Design and synthesis of novel sulphamide tethered quinazolinone hybrids as potential antitumor agents

Article abstract of DOI:10.1016/j.molstruc.2018.12.098

In an attempt to develop potential and selective antitumor agents, a series of novel sulphamide tethered quinazolinone hybrids were efficiently synthesized and evaluated for antitumor activity against four cancer cell lines such as HeLa (cervical), MDA-MB-231 (breast), PANC-1 (pancreatic), and A549 (lung) in vitro. All the compounds (5a-j, 6a-g) exhibited significant anti-proliferative activity with GI₅₀ values ranging from 0.045 to 6.94 μM, while compound 10c showed potent activity against all the cell lines (He La, MDA-MB-231, PANC-1 and A549) with GI₅₀ values ranging from 0.09 to 0.21 μM. We have explored the binding mode and key active site interactions in HDAC8 and EHMT2 proteins. The docking results are complementary to the experimental results.

Full text of DOI:10.1016/j.molstruc.2018.12.098

Accepted Manuscript
Design and synthesis of novel sulphamide tethered quinazolinone hybrids as potential
antitumor agents
Ramineni Venkatesh, Suresh Kasaboina, Nishant Jain, Sridhara Janardhan, Uma
Devi Holagunda, Lingaiah Nagarapu
PII:
S0022-2860(18)31532-1
DOI:
https://doi.org/10.1016/j.molstruc.2018.12.098
MOLSTR 26035
Reference:
To appear in: Journal of Molecular Structure
Received Date: 23 August 2018
Revised Date: 26 November 2018
Accepted Date: 23 December 2018
Please cite this article as: R. Venkatesh, S. Kasaboina, N. Jain, S. Janardhan, U.D. Holagunda,
L. Nagarapu, Design and synthesis of novel sulphamide tethered quinazolinone hybrids as
potential antitumor agents, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2018.12.098.
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Graphical Abstract
___________________________________________________________________________
Design and synthesis of novel sulphamide tethered quinazolinone hybrids
as potential antitumor agents
Ramineni Venkatesha, Suresh Kasaboinaa, Nishant Jainb, Sridhara Janardhanc
Holagundaa Lingaiah Nagarapua*
, Uma Devi
,
A series of novel sulphamide tethered quinazolinone derivatives were efficiently synthesized
and evaluated for antitumor activity against four cancer cell lines.
_______________________________________________________________________________________

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Design and synthesis of novel sulphamide tethered quinazolinone hybrids
as potential antitumor agents
Ramineni Venkatesha, Suresh Kasaboinaa, Nishant Jainb, Sridhara Janardhanc, Uma Devi
Holagundaa, Lingaiah Nagarapua*
aFluoro-Agrochemicals Division-II, CSIR-Indian Institute of Chemical Technology, Tarnaka,
Hyderabad- 500007, India.
bCenter for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka,
Hyderabad-500007, India.
cCenter for Molecular Modeling, CSIR-Indian Institute of Chemical Technology, Tarnaka,
Hyderabad 500007, India.
*Corresponding author: e-mail: lnagarapuiict@yahoo.com/nagarapu@csiriict.in
Tel: +91 40 27191509, Fax; ++ 91 40 27193382. IICT/Pubs./2018/049
Abstract: In an attempt to develop potential and selective antitumor agents, a series of novel
sulphamide tethered quinazolinone hybrids were efficiently synthesized and evaluated for
antitumor activity against four cancer cell lines such as HeLa (cervical), MDA-MB-231
(breast), PANC-1 (pancreatic), and A549 (lung) in vitro. All the compounds (5a-j, 6a-g)
exhibited significant anti-proliferative activity with GI50 values ranging from 0.045 to 6.94
µM, while compound 10c showed potent activity against all the cell lines (He La,
MDA-MB-231, PANC-1 and A549) with GI50 values ranging from 0.09 to 0.21 µM. We
have explored the binding mode and key active site interactions in HDAC8 and EHMT2
proteins. The docking results are complementary to the experimental results.
Keywords: Quinazolinones scaffolds, sulphamides, antitumor activity, docking studies.
Introduction
The potent antitumor activities and clinical applications of quinazolinones scaffold
containing alkaloids have attracted worldwide. Their parent representative analogues serves
as an excellent lead drug in anticancer research because of its potent antitumor activity [1,2].
Quinazolinones have been reported to have diverse range of biological activities such as
anticancer, anti-HIV, anti tubercular, diuretic, anti-inflammatory, anticonvulsant, antiviral,
anti malarial, antibacterial, Antifungal activity and antihypertensive agents [3-10]. Recently,
literature reports revealed that the importance of sulphones and their derivatives in the
medicinal chemistry due to their use as precursors in the synthesis of various biologically
active heterocyclic compounds. They are used as significant pharmacological activities such

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as antifungal, antibacterial, anti cancer and antimicrobial [11-16] activities. Chemically,
sulfonyl and sulphanamide functional groups are well established as activating moiety in a
number of intermediates for the construction of C-S and C-N hetero atoms bond formation
during organic transformations. Recently, they have been utilized in total synthesis and in the
generation of database of functionalized compounds. Moreover, these moieties have potential
to stabilize carnations, radicals and facilitate conjugate addition by activation of the olefins
with Lewis acids. In past decades, numerous quinazolinones embedded natural products have
been identified, such as cytotoxic alkaloids Luotonin A, Camptothecin (CPT), Aromathecin,
Rosettacin, 22-Hydroxyacuminatine, Rutaecarpineetc, whose derivatives are clinically proven
anticancer agents [17-25]. Additionally, some therapeutic agents are in the market and some
are in clinical trials for the treatment of cancer [26]. A literature survey revealed that
modification on quinazolinone pharmacophore may result increase in its biological potencies.
Structural variations bring about new physical and biological properties. The molecular
modification of a promising lead compound is still a major line of approach for the discovery
of new drugs. As a part of synthesis of new class of quinazolinones derivatives, we have
introduced sulphamide group with several secondary amines which might be a potential
tumor growth inhibitor. Recently, in an on-going programme to discover and develop
potential new anticancer agents, we have identified several classes of molecules as novel
tumor growth inhibitors [27-31]. In an attempt to design new anticancer agents, we
discovered a novel series of quinazolinone derivatives (Fig. 1) to elicit combined antitumor
efficacy/cytotoxicity against different cancer cell lines in vitro. The computational techniques
play a vital role in lead molecule identification and its optimization. Both structure based
(homology modeling, docking) and ligand based (pharmacophore, QSAR) drug design
approaches have been used for the design of better ligands in order to enhance potency,
selectivity, pharmacokinetic parameters. Hence, in the current study, we have applied both
ligand based and structure based techniques on a series of quinazolinone hybrids in both
EHMT2 and HDAC8 proteins. The crystal structures of EHMT2 and HDAC8 are known and
hence we have downloaded from the protein databank for further analysis. The docking
studies have been performed to understand the key active site residues of most potent
quinazolinone series of compounds in all EHMT2 and HDAC8 proteins and explored all
probable binding modes. Ligand based pharmacophore model will be helpful in identifying
drug-like features for responsible for better ligand binding. The pharmacophore models
provide the hypothetical active site and can be used for virtual screening [32-35] to obtain
diverse chemical classes of new compounds. The identified structural features from ligand

