Synthesis and anticancer activity of novel quinazolinone and benzamide derivatives

Article abstract of DOI:10.1007/s11164-017-3245-4

In trying to develop new anticancer agents, a series of quinazolinone and benzamide derivatives were synthesized via reaction of 6-iodo-2-phenyl-4H-benzoxazin-4-one with nitrogen nucleophiles, namely, formamide, ammonium acetate, hydrazine hydrate, hydroxylamine hydrochloride, substituted aromatic amines, benzyl amine, and/or thiocarbonohydrazide. All compounds were fully characterized by means of IR, MS, and ¹H-NMR spectra. Some of the synthesized compounds were evaluated in vitro for their anti-proliferative activity against HePG-2 and MCF-7 cell lines. 2-(Benzoylamino)-N-(4-hydroxyphenyl)-5-iodobenzamide and tetrazino[1,6-c]quinazoline-3(4H)-thione derivative were the most potent against the two cancer cells comparable to that of doxorubicin. Most of the synthesized compounds also exhibited good cytotoxic activity.

Full text of DOI:10.1007/s11164-017-3245-4

Res Chem Intermed
https://doi.org/10.1007/s11164-017-3245-4
Synthesis and anticancer activity of novel quinazolinone
and benzamide derivatives
1
1
Maher Abd El‑Aziz Mahmoud El‑Hashash · Marwa Sayed Salem ·
2
Selima Ali Mohamed Al‑Mabrook
Received: 25 May 2017 / Accepted: 29 December 2017
©
Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract In trying to develop new anticancer agents, a series of quinazolinone and
benzamide derivatives were synthesized via reaction of 6-iodo-2-phenyl-4H-ben-
zoxazin-4-one with nitrogen nucleophiles, namely, formamide, ammonium acetate,
hydrazine hydrate, hydroxylamine hydrochloride, substituted aromatic amines, ben-
zyl amine, and/or thiocarbonohydrazide. All compounds were fully characterized by
1
means of IR, MS, and H-NMR spectra. Some of the synthesized compounds were
evaluated in vitro for their anti-proliferative activity against HePG-2 and MCF-7 cell
lines. 2-(Benzoylamino)-N-(4-hydroxyphenyl)-5-iodobenzamide and tetrazino[1,6-
c]quinazoline-3(4H)-thione derivative were the most potent against the two cancer
cells comparable to that of doxorubicin. Most of the synthesized compounds also
exhibited good cytotoxic activity.
Keywords Anticancer activity · Quinazolin-4(3H)-one · Benzoxazinone ·
Hepatocellular carcinoma · Michigan Cancer Foundation-7
Introduction
Pharmacologically, quinazolin-4-ones are among the most important classes of
heterocyclic compounds. The stability of the quinazolinone nucleus has inspired
medicinal chemists to introduce many bioactive moieties to this nucleus to syn-
thesize new potential medicinal agents. The quinazolinone skeleton is a frequently
*
Marwa Sayed Salem
marwa_k@sci.asu.edu.eg
1
2
Synthetic Organic Chemistry Laboratory, Chemistry Department, Faculty of Science, Ain
Shams University, Abbassia, Cairo 11566, Egypt
Chemistry Department, Faculty of Science, El-Margeb University, Zliten, Libya
1
3

M. A. E.-A. M. El-Hashash et al.
encountered heterocycle in medicinal chemistry literature with applications includ-
ing antibacterial [1], analgesic, antiinꢀammatory [2], antifungal [3, 4], antimalarial,
antihypertensive [5], central nervous system (CNS) depressant [6], anticonvulsant
[
7], antihistaminic, antiparkinsonism, [8] antiviral and anticancer activities [9, 10].
The discovery and development of novel therapeutic DNA intercalators for the
treatment of malignancy is one of the most important goals in modern medicinal
chemistry. DNA intercalators including doxorubicin were originally isolated from
Streptomyces peucetius and are most widely used of the anthracycline antibiotic
group of anticancer agents [11, 12]. The precise mechanism of action of doxorubicin
is known to intercalate between base pairs in the DNA helix. Intercalation leads to
single- and double-strand breakages, thereby preventing DNA replication and ulti-
mately inhibiting protein synthesis. Scission of DNA is believed to be mediated
through the action of topoisomerase II or by the iron-catalyzed generation of free
radicals, both hydrogen peroxide and hydroxyl, which are highly destructive to cells.
