Synthesis and screening of 2-(2-(4-substituted piperazine-1-yl)-5- phenylthiazol-4-yl)-3-aryl quinazolinone derivatives as anticancer agents

Article abstract of DOI:10.1007/s00044-012-0260-2

Synthesis of novel quinazolinone derivatives was performed from the reaction of N-benzoyl substituted piperazine-1-carbothioamide with 2-chloromethyl quinazolinone derivatives and screened for their in vitro cytotoxic activity by MTT assay. The cell lines

Full text of DOI:10.1007/s00044-012-0260-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2788–2794
DOI 10.1007/s00044-012-0260-2
ORIGINAL RESEARCH
Synthesis and screening of 2-(2-(4-substituted piperazine-1-yl)-5-
phenylthiazol-4-yl)-3-aryl quinazolinone derivatives
as anticancer agents
•
Ritesh N. Sharma Rasik Ravani
Received: 24 November 2011 / Accepted: 22 September 2012 / Published online: 23 October 2012
Ó Springer Science+Business Media New York 2012
Abstract Synthesis of novel quinazolinone derivatives
was performed from the reaction of N-benzoyl substituted
piperazine-1-carbothioamide with 2-chloromethyl quinaz-
olinone derivatives and screened for their in vitro cytotoxic
activity by MTT assay. The cell lines used were NCI (human
lung cancer cell), MCF 7 (Breast cancer cell), and HEK 293
(Normal epidermal kidney cell). Result of screening on
cell line showed moderate to good anticancer activity for
all the compounds. Compound 3d (IC50 = 1.1 0.03 lM)
was found to be the most active compared to standard
methotrexate (IC50 = 2.20 0.18 lM) and 5-florouracil
(IC50 = 2.30 0.49 lM). Structure activity relationship of
synthesized analogs suggested that the presence of NH linker
with aryl moiety at the third position of quinazolinone ring
was important for potent anticancer activity. Electron
donating group on phenyl ring at the third position of qui-
nazolinone ring gave better anticancer activity then unsub-
stituted phenyl and electron withdrawing group. Activity by
substituted piperazine at 2nd position of thiazole linked with
quinazolinone scaffold gave better activity in the order of
H [ CH3 [ CO–C6H5. Our findings may impart new
direction to medicinal chemists and biochemists for further
investigations of quinazolinone-thiazole containing anti-
cancer agents.
Introduction
Cancer is a term used for diseases in which abnormal cells
divide without control and are able to invade other tissues.
Number of cancer patients is increasing rapidly from day to
day and good protection from cancer and with reduced
adverse effects is the requirement of present scenario.
Quinazolinones are among the most useful heterocyclic
compound from both synthetic and medicinal chemistry
aspects. Most of the researchers focused on synthesis of
quinazolinone and their anticancer property (Ahmed et al.,
2010; Shang et al., 2005). The structural design of quinaz-
olinone have attracted a great deal of attention because of
their ready accessibility, diverse chemical reactivity, and
biological activities like anti-inflammatory (Ashok and
Chatrasal, 2009), antimalarial (Shuren et al., 2010), anthel-
mintic (Rajiv and Anil, 2008), muscle relaxant (Buyuktimkin
and Ekinici, 1992), antihyperlipidemic (Fawzia and Amry,
2005), antitubercular (Kunes et al., 2000), antimicrobial
(Wasfy, 2003), and antihypertensive (Ashok and Chatrasal,
2009) activities. The synthetic flexibility of quinazolinone
scaffold led to the synthesis of variety of its substituted
analogs. Raltitrexed and thymitaq are now clinically used as
anticancer drugs having quinazolinone moiety.
Thiazole, as a part of our designed molecule is a five
member heterocyclic moiety having various pharmaco-
logical activities like anticancer, anti-inflammatory, and
antibacterial. Thiazole is found in certain natural product
like vitamin B1 and penicillins. There are many reports
available on the anticancer activity of 2-aminothiazole and
benzothiazole derivatives.
Keywords Anticancer Á Quinazolinone Á Thiazole Á
Piperazine Á MTT assay
R. N. Sharma (&) Á R. Ravani
Department of Pharmaceutical Chemistry, S. K. Patel College of
Pharmaceutical Education and Research, Ganpat University,
Mahesana-Gozaria Highway, Ganpat Vidyanagar Dist.,
Mahesana 384012, Gujarat, India
In the design of new anticancer agent, the development
of hybrid molecules through the combination of different
pharmacophore i.e., quinazolinone and thiazole moiety in
one frame may lead to compounds with interesting
e-mail: riteshn.sharma@gmail.com
123

Med Chem Res (2013) 22:2788–2794
2789
anticancer profiles (Rajan et al., 2010a, b; Al Obaid et al.,
2009). Quinazolinone and thiazole pharmacophore may
serve as an important scaffold to develop new anticancer
agents with improved activity.
without Ca??/Mg?? using a volume equivalent to half the
volume of culture medium. Trypsin/EDTA was added on to
the washed cell monolayer using 1 ml per 25 cm2 of sur-
face area. Flask was rotated to cover monolayer with
trypsin and moved to the incubator and left for 2–4 min.