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based and structure based approaches could help in rational design of quinazolinone series of
compounds with enhanced potency in EHMT2 and HDAC8 proteins.
Figure 1. Structures of Luotonin A, Camptothecin, Chloroqualone, Alloqualone and
quinazolinone derivatives.
Results and discussion:
Chemistry
The synthesis of 2-(2,4-dichlorophenyl)-4-oxo-1,2,3,4-tetrahydroquinazoline-6-sulfonyl
chloride (4) was done by the condensation of anthranilamide (1) with 2,4-dichloro
benzaldehyde (2) and a catalytic amount of tetrabutyl ammonium hydrogensulphate
(TBAHS) in methanol under reflux temperature for 2 h followed by the treatment with
chlorosulphonicacid at -10 oC for 25min to produce compound (4) in good yield (Scheme 1).
The synthesis of sulphamide tethered quinazolinone-piperzine hybrids (5a-j, 6a-g) have been
achieved in moderate to excellent yields by the coupling of compound 4 with N-phenyl
piperzine derivatives and 3-(4-phenylpiperazin-1-yl)propan-1-amine derivatives using
diisopropylethyl amine (DIEA) in DMF under room temperature for 1h as shown in Scheme
3.
Scheme-1. Synthesis of compound 4.

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O
S
O
NH
X
R
N
NH Cl
O
X
R
N
H
O
S
O
Cl
5a-j
DIEA, DMF,
Cl
NH Cl
O
1 h, rt,
82-88%
N
H
O
S
O
Cl
4
N
N
H
NH Cl
O
X
R
N
H
N
X
R
NH2
6a-g
Cl
for 6a-g, where
for 5a-j, where
5a, X = N, R =
O
F
NO2
6a, X = N, R =
6b, X = N, R =
6c, X = N, R =
O
5b, X = N, R =
F
5c, X = N, R =
5d, X = N, R =
5e, X = N, R =
5f, X = N, R =
5g, X = N, R =
5h, X = H, R =
5i, X = S
CF3
F
6d, X = N, R =
6e, X = S
CH3
CF3
6f, X = H, R =
O
O
NO2
6g, X = N, R =
5j, X = N, R =
Scheme-2. Synthesis of compounds 5a-j, 6a-g.
Pharmacology:
Cytotoxicity of all the compounds were evaluated against human cancer cell lines He
La (cervical), MDA-MB-231 (breast), PANC-1 (pancreatic), and A549 (lung) by the standard
MTT assay method [36-38]. The various human tumor cell growth inhibitor potential was
determined as described [39] The compounds were tested for an advanced assay against these
four human cancer cell lines at five different concentrations (0.01, 0.1, 1, 10, 100 µM). GI50

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(growth inhibitory activity) was calculated and these values corresponded to the
concentration of the compound causing 50% decrease in the net cell growth as compared to
the standard drugs, Doxorubicin, Nocadazole, Colchicine, Cambretostatin (CA4) and
Paclitaxel (Table 2). Results were calculated for each of these parameters if the level of
activity was reached; however, if the effect was not achieved, the value was expressed as
greater or less than the maximum or minimum concentration tested.
Based on Table 2, all the synthesized compounds showed significant cancer cell
growth inhibition with GI50 values ranging from 0.045 to 6.94 µM. In particular, the
compounds 5a, 5c, 5f, 5g, 5i, 6c, 6e, 6f, and 6g showed promising anti-proliferative activity
against the four human cancer cell lines. Among them, compounds 5c showed potent
anti-proliferative activity against all the four human cancer cell lines with GI50 values ranging
from 0.09 to 0.21 µM. The compounds 5i and 6f showed potent anti-proliferative activity
against the three human cancer cell lines He La, MDA-MB-231, and A549 with GI50 values
ranging from 0.045 to 0.35 µM. The compounds 6e showed significant anti-proliferative
activity against the two human cancer cell lines He La, and PNAC-1 with GI50 values 0.44
and 0.48 µM respectively. The selective anti-proliferative activity of compounds are as
follows, 5a against He La (GI50 0.67 µM), 5f against MDA-MB-231 (GI50 0.35 µM), 5g
against MDA-MB-231 (GI50 0.29 µM), 6c against HeLa (GI50 0.31 µM) and 6g against
MDA-MB-231 (GI50 0.61 µM). The effect of various substituents on the sulphonamide
moiety was examined. The structure-activity relationship (SAR) study revealed that not only
the substituent on sulphamide but also the sulphamide along with propyl linker is required for
inducing the anti-proliferative activity against these cancer cell lines. The substituents on
sulphamide such as o-fluorophenyl (5c), thiopiperidine (5i) and propyl linker substitution on
thiopiperidine (6e) and benzyl (6f) were associated with a significant increase in the growth
inhibitory effect against He La, MDA-MB-231, PANC-1 and A549 human cancer cell.
Molecular modeling:
Pharmacophore modeling
In order to identify the essential structural features of the ligand responsible for its
potency, we have generated a ligand based pharmacophore model from a dataset comprising
of (5a-j, 6a-g) quinazolinone compounds using A549 cell-line activity. A total of 428
conformations thus obtained were used to generate pharmacophore hypotheses. The series of
compounds having maximum variation in their growth inhibition (pGI50: 5.34 to 7.34). The
common pharmacophore hypothesis (CPH) was obtained based 6 highly active compounds

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(pIC50 ≥ 6) from the dataset and then inactive molecules (pIC50 < 6) were aligned with it. The
identified best CPH consists of one acceptor (A), one donor (D), one hydrophobic (H) and
two aromatic (R) features i.e ADHRR. Alignment of the set of active compounds from the
dataset using pharmacophore hypothesis (ADHRR) is shown in Figure 2.
Figure 2. A: Common pharmacophore hypothesis (ADHRR) and its pharmacophoric
distances (Å) are shown in pink colour. B: Alignment of active data set compounds on
ADHRR.
Molecular Docking:
In order to explore the binding mode and understanding of key active site residues,
molecular docking study has been performed. The synthesized quinazolinone series of
compounds were docked into the active site of HDAC8 and EHMT2 proteins. In the present
study, the docking result of compound (5c) in HDAC8 and EHMT2 proteins are discussed.
HDAC8: From the docking study, we observed that the non-covalent hydrophobic
interactions as well as electrostatic interactions are more predominating for strong binding
towards HDAC8 protein. The oxygen of 2,3-dihydro-1H-quinazolinone moiety formed salt
bridge with aspartic acid (Asp178) and histidine residue (His180) through Zn+2. Also the
same oxygen atom showed hydrogen bond interaction with methionine (Met274, 3.40Å). The
nitrogen atom of 2,3-dihydro-1H-quinazolinone showed hydrogen bond interaction with
carbonyl oxygen of glycine (Gly151, 3.16 Å) backbone residue. The phenyl ring of
2,3-dihydro-1H-quinazolinone moiety and sulfonyl piperazine moieties showed strong
noncovalent hydrophobic interactions with two phenylalanine residues (Phe152, Phe208) and
methionine (Met274) residue. 2,4-dichloro-phenyl moiety is oriented towards hydrophobic