Doxorubicin also induces the formation of covalent topoisomerase-DNA complexes,
resulting in inhibition of the religation portion of the ligation–religation reaction in
replicating DNA. Although it is active throughout the cell cycle, the maximal toxic-
ity occurs during the DNA synthesis (S) phase. At low drug concentrations, cells
will continue through the S phase and die in the G2 phase [13].
Based on the earlier ꢁndings, molecular hybridization between quinazolinone and
other eꢂective antitumor moieties was carried out in an attempt to obtain new eꢂec-
tive molecules with antitumor activity. Furthermore, in continuation of our previous
work, [14–21], the present work aimed at utilization of 6-iodo-2-phenyl-4H-benzox-
azin-4-one 1 for design and synthesis of diꢂerent quinazolin-4(3H)-ones in order to
have the main pharmacophoric features of DNA intercalating agents with the major
objective of developing agents with potential anti-proliferative activity that could be
highly eꢂective, safe, and devoid of side eꢂects.
Results and discussion
Chemistry
6
-Iodo-2-phenyl-4H-benzoxazin-4-one 1 was prepared from stirring 5-iodoan-
thranilic acid with benzoyl chloride in dry pyridine at room temperature [22]. Ami-
nolysis of benzoxazinone 1 was conducted through its reaction with formamide and/
or with ammonium acetate to aꢂord quinazolinone 2. The structure of compound 2
was assigned from its IR spectrum which exhibited strong absorption bands at 3165
−1
and 1675 cm corresponding to ν and ν , respectively (cf. Scheme 1).
NH
C=O
When benzoxazinone 1 reacted with hydroxylamine hydrochloride in pyridine,
3
-hydroxy-6-iodo-2-phenylquniazolin-4(3H)-one (3) was obtained. The structure of
compound 3 is conꢁrmed by IR spectrum which showed strong absorption bands
−1
1
at 3425 and 1670 cm due to ν and ν , respectively. The H NMR spectrum
OH
C=O
showed the D O exchangeable proton for the OH group at 12.07 ppm. Hydrazinoly-
2
sis of benzoxazinone 1 with hydrazine hydrate in boiling ethanol aꢂorded 3-amino-
6
-iodo-2-phenylquinazolin-4(3H)-one (4) [22].
1
3

Synthesis and anticancer activity of novel quinazolinone…
O
I
HCONH2
NH
Ph
or CH COONH
3
4
N
2
O
I
OH
Ph
NH OH.HCl
N
2
dry pyridine
N
3
O
I
NH NH H O
2
2.
2
NNH2
EtOH
N
Ph
4
H
N
S
N
S
NH
I
N
H NHN
NHNH2
EtOH
NH2
2
N
Ph
5
O
N
NH2
O
I
N
I
NH2
EtOH
O
Ph
6
N
1
Ph
R1
R1
HN
dry pyridine X
COOEt
R2
I
R
2
O
NH2
7, R
1
= Br, R
1 3 2
2
= H
8
, R = OCH , R =H
O
NH
EtOH
9, R =COCH , R = H
1 3 2
I
COOEt
10, R =H, R = OH
1
2
Ph
O
1
1, R =OH, R = H
1
2
NH2
X
O
N
NH
I
N
Ph
O
H
N
1
4, X= CN
5, X= COCH
NH
EtOH
12
1
3
Ph
O
NH2
O
Ph
I
N
H
EtOH
NH
Ph
1
O
3
Scheme 1 Reactions of benzoxazinone 1 with diꢂerent nitrogen and carbon nucleophiles
1
3

