorescence as compared to nuclei of
healthy cells [33]. Additionally, condensed chromosome and nu-
clear fragmentation are apparent in apoptotic cells. The results
from the Fig. 6 clearly demonstrated that the nuclear structure of
untreated cells showed normal round nuclei whereas 22a treated
cells exhibited nuclear fragmentation, condensed chromosome and
the characteristic pyknotic, horse shoe pattern of the nuclei. The
high concentration treated cells showed higher degree of apoptosis
as evidenced by the increased number of horseshoe shaped nuclei.
16g
16h
16i
16j
17a
17b
22a
22b
22’a
23a
23b
23c
23d
DOX
3.5. Colony forming assay
Colony formation assay or clonogenic assay is an in vitro cell
survival assay which is based on the ability of a single cell to form
into colony of cells through unhampered cell proliferation [34].
Clonogenic assay also evaluated the retention/loss of reproductive
integrity of a cell upon treatment with a cytotoxic compound. From
the Fig. 7, treatment with compound 22a at different
IC50 values are the concentrations that cause 50% inhibition of cancer cell growth.
breast (MDA-MB-231 and MCF-7), colon (HCT-116 and HT-29), lung (A549) DOX:
Doxorubicin as reference standard.

6
S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
Fig. 4. (A) Representative microphotographs of cell migration assay of all experimental groups at 0 h and 24 h (Magnification 10ꢁ). Treated wells show reduction in migration of
cells into the artificially created wound (B) Graphical representation of inhibition in cell migration upon treatment with 22a at different concentrations. *p < 0.05 and **p < 0.01 of
control vs. 22a treatment.
Fig. 5. Representative microphotographs of apoptosis induction by 22a in MDA-MB-231 cells using AO/EB staining (Magnification 20ꢁ). Untreated cells exhibiting normal green
structure with distinct nucleus, whereas treatment with 22a showed dose dependent induction of apoptosis as evident from membrane blebbing and apoptotic bodies. White
arrows indicate the altered cell/nuclei morphological changes.
Fig. 6. Representative microphotographs of morphological changes in nuclei of untreated cells and as a result of treatment with 22a in MDA-MB-231 cells as indicated by DAPI
staining (Magnification 20ꢁ). Untreated cells show normal oblong and distinct nuclei. 22a treated cells indicating shrinked, horse shoe nucleus, fragmentation and pyknotic nucleus
of the cells. The arrows indicate the nuclear morphological changes induced by 22a.
Fig. 7. (A) Image displaying the effect of 22a at various concentrations of 2, 4 and 6
mM on colony formation of MDA-MB-231 cells (B) Graphical representation of number of colonies
of MDA-MB-231 cells in control and treated culture wells. **p < 0.01 of control vs. 22a treatment.
concentrations of 2, 4 and 6
m
M exhibited a concentration depen-
3.6. Cell cycle analysis
dent inhibition of proliferation and colony formation. The total
colonies were represented as a total number of colonies vs.
concentration.
Most of the chemotherapeutic compounds exert their cytotoxic
effect either by induction of apoptosis or by arresting cell cycle at a

S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
7
specific checkpoint [35]. The inappropriate regulation of cell cycle
by anticancer agent has been considered to be an effective strategy
in the development of new cancer therapeutics. Therefore, it was
our interest to examine the effect of compound 22a on cell cycle
progression of MDA-MB-231 cell by using flow cytometry analysis
method [36]. The mechanism by which 22a inhibited cell growth
was studied by performing cell cycle analysis. Cells were treated
act as EGFR Kinase inhibitors. The ease of synthesis and good anti-
cancer potential of these compounds makes them acceptable
starting points for the development of new chemotherapeutic
agents.
6. Materials & methods
with 22a for 24 h at concentrations of 4 and 6 mM. Results from the
Fig. 8 clearly demonstrated the accumulation of cells in G0/G1
phase from 61.88% in untreated cells to 67.61% and 68.43% in 4 and
All the chemicals, reagents and starting materials were obtained
from commercial available providers and were used as such. The
monitoring of all the reactions were performed by thin layer
chromatography (TLC-MERCK pre-coated silica gel 60-F254
aluminium plates) under UV light. Melting points were checked
using Stuart® SMP30 apparatus and are uncorrected. 1H and 13C
NMR were performed on Bruker Advance 500 MHz spectrometer
using tetramethylsilane (TMS) as the internal standard and chem-
ical shifts are reported in ppm. Chemical shifts are referenced to
6 mM respectively. These results clearly indicated that the treat-
ment of 22a led to G0/G1phase of cell cycle arrest. Though there
was apparent increase in the percentage of cells arrested in G0/G1
phase upon treatment with 22a at both the doses, the difference
was not significant.
TMS (
d
0.00 for 1H NMR and 13C NMR), CDCl3 (
d
7.26 for 1H NMR and
4. Molecular docking studies
77.00 or 77.16 for 13C NMR) or DMSO/d6 2.50 for 1H NMR and
(d
39.7 for 13C NMR) or combination of CDCl3 and DMSO‑d6, in which
DMSO‑d6 was used as an internal reference or trifluoroacetic acid-
With a view to throw light on the probable protein target of the
newly synthesized quinazolinone derivatives, we have performed
their interactions studies with EGFR (PDB ID: 1XKK) binding site
[37]. The new representative compounds 11d, 16h, 22a and 23b
were docked against EGFR protein. Ligand interactions showed that
the compounds exhibited good interactions with glide scores
ranging from ꢀ6.033 to ꢀ7.386 against EGFR target protein. The
studies revealed that the 4-bromo of 3-N-benzyl moiety in the most
potent compound 22a exhibited hydrogen bonding interaction
with active site residue TYR998 and aromatic hydrogen bonding
interaction between phenyl ring system and ASP855 with good
docking score. Compound 23b exhibited aromatic hydrogen
bonding interactions between aromatic phenyl moieties and resi-
dues VAL717, LEU718 and ASP 855 of EGFR target protein (Fig. 9).
d (d 164.2 and d
116.6 for 13C NMR). Spin multiplicities are reported
as s (singlet), d (doublet), dd (double doublet), t (triplet) and m
(multiplet). Coupling constant (J) values are reported in hertz (Hz).
HRMS were determined with Agilent QTOF mass spectrometer
6540 series instrument and were performed in the ESI techniques
at 70 eV. Column chromatography was performed using silica gel
60e120 or 100e200 mesh.