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pocket containing tyrosine (Tyr306) and tryptophan (Trp141) residues.
EHMT2: From the docking results, we observed that the non-covalent hydrophobic
interactions are more predominating as compared to electrostatic interactions for binding of
the molecules. The active site comprises mostly the hydrophobic residues include tyrosine
(Tyr1085, Tyr1067), phenyl alanine (Phe1110, Phe1138, Phe1158, Phe1087) and proline
(Pro1121, Pro1011) and isoleucine (Ile1136, Ile1009, Ile1120). The nitrogen atom of
2,3-dihydro-1H-quinazolinone showed hydrogen bond interaction with aspartic acid
(Asp1088, 3.16 Å). The fluoro atom of fluorophenyl moiety showed electrostatic interaction
with positively charged arginine residue (Arg1157, 3.10 Å). The oxygen atom of
2,3-dihydro-1H-quinazolinone moiety oriented towards positively charged lysine residue
(Lys1162). Hydrophobic 2,4-dichloro-phenyl moiety showed strong non-covalent
hydrophobic interactions with phenyl alanine residues (Phe1087, Phe1152, Phe1158) and
tyrosine residue (Tyr1154) in the active site of EHMT2. The probable binding mode of most
potent compound (5c) of the quinazolinone series of compounds in EHMT2 and HDAC8 is
shown in Figure 3. The active site residues around 4Å of the most potent compound (5c) of
the series are shown in Table 1.

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Figure 3. The probable binding mode compound (5c, orange) in the active site of HDAC8
and EHMT2 proteins (helix: cyan, sheet: red and loop: purple). The hydrogen bond
interactions are shown in hot pink colored dotted lines, metal Zn+2 is shown in yellow color,
the important residues are shown in green color.
Target
Compound 10c (Active site residues around 4Å)
ARG37, GLY139, GLY140, TRP141, HIS142, HIS143, GLY151, PHE152,
CYS153, HIS180, PHE208, GLN263, ASP267, MET274, GLY303, GLY304,
GLY305, TYR306, ARG37, GLY139, GLY140, TRP141, HIS142, HIS143,
GLY151, PHE152, CYS153, HIS180, PHE208, GLN263, ASP267, MET274,
GLY303, GLY304, GLY305, TYR306
HDAC8
ASP1074, ALA1077, ASP1078, ASP1083, SER1084, LEU1086, PHE1087,
ASP1088, PRO1121, PHE1152, ASP1153, TYR1154, ARG1157, PHE1158,
ILE1161, LYS1162
EHMT2
Table 1: The active site residues around 4Å of the most potent compound (5c)
Table-2: (GI50/µM)a values of the tested compounds against four human cancer cell lines.
Sr. No.
Compounds
HeLa
MDA-MB-23 PANC-1
1
A549
1
0.675 ± 0.02
6.325 ± 0.01
0.324 ± 0.02
2.955 ± 0.01
2.362 ± 0.02
3.455 ± 0.02
5a
2
1.286 ± 0.01
0.124 ± 0.02
2.2 ± 0.01
2.2 ± 0.01
5b
5c
5d
5e
5f
3
0.095 ± 0.001 0.212 ± 0.02
0.115 ± 0.01
1.946 ± 0.02
4.324 ± 0.02
4
1.162 ± 0.02
6.94 ± 0.02
0.356 ± 0.01
0.297 ± 0.03
2.371 ± 0.01
3.697 ± 0.01
6.854 ± 0.01
5
3.344 ± 0.03
1.333 ± 0.02
2.534 ± 0.01
1.858 ± 0.01
6
1.412 ± 0.01 1.772 ± 0.01
7
1.778 ± 0.02
2.894 ± 0.02
6.737 ± 0.03
7.257 ± 0.02
6.128 ± 0.01
2.358 ± 0.02
1.864 ± 0.02
0.358 ± 0.01
4.736 ± 0.02
2.128 ± 0.03
5g
5h
5i
8
9
0.045 ± 0.003 0.194 ± 0.02
10
11
1.661 ± 0.02
2.863 ± 0.01
1.296 ± 0.02
5.963 ± 0.01
5j
6a

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12
13
14
15
16
17
18
19
20
1.683 ± 0.02
0.316 ± 0.01
2.414 ± 0.02
0.446 ± 0.02
0.296 ± 0.01
4.521 ± 0.02
0.02 ± 0.002
8.317 ± 0.03
1.743 ± 0.01
4.283 ± 0.01
4.337 ± 0.02
0.795 ± 0.01
0.617 ± 0.02
0.03 ± 0.001
6.422 ± 0.02
1.582 ± 0.02
3.452 ± 0.02
0.483 ± 0.02
1.743 ± 0.02
1.895 ± 0.02
2.783 ± 0.03
3.783 ± 0.01
4.572 ± 0.01
3.542 ± 0.03
0.644 ± 0.02
2.764 ± 0.02
6b
6c
6d
6e
6f
6g
Paclitaxelb
Nocadazoleb
Colchicineb
< 0.01 ± 0.003 0.02 ± 0.002
<0.01 ± 0.001 <0.01 ± 0.001 < 0.01 ± 0.003 0.03 ± 0.001
<0.01 ± 0.002 0.02 ± 0.003
0.02 ± 0.002
<0.01
0.002
±
21
22
Cambretostatin
(CA4)b
Doxorubicinb
0.02 ± 0.002
0.03 ± 0.003
<0.01 ± 0.001 <0.01 ± 0.002 0.01 ± 0.002
0.01 ± 0.002
< 0.01 ± 0.003 <0.01
0.003
±
aGI50: 50% Growth inhibition, concentration of drug resulting in a 50% reduction in net rotein
increase compared with control cells. bPositive control.
Conclusion
In conclusion, a novel sulphamide tethered quinazolinones hybrids as antitumor
agents have been synthesized. Compounds 5a-j, 6a-g were synthesized and screened for
viability assays against human HeLa (cervical), MDA-MB-231 (breast), PANC-1
(pancreatic), and A549 (lung carcinoma) cell lines in vitro. In general, most of the prepared
compounds showed significant cytotoxic effect against human tumor cell lines. Compounds
5c showed potent cytotoxic activity against all the four human cancer cell lines with GI50
values ranging from 0.09 to 0.21 µM. Also compounds 5i and 6f showed potent
anti-cytotoxic activity against the three human cancer cell lines HeLa, MDA-MB-231, and
A549 with GI50 values ranging from 0.045 to 0.35 µM. We succeeded in the substituent
change on sulphamide and propyl linker which played a critical role in exhibiting promising
anti-proliferative activities. This study provides that these compounds would be very useful
for further investigation of tumor cell metastasis, ant-proliferative mechanism and
angiogenesis in our next research project and one of them could be potential antitumor agent.
We have identified five featured pharmacophore (ADHRR) and from the docking study, we
have explored the probable binding mode and identified key active site residues around
potent compound (5c) in HDAC8 and EHMT2 proteins.