The cells were examined using an inverted microscope to
ensure that all the cells were detached and floated. The
cells were resuspended in a small volume of fresh serum
containing HEK-293 medium and 100–200 ll was
removed to perform cell count. The required number of
cells were transferred to a new labeled flask containing pre-
warmed HEK-293 medium and incubated as appropriate
for the cell line (Rathi et al., 2009; Freshney, 2005). All the
cytotoxicity experiments were carried out in 96-well plates.
Methotrexate and 5-flourouracil was used as a reference
standard for cytotoxic activity. All solutions of test com-
pounds were prepared using DMSO. IC50 values were
calculated, it is a drug concentration causing a 50 %
inhibition of cell proliferation (Prakash et al., 2011).
We hypothesized that the designing of molecule with
quinazolinone moiety as molecular scaffold by using
strategies like linking with thiazole and by varying chain
length using piperazine as linker and substituted phenyl
group attach at position the third of quinazolinone ring in
the target molecules for better anticancer activity.
Chemistry
This synthetic strategy began with the synthesis of quinaz-
olinone by the reaction of 2-(2-chloroacetamido)-benzoic
acid with aromatic amines in the presence of phosphorus
oxychloride. In the IR spectrum of quinazolinone, the car-
bonyl frequency was observed at 1,729 cm-1 (Hui et al.,
2009; Shashikant et al., 2006). The synthesis of isothiocy-
anate oil by the reaction of benzoyl chloride with potassium
thiocyanate in the presence of PEG 400 as phase separation
catalyst was carried out and characterized by boiling point
and TLC. The synthesis of N-(4-substituted piperazine-1-
thioyl)-benzamide was carried out using a reported method
(Rajan et al., 2010a, b). The IR spectrum of N-(4-substituted
piperazine-1-thioyl)-benzamide showed C–N stretch, C–S
Results and discussion
The target molecules were designed by joining two dif-
ferent moieties i.e., quinazolinones and thiazoles. These
novel quinazolinone-thiazole derivatives were synthesized
with different aryl substitutions at the third position of
quinazolinone ring and different substituted piperazines at
2nd position of thiazole ring. Synthesis of target molecule
was carried out as per Scheme 1. Different piperazines
were reacted with benzoyl isothiocyanate to give adducts
(1a–1c) that were further subjected to reaction with dif-
ferent quinazolinones (2a–2f) to provide target molecules
(3a–3o). The structure of all synthesized compound were
confirmed by physical characterization i.e., melting point,
Rf value, elemental analysis, and spectral characterization
i.e., IR, MASS, and NMR spectroscopy.
stretch, and C=O stretch at 1,437, 1,555, and 1,695 cm-1
,
respectively. Final product was synthesized by the reaction
of different quinazolinones and N-(4-substituted piperazine-
1-thioyl)-benzamides in the presence of triethylamine and
acetonitrile as solvent. Reaction mixture was refluxed at
temperature of 80 °C and monitored with TLC for comple-
tion. The yields of synthesized compound are given in
Table 1. We reported the synthesis of novel quinazolinone
derivatives and their cytotoxic potentials.
In the target molecules, optimization was done at the
third position of quinazolinone and the second position of
thiazole ring. The third position of quinazolinone was
substituted with different aryl groups (electron withdraw-
ing, electron releasing group with and without NH as lin-
ker) and the second position of thiazole was substituted
with different piperazines (H, CH3, and COC6H5). All the
synthesized compounds were subjected to in vitro cyto-
toxicity activity on MCF-7 (breast cancer), NCI (lung
cancer), and HEK-293 (normal cell of epidermal kidney)
cell lines by MTT assay for anticancer therapy. IC50 values
were calculated for test and standard compounds.
Biology
All the synthesized compounds were screened for their
cytotoxic activity on MCF-7 (human mammary gland
adrenocarcinoma cell line), NCI (lung cancer cell line), and
HEK-293 (human epidermal kidney cell line as normal
cell line) by MTT assay. MCF-7, NCI, and HEK-293 cell
cultures were procured from National Centre for Cell
Sciences, Pune, India. The screening experiments were
carried out at Department of Biotechnology, S.K. Patel
College of Pharmaceutical Education and Research, Gan-
pat University, Gujarat, India. Cultures were observed
using an inverted microscope to assess the degree of via-
bility, and the absence of bacterial and fungal contaminants
was confirmed. Cell monolayer was washed with PBS
Statistical significance of the data (expressed as
mean SEM) was demonstrated by performing one-way
ANOVA test followed by Dennett comparison of IC50 of
the entire compounds against methotrexate and
123

2790
Med Chem Res (2013) 22:2788–2794
Table1 In vitro cytotoxicity screening data of synthesized quinazolinone derivatives compared against the standard drugs
O
Ar
N
S
N
N
NR1
N
Compound No.