M. A. E.-A. M. El-Hashash et al.
A new fused tricyclic system containing the tetrazino moiety 5 was obtained
through the eꢂect of thiocarbonohydrazide on benzoxazinone 1. The IR spectrum
of tetrazino[1,6-c]quinazolinethione derivative 5 revealed the disappearance of the
1
carbonyl band, and the presence of absorption band at 1141 ν . H NMR revealed
C=S
the appearance of two D O exchangeable protons for −2NH signals. According to
2
our speculation, the latter product was formed as a result of nucleophilic attack of
the amino group of thiocarbonohydrazide on the electronically deꢁcient carbon
atom of benzoxazinone 1 via a tetrahedral mechanism, resulting in ring opening fol-
lowed by ring closure to aꢂord quinazolinylthiosemicarbazide derivative which was
subjected to dehydration to give tetrazino[1,6-c]quinazoline-thione derivative 5 (cf.
Scheme 2).
Reaction of benzoxazinone
1 with 3′-amino-4,4′-dimethyl-1,1′-biphenyl-
3
-ylamine under reꢀux gave 6-iodo-2-phenyl-N-(3′-amino-4,4′-dimethyl-1,1′-
biphenyl-3-yl)-quinazolin-4(3H)-one (6). On the other hand, treatment of ben-
zoxazinone 1 with nitrogen nucleophiles, namely p-bromoaniline, p-anisidine,
p-aminoacetophenone, p-aminophenol, o-aminophenol, p-aminopyridine, and/or
benzylamine, in ethanol under reꢀux aꢂorded 5-iodobenzamide derivatives 7–13.
The present investigation was extended to study the behaviour of benzoxazinone 1
towards carbon nucleophiles exempliꢁed by ethyl cyanoacetate and/or ethyl acetoac-
etate in dry pyridine to yield ethyl 3-(2-benzamido-5-iodophenyl)-2-cyano-3-oxo-
propanoate (14) and ethyl 2-(2-benzamido-5-iodobenzoyl)-3-oxobutanoate (15),
respectively (cf. Scheme 1).
Glycosylation of quinazolinone 2 with α-d-glucose pentaacetate and/or β-d-
glucose pentaacetate in ethanol gave glycosides 16 and 17, respectively. The IR
−
1
spectrum for glycosides 16 and 17 showed absorption bands at 1746 and 1751 cm
1
for acetoxy carbonyl groups. H NMR spectra of glycosides 16 and 17 showed a
H
S
N
H N
HN
2
S
H N
H
O
NH
2
HN
NH
O
O
I
N
S
S
H
I
I
O
O
+
H NHN
NHNH2
NH
2
N
1
Ph
N
Ph
Ph
O
H N NH
H
N
2
H N
2
NH
S
O
HN
O
N
N
S
I
NH
HO
I
NH
N
H
I
NH
Ph
-
H O
N
2
N
N
Ph
Ph
OH
N
H
N
S
NH
I
-
H O
2
N
N
Ph
5
Scheme 2 Proposed mechanism of formation of compound 5
1
3

Synthesis and anticancer activity of novel quinazolinone…
singlet at δ 6.33 and 5.72 ppm characteristic for anomeric protons of α-conꢁguration
and β-conꢁguration, respectively. The observation that an equatorial proton on C2
in the diastereomer 16 in consistently found further down ꢁeld by 0.61 ppm than
the axial one on the same carbon in the diastereomer 17 is due to its existence in
the deshielding cones of C3–C4 and O–C bonds. Also, when quinazolinone 2 was
submitted to react with benzyl chloride in ethanol under reꢀuxing temperature, the
3
-benzyl-4(3H)-quinazolinone 18 was isolated (cf. Scheme 3).
