Synthesis and evaluation of antiproliferative activity of novel quinazolin-4(3H)-one derivatives

Article abstract of DOI:10.1007/s00044-016-1632-9

Two series of novel quinazolin-4(3H)-one derivatives (10a–g and 11a–g) have been synthesized and evaluated for their in vitro antiproliferative activity against human HeLa, MIAPACA, MDA-MB-231, and IMR-31 cancer cell lines. The synthesized compounds were characterized by spectral (Fourier transform infrared, ¹H nuclear magnetic resonance, ¹³C nuclear magnetic resonance, high-resolution mass spectra) methods. Among them, compounds 11e and 11g exhibited potent in vitro antiproliferative activity with GI₅₀ values 0.02, less than 0.01 μM against MIAPACA human cancer cell line. Significantly, compounds 10a, 10b, 10c, 10g, 11b, 11c, 11d, 11e, 11f showed activity with GI₅₀ values ranging from 0.1 to 0.87 μM against human cancer cell lines MIAPACA, MDA-MB-231, and IMR-31. We have explored the probable 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:10.1007/s00044-016-1632-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1632-9
ORIGINAL RESEARCH
Synthesis and evaluation of antiproliferative activity of novel quinazolin-
4(3H)-one derivatives
Ramineni Venkatesh1 Suresh Kasaboina1 Sridhara Janardhan2 Nishant Jain3
●
●
●
●
Rajashaker Bantu1 Lingaiah Nagarapu1
●
Received: 19 March 2015 / Accepted: 27 June 2016
© Springer Science+Business Media New York 2016
Abstract Two series of novel quinazolin-4(3H)-one deri-
vatives (10a–g and 11a–g) have been synthesized and
evaluated for their in vitro antiproliferative activity against
human HeLa, MIAPACA, MDA-MB-231, and IMR-31
cancer cell lines. The synthesized compounds were char-
acterized by spectral (Fourier transform infrared, 1H nuclear
magnetic resonance, 13C nuclear magnetic resonance,
high-resolution mass spectra) methods. Among them,
compounds 11e and 11g exhibited potent in vitro anti-
proliferative activity with GI50 values 0.02, less than
0.01 μM against MIAPACA human cancer cell line. Sig-
nificantly, compounds 10a, 10b, 10c, 10g, 11b, 11c, 11d,
11e, 11f showed activity with GI50 values ranging from 0.1
to 0.87 μM against human cancer cell lines MIAPACA,
MDA-MB-231, and IMR-31. We have explored the prob-
able binding mode and key active site interactions in
HDAC8 and EHMT2 proteins. The docking results are
complementary to the experimental results.
Introduction
The concept of “privileged structure” was first introduced
by Evans in the late 80s (Evans et al., 1988; Horton et al.,
2003). The potent anticancer activities of camptothecin and
luotonin family natural alkaloids have drawn extreme
interest worldwide. Their parent representative 2-substituted
quinazolin-4(3H)-one analog acts as an excellent lead drug
in anticancer research (Wall et al., 1966). luotonin A is one
more camptothecin-family alkaloid isolated from Chinese
medicinal plant (Peganum nigellastrum) in 1997. 2-
substituted quinazolin-4(3H)-one is a principal constitute
of natural alkaloid luotonin A and their derivatives have
shown a wide range of biological activities like anticancer
(Xia et al., 2001), anti-inflammatory (Alagarsamy et al.,
2007), antibacterial (Selvam et al., 2005), antihypertensive
(Alagarsamy et al., 2007), vasopressin V3 receptor
antagonist (Letourneau et al., 2006), antitubercular agent
(Kuneš et al., 2000), and nonpeptide cholecystokinin B
receptor antagonistic activity (Padia et al., 1998). As a part
of our ongoing research program (Nagarapu et al., 2011;
Nagarapu et al., 2013a; Nagarapu et al., 2013b; Venkatesh
et al., 2015a; Venkatesh et al., 2015b; Yadagiri et al., 2014;
Nagarapu et al., 2015) to discover and develop tumor
growth inhibitors and apoptosis inducers as potential new
anticancer agents, we have designed and synthesized novel
quinazolin-4(3H)-one derivatives (Fig. 1) and evaluated
their in vitro antiproliferative activity against human HeLa,
MIAPACA, MDA-MB-231, and IMR-31 cancer cell lines.
The compounds 11g and 11e showed potent anti-
proliferative activity against the MIAPACA human cancer
cell lines with GI50 values less than 0.01, 0.02, respectively.
The computational techniques play a vital role in lead
molecule identification and its optimization. Both structure-
based (homology modeling, docking) and ligand-based
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Keywords Piperazine Quinazolin-4(3H)-one
Antiproliferative activity
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1632-9) contains supplementary material,
which is available to authorized users.
* Lingaiah Nagarapu
lnagarapuiict@yahoo.com
1
Organic Chemistry Division-II (CPC), CSIR-Indian Institute of
Chemical Technology, Tarnaka, Hyderabad 500007, India
2
Centre for Molecular Modeling, CSIR-Indian Institute of
Chemical Technology, Tarnaka, Hyderabad 500607, India
3
Centre for Chemical Biology, CSIR-Indian Institute of Chemical
Technology, Tarnaka, Hyderabad 500007, India

Med Chem Res
Fig. 1 Structures of luotonin A, camptothecin, chloroqualone, alloqualone, and quinazolin-4(3H)-one derivatives
(pharmacophore, Quantitative Structure Activity Relation-
ship) 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 quinazolin-4(3H)-one compounds
in both EHMT2 and HDAC8 proteins. The crystal struc-
tures 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 phar-
macophore model will be helpful in identifying drug-like
features responsible for better ligand binding. The phar-
macophore models provide the hypothetical active site and
can be used for virtual screening (Janardhan et al., 2006a,b)
to obtain diverse chemical classes of new compounds. The
identified structural features from ligand-based and
structure-based approaches could help in rational design of
quinazolin-4(3H)-one series of compounds with enhanced
potency in EHMT2 and HDAC8 proteins.
points were determined in capillaries and are uncorrected.
1H NMR spectra was recorded on Bruker DRX-300 and
Varian-500 NMR spectrometers. 13C 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 are reported in wave numbers (cm−1). Mass spectra
were recorded on Agilent Technologies 1100 Series (Agi-
lent Chemistation Software). High-resolution mass spectra
(HRMS) were obtained by using ESI-QTOF mass
spectrometry.