Ar
R1
Mean IC50(lM) SEM*
NCI
MCF-7
HEK-293
3a
C6H5
CH3
9.89 0.21aaa,bbb
13.40 0.14aaa,bbb
2.66 0.12NS,NS
1.11 0.03aa,bb
5.59 0.10aaa,bbb
2.92 0.15NS,NS
15.57 0.16aaa,bbb
[100aaa,bbb
6.10 0.09aaa,bbb
9.43 0.21aaa,bbb
4.33 0.98aaa,bbb
5.93 0.29NS,b
6.72 0.05aaa,bbb
8.39 0.07aaa,bbb
11.03 0.16aaa,bbb
2.20 0.18
5.84 0.12aaa,bbb
19.92 0.06aaa,bbb
46.72 0.24aaa,bbb
0.16 0.16aaa,NS
2.49 0.11NS,NS
11.40 0.19aaa,bbb
[100aaa,bbb
0.86 0.04NS,bbb
0.42 0.02aaa,bbb
0.74 0.03aa,bbb
3.03 0.09aaa,bbb
0.80 0.03NS,bbb
1.11 0.04NS,bbb
0.69 0.03aa,bbb
0.94 0.06NS,bbb
14.36 0.73aaa,bbb
0.67 0.03aa,bbb
0.55 0.04aaa,bbb
2.29 0.10aaa,NS
0.81 0.05NS,bbb
1.07 0.04NS,bbb
0.73 0.02aa,bbb
1.27 0.06
3b
C6H5
C6H5CO
3c
C6H5
H
3d
C6H5 NH
C6H5 NH
p-OCH3C6H4NH
p-Cl C6H4
p-Cl C6H4
p-Cl C6H4
o-OCH3C6H4
o-OCH3C6H4
o-OCH3C6H4
o-NO2C6H5
o-NO2C6H5
o-NO2C6H5
–
CH3
3e
C6H5CO
3f
H
3g
C6H5CO
3h
CH3
1.97 0.13NS,NS
[100aaa,bbb
3i
H
3j
CH3
1.50 0.09NS,NS
3.70 0.08a,bbb
0.66 0.02NS,NS
8.90 0.53aaa,bbb
5.86 0.31aaa,bbb
6.49 0.27aaa,bbb
2.26 0.11
3k
H
3l
C6H5CO
3m
CH3
3n
H
3o
C6H5CO
Methotrexate
5-Florouracil
–
–
–
2.30 0.49
1.25 0.07
2.15 0.13
NS Not Significant
* N = 3; One-way ANOVA was performed using Dennett Test aaa \0.0001; aa \0.001
a
\0.05 (Methotraxate); bbb \0.0001; bb \0.001; b \0.05 (5-florouracil)
Scheme 1 Schematic
representation for the synthesis
of quinazolinone derivatives
O
Ar
N
R1N
NH
S
O
R1N
N
+
NH
Cl
N
NCS
Toluene
R.T
O
(1a-1c)
(2a-2f)
Benzoyl
isothiocyanate
Reflux
Triethylamine,
Acetonitrile
O
N
N
No. Ar
No. R1
Ar
-
2a C6H5
1a H-
1b CH3-
1c C6H5CO-
2b C6H5NH-
2c p-Cl C6H4
S
-
N
NR1
2d p-OCH3C6H4NH-
N
-
2e o-NO2C6H4
-
2f o-OCH3 C6H4
(3a-3o)
123

Med Chem Res (2013) 22:2788–2794
2791
5-flourouracil using Graph pad Prism (Version 5.0) soft-
ware. Results of the biological screening and statistical
analysis are shown in Table 1.
unsubstituted piperazines were more active than substituted
piperazines on the compounds having thiazole ring.
Finally, the substitution C6H5NH at the third position of
quinazolinone ring, methyl substituted piperazines at the
second position of thiazole ring, and phenyl group at the
fourth position of thiazole ring as in compound 3d can be
considered as a four-point pharmacophore for designing
better anticancer agents.
Biological screening data suggested that compounds 3c
(IC50 = 2.66 0.12 lM), 3d (IC50 = 1.11 0.03 lM),
and 3e (IC50 = 2.92 0.10 lM) show high activity on
NCI (lung cancer) cell line. Compound 3d (p \ 0.001, for
both the standard drugs) was found to be more potent than
other compounds and compared to standard drug metho-
trexate (IC50 = 2.20 0.18 lM) and 5-florouracil
(IC50 = 2.30 0.49 lM) on this cell line activity. Com-
pounds having NH as linker between the third position of
quinazolinone and phenyl group gave high activity.
Screening on MCF-7 (breast caner cell line), compound 3d
(IC50 = 0.16 0.16 lM), 3l (IC50 = 0.66 0.02 lM),
and 3j (IC50 = 1.50 0.09 lM) showed high activity.