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Experimental
General chemistry:
All the chemicals and reagents were purchased from Sigma-Aldrich and SD
Fine-Chemicals, Pvt. Ltd. India, and used as received. The reactions were monitored and Rf
value was determined using analytical thin layer chromatography (TLC) with Merck Silica
gel 60-120 and F254 pre-coated plates (0.25 mm thickness). Spot on the TLC plates were
visualized using ultraviolet light (254 nm). Flash column chromatography was performed
with Merck silica gel (100-200 mesh). Melting points were determined in capillaries and are
1
uncorrected. H NMR spectra were recorded on Bruker DRX-300, Avance-300and Avance
13
new-500 NMR spectrometers. C NMR spectra were recorded on Bruker DRX-300. Proton
chemical shifts are reported in ppm (δ) relative to internal tetramethylsilane (TMS, δ 0.00 or
with the solvent reference relative to TMS employed as the internal standard (CDCl3, δ 7.26;
DMSO-d6 δ 2.54) and multiplicities of NMR signals are designated as s (singlet), d (doublet),
t (triplet), q (quartet), br (broad), m (multiplet, for unresolved lines). Infrared (IR) spectra
were recorded on a Perkin Elmer FT-IR 400 spectrometer; data is reported in wave numbers
(cm-1). Mass spectra were recorded on Agilent Technologies 1100 Series (Agilent
Chemistation Software). High-resolution mass spectra (HRMS) were obtained by using
ESI-QTOF mass spectrometry.
Preparation of 2-(2,4-dichlorophenyl)-4-oxo-1,2,3,4-tetrahydroquinazoline-6-sulfonyl
chloride (4).
A solution of 2-aminobenzamide (3g, 0.02 mol), 2, 4-dichloro benzaldehyde (3.7g,
0.02 mol) and a catalytic amount of TBAHS (1 mol%) in MeOH was made to react at reflux
temperature for 2 h. The reaction mixture was then cooled to rt and small amount of water
(15 mL) was added to the mixture, and then generated solid was filtered to give cyclic
compound 3. The intermediate 3 converted into 4 by the drop wise addition of Chloro
sulphonic acid (15 mL) and stirred for 25 min at -10°C. The reaction mixture was carefully
poured into ice. The cold water was washed with CH2Cl2. CH2Cl2 was separated, dried over
Na2SO4 and was concentrated by evaporation under vacuum to furnish a light yellow solid.
Synthesis of 2-(3-bromopropyl) isoindoline-1, 3-dione (7).
To a stirred solution of isoindoline-1, 3-dione (10g, 1mmol) in DMF (40 mL),
1,3-dibromo propane (14g, 1 mmol), K2CO3 (9.3 g, 3 mmol) were added at rt. The reaction

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mixture was stirred at rt for 3 h. After completion of the reaction, as indicated by TLC, The
resulting solution was poured into ice cold water, which was then extracted with ethyl acetate.
Ethyl acetate was separated, dried over Na2SO4 and concentrated under vacuum to afford a
white solid. Yield 85%; 1H-NMR (300 MHz, CDCl3): δ 2.23-2.33 (m, 2H, CH2), 3.42 (t, J =
6.4 Hz, 2H, CH2), 3.86 (t, J = 6.4, 6.9 Hz, 2H, CH2), 7.74 (m, 2H, Ar-H), 7.86 (m, 2H, Ar-H);
ESI MS : m/z = 268 [M+H]+
.
General procedure for the synthesis of compounds 5a-j, 6a-g.
2-(2,4-dichlorophenyl)-4-oxo-1,2,3,4-tetrahydroquina zoline-6-sulfonyl chloride
(0.1g, 0.0002 mol) was added to a round bottom flask containing N,N-dimethylformamide (4
mL) under nitrogen atmosphere, N-phenyl piperzine derivatives (0.0002 mol) or
3-(4-phenylpiperazin-1-yl)propan-1-amine derivatives (0.0002 mol) was added at rt with
stirring, followed by the addition of DIEA (1 mL). The stirring was continued for 1 h. After
completion of the reaction, as indicated by TLC, The ice cold water was added to the reaction
mixture stirred it for 5 min and then extract with Chloroform (10 mL). The chloroform layer
was separated, dried over Na2SO4 and evaporated under vacuum to give corresponding
sulphamide derivatives (5a-j, 6a-g) in good yields.
2-(2,4-Dichlorophenyl)-6-((4-(4-nitrophenyl)piperazin-1-yl)sulfonyl)-2,3-dihydroquinazoli
o
n-4(1H)-one (5a).Yield 85%; mp 138-141 C; IR (KBr) υ in cm-1: 1134, 1239, 1372, 1634,
1
3607; H NMR(300 MHz, CDCl3): δ 2.96 (s, 4H, 2xCH2), 3.52 (s, 4H, 2xCH2), 6.33 (s, 1H,
CH), 6.78-6.90 (m, 3H, Ar-H), 7.30 (d, J = 9.0 Hz, 1H, Ar-H), 7.42 (s, 1H, Ar-H), 7.57 (s, 2H,
Ar-H), 7.62 (d, J = 8.2 Hz, 1H, Ar-H), 7.92 (bs, 1H, NH), 8.07 (d, J = 9.0 Hz, 1H, Ar-H),
13
8.19 (s, 1H, Ar-H); C NMR (125 MHz, CDCl3) : δ 44.3, 45.3, 62.3, 112.2, 113.9, 121.1,
124.4, 126.3, 127.3, 128.1, 128.5, 131.4, 131.8, 133.7, 135.2, 137.1, 149.5, 152.9, 161.3; ESI
MS : m/z = 562 [M+H]+.
2-(2,4-Dichlorophenyl)-6-((4-(2-methoxyphenyl)piperazin-1-yl)sulfonyl)-2,3-dihydroquinaz
olin-4(1H)-one (5b).Yield 81%; mp 147-150 oC; IR (KBr) υ in cm-1: 1162, 1243, 1344, 1662,
3360; 1H NMR (300 MHz, CDCl3): δ 1.75-2.02 (bs, 8H, 4xCH2), 2.59 (s, 3H, OCH3), 5.11 (s,
1H, CH), 5.56-5.71 (bs, 3H, Ar-H), 5.72-5.82 (m, 1H, Ar-H), 6.06-6.16 (m, 1H, Ar-H), 6.22
(s, 1H, Ar-H), 6.34 (s, 1H, Ar-H), 6.43 (d, J = 8.3 Hz, 1H, Ar-H), 6.50 (s, 1H, Ar-H), 6.94 (d,
J = 11.5 Hz, 1H, NH), 7.41 (s, 1H, Ar-H); 13C NMR (125 MHz, CDCl3) : δ 44.9, 48.3, 54.0,
62.2, 109.9, 112.1, 113.4, 116.9, 121.3, 122.0, 126.3, 127.3, 127.9, 128.4, 131.4, 131.7, 133.6,