Preparation of 4-oxo-3,4-dihydroquinazoline-2-carboxylic
acid (4)
To the solid compound, 2-aminobenzamide (5 g, 0.037
mol), an excess of diethyl oxalate (15 mL, 0.11 mol) was
added carefully and the mixture was stirred at reflux tem-
perature for 8 h. After completion of the reaction [thin layer
chromatography (TLC) analysis], the solution was allowed
to cool to room temperature and the excess diethyl oxalate
was removed under vacuum. The remaining solid was
suspended in EtOH and filtered to produce compound 3 as
white solid.
Materials and methods
Chemistry
General
The intermediate 3 converted into 4 by treating with 1N
KOH: EtOH (30 mL, 1:1) under reflux condition for 1 h.
After completion of the reaction (TLC analysis), the solu-
tion was allowed to cool to room temperature and ethanol
was removed under vacuum, the obtained solid was filtered
All the chemicals and reagents are purchased from Sigma-
Aldrich and SD Fine-Chemicals, Pvt. Ltd., India, and are
used as received. Flash column chromatography was per-
formed with Merck silica gel (100–200 mesh). Melting

Med Chem Res
to give 4-oxo-3,4-dihydroquinazoline-2-carboxylic acid
(3.48 g, 69 %) as white solid.
concentrated under vacuum to yield 9a–g (Nagarapu et al.,
2015), as a semi solid.
Ethyl 4-oxo-3,4-dihydroquinazoline-2-carboxylate (3)
Yield 92 %; mp 190–193 °C; 1H NMR(300 MHz, CDCl3): δ
1.40 (t, J = 7.1 Hz, 3H, CH3, H-3′), 4.29–4.43 (q, J = 7.1 Hz,
2H, CH2, H-2′), 7.19 (t, J = 7.5 Hz, 1H, Ar–H), 7.45–7.60
(m, 1H, Ar–H), 7.89 (d, J = 7.7 Hz, 1H, Ar–H), 8.24 (br,
1H, NH), 8.60 (d, J = 8.1 Hz, 1H, Ar–H); 13C NMR (125
MHz, CDCl3): δ 12.3 (C-3′), 61.2 (C-2′), 118.6 (C-10),
122.1 (C-5, C-8), 127.1 (C-6), 130.7 (C-7), 136.6 (C-9),
152.7 (C-2), 158.4 (C-1′), 169.0 (C-4).
General procedure for the synthesis of compounds
10a–g, 11a–g
To the stirred solution of 4-oxo-3, 4-dihydroquinazoline-2-
carboxylic acid (0.1 g, 0.0005 mol) in N,N-dimethyl for-
mamide DMF, N-phenyl piperzine derivatives (0.0005 mol),
or 3-(4-phenylpiperazin-1-yl)propan-1-amine derivatives
(9a–g, 0.0005 mol) were added at room temperature, fol-
lowed by the addition of EDC HCl (0.0006 mol) and HOBt
(0.0006 mol) and the mixture was stirred for 1 h. After
completion of the reaction (TLC analysis), small amount of
ice cold water (10 mL) was added to the reaction mixture
and then extracted with chloroform (2 × 10 mL). The
chloroform layer was separated, dried over Na2SO4, and
evaporated under vacuum to give corresponding hybrids
(10a–g, 11a–g) in good yields.
4-oxo-3,4-dihydroquinazoline-2-carboxylic acid (4) Yield
1
83 %; H NMR(300 MHz, CDCl3): δ 6.84–6.98 (m, 1H,
Ar–H), 7.43–7.58 (m, 1H, Ar–H), 7.78 (t, J = 8.1, 11.2 Hz,
1H, Ar–H), 8.10 (bs, 1H, NH), 8.29 (dd, J = 7.3, 14.9 Hz,
1H, Ar–H); 13C NMR (75 MHz, CDCl3): δ 118.2 (C-10),
118.3 (C-5), 121.8 (C-8), 126.9 (C-6), 130.4 (C-7), 136.4
(C-9), 152.3 (C-2), 158.1 (C-4), 168.6 (C-1′).
2-(4-(4-nitrophenyl)piperazine-1-carbonyl)quinazolin-4
(3H)-one (10a) Yield 78 %; mp 121–124 °C; IR (KBr) υ
in cm–1: 931, 1194, 1386, 1553, 2039; 1H NMR (300 MHz,
CDCl3): δ 1.68 (bs, 8H, 4CH2, H-2′, H-3′), 6.19 (t, J = 9.6 Hz,
2H, Ar–H), 6.76 (t, J = 7.5 Hz, 1H, Ar–H), 6.87 (d, J = 7.9
Hz, 1H, Ar–H), 7.04 (t, J = 7.3 Hz, 1H, Ar–H), 7.20–7.28
(m, 2H, Ar–H), 7.33 (d, J = 7.9 Hz, 1H, Ar–H), 9.09 (bs,
1H, NH); 13C NMR (75 MHz, CDCl3): δ 46.1 (C-2′), 58.7
(C-3′), 116.9 (C-5′), 124.0 (C-10), 126.8 (C-6′, C-6), 127.2
(C-6), 128.8 (C-7′), 137.5 (C-5), 149.0 (C-8), 149.3 (C-7),
150.1 (C-9), 150.4 (C-4′), 159.7 (C-2), 163.3 (C-4), 165.1
(C-1′); HRMS calculated for C19H17N5NaO4: 402.1173,
found: 402.1137.
Synthesis of 2-(3-bromopropyl) isoindoline-1,3-dione (7)
To a stirred solution of isoindoline-1,3-dione (10 g, 1 mmol)
in N,N-dimethyl formamide (DMF) (40 mL), 1,3-dibromo
propane (14 g, 1 mmol), K2CO3 (9.3 g, 3 mmol) were added
at room temperature. The reaction mixture was stirred at
room temperature 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 con-
centrated under vacuum to afford a white solid.
Synthesis of compounds 8a–g
2-(4-(4-(trifluoromethyl) phenyl) piperazine-1-carbonyl)
quinazolin-4(3H)-one (10b) Yield 75 %; mp 130–133 °C;
IR (KBr) υ in cm−1: 931, 1194, 1386, 1553, 2039; 1H NMR
(500 MHz, CDCl3): δ 3.20–3.37 (m, 4H, 2CH2, H-3′), 3.63
(bs, 2H, CH2, H-2′), 3.78 (bs, 2H, CH2, H-2′), 6.93 (d, J =
8.7 Hz, 1H, Ar–H), 7.37 (dd, J = 9.1 Hz, 10.8 Hz, 1H,
Ar–H), 7.50 (d, J = 8.7 Hz, 1H, Ar–H), 7.58–7.63 (m, 1H,
Ar–H), 7.69 (d, J = 8.4 Hz, 1H, Ar–H), 7.74–7.86 (m, 1H,
Ar–H), 8.03 (br, 1H, NH), 8.33 (dd, J = 9.1 Hz, 21.2 Hz,
1H, Ar–H), 8.50 (s, 1H, Ar–H); 13C NMR (125 MHz,
CDCl3): δ 47.7 (C-2′), 53.0 (C-3′), 114.5 (C-5′), 122.8 (C-
10), 126.4 (CF3), 126.9 (C-6′, C-6), 127.7 (C-7′), 128.3 (C-
5, C-8), 134.5 (C-7), 143.8 (C-9), 147.2(C-4′), 153.2 (C-2),
159.0 (C-4), 160.7 (C-1′); HRMS calculated for
C20H17O2N4NaF3: 425.1196, found: 425.1207.