The comparison of compounds (3d, 3l, and 3j) also showed
comparable activity than methotrexate (IC50 = 2.26
0.11 lM) and 5-florouracil (IC50 = 1.25 0.07 lM). The
statistical analysis of compound 3l with the standard drug
suggested that the compound is nearly equally active as
both the standard drugs for MCF-7 cell lines and inactive
for NCI cell lines. Compound 3d substituted with methyl
piperazine on thiazole and phenyl amine on quinazolinone
ring is attributed to the highest activity on MCF-7 cancer
cell line. Assay on normal cell line HEK-293 suggested
Conclusion
A series of quinazolinone derivatives was synthesized and
screened for their in vitro cytotoxic activity. Results of
assay indicated that all the compounds were found to have
good to moderate activity. Compound 3d possesses higher
activity than the standard drug (methotrexate and
5-flourouracil). The compound 3d was particularly prom-
ising, since it was able to kill cancer cells more effectively
than the non-cancerous cell which was observed from the
result of HEK-293 cell line. Furthermore, it concluded that
compound with NH linker between aryl moiety and the
third position of quinazolinone ring has been recognized as
potent anticancer agent. Therefore, this type of compound
may further be optimized and evaluated with enzymatic
assay and in vivo animal models in the line of the devel-
opment and also can serve as a prototype molecule of new
class of anticancer agents.
that compound, 3d (IC50 = 3.03 0.09 lM), 3l (IC50
=
2.29 0.10), and 3i (IC50 = 14.36 0.73 lM) kill less to
normal cells compared to methotrexate (IC50 = 1.27
0.06 lM) and 5-florouracil (IC50 = 2.15 0.13 lM).
From the above results from the entire cell lines, one can
conclude that compound 3d is more potent among all the
synthesized compounds and also as compared to the stan-
dard drug. Compounds 3g, 3h, and 3i are almost inactive
among all the synthesized compounds and more toxic to
normal human cells also.
Experimental protocol
Chemistry
Melting points of all synthesized compounds were deter-
mined in open capillaries using Veego melting point
apparatus, Model VMP-D (Veego India ltd., Mumbai,
India) and were uncorrected. Infrared spectra were recor-
ded using KBr pellets on SHIMADZU-FT-IR 8400S
instrument. Mass spectra were recorded on PerkinElmer
LC–MS PE Sciex API/65 Spectrophotometer. The 1H
NMR spectra were recorded on Brucker Avance-300
(300 MHz) model spectrophotometer in CDCl3 using
Structure activity relationship suggested that compound
with NH linker on the third position of quinazolinone
moiety was more active throughout the series. On the other
hand, compound with p-Cl was found to be the least active
in the series. It is also suggested that electron withdrawing
group on phenyl ring at the third position of quinazolinone
ring may be responsible for less activity and also more
toxicity to normal human cell line. Compound having
electron donating group on phenyl ring at the third position
of quinazolinone ring i.e., 3j, 3k, and 3l have moderate
activity. Compounds 3a, 3b, and 3c which has only un-
substituted phenyl ring at the third position of quinazoli-
none ring also gave moderate activity but their activity is
less than compound with electron donating group. The
substitution on piperazine of thiazole ring did not make
much difference on the activity of the whole series. Even
1
DMSO as solvent and TMSi as internal standard with H
resonant frequency of 300 MHz. The TLC was performed
on precoated alumina silica gel 60 F254 (Merck). The
mobile phase was benzene:methanol (9:1) and detection
was made using UV light. The resulting compounds were
purified by recrystallization using suitable solvent. The
elemental analyses were done on elementar Vario EL 3
Carlo erba 1108 and were well in accordance with the
structures assigned to the compound. The entire compound
gave C, H, and N analysis within 0.4 % of the theoretical
123

2792
Med Chem Res (2013) 22:2788–2794
values. Synthetic grade chemicals procured from SD fine
chemicals, Baroda, India were used for the synthesis of the
target compounds. All the compounds of step I and 2
(1a–1c and 2a–2e) were prepared according to the litera-
ture procedures with some minor modifications (Hui et al.,
2009; Rajan et al., 2010a, b). General synthetic procedures
used for the preparation of the target compound are as
follows:
14.57. IR (KBr, tmax, cm-1): 1,680 (C=O), 1,579 (C–N
Stretch), 3,149 (C–H Stretch); 1H NMR (300 MHz, d ppm,
DMSO): 7.92–6.88 (m, 14H, Ar–H), 4.34 (s, 3H, CH3),
3.30–2.49 (m, 8H, CH–piperazine) ppm; and MS: m/z
480.6 (M ? 1).
2-(2-(4-Benzoylpiperazin-1-yl)-5-phenylthiazol-4-yl)-3-
phenylquinazolin-4(3H)-one (3b)
Synthesis of N-benzoyl 4-substituted piperazine-1-
carbothiomide (1a–1c)
Brown product; Rf value 0.54, Yield 64 %; M.p.