Thiation of quinazolinone 2 with phosphorus pentasulꢁde in dry toluene gave
-iodo-2-phenylquinazoline-4(3H)-thione (19). The behavior of quinazolinone 2
6
and/or quinazolinthione 19 towards carbon electrophile, namely ethyl chloroacetate,
in the presence of anhydrous K CO in dry acetone has also been investigated to
2
3
aꢂord the corresponding N-alkyl derivatives 20 and 21, respectively. This reaction
O
AcO
OAc
OAc
α-D-glucose
I
pentacetate
N
N
H O
Ph
EtOH
N
OAc
1
6
O
H
OAc
OAc
OAc
I
β-D-glucose
O
pentacetate
N
Ph
EtOH
AcO
O
1
7
O
I
NH
Ph
PhCH Cl
2
I
N
Ph
N
EtOH
N
Ph
2
18
S
P
2
S
5
CH COOEt
2
I
NH
Ph
Cl
toluene
anhydrous K
2 3
CO
N
S
dry acetone
COOEt
19
I
N
O
CH
Cl
2
COOEt
N
21
Ph
I
N
COOEt
P2S5
toluene
dry acetone
anhydrous K CO
2
3
N
Ph
20
EtOH N H H O
2
4. 2
CH3
N
N
Ar
H
N
O
O
COCH3
O
O
N
O
N
N
I
Ar CHO
N
COCH3
I
I
CONHNH2
CH3
N
N
EtOH
N
Ph
EtOH
Ph
22
2
3
2
4
Ar =
Cl
Scheme 3 Reactions of quinazolinone derivative 2 with diꢂerent electrophile reagents
1
3

M. A. E.-A. M. El-Hashash et al.
takes place via a direct nucleophilic displacement mechanism by a nitrogen nucleo-
phile of the predominant lactam of the partially positive saturated carbon on ethyl
2
chloroacetate via the S mechanism. The conꢁrmatory evidence of N-alkyl deriv-
N
atives 20 and 21 were obtained from the IR spectrum which exhibited absorption
bands at 1754 ν for
ester, 1680 ν for
, 1731 ν for
ester, and 1203 ν for
CO
CO
C=O
,
respectively, and disappearance of the NH band. It was found that the thiation
C=S
proceeded via reaction of N-alkyl derivative 20 with phosphorus pentasulꢁde in dry
toluene to aꢂord the corresponding thiated compound 21.
Hydrazinolysis of N-alkyl derivative 20 with hydrazine hydrate in ethanol
gave acetohydrazide 22. The pronounced biological activity [23, 24] of pyrazoles
prompted the authors to utilize the amino functionality of acetohydrazide 22 in
the design and preparation of new heterocyclic systems incorporating a pyrazole
nucleus. Thus, acetohydrazide 22 was allowed to react with acetylacetone to aꢂord
pyrazole derivative 23. On the other hand, condensation of acetohydrazide 22 with
p-chlorobenzaldehyde in ethanol yielded the acetohydrazide derivative 24 (cf.
Scheme 3).
Cytotoxicity and antitumor evaluation
Most of the synthesized compounds were screened for their anticancer activity
in vitro against two representative cell lines, hepatocellular carcinoma (HePG-2)
and mammary gland breast cancer (MCF-7). The results were expressed as growth
inhibitory concentration (IC ) values, which represent the compounds concentra-
5
0
tions required to produce a 50% inhibition of cell growth after 72 h of incubation,
compared to untreated controls (Table 1). The obtained results revealed that com-
pounds 10 and 5 were the most cytotoxic agents against the two cell lines. As for
activity against the HepG2 cell line, compound 10 was the most potent derivative
as active as doxorubicin which showed the percentage viability IC at 5.47 µg/ml,
5
0
whereas, the highest cytotoxic activities were displayed by compounds 5, 11, and 6
with IC values at 7.78, 10.02, and 10.80 µg/ml, respectively. The cytotoxic activity
5
0
against HepG2 and MCF-7 cell lines were enhanced in compound 10 when com-
pared to compounds 11 and 8. The high anticancer activity of compound 10 is due
to the presence of di-active groups (OH and NH) in the 1,2 position in its molecu-
lar structure. This structure feature has the ability to form an intramolecular hydro-
gen bonding (iHB). The H atom which is not involved in this bond will then be
abstracted by free radicals, resulting in a stable phenoxy radical [25]. On the other
hand, compound 5 exhibited cytotoxic activity against HepG2 and MCF-7 cell lines
higher than compound 6 because of the presence of two basic centers (2NH groups)
in compound 5, whereas compound 6 has only one center (NH group; cf. Fig. 1).