To a stirred solution of 2-(3-bromopropyl) isoindoline-1,3-
dione (1.0 g, 1 mmol) in N,N-dimethyl formamide DMF
(5 mL), N-phenyl piperazine derivatives (1 mmol) and
K2CO3 (1.1 g, 3 mmol) were added at room temperature.
The reaction mixture was stirred at room temperature for 24
h. 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 corresponding compounds 8a–g.
Synthesis of compounds 9a–g
To a stirred solution of compounds 8a–g in methanol (20
mL), hydrazine hydrate (4 mL) was added at room tem-
perature. The reaction was continued for 12 h. The pre-
cipitate obtained was filtered, and the filtrate was evaporated
under vacuum. Chloroform (30 mL) was added to the resi-
due obtained and washed with water (2 × 20 mL). The
organic layer was separated, dried over Na2SO4, and
Tert-butyl
4-(4-oxo-3,4-dihydroquinazoline-2-carbonyl)
piperazine-1-carboxylate (10c) Yield 81 %; mp 134–137
°C; IR (KBr) υ in cm−1: 764, 1160, 1416, 1633, 1691, 3201;
1H NMR (300 MHz, CDCl3): δ 1.50 (s, 9H, 3CH3, H-6′),

Med Chem Res
3.55–3.64 (m, 4H, 2CH2, H-3′), 3.81 (t, J = 4.9 Hz, 2H,
CH2, H-2′), 4.45 (t, J = 4.9 Hz, 2H, CH2, H-2′), 6.74 (s, 1H,
Ar–H), 7.60 (td, J = 6.6 Hz, 8.1 Hz, 1H, Ar–H), 7.73–7.87
(m, 1H, Ar–H), 8.35 (dd, J = 6.9 Hz, 7.9 Hz, 1H, Ar–H),
10.33 (br, 1H, NH); 13C NMR (125 MHz, CDCl3): δ 28.3
(C-6′), 45.8 (C-3′), 47.2 (C-2′), 80.4 (C-5′), 116.0 (C-10),
122.6 (C-5), 126.7 (C-8), 128.4 (C-6), 128.6 (C-7), 134.7
(C-9), 147.0 (C-4′), 154.4 (C-2), 159.7 (C-4′), 161.0 (C-1′);
HRMS calculated for C18H23O4N4: 359.1713, found:
359.1717.
159.6 (C-4) 161.0 (C-1′); HRMS calculated for
C19H18O2N4F: 353.1408, found: 353.1409.
2-(4-(2-methoxyphenyl) piperazine-1-carbonyl) quinazolin-
4(3H)-one (10g) Yield 82 %; mp 137–140 °C; IR (KBr) υ
in cm−1: 759, 1241, 1633, 1694, 3195; 1H NMR (300 MHz,
CDCl3): δ 3.18 (bs, 4H, 2CH2, H-3′), 3.91 (s, 3H, OCH3, H-
10′), 4.03 (t, J = 5.0 Hz, 2H, CH2, H-2′), 4.68 (t, J = 5.0 Hz,
2H, CH2, H-2′), 6.93 (dd, J = 12.0 Hz, 16.0 Hz, 3H, Ar–H),
7.03–7.08 (m, 1H, Ar–H), 7.59 (td, J = 6.7 Hz, 7.9 Hz, 1H,
Ar–H), 7.77–7.84 (m, 2H, Ar–H), 8.36 (dd, J = 6.8 Hz, 7.7
Hz, 1H, Ar–H), 10.24 (br, 1H, NH); 13C NMR (125 MHz,
CDCl3): δ 42.6 (C-2′), 46.8 (C-2′), 49.8 (C-3′), 50.3 (C-3′),
54.9 (C-10′), 110.8 (C-6′), 117.8 (C-10), 120.4 (C-7′),
122.0 (C-8′), 123.0 (C-9′), 125.8 (C-5), 127.4 (C-8), 127.5
(C-6), 134.0 (C-7), 139.9 (C-5′), 146.3 (C-9), 146.9 (C-2),
151.6 (C-4), 159.9 (C-4′), 161.0 (C-1′); HRMS calculated
for C20H21O3N4: 365.1608, found: 365.1611.
2-(4-benzylpiperidine-1-carbonyl)quinazolin-4(3H)-one
(10d) Yield 72 %; mp 152–155 °C; IR (KBr) υ in cm−1:
749, 1470, 1660, 1694, 2923; 1H NMR (500 MHz, CDCl3):
δ 2.50–2.67 (m, 4H, 2CH2, H-3′), 2.86 (d, J = 10.8 Hz, 2H,
CH2, H-5′), 3.06–3.22 (m, 4H, 2CH2, H-2′), 4.58–4.75 (m,
J = 13.2 Hz, 2H, CH2, H-4′), 7.10–7.24 (m, 4H, Ar–H),
7.28–7.33 (m, 1H, Ar–H), 7.57 (t, J = 7.4 Hz, 1H, Ar–H),
7.73–7.82 (m, 2H, Ar–H), 8.34 (d, J = 7.9 Hz, 1H, Ar–H),
10.31 (br, 1H, NH); 13C NMR (125 MHz, CDCl3): δ 31.7
(C-3′), 32.5 (C-4′), 38.0 (C-5′), 47.5 (C-2′), 122.4 (C-7′),
125.5 (C-8′), 125.9 (C-10), 126.5 (C-5), 128.2 (C-8), 128.9
(C-6), 134.5 (C-7), 139.6 (C-7′), 145.7 (C-6′), 147.2 (C-9),
159.8 (C-2), 160.7 (C-4), 161.2 (C-1′); HRMS calculated
for C21H22O2N3: 348.1706, found: 348.1707.