105–109 °C; Elem. analysis Calcd. for C34H27N5O2S: C,
71.68; H, 4.78; N, 12.29 %. Found: C, 71.63; H, 4.74; N,
12.25; IR (KBr, tmax, cm-1): 1,643 (C=O), 1,571 (C–N
Stretch), 3,117 (C–H Stretch), 1,582 (NH); 1H NMR
(300 MHz, d ppm, DMSO): 7.95–6.97 (m, 19H, Ar–H),
3.62–3.45 (m, 8H, CH–piperazine) ppm; and MS: m/z
579.9 (M ? 1).
Equimolar quantity of benzoyl isothiocyanate was added
from the dropping funnel to N-substituted piperazines in
toluene. Stirring was continued for 2 h at room tempera-
ture. Crude solid product of N-benzoyl substituted piper-
azine-1-carbothioamide (1a–1c) was precipitated out. The
collected precipitate was repeatedly washed with small
portions of toluene and recrystallized with ethyl acetate to
yield product.
3-Phenyl-2-(5-phenyl-2-(piperazin-1-yl)thiazol-4-
yl)quinazolin-4(3H)-one (3c)
Synthesis of 2-chloro methyl quinazolinone derivatives
Brown product; Rf value 0.50, Yield 72 %; M.p.
144–146 °C; Elem. analysis Calcd. for C27H23N5OS: C,
69.65; H, 4.98; N, 15.04 %. Found: C, 69.62; H, 4.99; N,
14.99; IR (KBr, tmax, cm-1): 1,645 (C=O), 1,556 (C–N
Stretch), 3,086 (C–H Stretch); 1H NMR (300 MHz, d ppm,
DMSO): 7.73–6.90 (m, 14H, Ar–H), 3.24–3.16 (m, 8H,
CH–piperazine), 2.17 (s, 1H, NH) ppm; and MS: m/z 466.8
(M ? 1).
(2a–2f)
Addition of 2-(2-chloroacetamido)-benzoic acid to the
equal mole of different substituted aromatic amines in
acetonitrile in the presence of phosphorus oxychloride was
carried out at room temperature and reaction was allowed
to reflux. Completion of reaction was monitored with TLC.
Then, sodium bicarbonate was added to neutralize reaction
mixture. Aqueous layer was extracted with chloroform.
The chloroform was evaporated under reduced pressure
and product (2a–2f) was collected. Crude compound was
recrystallized using toluene.
3-(4-Methoxy phenylamino)-2-(5-phenyl-2-(piperazin-
1-yl)-thiazol-4-yl)quinazolinone (3d)
Dark brown product; Rf value 0.56, Yield 64 %; M.p.
105–109 °C; Elem. analysis Calcd. for C28H26N6OS: C,
67.99; H, 5.30; N, 16.99 %. Found: C, 67.80; H, 5.27; N,
16.97; IR (KBr, tmax, cm-1): 1,662 (C=O), 1,520 (C–N
Stretch), 3,187 (C–H Stretch), 1,590 (NH); 1H NMR
(300 MHz, d ppm, DMSO):, 7.55–6.89 (m, 14H, Ar–H),
4.19 (s, 1H, PhNH), 3.36–3.11 (m, 8H, CH–piperazine),
2.38 (s, 3H, CH3) ppm; and MS: m/z 495.2 (M ? 1).
Synthesis of 2-(2-(4-substituted piperazine-1-yl)-5-
phenylthiazol-4-yl)-3-aryl quinazolinone (3a–3o)
Equimolar quantity of N-benzoyl substituted piperazine-1-
carbothioamide (1a–1c) and 2-chloromethyl quinazolinone
derivatives (2a–2e) in acetonitrile were reacted in the
presence of triethylamine. The reaction mixture was
allowed to reflux and completion of reaction was checked
by TLC. Precipitate was obtained after pouring the reaction
mixture to crushed ice and finally compounds were
recrystallized using methanol.
2-(2-(4-Benzoylpiperazin-1-yl)-5-phenylthiazol-4-yl)-3-
(phenylamino)quinazolin-4(3H)-one (3e)
Yellowish green product; Rf value 0.52, Yield 66 %; M.p.
130–132 °C; Elem. analysis Calcd. for C34H28N6O2S: C,
69.84; H, 4.83; N, 14.37 %. Found: C, 69.80; H, 4.80; N,
14.36, IR (KBr, tmax, cm-1): 1,680 (C=O), 1,527 (C–N
Stretch), 3,110 (C–H Stretch), 1,418 (N=O); 1H NMR
(300 MHz, d ppm, DMSO): 7.96–6.31 (m, 19H, Ar–H),
4.09 (s, 1H, PhNH), 3.46–2.86 (m, 8H, CH–pipera-
zine) ppm; and MS: m/z 585.73 (M ? 1).
2-(2-(4-Methylpiperazin-1-yl)-5-phenylthiazol-4-yl)-3-
phenylquinazolin-4(3H)-one (3a)
Light brown product; Rf value 0.60, Yield 66 %; M.p.