Intermediates 3, 4, 7, 8, 14 (Scheme 1 & 2) were prepared ac-
cording to the procedures described in literature [27,28].
6.1. General reaction procedure for the synthesis of 4-(4-phenyl-
1H-1,2,3-triazol-1-yl)benzamide derivatives 5a-5b
Compound (4, 2 mmol) (Scheme 1) and phenylacetylene
(2 mmol) were suspended in 10 mL of a 1:1 tert-BuOH/H2O mixture.
To the heterogeneous mixture, sodium ascorbate (5 mol%), cop-
per(II)sulfate pentahydrate (2 mol%) were added and the reaction
mixture was allowed to stir for 8 h to give crude white or pale
yellow precipitates, which were filtered, washed with water, fol-
lowed by hexane and finally purified by using column chroma-
tography to obtain pure solid compounds 5a-5b in 65e69% yields.
All the newly synthesized compounds were characterized by 1H
NMR, 13C NMR and HRMS (ESI).
5. Conclusion
In conclusion, a series of new quinazolin-4(3H)-ones have been
synthesized and evaluated for their cytotoxic activity against
selected human cancer cell lines viz. colon (HCT-116 and HT-29),
breast (MDA-MB-231 and MCF-7) and lung (A549). Among all the
tested compounds, 22a exhibited potent cytotoxic activity with IC50
value of 3.21 mM against MDA-MB-231 cell line. The exposure of
compound 22a to MDA-MB-231 cells resulted in G0/G1 phase of
cell cycle arrest and inhibition of in vitro cell migration. The
apoptosis inducing effect of 22a was further analysed by acridine
orange/ethidium bromide (AO-EB) staining and DAPI staining.
Molecular docking studies of these compounds with EGFR protein
showed good binding affinities, suggesting that these compounds
6.1.1. Ethyl 3-phenyl-2-(4-(4-phenyl-1H-1,2,3-triazol-1-yl)
benzamido)propanoate (5a)
White solid, Yield 65%; mp: 118e120 ꢂC. 1H NMR (500 MHz,
Fig. 8. Effect of 22a on progression of MDA-MB-231 cell cycle as analysed by flow cytometry. Cells treated with 22a at concentrations of 4 and 6 mM show G0/G1 phase arrest of cell
cycle.

8
S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
Fig. 9. (A) Interactions of compound 22a with active site residue TYR998 and ASP855 of EGFR protein (PDB ID: 1XKK) (B) Interactions of compound 23b with VAL717, LEU718 and
ASP 855 of EGFR protein (PDB ID: 1XKK).
DMSOed6)
d
9.40 (s, 1H), 9.02 (d, J ¼ 7.7 Hz, 1H), 8.11e8.03 (m, 4H),
6.2.2. Methyl2-(2-methyl-4-oxo-6-((3-(p-tolyl)isoxazol-5-yl)
methoxy)quinazolin-3(4H)-yl)acetate (10b)
7.99e7.94 (m, 2H), 7.55e7.48 (m, 2H), 7.43e7.38 (m, 1H), 7.35e7.27
(m, 4H), 7.24e7.18 (m, 1H), 4.74e7.64 (m,1H), 4.11 (q, J ¼ 7.1 Hz, 2H),
3.23e3.09 (m, 2H), 1.15 (t, J ¼ 7.1 Hz, 3H) ppm; 13C NMR (125 MHz,
White solid, Yield 74%; mp: 177e179 ꢂC. 1H NMR (500 MHz,
DMSOed6)
d 7.81e7.75 (m, 2H), 7.66e7.60 (m, 2H), 7.57e7.53 (m,
DMSOed6)
d
172.05, 165.84, 148.01, 139.09, 138.06, 133.97, 130.57,
1H), 7.35e7.30 (m, 2H), 7.18 (s, 1H), 5.48 (s, 2H), 4.94 (s, 2H), 3.73 (s,
129.67, 129.56, 129.51, 128.84, 128.73, 126.99, 125.88, 120.15, 120.02,
61.09, 54.96, 36.83, 14.48 ppm; HRMS (ESI): m/z calcd for [MþH]þ
C26H25N4O3 441.1926; found 441.1935.
3H), 2.50 (s, 3H), 2.37 (s, 3H) ppm; 13C NMR (125 MHz, DMSOed6)
d
169.07, 168.52, 162.36, 161.28, 156.30, 153.27, 142.60, 140.59,
130.18, 129.11, 127.04, 125.91, 125.11, 120.65, 108.13, 102.84, 61.43,
52.99, 45.97, 23.05, 21.43 ppm; HRMS (ESI): m/z calcd for [MþH]þ
C23H22N3O5 420.1559; found 420.1560.
6.1.2. Methyl 3-methyl-2-(4-(4-phenyl-1H-1,2,3-triazol-1-yl)
benzamido)butanoate (5b)
White solid, Yield 69%; mp: 255e257 ꢂC. 1H NMR (500 MHz,
6.2.3. Methyl 2-(6-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)
methoxy)-2-methyl-4-oxoquinazolin-3(4H)-yl)acetate (11a)
Yellow solid, Yield 74%; mp: 255e257 ꢂC 1H NMR (500 MHz,
DMSOed6)
d
9.42 (s, 1H), 8.80 (d, J ¼ 7.7 Hz, 1H), 8.18e8.13 (m, 2H),
8.12e8.07 (m, 2H), 8.00e7.95 (m, 2H), 7.55e7.49 (m, 2H), 7.44e7.38
(m, 1H), 4.38e4.33 (m, 1H), 3.68 (s, 3H), 2.27e2.17 (m, 1H), 1.02 (d,
J ¼ 6.7 Hz, 3H), 0.97 (d, J ¼ 6.8 Hz, 3H) ppm; 13C NMR (125 MHz,
DMSOed6)
d
9.06 (s, 1H), 8.07 (t, J ¼ 2.0 Hz, 1H), 7.96e7.92 (m, 1H),
7.68 (d, J ¼ 2.9 Hz, 1H), 7.66e7.59 (m, 2H), 7.59e7.55 (m, 1H),
7.54e7.50 (m, 1H), 5.38 (s, 2H), 4.95 (s, 2H), 3.73 (s, 3H), 2.51 (s, 3H)
DMSOed6)
d 172.59, 166.37, 148.03, 139.06, 134.14, 130.58, 129.97,
129.49, 128.84, 125.89, 120.15, 119.88, 59.22, 52.15, 30.05, 19.62,
19.53 ppm; HRMS (ESI): m/z calcd for [MþH]þ C21H23N4O3
379.1770; found 379.1777.
ppm; 13C NMR (125 MHz, DMSOed6)
d 169.11, 161.31, 156.71, 153.03,
144.23, 142.34, 138.03, 134.69, 132.12, 129.08, 128.99, 125.08,
123.62, 120.68, 120.44, 119.22, 108.03, 62.02, 52.98, 45.95,
23.03 ppm; HRMS (ESI): m/z calcd for [MþH]þ C22H19ClN3O5
440.1013; found 440.1019.