N-(3-(4-(4-nitrophenyl)piperazin-1-yl)propyl)-4-oxo-3,4-
dihydroquinazoline-2-carboxamide (11a) Yield 67 %; mp
141–144 °C; IR (KBr) υ in cm−1: 930, 1089, 1384, 1681,
1
2038, 2925; H NMR (300 MHz, CDCl3): δ 1.92–2.08 (m,
2H, CH2, H-3′), 2.35 (t, J = 7.5 Hz, 2H, CH2, H-4′),
3.29–3.37 (m, 2H, CH2, H-2′), 3.44 (t, J = 10.1 Hz, 4H,
2CH2, H-5′), 3.67 (t, J = 10.1 Hz, 4H, 2CH2, H-6′),
6.87–6.99 (m, 3H, Ar–H), 7.03–7.07 (m, 1H, Ar–H), 7.53
(t, J = 7.6 Hz, 1H, Ar–H), 7.60 (t, J = 7.4 Hz, 1H, Ar–H),
7.73 (d, J = 8.0 Hz, 1H, Ar–H), 8.32 (d, J = 7.7 Hz, 1H,
Ar–H), 9.40 (br, 1H, NH), 10.23 (br, 1H, NH); 13C NMR
(125 MHz, CDCl3): δ 27.1 (C-3′), 29.3 (C-2′), 61.1 (C-4′),
61.2 (C-5′), 61.3 (C-6′), 119.3 (C-8′), 124.5 (C-10), 127.7
(C-9′), 128.1 (C-5), 128.6 (C-8), 130.2 (C-7, C-10′), 131.5
(C-6), 132.6 (C-9), 144.2 (C-7′), 158.5 (C-2), 160.4 (C-4),
163.9 (C-1′); ESI MS: m/z = 437 [M+H]+.
2-(4-(4-fluorophenyl) piperazine-1-carbonyl) quinazolin-4
(3H)-one (10e) Yield 77 %; mp 139–142 °C; IR (KBr) υ in
1
cm−1: 817, 1235, 1511, 1647, 1710, 3215; H NMR (300
MHz, CDCl3): δ 3.22 (bs, 4H, 2CH2, H-3′), 4.00 (t, J = 5.1
Hz, 2H, CH2, H-2′), 4.67 (t, J = 5.1 Hz, 2H, CH2 H-2′),
6.90–6.95 (m, 2H, Ar–H), 6.97–7.03 (m, 2H, Ar–H), 7.60
(td, J = 6.8 Hz, 8.0 Hz, 1H, Ar–H), 7.76–7.85 (m, 2H,
Ar–H), 8.36 (dd, J = 6.8 Hz, 7.9 Hz, 1H, Ar–H), 10.20 (br,
1H, NH); 13C NMR (125 MHz, CDCl3): δ 44.0 (C-2′), 44.5
(C-2′), 50.3 (C-3′), 51.0 (C-3′), 116.3 (C-5′), 119.2 (C-6′),
122.6 (C-10), 123.2 (C-5), 124.5 (C-8), 126.7 (C-6), 128.5
(C-7), 134.7 (C-4′), 145.0 (C-9), 147.1 (C-7′), 156.7 (C-2),
159.6 (C-4), 161.0 (C-1′); HRMS calculated for
C19H18O2N4F: 353.1408, found: 353.1409.
N-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-4-oxo-3,
4-dihydroquinazoline-2-carboxamide (11b) Yield 69 %;
mp 152–155 °C; IR (KBr) υ in cm−1: 930, 1089, 1384,
1681, 2038, 2925; 1H NMR(500 MHz, CDCl3): δ 0.88–0.94
(m, 2H, CH2, H-3′), 1.25 (bs, 4H, 2CH2, H-2′, H-4′), 1.93
(bs, 2H, CH2, H-5′), 2.69 (bs, 2H, CH2, H-5′), 3.27 (bs, 2H,
CH2, H-6′), 3.61 (bs, 2H, CH2, H-6′), 3.88 (s, 3H, OCH3,
H-13′), 6.87–6.99 (m, 3H, Ar–H), 7.03–7.07 (m, 1H,
Ar–H), 7.53 (t, J = 7.6 Hz, 1H, Ar–H), 7.60 (t, J = 7.4 Hz,
1H, Ar–H), 7.73 (d, J = 8.0 Hz, 1H, Ar–H), 8.32 (d, J = 7.7
2-(4-(2-fluorophenyl) piperazine-1-carbonyl) quinazolin-4
(3H)-one (10f) Yield 78 %; mp 132–135 °C; IR (KBr) υ in
1
cm−1: 856, 1217, 1632, 1686, 3173; H NMR (500 MHz,
CDCl3): δ 3.24 (bs, 4H, 2CH2, H-3′), 4.02 (t, J = 5.0 Hz,
2H, CH2, H-2′), 4.67 (t, J = 5.0 Hz, 2H, CH2, H-2′),
6.92–7.03 (m, 2H, Ar–H), 7.04–7.11 (m, 2H, Ar–H),
7.56–7.62 (m, 1H, Ar–H), 7.76–7.85 (m, 2H, Ar–H), 8.35
(dd, J = 1.2 Hz, 7.9 Hz, 1H, Ar–H); 13C NMR (125 MHz,
CDCl3): δ 44.0 (C-2′), 44.5 (C-2′), 50.3 (C-3′), 51.0 (C-3′),
116.1 (C-9′), 116.3 (C-6′), 119.2 (C-10), 122.6 (C-7′),
123.2 (C-8′), 123.2 (C-5), 124.5 (C-8), 126.7 (C-6), 128.4
(C-7), 128.5 (C-4′), 134.7 (C-9), 145.0 (C-5′), 147.1 (C-2),
Hz, 1H, Ar–H), 9.40 (br, 1H, NH), 10.23 (br, 1H, NH); 13
C
NMR (125 MHz, CDCl3): δ 24.4 (C-3′), 29.6 (C-2′), 50.0
(C-5′), 53.6 (C-4′, C-6′), 55.3 (C-13′), 111.2 (C-9′), 118.3
(C-10), 120.8 (C-10′), 122.4 (C-11′), 122.8 (C-12′), 123.1
(C-5, C-8), 126.8 (C-6), 128.3 (C-7), 128.3 (C-8′), 134.7
(C-9), 147.4 (C-2), 152.1 (C-4), 159.1 (C-7′), 160.7 (C-1′);
HRMS calculated for C23H28O3N5: 422.2186, found:
422.2188.