ACCEPTED MANUSCRIPT
135.2, 138.9, 149.3, 150.6, 161.4; ESI MS : m/z = 547 [M+H]+; HRMS calculated for
C25H25Cl2N4O4S : 547.0900, Found : 547.0974.
2-(2,4-Dichlorophenyl)-6-((4-(2-fluorophenyl)piperazin-1-yl)sulfonyl)-2,3-dihydroquinazol
o
in-4(1H)-one (5c).Yield 87%; mp 142-145 C; IR (KBr) υ in cm-1: 1134, 1237, 1370, 1669,
3653; 1H NMR(300 MHz, CDCl3): δ 3.05 (t, J = 5.1, 10.2 Hz, 2H, CH2), 3.10 (t, J = 5.0, 10.0
Hz, 2H, CH2), 3.55 (t, J = 4.8, 10.0 Hz, 2H, CH2), 3.74 (t, J = 4.8, 10.2 Hz, 2H, CH2),
6.91-6.96 (m, 1H, Ar-H), 6.97-7.02 (m, 1H, Ar-H), 7.03 (s, 1H, Ar-H), 7.04-7.10 (m, 2H,
Ar-H), 7.29-7.38 (m, 1H, Ar-H), 7.40-7.48 (m, 1H, Ar-H), 7.50-7.56 (m, 1H, Ar-H),
7.61-7.74 (m, 1H, Ar-H), 8.04 (dd, J = 7.7, 9.1 Hz, 1H, Ar-H), 8.10 (s, 1H, Ar-H); 13C NMR
(125 MHz, CDCl3) : δ 40.1, 45.7, 50.1, 116.2, 116.3, 119.3, 123.3, 123.4, 124.5, 126.8, 128.8,
130.9, 133.3, 134.0, 137.0, 139.5, 144.4, 147.8, 151.0, 154.6, 156.7, 160.8; ESI MS : m/z =
532 [M-2H]+.
2-(2,4-Dichlorophenyl)-6-((4-(4-fluorophenyl)piperazin-1-yl)sulfonyl)-2,3-dihydroquinazol
o
in-4(1H)-one (5d).Yield 89%; mp 136-139 C; IR (KBr) υ in cm-1: 1191, 1293, 1315, 1616,
3661; 1H NMR(300 MHz, CDCl3): δ 1.25 (bs, 4H, 2xCH2), 1.81 (bs, 4H, 2xCH2), 5.46 (s, 1H,
CH), 7.33-7.43 (m, 2H, Ar-H), 7.50-7.61 (m, 2H, Ar-H), 7.74 (t, J = 7.3, 14.1 Hz, 1H, Ar-H),
7.89 (t, J = 7.5, 15.1 Hz, 1H, Ar-H), 7.98 (d, J = 7.5 Hz, 1H, Ar-H), 8.61 (d, J = 4.1 Hz, 1H,
Ar-H), 8.81 (d, J = 4.5 Hz, 1H, Ar-H), 10.09 (s, 1H, Ar-H); 13C NMR (125 MHz, CDCl3) : δ
51.6, 52.7, 58.6, 107.7, 109.0, 110.5, 111.9, 114.4, 120.4, 123.2, 126.7, 128.8, 129.5, 131.6,
134.0, 135.8, 137.4, 140.9, 142.6, 148.6, 153.2, 160.3; ESI MS : m/z = 535 [M+H]+.
2-(2,4-Dichlorophenyl)-6-((4-methylpiperazin-1-yl)
o
sulfonyl)-2,3-dihydroquinazolin-4(1H)-one (5e). Yield 82%; mp 137-140 C; IR (KBr) υ in
1
cm-1: 1239, 1372, 1740, 2920; H NMR(300 MHz, CDCl3): δ 2.06 (s, 3H, CH3), 2.18 (t, J =
7.3, 14.6 Hz, 4H, 2xCH2), 3.01 (t, J = 5.1, 10.6 Hz, 4H, 2xCH2), 5.73 (s, 1H, CH), 7.16-7.91
13
(m, 5H, Ar-H), 8.30 (bs, 1H, Ar-H); C NMR (125 MHz, CDCl3) : δ 28.1, 44.5, 45.6, 58.6,
111.5, 113.8, 116.7, 124.9, 126.3, 127.8, 130.6, 131.5, 135.1, 150.9, 164.9, 168.9; ESI MS :
m/z = 455 [M+H]+.
2-(2,4-Dichlorophenyl)-6-((4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)sulfonyl)-2,3-dihyd
o
roquinazolin-4(1H)-one (5f). Yield 89%; mp 142-145 C; IR (KBr) υ in cm-1: 1134, 1293,
1323, 1667, 3672; 1H NMR(300 MHz, CDCl3): δ 2.50 (t, J = 1.8, 3.6 Hz, 2H, CH2), 2.99 (t, J