6.2. General reaction procedure for the synthesis of methyl 2-(2-
methyl-4-oxoquinazolin-3(4H)-yl)acetate 10a-10b, 11a-11d
derivatives
6.2.4. Methyl 2-(6-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)
methoxy)-2-methyl-4-oxoquinazolin-3(4H)-yl)acetate (11b)
White solid, Yield 55%; mp: 135e137 ꢂC. 1H NMR (500 MHz,
Methyl 2-(2-methyl-4-oxo-6-(prop-2-yn-1-yloxy)quinazolin-
3(4H)-yl)acetate (9, 2 mmol) and azides or oximes (2 mmol)
(Scheme 1) were mixed in a 10 mL volume of 1:1 tert-BuOH/H2O
mixture. To the heterogeneous mixture, sodium ascorbate (10 mol
%), followed by copper(II)sulfate pentahydrate (2 mol%) were added
and the reaction mixture was allowed to stir for 10-14 h to give
crude white precipitates, which were filtered, washed with water,
followed by hexane and finally purified by using column chroma-
tography to obtain as pure white solid compounds 10a-10b, 11a-
11d in 84e90% yields. All the newly synthesized compounds were
characterized by 1H NMR, 13C NMR and HRMS (ESI).
DMSOed6)
d
8.86 (s, 1H), 7.90e7.75 (m, 2H), 7.68 (d, J ¼ 2.9 Hz, 1H),
7.60 (d, J ¼ 8.9 Hz, 1H), 7.55e7.50 (m, 1H), 7.17e7.09 (m, 2H), 5.35 (s,
2H), 4.94 (s, 2H), 3.83 (s, 3H), 3.73 (s, 3H), 2.51 (s, 3H) ppm; 13C NMR
(125 MHz, DMSOed6)
d 169.08, 161.33, 159.84, 156.81, 152.98,
143.79, 142.33,130.46, 128.97, 125.08, 123.40, 122.34, 120.70, 115.38,
108.11, 62.15, 56.05, 52.95, 45.94, 23.01 ppm; HRMS (ESI): m/z calcd
for [MþH]þ C22H22N5O5 436.1621; found 436.1629.
6.2.5. Methyl 2-(2-methyl-4-oxo-6-((1-(m-tolyl)-1H-1,2,3-triazol-
4-yl)methoxy)quinazolin-3(4H)-yl)acetate (11c)
6.2.1. Methyl 2-(6-((3-(4-chlorophenyl)isoxazol-5-yl)methoxy)-2-
methyl-4-oxoquinazolin-3(4H)-yl) acetate (10a)
White solid, Yield 74%; mp: 188e190 ꢂC. 1H NMR (500 MHz,
White solid, Yield 51%; mp: 125e127 ꢂC 1H NMR (500 MHz,
DMSOed6) d 8.95 (s, 1H), 7.78e7.75 (m, 1H), 7.72e7.69 (m, 1H), 7.68
DMSOed6)
d
7.94e7.90 (m, 2H), 7.64 (d, J ¼ 2.9 Hz, 1H), 7.62e7.57
(d, J ¼ 2.9 Hz, 1H), 7.61 (d, J ¼ 8.9 Hz, 1H), 7.55e7.51 (m, 1H), 7.49 (t,
J ¼ 7.8 Hz, 1H), 7.34e7.31 (m, 1H), 5.37 (s, 2H), 4.95 (s, 2H), 3.73 (s,
3H), 2.50 (s, 3H), 2.42 (s, 3H) ppm; 13C NMR (125 MHz, DMSOed6)
(m, 3H), 7.57e7.53 (m, 1H), 7.24 (s, 1H), 5.49 (s, 2H), 4.94 (s, 2H),
3.72 (s, 3H), 2.50 (s, 3H) ppm; 13C NMR (125 MHz, DMSOed6)
d
169.05, 169.04, 161.58, 161.28, 156.29, 153.27, 142.65, 135.54,
d 169.11, 161.32, 156.76, 153.03, 143.96, 142.32, 140.17, 136.97, 130.19,
129.73, 129.12, 128.96, 127.61, 125.07, 120.69, 108.30, 102.93, 61.49,
52.96, 45.96, 23.03 ppm; HRMS (ESI): m/z calcd for [MþH]þ
C22H19ClN3O5 440.1013; found 440.1016.
129.86, 128.99, 125.12, 123.39, 121.07, 120.68, 117.72, 108.01, 62.08,
52.98, 45.95, 23.03, 21.38 ppm; HRMS (ESI): m/z calcd for [MþH]þ
C22H22N5O4 420.1672; found 420.1670.

S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
9
6.2.6. Methyl-2-(2-methyl-4-oxo-6-((1-(3,4,5-trimethylphenyl)-
438.1931.
1H-1,2,3-triazol-4-yl)methoxy)quinazolin-3(4H)-yl)acetate (11d)
White solid, Yield 64%; mp: 139e141. ꢂC 1H NMR (500 MHz,
6.3.4. 3-benzyl-6-((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-
4-yl)methoxy)-2-methylquinazolin-4(3H)-one (16d)
DMSOed6)
d
8.45 (s, 1H), 7.66 (d, J ¼ 2.9 Hz, 1H), 7.59 (d, J ¼ 8.9 Hz,
1H), 7.53 (dd, J ¼ 8.9, 2.9 Hz, 1H), 7.09 (s, 2H), 5.38 (s, 2H), 4.94 (s,
White solid, Yield 62%; mp: 195e197 ꢂC. 1H NMR (500 MHz,
2H), 3.73 (s, 3H), 2.50 (s, 3H), 2.33 (s, 3H), 1.86 (s, 6H) ppm; 13C NMR
DMSO‑d6)
d
9.02 (s, 1H), 8.25 (dd, J ¼ 6.3, 2.6 Hz, 1H), 8.02e7.96 (m,
(125 MHz, DMSOed6))
d
169.07, 161.30, 156.76, 153.00, 142.94,
1H), 7.75 (d, J ¼ 2.8 Hz, 1H), 7.72e7.66 (m, 1H), 7.63e7.58 (m, 1H),
7.54e7.50 (m, 1H), 7.39e7.32 (m, 2H), 7.31e7.26 (m, 1H), 7.20 (d,
J ¼ 7.5 Hz, 2H), 5.39 (s, 4H), 2.47 (s, 3H) ppm; 13C NMR (125 MHz,
142.32, 140.02, 134.95, 133.79, 129.34, 128.91, 127.14, 125.28, 120.66,
108.49, 62.28, 52.95, 45.94, 23.01, 21.09, 17.26 ppm; HRMS (ESI): m/
z calcd for [MþH]þ C24H26N5O4 448.1985; found 448.1990.