2
Compounds 8, 20, 21, and 22 also possessed strong anti-proliferative activities
against HepG2 cells. Furthermore, moderate inhibitory activity was also demon-
strated by compounds 4, 7, 9, 13, 15, and 23. On the other hand, several compounds
showed weak antiproliferative activities as compounds 2, 3, 12, 16, and 17. Finally,
compound 14 appeared to be inactive.
1
3

Synthesis and anticancer activity of novel quinazolinone…
Table 1 Cytotoxic activity of
Compounds
In vitro cytotoxicity IC50 (µg/ml)
synthesized compounds against
human tumor cells
HePG2
MCF-7
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
2
2
2
2
86.96 ± 3.9
83.11 ± 3.6
49.11 ± 2.5
7.78 ± 0.5
10.80 ± 1.0
44.44 ± 2.3
13.34 ± 1.3
20.50 ± 1.6
5.47 ± 0.3
10.02 ± 0.9
62.23 ± 2.7
37.40 ± 2.1
> 100
31.36 ± 1.9
79.60 ± 3.6
70.91 ± 3.1
19.48 ± 1.6
11.68 ± 1.2
16.29 ± 1.5
26.34 ± 1.8
4.50 ± 0.2
82.26 ± 4.1
90.39 ± 4.2
68.29 ± 3.8
9.26 ± 0.8
28.78 ± 2.0
57.97 ± 3.2
19.91 ± 1.7
47.56 ± 3.1
8.94 ± 0.6
14.05 ± 1.2
86.81 ± 4.2
54.76 ± 3.2
92.87 ± 4.6
43.89 ± 2.8
65.42 ± 3.5
71.72 ± 3.9
33.66 ± 2.3
26.35 ± 1.8
18.14 ± 1.5
39.68 ± 2.6
4.17 ± 0.2
0
1
2
3
4
5
6
7
0
1
2
3
DOX
IC (µg/ml): 1–10 (very strong), 11–20 (strong), 21–50 (moderate),
5
0
5
1–100 (weak), and above 100 (non-cytotoxic)
DOX doxorubicin
On the other hand, compounds 8, 11, and 22 have strong cytotoxic activity against
MCF-7, but compound 8 has lower cytotoxic activity compared to compound 11, pos-
sibly due to the introduction of an electron-donating group (OCH ) at position 4 of the
3
benzene ring, suggesting that the electronic inꢀuence of the substituents in the benzene
ring appears to play an important role in the antiproliferative activity. Compound 22
exhibited strong activity with IC at 18.14 µg/ml, and has two basic centers (NH and
5
0
NH groups; cf. Fig. 1).
2
Furthermore, interpretation of the results revealed that compounds 6, 9, 15, 20,
2
1, and 23 have moderate anticancer activity against the MCF-7 cell line with per-
centage inhibition IC at 28.78, 47.56, 43.89, 33.66, 26.35, and 39.68 µg/ml, respec-
50
tively, whereas, compounds 2, 3, 4, 7, 12, 13, 14, 16, and 17 displayed weak cytotoxic
activities.
1
3

M. A. E.-A. M. El-Hashash et al.
O
O
OH
OH
OH
OCH O
O
3
O
H C
3
NH2
OH
Doxorubicin
OCH3
OH
HN
O
HN
HN
O
I
I
OH
I
O
NH
NH
NH
Ph
O
Ph
O
0
Ph
O
8
1
11
H
N
O
S
O
N
H
N
N
I
I
NH2
N
NH2
I
NH
Ph
N
N
O
N
Ph
Ph
N
6
22
5
Fig. 1 Structure of doxorubicin and some of the designed target compounds
Experimental
All melting points were measured on a Gallenkamp melting point apparatus and
are uncorrected. The IR spectra were recorded using potassium bromide disks
1
on a Pye Unicam SP-3-300 IR spectrophotometer. H-NMR experiments were
run at 300 and 400 MHz on a Varian Mercury VX-300 NMR spectrometer using
tetramethylsilane (TMS) as internal standard in CDCl or dimethyl sulfoxide
3
(
DMSO-d ). Chemical shifts are quoted as δ. The mass spectra were recorded on
6
a Shimadzu GCMS-QP-1000EX mass spectrometers at 70 eV. All spectral meas-
urements were carried out at the Central Laboratory of Ain-Shams University and
the Main Defense Chemical Laboratory, Egypt. Biological activity was carried
in the Pharmacognosy Department, Faculty of Pharmacy, Mansoura University,
Mansoura, Egypt. All the newly synthesized compounds gave satisfactory ele-
mental analyses. The purity of the synthesized compounds were checked by thin-
layer chromatography (TLC).