ACCEPTED MANUSCRIPT
= 4.7, 9.6 Hz, 2H, CH2), 3.38 (bs, 4H, 2xCH2), 6.31 (s, 1H, CH), 6.90 (d, J = 8.6 Hz, 1H,
Ar-H), 7.04 (d, J = 8.6 Hz, 1H, Ar-H), 7.50 (d, J = 8.5 Hz, 1H, Ar-H), 7.60 (dd, J = 8.6, 10.9
Hz, 1H, Ar-H), 7.63 (s, 1H, Ar-H), 7.65 (s, 1H, Ar-H), 7.70 (d, J = 2.1 Hz, 1H, Ar-H), 7.97 (d,
J = 2.1 Hz, 1H, Ar-H), 8.01 (bs, 1H, Ar-H), 8.56 (bs, 1H, Ar-H); 13C NMR (125 MHz,
CDCl3) : δ 51.6, 52.4, 62.3, 112.2, 114.5, 124.4, 126.3, 128.1, 128.5, 131.3, 133.7, 135.2,
137.2, 141.1, 143.1, 149.1, 152.9, 161.2; ESI MS : m/z = 585 [M+H]+; HRMS calculated for
C25H22Cl2F3N4O3S : 585.0700, Found : 585.0749.
tert-Butyl4-((2-(2,4-dichlorophenyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-6-yl)sulfonyl)piper
o
azine-1-carboxylate (5g).Yield 85%; mp 139-142 C; IR (KBr) υ in cm-1: 1173, 1321, 1662,
3284; 1H NMR(300 MHz, CDCl3): δ 1.41 (s, 9H, 3xCH3), 2.92 (bs, 4H, 2xCH2), 3.48 (bs, 4H,
2xCH2), 6.35 (s, 1H, CH), 6.89 (d, J = 8.5 Hz, 1H, Ar-H), 7.45 (s, 1H, Ar-H), 7.53 (d, J = 7.8
Hz, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.65 (s, 1H, Ar-H), 8.12 (s, 1H, NH), 8.21 (s, 1H, Ar-H);
13C NMR (125 MHz, CDCl3) : δ 26.5, 44.1, 61.8, 78.0, 111.6, 113.3, 120.8, 126.0, 126.7,
127.5, 128.2, 130.9, 131.4, 133.1, 134.9, 149.1, 152.1, 160.8; ESI MS : m/z = 541 [M+H]+.
6-((4-Benzylpiperidin-1-yl)sulfonyl)-2-(2,4-dichlorophenyl)-2,3-dihydroquinazolin-4(1H)-o
o
1
ne (5h).Yield 85%; mp 142-145 C; IR (KBr) υ in cm-1: 1161, 1336, 1655, 3367; H
NMR(300 MHz, CDCl3): δ 1.66 (d, J = 12.4 Hz, 2H, CH2), 2.19 (t, J = 10.5, 11.7 Hz, 2H,
CH2), 2.50 (d, J = 6.8 Hz, 2H, Ph-CH2), 2.58 (bs, 1H, CH), 3.16 (bs, 4H, 2xCH2), 6.33 (s, 1H,
CH), 6.85 (d, J = 8.5 Hz, 1H, Ar-H), 7.08 (d, J = 7.3 Hz, 1H, Ar-H), 7.16 (d, J = 6.4 Hz, 1H,
Ar-H), 7.24 (t, J = 7.5, 14.7 Hz, 2H, Ar-H), 7.35 (dd, J = 8.4, 10.1 Hz, 1H, Ar-H), 7.45 (bs,
1H, Ar-H), 7.49 (bs, 1H, Ar-H), 7.65 (d, J = 8.3 Hz, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 8.09 (s,
13
1H, NH), 8.15 (s, 1H, Ar-H); C NMR (125 MHz, CDCl3) : δ 29.4, 35.2, 40.6, 44.7, 61.9,
111.6, 113.1, 121.8, 124.3, 126.1, 126.5, 127.3, 127.6, 128.3, 130.9, 131.5, 133.1, 135.0,
138.1, 148.8; ESI MS : m/z = 530 [M+H]+.
2-(2,4-Dichlorophenyl)-6-(thiomorpholinosulfonyl)-2,3-dihydroquinazolin-4(1H)-one
o
1
(5i).Yield 82%; mp 148-151 C; IR (KBr) υ in cm-1: 1134, 1317, 1635, 3478; H NMR(300
MHz, CDCl3): δ 2.74 (bs, 4H, 2xCH2), 3.31 (bs, 4H, 2xCH2), 6.37 (d, J = 6.0 Hz, 1H, CH),
6.87 (d, J = 8.6 Hz, 1H, Ar-H), 7.34 (d, J = 7.7 Hz, 1H, Ar-H), 7.46 (d, J = 11.1 Hz, 1H,
Ar-H), 7.54-7.62 (m, 1H, Ar-H), 7.65 (bs, 1H, NH), 8.22 (d, J = 12.8 Hz, 1H, Ar-H); 13C
NMR (125 MHz, CDCl3) : δ 28.0, 46.3, 58.5, 111.5, 113.7, 116.7, 123.1, 124.9, 126.3, 127.8,
130.6, 131.5, 135.1, 140.3, 164.8, 168.9; ESI MS : m/z = 458 [M+H]+.

ACCEPTED MANUSCRIPT
2-(2,4-Dichlorophenyl)-6-((4-phenylpiperazin-1-yl)sulfonyl)-2,3-dihydroquinazolin-4(1H)-
o
1
one (5j). Yield 81%; mp 147-150 C; IR (KBr) υ in cm-1: 1134, 1292, 1540, 1794, 3630; H
NMR(300 MHz, CDCl3): δ 2.37 (bs, 4H, 2xCH2), 2.64 (bs, 4H, 2xCH2), 5.07 (s, 1H, CH),
6.11 (d, J = 15.2 Hz, 1H, Ar-H), 6.63 (d, J = 8.6 Hz, 1H, Ar-H), 6.92 (d, J = 9.7 Hz, 2H,
Ar-H), 7.09 (s, 1H, Ar-H), 7.12-7.18 (m, 2H, Ar-H), 7.31 (t, J = 8.0, 16.0 Hz, 1H, Ar-H),
13
7.36-7.46 (m, 2H, Ar-H), 7.95 (d, J = 8.8 Hz, 1H, Ar-H); C NMR (125 MHz, CDCl3) : δ
55.5, 56.1, 62.3, 112.2, 114.0, 121.1, 124.5, 126.3, 127.3, 128.1, 128.6, 131.4, 131.8, 133.8,
135.2, 137.1, 143.1, 149.6, 153.0, 161.4; ESI MS : m/z = 517 [M+H]+.
2-(2,4-Dichlorophenyl)-N-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-4-oxo-1,2,3,4-tetr
ahydroquinazoline-6-sulfonamide (6a).Yield 79%; mp 141-144 oC; IR (KBr) υ in cm-1: 1140,
1241, 1384, 1665, 3673; 1H NMR(300 MHz, CDCl3): δ 1.63-1.82 (m, 2H, CH2), 2.60 (t, J =
6.0, 11.7 Hz, 2H, CH2), 2.75 (bs, 4H, 2xCH2), 3.03 (t, J = 6.0, 11.1 Hz, 2H, CH2), 3.12 (bs,
4H, 2xCH2), 3.84 (s, 3H, OCH3), 5.54 (bs, 1H, NH), 6.33 (s, 1H, CH), 6.67-7.06 (m, 6H,
Ar-H), 7.24 (d, J = 1.8 Hz, 1H, Ar-H), 7.39 (d, J = 1.8 Hz, 1H, Ar-H), 7.55 (d, J = 8.5 Hz, 1H,
Ar-H), 7.73 (dd, J = 6.6, 8.5 Hz, 1H, SO2NH), 8.00 (bs, 1H, NH), 8.33 (d, J = 1.8 Hz, 1H,
Ar-H); 13C NMR (125 MHz, CDCl3) : δ 29.0, 49.9, 52.7, 54.8, 56.6, 63.3, 110.8, 113.1, 114.2,
117.7, 120.4, 122.3, 127.1, 127.4, 128.5, 128.9, 131.7, 132.4, 134.8, 135.7, 140.5, 141.3,
149.5, 151.7, 162.5; ESI MS : m/z = 604 [M+H]+.
2-(2,4-Dichlorophenyl)-N-(3-(4-(2-fluorophenyl)piperazin-1-yl)propyl)-4-oxo-1,2,3,4-tetrah
o
ydroquinazoline-6-sulfonamide (6b).Yield 78%; mp 151-154 C; IR (KBr) υ in cm-1: 1154,
1
1238, 1384, 1651, 3451; H NMR(300 MHz, CDCl3): δ 1.71 (bs, 2H, CH2), 2.54 (bs, 2H,
CH2), 2.65 (bs, 4H, 2xCH2), 3.03 (bs, 2H, CH2), 3.14 (bs, 4H, 2xCH2), 6.35 (s, 1H, CH), 6.74
(d, J = 8.5 Hz, 1H, Ar-H), 6.88 (bs, 3H, Ar-H), 6.91-6.99 (m, 3H, Ar-H), 7.40 (d, J = 16.0 Hz,
1H, Ar-H), 7.56 (t, J = 8.3, 16.7 Hz, 1H, Ar-H), 7.72 (br, 1H, SO2NH), 7.99 (br, 1H, NH),
8.31 (d, J = 10.0 Hz, 1H, Ar-H); 13C NMR (125 MHz, CDCl3) : δ 50.0, 53.2, 54.8, 56.1, 63.3,
110.9, 113.1, 114.2, 117.7, 120.5, 122.3, 127.1, 127.4, 128.5, 129.0, 131.7, 132.5, 134.9,
135.2, 140.5, 149.5, 151.7, 162.4; ESI MS : m/z = 592 [M+H]+.
2-(2,4-Dichlorophenyl)-4-oxo-N-(3-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)propyl)-1,
o
2,3,4-tetrahydroquinazoline-6-sulfonamide (6c).Yield 79%; mp 145-148 C; IR (KBr) υ in
1
cm-1: 1134, 1323, 1667, 2965, 3672; H NMR(300 MHz, CDCl3): δ 1.62-1.75 (m, 2H, CH2),