DMSO‑d6)
d 161.73, 158.42, 156.72, 156.45, 153.38, 144.32, 142.47,
137.04, 134.04, 129.26, 128.96, 127.77, 126.77, 124.89, 123.78, 123.04,
121.57, 121.13, 118.71, 108.39, 62.10, 46.92, 23.16. HRMS (ESI): m/z
calcd for [MþH]þ C25H20FClN5O2 476.1289; found 476.1294.
6.3. General reaction procedure for the synthesis of 6-((1H-1,2,3-
triazol-4-yl)methoxy)-3-benzyl-2-methylquinazolin-4(3H)-one
16a-16j, and (E)-4-(2-(6-((1H-1,2,3-triazol-4-yl)methoxy)-3-
benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile
derivatives 17a-17b
6.3.5. 3-benzyl-2-methyl-6-((1-(2-methyl-4-nitrophenyl)-1H-
1,2,3-triazol-4-yl)methoxy)quinazolin-4(3H)-one (16e)
Pale yellow solid, Yield 79%; mp: 205e207 ꢂC. 1H NMR
Compounds 16a-16j were prepared in a similar way as pro-
cedure described above. Compound (16, 1 mmol) was dissolved in
glacial acetic acid, to which 4-cyano benzaldehyde (1 mmol)
(Scheme 2) was added and the reaction mixture was allowed to
reflux for 16e18 h. The reaction was monitored by TLC. After
completion of reaction, water was added to afford crude pre-
cipitates, which were filtered, washed with water, followed by cold
methanol and hexane to afford a pure yellow to pale yellow
products of (E)-4-(2-(6-((1H-1,2,3-triazol-4-yl)methoxy)-3-benzyl-
4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile derivatives 17a-
17b in 71e83% yields. All the newly synthesized compounds were
characterized by 1H NMR, 13C NMR and HRMS (ESI).
(500 MHz, DMSO‑d6)
d
8.81 (s, 1H), 8.40 (d, J ¼ 11.3 Hz, 1H),
8.30e8.19 (m, 1H), 7.84e7.72 (m, 2H), 7.66e7.49 (m, 2H), 7.40e7.32
(m, 3H), 7.32e7.25 (m, 1H), 7.20 (d, J ¼ 7.3 Hz, 1H), 5.42 (s, 2H), 5.39
(s, 2H), 2.47 (s, 3H), 2.35 (s, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6)
d
13C NMR (125 MHz, DMSO)
d 161.74, 156.73, 153.39, 148.06, 143.51,
142.46, 141.30, 137.03, 135.61, 129.26, 128.94, 127.78, 127.76, 126.91,
126.87, 126.77, 125.00, 122.70, 121.10, 108.40, 62.00, 46.91, 23.16,
18.30 ppm; HRMS (ESI): m/z calcd for [MþH]þ C26H23N6O4
483.1781; found 483.1785.
6.3.6. 3-benzyl-2-methyl-6-((1-(3,4,5-trimethylphenyl)-1H-1,2,3-
triazol-4-yl)methoxy)quinazolin-4(3H)-one (16f)
White solid, Yield 75%; mp: 165e167 ꢂC. 1H NMR (500 MHz,
6.3.1. 3-benzyl-6-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)
methoxy)-2-methylquinazolin-4(3H)-one (16a)
DMSO‑d6)
d
8.46 (s, 1H), 7.74 (d, J ¼ 2.6 Hz, 1H), 7.59 (d, J ¼ 8.9 Hz,
1H), 7.53 (dd, J ¼ 8.9, 2.7 Hz, 1H), 7.39e7.32 (m, 2H), 7.32e7.26 (m,
1H), 7.19 (d, J ¼ 7.5 Hz, 2H), 7.09 (s, 2H), 5.40 (s, 2H), 5.39 (s, 2H),
2.47 (s, 3H), 2.33 (s, 3H), 1.87 (s, 6H) ppm; 13C NMR (125 MHz,
White solid, Yield 70%; mp: 240e242 ꢂC. 1H NMR (500 MHz,
DMSOed6)
d 9.06 (s, 1H), 8.08 (s, 1H), 7.97e7.93 (m, 1H), 7.75 (d,
J ¼ 2.8 Hz, 1H), 7.66e7.59 (m, 2H), 7.59e7.56 (m, 1H), 7.55e7.50 (m,
1H), 7.40e7.32 (m, 2H), 7.31e7.25 (m, 1H), 7.20 (d, J ¼ 7.6 Hz, 2H),
5.39 (s, 4H), 2.47 (s, 3H) ppm; 13C NMR (125 MHz, DMSOed6)
DMSO‑d6) d 161.71, 156.70, 153.34, 142.99, 142.41, 140.01, 137.04,
134.94, 133.78, 129.33, 129.25, 128.87, 127.78, 127.12, 126.76, 125.15,
121.06, 108.66, 62.27, 46.89, 23.15, 21.09, 17.26 ppm; HRMS (ESI): m/
z calcd for [MþH]þ C28H28N5O2 466.2243; found 466.2243.
d
161.74, 156.72, 153.38, 144.31, 142.47, 138.08, 137.04, 134.70,132.11,
129.26, 129.08, 128.96, 127.77, 126.77, 124.91, 123.60, 121.13, 120.49,
119.27, 108.39, 62.11, 46.91, 23.16 ppm; HRMS (ESI): m/z calcd for
[MþH]þ C25H21ClN5O2 458.1384; found 458.1384.