Med Chem Res
4-oxo-N-(3-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)pro-
2H, CH2, H-3′), 1.87–1.91 (m, 2H, CH2, H-4′), 2.33 (t, J =
7.6 Hz, 2H, CH2, H-3′), 2.68 (t, J = 5.7 Hz, 2H, CH2, H-5′),
2.74 (bs, 2H, CH2, H-5′), 3.46 (bs, 2H, CH2, H-6′),
3.61–3.70 (bs, 2H, CH2, H-6′), 6.91 (t, J = 7.1 Hz, 1H,
Ar–H), 6.98 (d, J = 7.9 Hz, 2H, Ar–H), 7.30 (t, J = 7.3 Hz,
2H, Ar–H), 7.36–7.41 (m, 1H, Ar–H), 7.47 (t, J = 5.6 Hz,
2H, Ar–H), 8.29 (dd, J = 6.8 Hz, 7.7 Hz, 1H, Ar–H), 9.56
(br, 1H, NH), 10.32 (br, 1H, NH); 13C NMR (125 MHz,
CDCl3): δ 29.6 (C-3′), 36.5 (C-2′), 38.2 (C-4′), 53.1 (C-5′),
57.2 (C-6′), 110.6 (C-8′), 111.2 (C-10), 116.3 (C-10′),
126.8 (C-5), 128.2 (C-8), 129.2 (C-6), 134.7 (C-9′), 141.8
(C-7), 142.8 (C-9), 149.2 (C-7′), 156.3 (C-2), 161.0 (C-4),
163.1 (C-1′); HRMS calculated for C22H26O2N5: 392.2081,
found: 392.2077.
pyl)-3,4-dihydroquinazoline-2-carboxamide
(11c) Yield
71 %; mp 149–152 °C; IR (KBr) υ in cm−1: 821, 1102,
1
1334, 1669, 1701, 2924; H NMR(500 MHz, CDCl3): δ
0.82–0.93 (m, 2H, CH2, H-3′), 1.26 (bs, 2H, CH2, H-4′),
1.82 (bs, 2H, CH2, H-2′), 2.68 (bs, 2H, CH2, H-5′), 2.74
(bs, 2H, CH2, H-5′), 3.54 (bs, 2H, CH2, H-6′), 3.65 (bs, 2H,
CH2, H-6′), 6.81 (d, J = 8.7 Hz, 1H, Ar–H), 6.97 (d, J = 8.7
Hz, 1H, Ar–H), 7.29–7.38 (m, 2H, Ar–H), 7.47 (td, J = 6.1
Hz, 6.2 Hz, 1H, Ar–H), 7.52 (d, J = 8.7 Hz, 2H, Ar–H),
8.29 (d, J = 8.4 Hz, 1H, Ar–H), 9.53 (br, 1H, NH), 10.23
(br, 1H, NH); 13C NMR (125 MHz, CDCl3): δ 29.6 (C-3′),
40.8 (C-2′), 47.6 (C-4′), 53.0 (C-5′), 58.2 (C-6′), 114.5 (C-
8′), 122.8 (C-10), 126.4 (C-11′), 126.9 (C-9′, C-10′), 127.7
(C-5, C-8), 128.3 (C-6), 134.5 (C-7), 143.8 (C-9), 147.2 (C-7′),
153.2 (C-2), 159.0 (C-4), 160.6 (C-1′); HRMS calculated
for C23H25O2N5F3: 460.1955, found: 460.1958.
N-(3-(4-benzylpiperidin-1-yl)propyl)-4-oxo-3,4-dihydro-
quinazoline-2-carboxamide (11g) Yield 75 %; mp
170–173 °C; IR (KBr) υ in cm−1: 752, 1172, 1397, 1534,
1
N-(3-(4-(2-fluorophenyl)piperazin-1-yl)propyl)-4-oxo-3,4-
dihydroquinazoline-2-carboxamide (11d) Yield 72 %; mp
154–157 °C; IR (KBr) υ in cm−1: 772, 1119, 1464, 1733,
1679, 2931; H NMR (500 MHz, CDCl3): δ 1.68–1.93 (m,
2H, CH2, H-3′), 2.49–2.73 (m, 2H, CH2, H-8′), 3.00–3.22
(m, 12H, 6CH2, H-2′, H-4′, H-5′, H-6′), 3.54–3.62 (m, 1H,
CH2, H-7′), 7.09–7.16 (m, 1H, Ar–H), 7.22 (d, J = 7.1 Hz,
1H, Ar–H), 7.27–7.32 (m, 2H, Ar–H), 7.58 (t, J = 7.9 Hz,
1H, Ar–H), 7.79–7.86 (m, 1H, Ar–H), 8.01 (s, 1H, Ar–H),
8.32 (d, J = 7.6 Hz, 1H, Ar–H), 8.58 (t, J = 5.9 Hz, 1H,
Ar–H), 10.22 (br, 1H, NH), 11.73 (br, 1H, NH); 13C NMR
(125 MHz, CDCl3): δ 31.6 (C-3′), 32.5 (C-6′), 38.3 (C-7′),
41.9 (C-4′), 42.7 (C-8′), 45.8 (C-2′), 53.2 (C-5′), 126.3 (C-
10), 128.4 (C-12′), 128.8 (C-5, C-8), 128.9 (C-6, C-10′, C-
11′), 134.8 (C-7), 138.8 (C-7′), 139.7 (C-9), 147.1 (C-2),
159.5 (C-4), 160.9 (C-1′); HRMS calculated for
C24H29O2N4: 405.2285, found: 405.3005.
1
2924; H NMR (500 MHz, CDCl3): δ 0.81–0.93 (m, 2H,
CH2, H-3′, H-4′), 2.79 (bs, 4H, 2CH2, H-5′), 3.37 (bs, 4H,
2CH2, H-6′), 3.60–3.68 (m, 2H, CH2, H-2′), 6.94–7.02 (m,
2H, Ar–H), 7.03–7.12 (m, 2H, Ar–H), 7.51–7.56 (m, 1H,
Ar–H), 7.59 (t, J = 8.0 Hz, 1H, Ar–H), 7.68 (d, J = 7.9 Hz,
1H, Ar–H), 8.33 (dd, J = 7.7 Hz, 9.3 Hz, 1H, Ar–H), 9.29
(br, 1H, NH), 10.23 (br, 1H, NH); 13C NMR (75 MHz,
CDCl3): δ 29.6 (C-3′), 53.4 (C-2′, C-4′), 55.6 (C-5′), 55.8
(C-6′), 124.3 (C-8′), 125.2 (C-11′), 126.8 (C-10), 126.9 (C-
10′), 131.1 (C-9′), 131.2 (C-5), 132.9 (C-8), 144.6 (C-6),
142.5 (C-7), 147.4 (C-7′), 155.1 (C-9, C-2), 158.8 (C-12′),
160.7 (C-4), 162.1 (C-1′); HRMS calculated for
C22H25O2N5F: 410.1986, found: 410.1988.