140–142 °C; Elem. analysis Calcd. for C28H25N5OS: C,
70.12; H, 5.25; N, 14.60 %. Found: C, 70.08; H, 5.27; N,
123

Med Chem Res (2013) 22:2788–2794
2793
3-(4-Methoxyphenylamino)-2-(5-phenyl-2-(piperazin-1-
yl)thiazol-4-yl)quinazolin-4(3H)-one (3f)
68.35; H, 5.35; N, 13.74 %. Found: C, 68.36; H, 5.32; N,
13.72, IR (KBr, tmax, cm-1): 1,723 (C=O), 1,577 (C–N
Stretch), 3,022 (C–H Stretch); 1H NMR (300 MHz, d ppm,
DMSO): 7.86–6.84 (m, 13H, Ar–H), 3.79 (s, OCH3),
3.14–2.88 (m, 8H, CH–piperazine), 2.62 (s, CH3) ppm; and
MS: m/z 510.2 (M ? 1).
Green product; Rf value 0.60, Yield 70 %; M.p.
129–133 °C; Elem. analysis Calcd. for C28H28N6O2S: C,
65.86; H, 5.13; N, 16.46 %. Found: C, 65.84; H, 5.10; N,
16.49, IR (KBr, tmax, cm-1): 1,749 (C=O), 1,556 (C–N
Stretch), 3,023 (C–H Stretch); 1H NMR (300 MHz, d ppm,
DMSO): 8.13–6.9 (m, 18H, Ar–H), 4.07 (s, 1H, PhNH),
3.79 (s, 3H, OCH3), 3.19–2.77 (m, 8H, CH–piperazine)
2.19 (s, 1H, NH) ppm; and MS: m/z 510.61 (M)
3-(2-Methoxyphenyl)-2-(5-phenyl-2-(piperazin-1-
yl)thiazol-4-yl)quinazolin-4(3H)-one (3k)
Brown product; Rf value 0.63, Yield 59 %; M.p.
101–103 °C; Elem. analysis Calcd. for C28H25N5O2S: C,
67.86; H, 5.08; N, 11.68 %. Found: C, 67.81; H, 5.10; N,
14.10, IR (KBr, tmax, cm-1): 1,666 (C=O), 1,543 (C–N
Stretch), 3,126 (C–H Stretch), 1,570 (NH); 1H NMR
(300 MHz, d ppm, DMSO 8.10–6.74 (m, 13H, Ar–H),
2.61–2.19 (m, 8H, CH–piperazine) 2.26 (s, 1H, NH) ppm;
and MS: m/z 496.3 (M ? 1).
2-(2-(4-Benzoylpiperazin-1-yl)-5-phenylthiazol-4-yl)-
3-(4-chlorophenyl)quinazolin-4(3H)-one (3g)
Yellowish green product; Rf value 0.60, Yield 72 %; M.p.
130–132 °C; Elem. analysis Calcd. for C34H26ClN5O2S: C,
67.60; H, 4.34; N, 11.59 %. Found: C, 67.55; H, 4.34; N,
11.61, IR (KBr, tmax, cm-1): 1,713 (C=O), 1,570 (C–N
Stretch), 3,043 (C–H Stretch), 1,566 (NH); 1H NMR
(300 MHz, d ppm, DMSO): 8.33–6.62 (m, 18H, Ar–H),
2.72–2.07 (m, 8H, piperazine) ppm; and MS: m/z 603.97
(M ? 1).
2-(2-(4-Benzoylpiperazin-1-yl)-5-phenylthiazol-4-yl)-
3-(2-methoxyphenyl)quinazolin-4(3H)-one (3l)
Brown product; Rf value 0.55, Yield 62 %; M.p.
125–128 °C; Elem. analysis Calcd. for C35H29N5O3S: C,
70.10; H, 4.87; N, 11.68 %. Found: C, 70.07; H, 4.83; N,
11.67, IR (KBr, tmax, cm-1): 1,670 (C=O), 1,556 (C–N
Stretch), 3,090 (C–H Stretch), 810 (C–Cl); 1H NMR
(300 MHz, d ppm, DMSO): 8.35–7.36 (m, 18H, Ar–H),
3.68–2.39 (m, 8H, CH–piperazine) ppm; and MS: m/z
601.2 (M ? 1).
3-(4-Chlorophenyl)-2-(2-(4-methylpiperazin-1-yl)-
5-phenylthiazol-4-yl)quinazolin-4(3H)-one (3h)
Green product; Rf value 0.52, Yield 55 %; M.p.