6.3.7. 3-benzyl-2-methyl-6-((1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazol-4-yl)methoxy)quinazolin-4(3H)-one (16g)
White solid, Yield 77%; mp: 255e257 ꢂC. 1H NMR (500 MHz,
6.3.2. 3-benzyl-6-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)
methoxy)-2-methylquinazolin-4(3H)-one (16b)
DMSO‑d6)
d
9.00 (s, 1H), 7.75 (d, J ¼ 2.9 Hz, 1H), 7.61 (d, J ¼ 8.9 Hz,
1H), 7.52 (dd, J ¼ 8.9, 2.9 Hz, 1H), 7.38e7.33 (m, 2H), 7.31e7.26 (m,
1H), 7.24 (s, 2H), 7.21e7.17 (m, 2H), 5.39 (s, 4H), 3.88 (s, 6H), 3.72 (s,
White solid, Yield 74%; mp: 235e237 ꢂC. 1H NMR (500 MHz,
DMSOed6)
d
9.01 (s, 1H), 8.02e7.93 (m, 2H), 7.75 (d, J ¼ 2.8 Hz, 1H),
3H), 2.47 (s, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6)
d 161.74,
7.71e7.66 (m, 2H), 7.63e7.58 (m, 1H), 7.52 (dd, J ¼ 8.9, 2.9 Hz, 1H),
156.76, 154.02, 153.38, 143.95, 142.46, 138.01, 137.04, 132.90, 129.26,
128.95, 127.78, 126.76, 124.95, 123.69, 121.12, 108.35, 98.78, 62.23,
60.68, 56.82, 46.92, 23.16 ppm; HRMS (ESI): m/z calcd for [MþH]þ
C28H28N5O5 514.2090; found 514.2095.
7.39e7.32 (m, 2H), 7.31e7.25 (m, 1H), 7.20 (d, J ¼ 7.4 Hz, 2H), 5.39 (s,
4H), 2.47 (s, 3H) ppm; 13C NMR (125 MHz, DMSOed6)
d 161.73,
156.73,153.36, 144.28,142.46,137.03, 135.84,133.56, 130.35,129.26,
128.95, 127.77, 126.76, 124.91, 123.51, 122.35, 121.12, 108.34, 62.08,
46.91, 23.16 ppm; HRMS (ESI): m/z calcd for [MþH]þ C25H21ClN5O2
458.1384; found 458.1387.
6.3.8. 3-benzyl-6-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)-2-
methylquinazolin-4(3H)-one (16h)
White solid, Yield 70%; mp: 155e157 ꢂC. 1H NMR (500 MHz,
6.3.3. 3-benzyl-2-methyl-6-((1-(m-tolyl)-1H-1,2,3-triazol-4-yl)
methoxy)quinazolin-4(3H)-one (16c)
DMSO‑d6)
d
8.31 (s, 1H), 7.69 (d, J ¼ 2.9 Hz, 1H), 7.58 (d, J ¼ 8.9 Hz,
1H), 7.48 (dd, J ¼ 8.9, 2.9 Hz, 1H), 7.39e7.27 (m, 8H), 7.22e7.17 (m,
White solid, Yield 58%; mp: 215e217 ꢂC. 1H NMR (500 MHz,
2H), 5.62 (s, 2H), 5.39 (s, 2H), 5.28 (s, 2H), 2.47 (s, 3H) ppm; 13C NMR
DMSO‑d6)
d
8.95 (s, 1H), 7.77 (s, 1H), 7.75 (d, J ¼ 2.8 Hz, 1H),
(125 MHz, DMSO‑d6) d 161.66, 156.82, 153.46, 143.16, 142.12, 136.99,
7.73e7.69 (m, 1H), 7.61 (d, J ¼ 8.9 Hz, 1H), 7.53 (dd, J ¼ 8.9, 2.8 Hz,
1H), 7.48 (t, J ¼ 7.8 Hz, 1H), 7.39e7.25 (m, 4H), 7.20 (d, J ¼ 7.6 Hz,
2H), 5.39 (s, 2H), 5.38 (s, 2H), 2.47 (s, 3H), 2.42 (s, 3H) ppm; 13C NMR
136.46, 129.25, 129.23, 128.70, 128.62, 128.42, 127.78, 126.78,
125.24,125.02,121.05,108.29, 62.17, 53.34, 46.94, 23.08 ppm; HRMS
(ESI): m/z calcd for [MþH]þ C26H24N5O2 438.1930; found 438.1934.
(125 MHz, DMSO‑d6) d 161.75, 156.77, 153.36, 144.04, 142.45, 140.16,
137.04, 137.02, 130.17, 129.84, 129.26, 128.95, 127.77, 126.77, 124.93,
123.35, 121.13, 121.10, 117.75, 108.33, 62.16, 46.91, 23.16, 21.37 ppm;
HRMS (ESI): m/z calcd for [MþH]þ C26H24N5O2 438.1930; found
6.3.9. 3-benzyl-6-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)
methoxy)-2-methylquinazolin-4(3H)-one (16i)
White solid, Yield 55%; mp: 175e177 ꢂC. 1H NMR (500 MHz,

10
S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
DMSO‑d6)
d
8.31 (s, 1H), 7.69 (d, J ¼ 2.9 Hz, 1H), 7.60e7.54 (m, 3H),
6.4.1. 3-(4-bromobenzyl)-7-(4-phenyl-1H-1,2,3-triazol-1-yl)
quinazolin-4(3H)-one (22a)
White solid, Yield 74%; mp: 215e217 ꢂC; 1H NMR (500 MHz,
DMSO‑d6)
7.47 (dd, J ¼ 8.9, 2.9 Hz, 1H), 7.38e7.33 (m, 2H), 7.31e7.26 (m, 3H),
7.21e7.17 (m, 2H), 5.61 (s, 2H), 5.38 (s, 2H), 5.28 (s, 2H), 2.46 (s, 3H)
ppm; 13C NMR (125 MHz, DMSO‑d6)
d
161.71, 156.77, 153.34, 143.22,
d
9.57 (s, 1H), 8.71 (s, 1H), 8.37 (d, J ¼ 8.7 Hz, 1H), 8.29 (d,
142.32, 137.02, 135.86, 132.17, 130.68, 129.26, 128.85, 127.78, 126.76,
125.32, 124.99, 121.91, 121.06, 108.25, 62.13, 52.58, 46.91,
23.14 ppm; HRMS (ESI): m/z calcd for [MþH]þ C26H23BrN5O2
516.1035; found 516.1037.