Materials and methods: cell cultures, maintenance, and
antiproliferative evaluation
4-oxo-N-(3-thiomorpholinopropyl)-3,4-dihydroquinazoline-
2-carboxamide (11e) Yield 68 %; mp 147–150 °C; IR
1
(KBr) υ in cm−1: 775, 1292, 1469, 1658, 2926; H NMR
The cell lines, He La, MIAPACA, MDA-MB-231, and
IMR-32 (cervical, pancreatic, breast, and neuroblastoma)
used in this study were procured from American Type
Culture Collection, USA. The synthesized test compounds
were evaluated for their in vitro antiproliferative activity in
these four different human cancer cell lines. A protocol of
48 h continuous drug exposure was used and an Sulforho-
damine B (SRB) cell proliferation assay was used to esti-
mate cell viability or growth. All the cell lines were grown
in Dulbecco’s modified Eagle’s medium (containing 10 %
fetal bovine serum in a humidified atmosphere of 5 % CO2
at 37 °C). Cells were trypsinized with sub-confluent from
T25 flasks/60 mm dishes and seeded in 96-well plates in
100 μL aliquots at plating densities depending on the dou-
bling time of individual cell lines. The micro titer plates
were incubated at 37 °C, 5 % CO2, 95 % air, and 100 %
relative humidity for 24 h prior to addition of experimental
(500 MHz, CDCl3): δ 0.98–1.07 (m, 2H, CH2, H-3′),
2.33–2.42 (m, 4H, 2CH2, H-2′, H-4′), 2.96 (bs, 4H, 2CH2,
H-6′), 3.79 (bs, 2H, CH2, H-5′), 4.36 (bs, 2H, CH2, H-5′),
7.53 (t, J = 8.0 Hz, 1H, Ar–H), 7.57–7.60 (m, 1H, Ar–H),
7.65 (t, J = 6.8 Hz, 1H, Ar–H), 7.74 (d, J = 8.0 Hz, 1H,
Ar–H), 8.30 (bs, 1H, NH), 8.41 (bs, 1H, NH); 13C NMR
(125 MHz, CDCl3): δ 28.9 (C-3′), 29.6 (C-6′), 36.9 (C-2′),
46.2 (C-4′), 62.8 (C-5′), 122.8 (C-10), 126.9 (C-5), 127.9
(C-8), 128.5 (C-6), 134.8 (C-7), 146.5 (C-9), 155.3 (C-2),
159.1 (C-4), 160.2 (C-1′); HRMS calculated for
C16H21O2N4S: 333.1379, found: 333.1377.
4-oxo-N-(3-(4-phenylpiperazin-1-yl)propyl)-3,4-dihydro-
quinazoline-2-carboxamide (11f) Yield 78 %; mp
148–151 °C; IR (KBr) υ in cm−1: 921, 1358, 1599, 1672,
1
2040, 2922; H NMR (500 MHz, CDCl3): δ 0.82–0.93 (m,

Med Chem Res
drugs and were incubated for 48 h with different doses
(0.01, 0.1, 1, 10, 100 µM) of prepared derivatives. After 48
h 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 determination at 510 nm using a micro
plate reader (Enspire, Perkin Elmer, 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 cal-
culated at each of the drug concentrations levels. Percentage
growth inhibition was calculated as:
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 modeling and docking studies.
Pharmacophore modeling
The pharmacophore model was generated using Phase
module of Schrödinger software v9.3. The set of pharma-
cophore features, such as hydrogen bond acceptor (A),
hydrogen bond donor (D), hydrophobic group (H), nega-
tively ionizable (N), positively ionizable (P), and aromatic
ring (R), were created for a series of compounds containing
HeLa cell-line activity.
ꢀ
ꢁ
ðTi ꢀ TzÞ
Protein preparation
´ 100 for concentrations for which Ti > = ¼ Tz;
ðC ꢀ ꢀTzÞ
These series of compounds have showed strong affinity,
intrinsic activity toward various cancer cell lines and
showed inhibition against Histone deacetylase 8 (HDAC8)
and Histone-lysine N-methyltransferase, H3 lysine-9 spe-
cific 3 (EHMT2). Hence in our current study, we have
carried out molecular docking study that has been per-
formed to understand the key active site residues HDAC8,
EHMT2 proteins, and explored all probable binding modes.
The crystal structure of human HDAC8 (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.
ꢀ
ꢁ
ðTi ꢀ TzÞ
´ 100 for concentrations for which Ti < Tz:
Tz
The dose response parameter, growth inhibition of 50 %
(GI50) was calculated from [(Ti−Tz)/(C−Tz)] × 100 = 50,
which is the drug concentration resulting in a 50 % reduc-
tion 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.
Data set preparation
A data set comprising of 14 quinazolin-4(3H)-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 (Guimaraes and Car-
dozo, minimization of molecules was carried out using
Polak–Ribiere Conjugate Gradient method with maximum
of 5000 iterations. Further for the generation of pharma-
cophore 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
Docking studies
The data set compounds were docked into the prepared
3SFH and 3K5K crystal structures using Glide module of
Schrödinger suite (Friesner et al., 2004). 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 flexible docking of ligands was
carried out with default value of ligand atom scaling (0.8).
Scheme 1 Reagents and
conditions: (i) diethyl oxalate (3
mol), reflux, 8 h, 92 %; (ii) 1N
KOH : EtOH (1: 1), reflux, 1 h,
85 %

Med Chem Res
Scheme 2 Reagents and
conditions: (i) K2CO3, DMF, rt,
3 h, 85 %; (ii) N-phenyl
piperzinederivatives, K2CO3,
DMF, rt, 24 h, 95 %; (iii)
NH2–NH2 H2O, methanol, rt,
12 h, 72 %
The post-docking minimization has been carried out and
maximum of 10 poses per ligand were saved. The obtained
docked complex structures were analyzed and the com-
pounds were prioritized by using docking score, interactions
with active site residues.
Effects of the compounds on the viability of human
cancer cells
The in vitro antiproliferative activity of the designed com-
pounds (10a–g and 11a–g) was evaluated against four dif-
ferent human cancer cell lines, HeLa (cervical), MIAPACA
(pancreatic), MDA-MB-231 (breast), and IMR-32 (neuro-
blastoma) by the standard SRB assay method (Denizot and
Lang, 1986; Alley et al., 1988; Van de Loosdrecht et al.,
1994). The compounds were picked for an advanced assay
against these four human cancer cell lines at five con-
centrations (0.01, 0.1, 1, 10, 100 µM). GI50 (growth inhi-
bitory activity) was calculated and these values
corresponded to the concentration of the compound causing
50 % decrease in the net cell growth as compared with the
standard drugs, doxorubicin, and paclitaxel. 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.