190–192 °C; Elem. analysis Calcd. for C28H24ClN5OS: C,
65.42; H, 4.71; N, 13.62 %. Found: C, 65.41; H, 4.69; N,
13.63, IR (KBr, tmax, cm-1): 1,664 (C=O), 1,511 (C–N
Stretch), 3,080 (C–H Stretch), 795 (C–Cl); 1H NMR
(300 MHz, d ppm, DMSO): 7.34–6.53 (m, 13H, Ar–H),
3.84 (s, 3H, CH3), 3.76–2.84 (m, 8H, CH–piperazine) ppm;
and MS: m/z 513.9 (M)
2-(2-(4-Methylpiperazin-1-yl)-5-phenylthiazol-4-yl)-
3-(2-nitrophenyl)quinazolin-4(3H)-one (3m)
3-(4-Chlorophenyl)-2-(5-phenyl-2-(piperazin-1-yl)
thiazol-4-yl)quinazolin-4(3H)-one (3i)
Yellowish green product; Rf value 0.58, Yield 62 %; M.p.
175–177 °C; Elem. analysis Calcd. for C28H24N6O3S: C,
64.11; H, 4.61; N, 16.02 %. Found: C, 64.09; H, 4.58; N,
15.98, IR (KBr, tmax, cm-1): 1,716 (C=O), 1,507 (C–N
Stretch), 2,978 (C–H Stretch), 1,580 (NH); 1H NMR
(300 MHz, d ppm, DMSO): 8.16 (d, H, NO2Ar–H),
7.95–7.33 (m, 12H, Ar–H), 3.36–2.61 (m, 8H, piperazine),
2.31 (s, 3H, CH3) and MS: m/z 524.29 (M).
Green product; Rf value 0.56, Yield 64 %; M.p.
122–126 °C; Elem. analysis Calcd. for C27H22ClN5OS: C,
64.86; H, 4.43; N, 14.01 %. Found: C, 64.83; H, 4.41; N,
13.98, IR (KBr, tmax, cm-1): 1,692 (C=O), 1,542 (C–N
Stretch), 3,072 (C–H Stretch), 814 (C–Cl), 1,577 (NH); 1H
NMR (300 MHz, d ppm, DMSO): 7.41–6.46 (m, 13H,
Ar–H), 3.84–2.71 (m, 8H, piperazine), 2.22 (s, 1H,
NH) ppm; and MS: m/z 500.1 (M)
3-(2-Nitrophenyl)-2-(5-phenyl-2-(piperazin-1-yl)thiazol-
4-yl)quinazolin-4(3H)-one (3n)
3-(2-Methoxyphenyl)-2-(2-(4-methylpiperazin-1-yl)-
5-phenylthiazol-4-yl)quinazolin-4(3H)-one (3j)
Pale yellow product; Rf value 0.54, Yield 64 %; M.p.
180–182 °C; Elem. analysis Calcd. for C27H22N6O3S: C,
63.52; H, 4.34; N, 16.46 %. Found: C, 63.81; H, 4.51; N,
16.18, IR (KBr, tmax, cm-1): 1,726 (C=O), 1,510
Yellow product; Rf value 0.59, Yield 66 %; M.p.
125–127 °C; Elem. analysis Calcd. for C29H27N5O2S: C,
123

2794
Med Chem Res (2013) 22:2788–2794
1
References
(C–N Stretch), 2,928 (C–H Stretch), 1,575 (NH); H NMR
(300 MHz, d ppm, DMSO): 8.21 (d, H, NO2Ar–H),
7.98–7.24 (m, 12H, Ar–H), 3.44–2.32 (m, 8H, CH–piper-
azine) 2.15 (s, 1H, NH) ppm; and MS: m/z 511.71
(M ? 1).
Ahmed K, Vijaya BE, Surekha Z, Aarti J (2010) Quinazolinone
linked pyrrolo [2,1-c][1,4] benzodiazepine (PBD) conjugates:
design, synthesis and biological evaluation as potential antican-
cer agents. Bioorg Med Chem 18:526–542
Al-Obaid AM, Abdel-Hamide SG, El-kashef HA, Abdel-Aziz AA, El-
Azab AS, Al-Khamees HA, El-Subbagh HI (2009) Substituted
quinazolines, part 3. Synthesis, in vitro antitumor activity and
molecular modeling study of certain 2-thieno-4(3H)-quinazoli-
none analogs. Eur J Med Chem 44:2379–2391
2-(2-(4-Benzoylpiperazin-1-yl)-5-phenylthiazol-4-yl)-
3-(2-nitrophenyl)quinazolin-4(3H)-one (3o)
Ashok K, Chatrasal SR (2009) Synthesis and anti-inflammatory
activity of newer quinazolin-4-one derivatives. Eur J Med Chem
44(1):83–90
Buyuktimkin S, Ekinici AC (1992) Pharmacological studies on
quarternized-4-(3H)-quinazolinones. J Pharm Sci 81(11):1092–
1094
Fawzia MR, Amry E (2005) The antihyperlipidemic activities of
4-(3H)-quinazolinone and two halogenated derivatives in rats.