1
3

Synthesis and anticancer activity of novel quinazolinone…
Synthesis
6
‑Iodo‑2‑phenylquinazolin‑4(3H)‑one (2) A solution of benzooxazinone deriva-
tive 1 (5 mmol, 1.75 g) in formamide (10 ml) was reꢀuxed for 2 h, left to cool,
then poured into ice. The crude solid product was collected by ꢁltration, dried, and
recrystallised from ethanol to give 2 as white crystal: mp > 300 °C, yield 76%.
−1
1
FT-IR (KBr, cm ): 3165 ν , 3092 ν aromatic, 1675 ν . HNMR (300 MHz,
NH
CH
C=O
DMSO-d ): 12.66 (s, 1H, NH, D O exchangeable), 8.49–7.44 (m, 8H, Ar–H). MS
6
2
+
̇
(
m/z (%)): 348 (M , 100.00), 245 (57.14), 193 (7.52), 104 (20.47), 90 (21.97), 77
(
23.29).
3
‑Hydroxy‑6‑iodo‑2‑phenylquniazolin‑4(3H)‑one (3) A mixture of benzoox-
azinone derivative 1 (5 mmol, 1.75 g) and hydroxylamine hydrochloride (5 mmol,
0
.35 g) in dry pyridine (20 ml) was reꢀuxed for 15 h, left to cool, and neutralized
by cold dilute hydrochloric acid. The precipitated solid was collected, washed with
cold water, dried, and recrystallised from toluene to aꢂord 3 as orange crystals: mp
−1
2
50 °C, yield 75%. FT-IR (KBr, cm ): 3425 ν , 3113 ν aromatic, 1670 ν
.
C=O
OH
CH
1
HNMR (300 MHz, DMSO-d ): 12.07 (s, 1H, OH, D O exchangeable), 8.53–7.51
6
2
+
̇
+̇
(
m, 8H, Ar–H). MS (m/z (%)): 366 (M + 2] , 4.08), 364 (M , 2.47), 245 (15.56),
2
17 (2.24), 140 (2.18), 105 (100.00), 77 (63.79).
1
0‑Iodo‑6‑phenyl‑2H‑[1,2,4,5]tetrazino[1,6‑c]quinazoline‑3(4H)‑thione
(5)
A
mixture of benzooxazinone derivative 1 (5 mmol, 1.75 g) and thiocarbonohydrazide
(
5 mmol, 0.53 g) in ethanol (20 ml) was heated at reꢀux temperature for 12 h, left
to cool, and the precipitated solid was collected, dried, and recrystallised from
−
1
ethanol to aꢂord 5 as yellow crystals: mp 170 °C, yield 71%. FT-IR (KBr, cm ):
1
3
8
304, 3275, 3207 ν , 1644 ν , 1141 ν . HNMR (300 MHz, DMSO-d ):
NH
C=N
C=S
6
.66–7.53 (m, 8H, Ar–H), 5.77 (s, 1H, =N–NHCS, D O exchangeable), 4.50 ( , 1H,
2
S
∔
CSNHN-, D O exchangeable). MS (m/z (%)): 419 (M , not observed), 349 (20.60),
2
2
56 (44.60), 224 (23.80), 192 (58.10), 160 (68.30), 145 (54.90), 128 (55.10), 105
(
83.20), 91 (12.40), 77 (70.60), 64 (100.00).