ACCEPTED MANUSCRIPT
2.49 (bs, 2H, CH2), 2.59 (bs, 4H, 2xCH2), 3.03 (bs, 2H, CH2), 3.28 (bs, 4H, 2xCH2), 5.50 (br,
1H, NH), 6.33 (s, 1H, CH), 6.73 (d, J = 8.5 Hz, 1H, Ar-H), 6.89-6.98 (m, 3H, Ar-H), 7.39 (bs,
1H, Ar-H), 7.46 (d, J = 8.5 Hz, 2H, Ar-H), 7.55 (d, J = 8.5 Hz, 2H, Ar-H), 7.72 (dd, J = 7.1,
8.4 Hz, 1H, SO2NH), 7.98 (br, 1H, NH), 8.31 (br, 1H, Ar-H); 13C NMR (125 MHz, CDCl3) :
δ 43.5, 47.8, 52.7, 57.4, 63.9, 114.0, 114.6, 114.7, 126.3, 126.3, 126.4, 128.0, 128.7, 128.8,
129.9, 130.3, 130.8, 132.8, 132.9, 135.0, 136.1, 148.9, 153.0, 163.1; ESI MS : m/z = 642
[M+H]+.
2-(2,4-Dichlorophenyl)-4-oxo-N-(3-(4-phenylpiperazin-1-yl)propyl)-1,2,3,4-tetrahydroquin
o
azoline-6-sulfonamide (6d).Yield 82%; mp 152-155 C; IR (KBr) υ in cm-1: 1157, 1319,
1
1667, 2926, 3640; H NMR(300 MHz, CDCl3): δ 1.55-1.75 (m, 2H, CH2), 2.45 (t, J = 6.2,
12.6 Hz, 2H, CH2), 2.58 (bs, 4H, 2xCH2), 2.94 (t, J = 6.4, 15.8 Hz, 2H, CH2), 3.18 (bs, 4H,
2xCH2), 6.33 (s, 1H, CH), 6.79-6.95 (m, 3H, Ar-H), 7.19-7.36 (m, 3H, Ar-H), 7.44 (d, J = 1.8
Hz, 1H, Ar-H), 7.60-7.69 (m, 3H, Ar-H), 7.86 (bs, 1H, SO2NH), 8.00 (bs, 1H, NH), 8.28 (d, J
13
= 2.0 Hz, 1H, Ar-H); C NMR (125 MHz, CDCl3) : δ 43.5, 48.9, 52.9, 57.3, 63.8, 113.8,
114.7, 116.1, 119.9, 128.0, 128.1, 128.7, 128.8, 129.0, 129.9, 130.0, 130.8, 132.8, 135.1,
136.0, 149.0, 150.8, 163.2; ESI MS : m/z = 574 [M+H]+.
2-(2,4-Dichlorophenyl)-4-oxo-N-(3-thiomorpholinopropyl)-1,2,3,4-tetrahydroquinazoline-6
o
-sulfonamide (6e).Yield 81%; mp 151-154 C; IR (KBr) υ in cm-1: 1005, 1318, 1667, 3526;
1H NMR(300 MHz, CDCl3): δ 1.58-1.70 (m, 2H, CH2), 2.44 (t, J = 6.0, 12.0 Hz, 2H, CH2),
2.69 (s, 8H, 4xCH2), 3.00 (t, J = 5.8, 11.7 Hz, 2H, CH2), 5.54 (s, 1H, NH), 6.36 (s, 1H, CH),
6.75 (d, J = 8.6 Hz, 1H, Ar-H), 6.88 (s, 1H, Ar-H), 7.29 (dd, J = 6.4, 8.5 Hz, 1H, Ar-H), 7.43
(d, J = 2.0 Hz, 1H, Ar-H), 7.59 (d, J = 8.3 Hz, 1H, SO2NH), 7.72 (dd, J = 6.4, 8.5 Hz, 1H,
13
Ar-H), 7.99 (bs, 1H, NH), 8.29 (d, J = 1.8 Hz, 1H, Ar-H); C NMR (125 MHz, CDCl3) : δ
26.6, 27.1, 45.9, 54.3, 56.7, 63.3, 113.1, 114.2, 121.2, 127.4, 128.5, 129.0, 131.9, 132.4,
134.9, 135.7, 137.3, 149.5, 162.5; ESI MS : m/z = 515 [M+H]+; HRMS calculated for
C21H25Cl2N4O3S2 : 515.0700, Found : 515.073.
N-(3-(4-Benzylpiperidin-1-yl)propyl)-2-(2,4-dichlorophenyl)-4-oxo-1,2,3,4-tetrahydroquina
zoline-6-sulfonamide (6f).Yield 78%; mp 149-153 oC; IR (KBr) υ in cm-1: 1156, 1321, 1666,
3239; 1H NMR(300 MHz, CDCl3): δ 1.31-1.48 (m, 2H, CH2), 1.49-1.79 (m, 5H, 2xCH2, CH),
2.10 (t, J = 10.3, 11.7 Hz, 2H, CH2), 2.52 (d, J = 6.6 Hz, 2H, PhCH2), 2.61 (bs, 4H, 2xCH2),
3.08 (d, J = 10.3 Hz, 2H, CH2), 5.74 (bs, 1H, NH), 6.33 (s, 1H, CH), 6.76 (d, J = 8.5 Hz, 1H,