Lipid Health Dis 4:22–24
Freshney IR (2005) Culture of animal cells: a manual of basic
technique, 5th edn. Wiley, New York, pp 359–370
Hui H, Jian FG, Ling HC, Duo ZW, Zhin BZ (2009) Synthesis of
novel thioglycoside derivatives containing quinazolinone. J Chi-
nese Chem Soc 56:419–424
Yellowish product; Rf value 0.51, Yield 67 %; M.p.
175–178 °C; Elem. analysis Calcd. for C34H26N6O4S: C,
66.44; H, 4.26; N, 13.67 %. Found: C, 66.47; H, 4.31; N,
13.61, IR (KBr, tmax, cm-1): 1,723 (C=O), 1,570 (C–N
Stretch), 3,013 (C–H Stretch), 1,546 (NH); 1H NMR
(300 MHz, d ppm, DMSO): 8.21 (d, H, NO2Ar–H),
7.92–7.31 (m, 17H, Ar–H), 3.36–3.44 (m, 8H, pipera-
zine) ppm; and MS: m/z 615.57 (M ? 1).
Cytotoxic assay
´
Kunes J, Bazant J, Pour M, Waisser K, Slosarek M, Janota J (2000)
Quinazoline derivatives with antitubercular activity. Farmaco
55(11–12):725–729
Prakash S, Poonam S, Sarav AD, Maulik PS (2011) In vitro
cytotoxicity evaluation of novel N-substituted bis-benzimidazole
derivatives for anti-lung and anti-breast cancer activity. Ann Biol
Res 2:51–59
Rajan G, Hardik T, Tony G, Williams J, Rogers D, Kamala V,
Vasudevan S (2010a) Design, synthesis and evaluation of novel
2-thiophen-5-yl-3H-quinazolin-4-one analogues as inhibitors of
transcription factors NF-rB and AP-1 mediated transcriptional
activation: Their possible utilization as anti-inflammatory and
anti-cancer agents. Bioorg Med Chem 18:2796–2808
Rajan G, Hardik T, Tony G, Bing C, Sam N, Kamala V, Vasudevan S
(2010b) Synthesis and evaluation of quinazolinone derivatives as
inhibitors of NF-kB, AP-1 mediated transcription and eIF-4E
mediated translational activation: inhibitors of multi-pathways
involve in cancer. Eur J Med Chem 45:3558–3563
Rajiv D, Anil K (2008) Synthesis and biological activity of peptide
derivatives of Iodoquinazolinones. Molecules 13(4):958–976
Rathi SG, Suthar M, Patel P, Bhaskar VH, Rajgor NB (2009) J Young
Pharm 1:239–243
Shang LC, Yu PF, Run TL, Guo YD, Yu YZ (2005) Synthesis and
in vitro antitumor activity of 4(3H)-quinazolinone derivatives
with dithiocarbamate side chains. Bioorg Med Chem Lett 15:
1915–1917
Shashikant RP, VV KrishnaR, Manvi FV, Desai BG, Bhatt AR (2006)
Synthesis of N-3(4-(4-chlorphenyl thiazole-2-yl)-(2-amino-
methyl)-quinazolinone and their derivatives as anti tubercular
activity. Indian J Chem 45b:1778–1781
Shuren Z, Joe W, Gudise C, Erika S, Xianjun L, Yongshen Z (2010)
Synthesis and evaluation of 4-quinazolinone compounds as
potential antimalarial agents. Eur J Med Chem 45(9):3864–3869
Wasfy AAF (2003) Synthesis and antimicrobial evaluation of some
2,2-disubstituted-3,3-biquinazolin-4-(3H)-ones. Indian J Chem B
42B:3102–3107
The cells were preincubated at a concentration of
1 9 106 cells/ml in culture medium for 3 h at 37 °C and
6.5 % CO2. Then, the cells were seeded at a concentration
of 5 9 104 cells/well in 100 ll culture medium and at
various concentrations (0.005–100 lM/ml) of standard
methotrexate and synthesized compounds [dissolved in
2 % DMSO (dimethylsulfoxide) solution] into microplates
(tissue culture grade, 96 wells, flat bottom) and incubated
for 24 h at 37 °C and 6.5 % CO2. The cell proliferation is
based on the ability of the mitochondrial succinate-
terazolium reductase system to convert 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a
blue-colored formazan. The test denotes the survival cells
after toxic exposure. Then, 10 ll MTT labeling mixture
was added and incubated for 4 h at 37 °C and 6.5 % CO2.
Each experiment was done in triplicate. Then, 100 ll of
solubilization solution was added into each well and
incubated overnight. The spectrophotometric absorbance of
the samples was measured using a microplate (ELISA)
reader. The wavelength to measure absorbance of the for-
mazan product is between 550 and 600 nm according to the
filters available for the ELISA reader was used. The ref-
erence wavelength should be more than 650 nm. IC50 was
then calculated as the drug concentration causing a 50 %
inhibition of cell proliferation. HEK 293 cell line was used
to find out cytotoxic effects of synthesized compound on
non-cancerous cells (Prakash et al., 2011; Rathi et al.,
2009).
123