J ¼ 2.0 Hz, 1H), 8.21 (dd, J ¼ 8.7, 2.1 Hz, 1H), 8.01e7.94 (m, 2H),
7.60e7.55 (m, 2H), 7.55e7.49 (m, 2H), 7.44e7.35 (m, 3H), 5.21 (s,
2H) ppm; 13C NMR (125 MHz, DMSO‑d6)
d 159.99, 150.00, 149.67,
148.21, 141.23, 136.51, 132.03, 130.54, 130.44, 129.53, 129.07, 128.94,
125.91, 121.64, 121.43, 120.41, 118.97, 117.42, 49.07 ppm; HRMS
(ESI): m/z calcd for [MþH]þ C23H17BrN5O 458.0616; found
458.0618.
6.3.10. 3-benzyl-2-methyl-6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-
4-yl)methoxy)quinazolin-4(3H)-one (16j)
White solid, Yield 76%; mp: 254e256 ꢂC. 1H NMR (500 MHz,
6.4.2. 3-(4-bromobenzyl)-7-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)
quinazolin-4(3H)-one (22b)
DMSO‑d6)
d
8.39 (s, 1H), 8.25e8.22 (m, 2H), 7.70 (d, J ¼ 2.9 Hz, 1H),
7.62e7.58 (m, 1H), 7.55e7.51 (m, 2H), 7.51e7.47 (m, 1H), 7.38e7.32
(m, 2H), 7.31e7.26 (m, 1H), 7.22e7.18 (m, 2H), 5.82 (s, 2H), 5.39 (s,
2H), 5.31 (s, 2H), 2.49 (s, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6)
White solid, Yield 64%; mp: 200e202 ꢂC. 1H NMR (500 MHz,
DMSO‑d6)
d
9.51 (s, 1H), 8.70 (s, 1H), 8.37 (d, J ¼ 8.7 Hz, 1H), 8.28 (d,
J ¼ 2.1 Hz, 1H), 8.20 (dd, J ¼ 8.7, 2.2 Hz, 1H), 7.88e7.82 (m, 2H),
7.59e7.54 (m, 2H), 7.40e7.36 (m, 2H), 7.36e7.31 (m, 2H), 5.21 (s,
d
161.51, 156.88, 153.92, 147.75, 143.84, 143.32, 141.48, 136.83,
129.51, 129.25, 128.26, 127.82, 126.82, 125.70, 125.15, 124.38, 120.98,
108.45, 62.17, 52.44, 47.03, 22.87 ppm; HRMS (ESI): m/z calcd for
[MþH]þ C22H23N6O4 483.1781; found 483.1785.
2H), 2.37 (s, 3H) ppm; 13C NMR (125 MHz, TFA)
d 159.37, 154.52,
148.54, 147.57, 143.18, 139.91, 135.14, 134.23, 132.97, 132.88, 132.18,
128.89, 126.95, 125.23, 124.10, 119.71, 118.26, 116.01, 55.01,
22.06 ppm; HRMS (ESI): m/z calcd for [MþH]þ C24H19BrN5O
472.0773; found 472.0774.
6.3.11. (E)-4-(2-(3-benzyl-6-((1-(3-chlorophenyl)-1H-1,2,3-triazol-
4-yl)methoxy)-4-oxo-3,4-dihydroquinazolin-2-yl)vinyl)benzonitrile
(17a)
6.4.3. 3-benzyl-6-(4-phenyl-1H-1,2,3-triazol-1-yl)quinazolin-
4(3H)-one (22’a)
Yellow solid, Yield 68%; mp: 200e202 ꢂC. 1H NMR (500 MHz,
DMSOed6)
d
9.08 (s, 1H), 8.08 (t, J ¼ 2.0 Hz, 1H), 7.98e7.93 (m, 1H),
White solid, Yield 75%; mp: 155e157 ꢂC. 1H NMR (500 MHz,
7.89e7.84 (m, 4H), 7.84e7.78 (m, 2H), 7.73 (d, J ¼ 8.9 Hz, 1H), 7.65 (t,
J ¼ 8.1 Hz,1H), 7.60e7.55 (m, 2H), 7.53 (d, J ¼ 15.4 Hz,1H), 7.35e7.30
(m, 2H), 7.29e7.21 (m, 3H), 5.68 (s, 2H), 5.42 (s, 2H) ppm; 13C NMR
DMSO‑d6)
d
9.55 (s, 1H), 8.69 (s, 1H), 8.66 (d, J ¼ 2.5 Hz, 1H), 8.45
(dd, J ¼ 8.8, 2.6 Hz, 1H), 8.02e7.93 (m, 3H), 7.54e7.48 (m, 2H),
7.44e7.35 (m, 5H), 7.34e7.29 (m, 1H), 5.27 (s, 2H) ppm; 13C NMR
(125 MHz, DMSOed6)
d 161.60, 157.20, 150.10, 144.22, 142.31,
(125 MHz, DMSO‑d6) d 160.25, 149.19, 148.07, 137.06, 135.39, 130.58,
140.20, 138.05, 137.71, 137.68, 134.70, 133.19, 132.13, 129.66, 129.22,
129.10, 128.95, 127.84, 127.04, 125.23, 123.94, 123.66, 121.45, 120.47,
119.25, 119.17, 111.86, 108.52, 62.11, 45.76 ppm; HRMS (ESI): m/z
calcd for [MþH]þ C33H24ClN6O2 571.1649; found 571.1650.
129.88, 129.48, 129.15, 128.80, 128.25, 128.23, 126.45, 125.86,
123.02, 120.60, 120.31, 116.86, 49.59 ppm; HRMS (ESI): m/z calcd for
[MþH]þ C23H18N5O 380.1511; found 380.1514.
6.4.4. 3-benzyl-7-(4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)
quinazolin-4(3H)-one (23a)
6.3.12. (E)-4-(2-(3-benzyl-6-((1-(3-chloro-4-fluorophenyl)-1H-
1,2,3-triazol-4-yl)methoxy)-4-oxo-3,4-dihydroquinazolin-2-yl)
vinyl)benzonitrile (17b)
White solid, Yield 79%; mp: 200e202 ꢂC. 1H NMR (500 MHz,
DMSO‑d6)
d
9.22 (s, 1H), 8.70 (s, 1H), 8.35 (d, J ¼ 8.7 Hz, 1H), 8.25 (d,
Yellow solid, Yield 64%; mp: 229e231 ꢂC; 1H NMR (500 MHz,
J ¼ 1.4 Hz, 1H), 8.17 (dd, J ¼ 8.7, 1.6 Hz, 1H), 7.43e7.28 (m, 7H), 7.10
(d, J ¼ 8.2 Hz, 2H), 6.98 (t, J ¼ 7.3 Hz, 1H), 5.27 (s, 2H), 5.24 (s, 2H)
DMSOed6)
d 9.04 (s, 1H), 8.28e8.24 (m, 1H), 8.03e7.97 (m, 1H),
ppm; 13C NMR (125 MHz, DMSO‑d6)
d
13C NMR (125 MHz, DMSO)
7.91e7.84 (m, 4H), 7.84e7.78 (m, 2H), 7.76e7.66 (m, 2H), 7.60e7.50
(m, 2H), 7.35e7.30 (m, 2H), 7.30e7.20 (m, 3H), 5.68 (s, 2H), 5.43 (s,
d
159.96, 158.45, 150.00, 149.59, 144.91, 141.10, 137.09, 130.03,
2H) ppm; 13C NMR (125 MHz, DMSOed6)
d 161.61, 157.20, 150.12,
129.14, 128.99, 128.22, 128.19, 123.65, 121.69, 121.49, 119.07, 117.62,
115.23, 61.39, 49.52 ppm; HRMS (ESI): m/z calcd for [MþH]þ
C24H20N5O2 410.1617; found 410.1619.