6
‑Iodo‑2‑phenyl‑N‑(3′‑amino‑4,4′‑dimethyl‑1,1′‑biphenyl‑3‑yl)‑quinazolin‑4(3H)‑
one (6) A mixture of benzooxazinone derivative 1 (5 mmol, 1.75 g) and 3′-amino-
4
,4′-dimethyl-1,1′-biphenyl-3-ylamine (5 mmol, 1.06 ml) in ethanol (20 ml)
was heated at reꢀux temperature for 10 h, left to cool, and the precipitated solid
was collected, dried, and recrystallised from DMF to yield 6 as brown crystals:
−1
mp > 300 °C, yield 90%. FT-IR (KBr, cm ): 3432, 3200 ν , 3060 ν aromatic,
NH2
CH
1
2
867 ν aliphatic, 1620 ν . HNMR (300 MHz, DMSO-d ): 7.59–7.30 (m, 14H,
CH C=O
6
Ar–H), 3.60 (s, 2H, NH , D O exchangeable), 2.49 ( , 6H, 3CH ). MS (m/z (%)):
2
2
S
3
∔
5
44 (M , 1.29), 465 (25.90), 334 (33.77), 204 (58.13), 127 (32.99), 104 (60.49), 77
(
100.00).
Reaction of 6‑iodo‑2phenyl‑4H‑benzo[d][1,3]oxazine‑4‑one (1) with substituted
amines; General procedure
1
3

M. A. E.-A. M. El-Hashash et al.
A mixture of benzooxazinone derivative 1 (5 mmol, 1.75 g) and substituted
amines, namely p-bromoaniline, p-anisidine, p-aminoacetophenone, o-aminophenol,
p-aminophenol, p-aminopyridine, and/or benzylamine (5 mmol), in ethanol (20 ml)
was heated at reꢀux temperature for 8–12 h, left to cool, and the solid product was
collected, dried, and recrystallised to give 7–13, respectively.
2
‑(Benzoylamino)‑N‑(4‑bromophenyl)‑5‑iodobenzamide (7) Yellow crystals, mp
−1
2
45 °C (toluene), yield 60%. FT-IR (KBr, cm ): 3278, 3113 ν , 3065 ν aro-
NH CH
1
matic, 1645 ν . HNMR (300 MHz, DMSO-d ): 11.42 (s, 1H, NH, D O exchange-
C=O
6
2
able), 10.66 (s, 1H, NH, D O exchangeable), 8.21–7.53 (m, 12H, Ar–H). MS (m/z
2
∔
∔
(
(
%)): 520 (M , not observed), 502 (M–H O] , 3.00), 349 (68.40), 305 (20.70), 178
2
21.10), 105 (100.00), 77 (94.00), 51 (14.60).
2
‑(Benzoylamino)‑5‑iodo‑N‑(4‑methoxyphenyl)benzamide (8) Yellow crystals, mp
−1
1
90 °C (toluene), yield 81%. FT-IR (KBr, cm ): 3270, 3135 ν , 3065 ν aro-
NH CH
1
matic, 2995 ν aliphatic, 1646 ν . HNMR (300 MHz, DMSO-d ): 11.80 (s, 1H,
CH
C=O
6
NH, D O exchangeable), 10.49 (s, 1H, NH, D O exchangeable), 8.52–6.63 (m, 12H,
2
2
∔
∔
Ar–H), 3.70 ( , 3H, OCH ). MS (m/z (%)): 472 (M , not observed), 454 (M–H O] ,
S
3
2
0
5
.34), 424 (10.20), 349 (26.30), 223 (44.30), 179 (35.10), 105 (100.00), 77 (74.90),
1 (18.40).
N‑(4‑Acetylphenyl)‑2‑(benzoylamino)‑5‑iodobenzamide (9) Pale yellow crys-
−
1
tals, mp 240 °C (ethanol), yield 60%. FT-IR (KBr, cm ): 3113 ν , 3058 ν aro-
NH
CH
1
matic, 2957 ν aliphatic, 1701 ν
ketone, 1683 ν
amide. HNMR (300 MHz,
CH
C=O
C=O
DMSO-d ): 12.12 (s, 2H, 2NH, D O exchangeable), 8.53–7.23 (m, 12H, Ar–H),
6
2
∔
∔
2
.59 ( , 3H, COCH ). MS (m/z (%)): 484 (M , not observed), 466 (M–H O] , 0.54),
S
3
2
4
40 (1.00), 349 (60.50), 223 (26.60), 179 (21.60), 105 (100.00), 77 (66.30), 51
(
16.90).