From Table 1, all the synthesized compounds showed
significant cancer cell growth inhibition with GI50 values
ranging from 0.01 to 92.1 µM. The significant anti-
proliferative activity of compounds are as follows, 10a
against MDA-MB-231 (GI50 = 0.01 µM), 11f against
MIAPACA (GI50 = 0.14 µM), and 10g against IMR-32
(GI50 = 0.19 µM), in particular, the compounds 10a, 10c,
11d (substituents at ring-A are p-NO2 and ester, ring-B are
o-F) showed promising antiproliferative activity against
the human breast cancer cell lines with GI50 values range
of 0.1–0.84 µM, compounds 11b, 11d, 11e, 11f, 11g
(substituents at ring-B are o-OMe, o-F, S, Phenyl, and
Results and discussion
Chemistry
4-oxo-3,4-dihydroquinazoline-2-carboxylic acid (4) was
synthesized by treating anthranilamide (1) with excess of
diethyl oxalate (2) under reflux temperature for 8 h followed
by treatment with 1N KOH: EtOH (1:1) for 1 h to produce
compound (4) in good yields (Scheme 1). The reaction of
pthalimide (5) with 1,3-dibromo propane (6) in the presence
of potassium carbonate furnished 2-(3-bromopropyl)isoindo-
line-1,3-dione (7). Treatment of compound 7 with various
piperazines in the presence of potassium carbonate in
N,N-dimethyl formamide DMF resulted in 8a–g inter-
mediates, which were then treated with hydrazine hydrate
in MeOH to give compounds 9a–g (Scheme 2). The
quinazolinone-piperazine hybrids (10a–g, 11a–g) have been
obtained in excellent yields by the coupling of compound
4 with N-phenyl piperazine derivatives with 3-(4-phenylpi-
perazin-1-yl)propan-1-amine derivatives (9a–g) using
EDC.HCl and HOBt in N,N-dimethyl formamide DMF under
room temperature for 1 h to give corresponding target
compounds (10a–g, 11a–g), respectively, as shown in
Scheme 3.

Med Chem Res
Scheme 3 Reagents and
conditions: (i) A or B, EDC.HCl,
HOBt, DMF, rt, 1 h
benzyl) showed potent activity against MIAPACA cancer
cell line with GI50 values of 0.01–0.83 µM, and the com-
pounds 10b, 10c, 10g (substituents at ring-A are p-CF3,
ester and o-OMe) showed promising antiproliferative
activity against the human IMR-32 cancer cell lines with
GI50 values range of 0.19–0.83 µM. Among the synthe-
sized compounds, only two compounds (11e and 11g)
showed potent antiproliferative activity with GI50 values
of 0.02, less than 0.01 μM against MIAPACA human
cancer cell line and some compounds showed significant
activity within the range of 0.1–0.87 μM against human
cancer cell lines. Potencies of all the synthesized com-
pounds were comparable to the standard drugs doxor-
ubicin and paclitaxel. As per the data from Table 1, we can
examine 11a–g series of the compounds were more potent
as compared with the series 10a–g. The effect of various
substituents at ring-A and ring-B on the quinazolinone
moiety was examined.
The substitution of p-NO2 (10a) on the quinazolinone
moiety showed good inhibitory potency than p-CF3 (10b) in
HeLa, MDAMB-231 cell lines, as electrostatic interactions
are more predominant than hydrophobic substitution. The
substitution of o-flouro (10f), o-OMe (10g) on the quina-
zolinone moiety showed least inhibitory potency in HeLa,
MIAPACA, MDA-MB-231 cell lines.
In comparison of o-F substitutions like 10f with
11d, propyl linker plays an important role and is more
active in HeLa, MIAPACA, MDA-MB-231 cell lines.

Med Chem Res
Table 1 (GI50/µM) values of
the tested compounds against
four human cancer cell lines
S. No
Code
HeLa
MIAPACA
MDA-MB-231
IMR-32
1.
2.
10a
5.1 0.7
>100
0.1 0.03
>100
1.0 0.08
0.2 0.03
0.59 0.01
1.2 0.03
>100
10b
>100
>100
3.
10c
1.0 0.03
1.8 0.08
1.6 0.09
16.8 0.9
92.1 0.55
3.5 0.4
>100
0.42 0.08
>100
4.
10d
>100
5.
10e
>100
22.3 0.6
68.1 0.2
25.1 0.1
2.5 0.09
3.8 0.03
10.1 0.7
0.84 0.05
91.9 0.5
2.1 0.09
7.2 0.4
6.
10f
61.7 0.53
15.7 0.2
> 100
5.0 0.09
0.19 0.04
5.1 0.1
7.
10g
8.
11a
9.
11b
>100
0.83 0.2
11.3 0.25
0.73 0.9
0.02 0.001
0.14 0.03
<0.01
0.1 0.02
0.87 0.03
1.7 0.1
10.
11.
12.
13.
14.
15.
16.
11c
1.3 0.4
11d
1.5 0.1
11e
1.6 0.4
3.1 0.06
1.5 0.09
14.1 0.5
005.029 0.0023
0.073 0.0026
11f
1.5 0.06
1.8 0.05
0.074 0.003
0.032 0.0012
11g
Doxorubicina
Paclitaxela
0.082 0.001
0.059 0.0032
0.065 0.0041
0.089 0.0052
GI50: 50 % growth inhibition, concentration of drug resulting in a 50 % reduction in net protein increase
compared with control cells
Bold indicating for significance of activity
a
Positive control
Comparison of benzyl substitutions like 10d (> 100)
with 11g (< 0.01), the compound shows selectivity against
MIAPACA cell line. In Comparison of o-OMe substitutions
like 10g with 11b, propyl linker plays an important role and
is more active in MIAPACA, IMR-32 cell lines.
The structure–activity relationship study revealed that not
only the electron accepting substituents on the quinazolinone
moiety but also the quinazolinone with a propyl linker is
required for inducing antiproliferative activity against the
MIAPACA as well as remaining human cancer cell line. The
substituent on rings “A” and “B” of quinazolinone moiety
showed mild antiproliferative activity against HeLa cancer
cell line compared to the remaining tested cancer cell lines.
The substituent on ring-B of quinazolinone moiety with
propyl linker of thiopiperazine (11e) and benzylpiperidine
(11g) were associated with a potent increase in the growth
inhibitory effect against MIAPACA.
cell line activity. A total of 5280 conformations thus
obtained were used to generate pharmacophore hypotheses.
The series of compounds having maximum variation in
their growth inhibition (pGI50: 4.00–6.00). The common
pharmacophore hypothesis (CPH) was obtained based on
eight highly active compounds (pIC50 ≥ 5.5) from the data
set and then inactive molecules (pIC50 < 5.5) were aligned
with it. The identified best CPH consists of two acceptor
(A), two hydrophobic (H), and two aromatic (R) features,
i.e., AAHHRR. Alignment of the set of active compounds
from the data set using pharmacophore hypothesis
(AAHHRR) is shown in Fig. 2.