144.24, 142.32, 140.21, 137.72, 137.68, 134.04, 134.01, 133.20, 129.68,
129.27, 129.22, 128.96,127.85,127.04, 125.24,123.96, 123.86, 123.04,
121.58, 121.52, 121.46, 121.36, 121.21, 119.17, 118.74, 118.56, 111.87,
108.54, 62.10, 45.76 ppm; HRMS (ESI): m/z calcd for [MþH]þ
C33H23ClFN6O2 589.1555; found 589.1563.
6.4.5. 3-(4-bromobenzyl)-7-(4-((2,5-dimethylphenoxy)methyl)-
1H-1,2,3-triazol-1-yl)quinazolin-4(3H)-one (23b)
White solid, Yield 61%; mp: 233e235 ꢂC. 1H NMR (500 MHz,
6.4. General reaction procedure for the synthesis of 2-methyl-3-1H-
1,2,3-triazol-1-yl)phenyl)quinazolin-4(3H)-one derivatives 22a-
22b, 22’a and 23a-23d
DMSO‑d6)
d
9.20 (s, 1H), 8.70 (s, 1H), 8.34 (d, J ¼ 8.7 Hz, 1H), 8.29 (d,
J ¼ 2.0 Hz, 1H), 8.19 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.59e7.53 (m, 2H),
7.40e7.34 (m, 2H), 7.02 (d, J ¼ 7.5 Hz, 1H), 7.00 (s, 1H), 6.69 (d,
J ¼ 7.4 Hz, 1H), 5.24 (s, 2H), 5.20 (s, 2H), 2.29 (s, 3H), 2.12 (s, 3H)
6 or 7-azido-3-benzylquinazolin-4(3H)-one (21, 2 mmol) and
phenylacetylene or phenoxyacetylenes (2 mmol) (Scheme 3) were
mixed in a 10 mL volume of 1:1 tert-BuOH/H2O mixture. To the
heterogeneous mixture, sodium ascorbate (10 mol%), followed by
copper(II)sulfate pentahydrate (2 mol%) were added and the reac-
tion mixture was allowed to stir for 12-14 h to give crude white
precipitates, which were filtered, washed with water, followed by
hexane and finally purified by using column chromatography to
obtain as pure white solid compounds 22a-22b, 22’a and 23a-23d
in 84e90% yields. All the newly synthesized compounds were
characterized by 1H NMR, 13C NMR and HRMS (ESI).
ppm; 13C NMR (125 MHz, DMSO‑d6)
d 159.99, 156.47, 149.94, 149.61,
145.37, 141.18, 136.66, 136.50, 132.02, 130.74, 130.53, 128.98, 123.36,
121.75, 121.67, 121.43, 119.15, 117.63, 113.27, 61.75, 49.07, 21.52,
16.15 ppm; HRMS (ESI): m/z calcd for [MþH]þ C26H23BrN5O2
516.1035; found 516.1039.
6.4.6. 3-(4-bromobenzyl)-7-(4-((5-ethyl-2-methoxyphenoxy)
methyl)-1H-1,2,3-triazol-1-yl)quinazolin-4(3H)-one (23c)
White solid, Yield 74%; mp: 265e267 ꢂC. 1H NMR (500 MHz,
DMSO‑d6)
d
9.19 (s, 1H), 8.69 (s, 1H), 8.34 (d, J ¼ 8.7 Hz, 1H), 8.26 (d,
J ¼ 1.6 Hz, 1H), 8.17 (dd, J ¼ 8.7, 1.8 Hz, 1H), 7.56 (d, J ¼ 8.3 Hz, 2H),

S. Gatadi et al. / Journal of Molecular Structure 1200 (2020) 127097
11
7.37 (d, J ¼ 8.3 Hz, 2H), 7.06 (d, J ¼ 8.1 Hz, 1H), 6.84 (s, 1H),
6.75e6.70 (m,1H), 5.20 (s, 2H), 5.18 (s, 2H), 3.75 (s, 3H), 2.55 (q, 2H),
phosphate buffer saline (PBS) to remove detached cells and fresh
culture media supplemented with Fetal bovine serum (FBS) was
added. Then, the cells were treated with 22a at doses of 2, 4 and
1.17 (t, J ¼ 7.6 Hz, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6)
d 159.97,
149.92, 149.69, 149.60, 145.90, 145.09, 141.16, 137.88, 136.48, 132.01,
130.51, 128.98, 123.71, 121.66, 121.42, 119.84, 119.11, 117.61, 114.98,
112.68, 62.40, 55.95, 49.06, 28.26, 16.20 ppm; HRMS (ESI): m/z calcd
for [MþH]þ C27H25BrN5O3 546.1141; found 546.1145.
6 mM accordingly. Representative photographs of the artificially
created gap were captured by camera (Carl Zeiss phase contrast
microscope) at 0 h and 24 h post incubation and were evaluated
using image analyzing software.