ACCEPTED MANUSCRIPT
Ar-H), 6.90 (bs, 1H, Ar-H), 7.12 (d, J = 7.3 Hz, 2H, Ar-H), 7.19 (d, J = 6.6 Hz, 1H, Ar-H),
7.22-7.31 (m, 3H, Ar-H), 7.38 (s, 1H, Ar-H), 7.55 (d, J = 8.5 Hz, 1H, Ar-H), 7.70 (d, J = 8.5
13
Hz, 1H, SO2NH), 8.14 (d, J = 15.9 Hz, 1H, NH), 8.30 (s, 1H, Ar-H); C NMR (125 MHz,
CDCl3) : δ 24.0, 29.7, 30.8, 37.0, 42.4, 53.4, 56.7, 63.9, 113.6, 115.0, 125.9, 127.9, 128.1,
128.2, 129.0, 129.6, 129.8, 132.7, 132.7, 135.3, 135.8, 139.9, 149.2, 163.1; ESI MS : m/z =
587 [M+H]+.
2-(2,4-Dichlorophenyl)-N-(3-(4-(4-nitrophenyl)piperazin-1-yl)propyl)-4-oxo-1,2,3,4-tetrahy
o
droquinazoline-6-sulfonamide (6g).Yield 79%; mp 148-151 C; IR (KBr) υ in cm-1: 1050,
1378, 1470, 1681, 3181; 1H NMR(300 MHz, CDCl3): δ 0.77-0.95 (m, 2H, CH2), 2.07 (s, 2H,
CH2), 2.59 (bs, 2H, CH2), 3.17 (bs, 8H, 4xCH2), 5.90 (bs, 1H, NH), 6.24 (d, J = 10.9 Hz, 1H,
Ar-H), 6.50 (s, 1H, CH), 6.76 (d, J = 8.6 Hz, 1H, Ar-H), 7.23-7.32 (m, 1H, Ar-H), 7.34-7.45
(m, 2H, Ar-H), 7.49-7.81 (m, 3H, Ar-H), 7.92 (d, J = 8.4 Hz, 1H, SO2NH), 8.08 (d, J = 8.6
Hz, 1H, Ar-H), 8.17-8.33 (m, 1H, Ar-H), 8.50 (s, 1H, NH); 13C NMR (125 MHz, CDCl3) : δ
43.6, 47.7, 52.8, 57.7, 64.0, 114.0, 114.7, 126.3, 126.4, 126.4, 128.1, 128.7, 128.8, 140.1,
142.7, 143.1, 149.2, 154.1, 163.1; ESI MS : m/z = 616 [M-2H]+.
Materials and Methods:
Cell Cultures, Maintenance and Anti proliferative Evaluation
The cell lines, HeLa (cervical), MDA-MB-231 (breast), PANC-1 (pancreatic), and
A549 (lung carcinoma) which were used in this study were procured from American Type
Culture Collection (ATCC), United States. The synthesized test compounds were evaluated
for their in vitro anti proliferative activity in these four different human cancer cell lines. A
protocol of 48 h continuous drug exposure was used and an SRB cell proliferation assay was
used to estimate cell viability or growth. All the cell lines were grown in Dulbecco's modified
Eagle's medium (containing 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C). Cells
were trypsinized when sub-confluent from T25 flasks/60 mm dishes and seeded in 96-well
plates in 100 µL aliquots at plating densities depending on the doubling time of individual
cell lines. The micro titre plates were incubated at 37 °C, 5% CO2, 95% air, and 100%
relative humidity for 24 h prior to addition of experimental drugs and were incubated for 48
hrs with different doses (0.01, 0.1, 1, 10, 100µM) of prepared derivatives. After 48 hours
incubation at 37 °C, cell mono layers were fixed by the addition of 10% (wt/ vol) cold
trichloroacetic acid and incubated at 4 °C for 1 h and were then stained with 0.057% SRB
dissolved in 1% acetic acid for 30 min at room temperature. Unbound SRB was washed with
1% acetic acid. The protein –bound dye was dissolved in 10 mM Tris base solution for OD

ACCEPTED MANUSCRIPT
determination at 510 nm using a micro plate reader (Enspire, Perkin Elmer, and USA). Using
the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in
the presence of drug at the five concentration levels (Ti)], the percentage growth was
calculated at each of the drug concentrations levels. Percentage growth inhibition was
calculated as:
[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz
[(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz.
The dose response parameter, growth inhibition of 50 % (GI50) was calculated
from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug concentration resulting in a 50%
reduction in the net protein increase (as measured by SRB staining) in control cells during the
drug incubation. Values were calculated for this parameter if the level of activity is reached;
however, if the effect is not reached or is exceeded, the value for that parameter was
expressed as greater or less than the maximum or minimum concentration tested.
Methadology for modelling:
Dataset preparation
A dataset comprising of 17 quinazolin-4-one series of compounds and these
compounds were drawn with all possible tautomers, protonated states and were minimized
with OPLS-2005 force field using water as solvent in the GB/SA continuum solvation model
[40]. The minimization of molecules was carried out using Polak-Ribiere Conjugate Gradient
(PRCG) method with maximum of 5000 iterations. Further for the generation of
pharmacophore model, the minimized structures were subjected to extensive conformational
search with Mixed torsional/Low-mode sampling method with the use of 100 steps per
rotatable bond, maximum number of steps 1000, energy window for saving structures with
5.02 kcal/mol, eliminate the redundant conformers with maximum atom deviation cut-off 0.5
Å and saved 250 structures for each search. The generated conformers were used for the
generation of pharmacophore modelling and docking studies.
Pharmacophore modeling:
The pharmacophore model was generated using Phase module of Schrödinger
software v9.3. The set of pharmacophore features, such as hydrogen bond acceptor (A),
hydrogen bond donor (D), hydrophobic group (H), negatively ionizable (N), positively
ionizable (P), and aromatic ring (R), were created for a series of compounds containing A549
cell-line activity.

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Protein preparation:
These series of compounds have showed strong affinity, intrinsic activity
towards various cancer cell-lines and showed inhibition against Histone deacetylase 8
(HDAC 8) and Histone-lysine N-methyltransferase, H3 lysine-9 specific 3 (EHMT2). Hence
in our current study, we have carried out molecular docking study have been performed to
understand the key active site residues HDAC8, EHMT2 proteins and explored all probable
binding modes. The crystal structure of human HDAC 8 (PDB ID: 3SFH) and human
EHMT2 (PDB ID: 3K5K) have been selected for the docking studies. The 3SFH and 3K5K
structures were prepared by adjusting bond orders, tautomers and adding hydrogen atoms
using protein preparation wizard of Schrödinger software graphical user interface Maestro
v9.3 [Maestro, version 9.3, Glide version 2.8, Schrödinger, LLC, New York, NY 2013.].
Further the protein was minimized by OPLS-2005 force field with converge heavy atoms to
RMSD 0.3 Ǻ relative to original protein structure.
Docking studies:
The dataset compounds were docked into the prepared 3SFH and 3K5K crystal
structures using Glide module of Schrödinger suite [41]. The prepared protein structures were
used for grid generation using the default value of protein atom scaling (1.0) within a cubic
box centered on the co-crystal ligand. No constraints were imposed and extra precision (XP)
flexible docking of ligands was carried out with default value of ligand atom scaling (0.8).
The post docking minimization has been carried out and maximum of 10 poses per ligand
was saved. The obtained docked complex structures were analyzed and the compounds were
prioritized by using docking score, interactions with active site residues.
Acknowledgments
The authors gratefully acknowledge the financial support through the project:
DST-SERB/EEQ/2017/095 RV thanks CSIR, New Delhi for award of research fellowship.
Supplementary data
Experimental section and Copies of the 1H, 13C NMR, ESI-MS and HRMS spectra for some of
the important compounds.

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Research highlights
ꢀ Novel sulphamide tethered quinazolinones hybrids were efficiently synthesized.
ꢀ Compounds 5a-j, 6a-g screened in viability assays against human cancer cell lines.
ꢀ 5c Showed potent cytotoxicity against four cancers cell lines with GI50 0.09 to 0.21
µM.
ꢀ 5i, 6f Showed cytotoxic against cancer cell lines with GI50 0.045 to 0.35 µM.
ꢀ 5c Showed Binding mode and identified key active site residues in HDAC8 and
EHMT2 proteins.