Molecular docking
In order to explore the binding mode and understanding of
key active site residues, molecular docking study has been
performed. The synthesized quinazolin-4(3H)-one series of
compounds were docked into the active site of HDAC8 and
EHMT2 proteins. In the present study, the docking result of
compound (11f) in HDAC8 and EHMT2 proteins are
discussed.
In conclusion, in the series 10a–g the para position
electron withdrawing groups are favorable, and in the series
11a–g propyl linker and substitution of electron donating
groups at ortho position are favorable in evaluation of
antiproliferative activity.
Pharmacophore modeling
HDAC8
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 data set com-
prising of quinazolin-4(3H)-one compounds using HeLa
From the docking study, we observed that the noncovalent
hydrophobic interactions as well as electrostatic interactions
are more predominating for strong binding toward HDAC8
protein. The oxygen atom of carboxylic acid amide linker

Med Chem Res
A
Aromatic
Hydrogen bond Acceptor
Hydrophobic
B
Fig. 2 a CPH (AAHHRR) and its pharmacophoric distances (Å) are shown in pink color. b Alignment of active data set compounds on AAHHRR
between 4-oxo-3,4-dihydro-quinazoline and phenyl piper-
azine formed salt bridge with aspartic acid (Asp178) and
histidine residue (His180) through Zn+2. It also showed
hydrogen bond interaction with aspartic acid (Asp267)
residue. The nitrogen atom of carboxylic acid amide
showed hydrogen bond interaction with carbonyl oxygen of
glycine (Gly151) backbone residue. The oxygen atom of 4-
oxo-3,4-dihydro-quinazoline ring showed electrostatic
interaction with tyrosine (Tyr306, 3.93 Å) and methionine
(Met274, 3.22 Å) residues. 4-oxo-3,4-dihydro-quinazoline
ring showed noncovalent interaction with phenylalanine (T-
shaped π–π stacking, Phe208, Phe152) residues. The phenyl
piperazine moiety showed strong hydrophobic interactions
with isoleucine (Ile108, Ile34), leucine (Leu31), alanine
(Ala32), tyrosine (T-shaped π–π stacking, Tyr151, Tyr306),
phenylalanine (T-shaped π–π stacking, Phe152), tryptophan
(T-shaped π–π stacking, Trp141).
compared to electrostatic interactions for binding of the
molecules. The active site comprises mostly the hydrophobic
residues include tyrosine (Tyr1085, Tyr1067), phenyl ala-
nine (Phe1110, Phe1138, Phe1158, Phe1087), proline
(Pro1121, Pro1011), and isoleucine (Ile1136, Ile1009,
Ile1120). The phenyl ring of the molecule showed hydro-
phobic contacts with isoleucine (Ile1161), and leucine
(Leu1086) residues. The piperazine moiety between phenyl
ring and 4-oxo-3,4-dihydro-quinazoline-2-carboxylic acid
amide ring showed non-covalent hydrophobic interactions
with phenylalanine (Phe1158) and leucine (Leu1086). The
propyl linker between 4-oxo-3, 4-dihydro-quinazoline-2-
carboxylic acid amide and phenyl piperazine showed
hydrophobic interactions with tyrosine (Tyr1154) and Phe-
nylalanine (Phe1152, Phe1087) residues. The oxygen atom
of carboxylic acid amide moiety showed hydrogen bond
interaction with aspartic acid (Asp1088) residue. Also 4-
oxo-3, 4-dihydro-quinazoline moiety showed hydrophobic
close contacts with proline (Pro1121) and its oxygen atom
showed electrostatic interaction with aspartic acid (Asp1019,
3.59 Å) residue. The probable binding mode of most potent
compound (11f) of the 4H-benzo [1,4]thiazin-3-one series of
EHMT2
From the docking results, we observed that the noncovalent
hydrophobic interactions are more predominating as

Med Chem Res
HDAC8
EHMT2
Fig. 3 The probable binding mode compound (11f, orange) in the
active site of HDAC8 and EHMT2 and the hydrogen bond interactions
are shown in pink colored dotted lines, metal Zn+2 in HDAC8 is
shown in yellow color, the important residues are shown in green
color, and protein cartoon is represented in wheat color. The 2D
protein-ligand interaction diagram is shown in the picture
Table 2 The active site residues around 4 Å of the most potent compound (11f)
Target Compound 11f (active site residues around 4 Å)
HDAC8 LEU31, ALA32, LYS33, ILE34, ARG37, ILE108, TYR111, TRP141, HIS142, HIS143, GLY151, PHE152, CYS153, TYR154,
HIS180, PHE208, ASP267, MET274, GLY303, GLY304, GLY305, TYR306, LEU31, LYS33, ILE34, ARG37, TRP141, HIS142,
HIS143, GLY151, PHE152, CYS153, HIS180, PHE208, ASP267, MET274, GLY303, GLY304, GLY305, TYR306
EHMT2 ASP1083, SER1084, LEU1086, PHE1087, ASP1088, ASP1090, PRO1121, PHE1152, ASP1153, TYR1154, ARG1157, PHE1158,
ILE1161
compounds in EHMT2 and HDAC8 is shown in Fig. 3. The
active site residues around 4 Å of the most potent compound
(11f) of the series are shown in Table 2.
MB-231, and IMR-32 human cancer cell lines. All the
synthesized compounds showed significant growth inhibi-
tory effects. We observed that the compounds 11e and 11g
exhibited potent in vitro antiproliferative activity with GI50
values 0.02, less than 0.01 μM against MIAPACA human
cancer cell lines. Moreover, the compounds (10a, 10b, 10c,
10g, 11b, 11c, 11d, 11e, 11f) showed a significant in vitro
antiproliferative activity against MIAPACA, MDA-MB-
231, and IMR-32 human cancer cell lines with GI50 values
ranging from 0.1 to 0.87 μM. Introduction of carbon chain
Conclusion
In summary, we have synthesized two series of novel qui-
nazolin-4(3H)-one derivatives and evaluated their in vitro
antiproliferative activity against HeLa, MIAPACA, MDA-

Med Chem Res
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in the series (11a–g) was associated with an increase in the
in vitro antiproliferative potency as compared with a series
of 10a–g. The computational techniques such as pharma-
cophore modeling and molecular docking studies have been
applied to support our experimental results. We have
identified five featured pharmacophore (AAHHRR) and
from the docking study, we have explored the probable
binding mode and identified key active site residues around
potent compound (11f) in HDAC8 and EHMT2 proteins.
This study provides valuable information for further
development of more potent anticancer agents.
Acknowledgments The authors acknowledge DST-SERB/EMEQ-
078/2013 for financial assistance. RV thanks CSIR, New Delhi, for
award of research fellowship.
Compliance with ethical standards
Conflict of interest The authors declare that they have no com-
peting interests.
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