6.4.7. 3-(4-bromobenzyl)-7-(4-((4-(tert-pentyl)phenoxy)methyl)-
1H-1,2,3-triazol-1- yl)quinazolin-4(3H)-one (23d)
7.4. Acridine orange-ethidium bromide (AO-EB) staining
White solid, Yield 66%; mp: 245e247 ꢂC. 1H NMR (500 MHz,
MDA-MB-231 cells were plated at a density of 5 ꢁ 103 cells in a
DMSO‑d6)
d
9.21 (s, 1H), 8.70 (s, 1H), 8.34 (d, J ¼ 8.7 Hz, 1H), 8.26 (d,
12 well plate and allowed to adhere overnight. The following day,
J ¼ 2.1 Hz, 1H), 8.17 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.64e7.51 (m, 2H),
7.39e7.35 (m, 2H), 7.28e7.24 (m, 1H), 7.04e7.00 (m, 1H), 5.23 (s,
2H), 5.20 (s, 2H), 1.58 (q, J ¼ 7.4 Hz, 2H), 1.22 (s, 6H), 0.62 (t,
cells were treated with 22a at concentration of 2, 4 and 6 mM and
incubated for 48 h. After 48 h, fluorescent dyes AO and EB each
were added into wells and cells were observed under fluorescence
microscope immediately. Representative images of all groups were
captured using microscope (Nikon, Inc. Japan) at excitation of
488 nm and emission of 550 nm using 20X objective lens.
J ¼ 7.4 Hz, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6)
d 159.97, 156.17,
149.95, 149.61, 145.10, 141.86, 141.16, 136.49, 132.01, 130.52, 128.99,
127.26, 123.59, 121.67, 121.42, 119.12, 117.62, 114.65, 61.43, 49.06,
37.41, 36.72, 28.89, 9.52 ppm; HRMS (ESI): m/z calcd for [MþH]þ
C29H29BrN5O2 558.1504; found 558.1508.
7.5. DAPI nuclear staining
7. Pharmacology
MDA-MB-231 cells were seeded at a density of 5 ꢁ 103 cells.
Next day, cells were treated with different concentrations of 22a (2,
7.1. Cell cultures
4 and 6 mM) and incubated for 48 h. Post incubation, cells were
washed with PBS and permeabilized using PBS containing 0.1%
Tween-20. Further, cells were fixed with 4% paraformaldehyde
followed by staining with DAPI. Representative images for nuclear
morphological changes induced by 22a were captured by fluores-
cence microscope (Nikon, Japan) with excitation of 359 nm and
emission of 461 nm using 20X objective lens.
MDA-MB-231, MCF-7 (breast cancer), HCT-116, HT-29 (colon
cancer) and A549 (lung cancer) were procured from National
Centre for Cell Science (NCCS) Pune, India. MDA-MB-231, MCF-7,
HCT-116 and HT-29 were grown in DMEM (Dulbecco Modified
Eagle medium, Himedia) and A549 was grown in RPMI (Roswell
Park Memorial Institute medium, Himedia) supplemented with 10%
fetal bovine serum and 1% antibiotic-antimycotic solution (Sigma)
in a CO2 incubator with 5% CO2 and 95% relative humidity at 37 ꢂC.
When the cells reached 70e75% confluence, they were treated with
0.25% trypsin/1 mM EDTA solution for further passage.
7.6. Cell cycle analysis
To perform cell cycle analysis assay, the following procedure was
followed. Briefly, MDA-MB-231 cells at a density of 1 ꢁ 106 cells/
well in 6 well plate followed by treatment with 22a at concentra-
7.2. Cytotoxicity assay
tions of 4 and 6 mM. 24 hrs post incubation with 22a, cells were
harvested by trypsinisation and fixed with 70% ethanol for
30 min at 4 ꢂC. After fixation, cells were again washed with PBS and
stained with propidium iodide staining buffer for 15 min in dark at
room temperature. Then, the samples were analysed for propidium
iodide fluorescence by flow cytometry using BD Accuri C6 flow-
cytometer.
Newly synthesized compounds were evaluated for cytotoxicity
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay in the above mentioned five cell lines. Doxorubicin was
used as a reference standard. Briefly, cells were seeded in 96 well
microculture plates at a density of 5 ꢁ 103 in 100
mL of media per
well. The following day, cells were treated with respective com-
pounds dissolved in DMSO for 48 h at 37 ꢂC in a CO2 incubator. Post
incubation, media was discarded and MTT was added to all the
7.7. Colony formation assay
wells at a concentration of 0.5 mg/mL (100
incubation. Further, MTT was discarded and the formazan crystals
so formed were dissolved by adding 200 L of DMSO to each well.
m
L/well) followed by 4 h
MDA-MB-231 cells were seeded at a density of 250 cells/well in
a 12 well plate. After cells adhered to the culture well plate, cells
were treated with different concentrations of 22a (2, 4 and 6 mM)
m
Absorbance was recorded at 570 nm wavelength using a spectro-
photometer and the percentage of growth inhibition was calcu-
lated. The tested compounds which exhibited >50% inhibition of
for 24 h. Post incubation, media was discarded and the cells were
maintained in media containing Fetal bovine serum (FBS) to form
colonies for a period of 10 days. After 10 days, the so called colonies
were stained with 0.5% crystal violet stain. Then, the plate was
imaged and colonies were counted using Vilber Fusion Fx software
(Vilber Lourmat, France). The results are represented as total colony
number vs. concentration.
cell viability at 10 mM in preliminary screening were further
selected to evaluate the drug response curve (DRC). IC50 values for
these selected compounds were determined from the DRC plot by
linear regression method: % cell inhibition (from control optical
density) versus different concentrations (in
mM).
8. Computational studies
7.3. Scratch wound assay/cell migration assay
The 3D crystal coordinates of EGFR were downloaded from the
Effect of 22a on MDA-MB-231 cell migration was determined by
scratch wound assay. Briefly, cells were seeded at a density of
1 ꢁ 105 in a 6 well plate. The following day, artificial wound was
PDB and were prepared according to protocols specified in Protein
€
Preparation Wizard of Schrodinger suite 2017. Briefly, hydrogens
were added, bond orders were assigned, water molecules were
removed within 5 Å distance and minimization done using OPLS-
2005 force field. Compounds 11b, 11d, 16h, 17b, 22a and 23b were
created by disrupting monolayer of cells with a sterile 200 mL tip
across the centre of the well. Wells were washed with sterile

12
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Acknowledgements
G. S. conveys cordial thanks to DoP, Ministry of Chemicals &
Fertilizers, Govt. of India, for the award of NIPER fellowship. GS and
SN gratefully acknowledges Department of science and technology
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CuAAC
DMF
DAPI
MTT
Cu(I)- catalyzed azide-alkyne cycloaddition;
Dimethylformamide
4,6-Diamidino-2-phenylindole
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
Phosphate Buffered Saline
Fetal bovine serum
acridine orange/ethidium bromide
Drug response curve
PBS
FBS
AO-EB
DRC
DOX
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