2
‑(Benzoylamino)‑N‑(2‑hydroxyphenyl)‑5‑iodobenzamide (10) Black crystals,
−
1
mp 180 °C (toluene), yield 70%. FT-IR (KBr, cm ): 3497 ν , 3385 ν , 3057
OH
NH
1
ν
aromatic, 1667 ν . HNMR (300 MHz, DMSO-d ): 10.03 (s, 1H, NH,
CH
C=O
6
D O exchangeable), 9.57 (s, 1H, NH, D O exchangeable), 9.04 (s, 1H, OH, D O
2
2
2
∔
exchangeable), 8.52–6.42 (m, 12H, Ar–H). MS (m/z (%)): 458 (M , not observed),
∔
4
40 (M–H O] , 30.00), 348 (92.50), 286 (84.00), 245 (100.00), 217 (10.20), 193
2
(
14.60), 105 (30.50), 90 (28.90), 77 (40.20), 63 (14.40).
2
‑(Benzoylamino)‑N‑(4‑hydroxyphenyl)‑5‑iodobenzamide (11) Yellow crystals,
−1
mp 186–190 °C (ethanol), yield 70%. FT-IR (KBr, cm ): 3414 ν , 3312 ν
,
OH
NH
1
3
062 ν aromatic, 1688 ν . HNMR (300 MHz, DMSO-d ): 11.87 (s, 1H, NH,
CH
C=O
6
D O exchangeable), 10.41 (s, 1H, NH, D O exchangeable), 9.35 (s, 1H, OH, D O
2
2
2
∔
exchangeable), 8.52–6.56 (m, 12H, Ar–H). MS (m/z (%)): 458 (M , not observed),
3
9
95 (29.80), 282 (2.20), 218 (2.30), 207 (18.40), 191 (2.90), 133 (2.20), 105 (99.90),
1 (4.40), 77 (39.10).
1
3

Synthesis and anticancer activity of novel quinazolinone…
2
‑(Benzoylamino)‑5‑iodo‑N‑pyridin‑4‑ylbenzamide (12) Black crystals, mp 140 °C
−1
(
toluene), yield 60%. FT-IR (KBr, cm ): 3249 ν , 3059 ν aromatic, 1688, 1669
NH
CH
1
ν
8
. HNMR (300 MHz, DMSO-d ): 11.48 (s, 2H, 2NH, D O-exchangeable),
C=O
6
2
∔
∔
.34–7.57 (m, 12H, Ar–H). MS (m/z (%)): 443 (M , not observed), 425 (M–H O] ,
2
0
.07), 396 (73.00), 349 (43.00), 105 (100.00), 91 (58.00), 77 (50.50), 63 (49.00).
2
‑(Benzoylamino)‑N‑benzyl‑5‑iodobenzamide (13) Yellow crystals, mp 150 °C
−1
(
toluene), yield 96%. FT-IR (KBr, cm ): 3298 ν , 2922 ν aliphatic, 1675 ν
.
C=O
NH
CH
1
HNMR (300 MHz, DMSO-d ): 12.45 (s, 2H, 2NH, D O-exchangeable), 8.49–7.24
6
2
∔
(
m, 13H, Ar–H), 4.03 ( , 2H, CH -NH). MS (m/z (%)): 456 (M , not observed), 438
S 2
∔
(
M–H O] , 2.15), 368 (44.96), 299 (40.29), 285 (73.79), 236 (100.00), 211 (49.32),
2
1
71 (21.15), 152 (42.01), 123 (44.79), 104 (24.85).
Reaction of 6‑iodo‑2phenyl‑4H‑benzo[d][1.3]oxazine 4‑one (1) with ethyl cyanoac‑
etate and/or ethyl acetoacetate; General procedure
To a solution of benzooxazinone derivative 1 (5 mmol, 1.75 g) in dry pyridine
(
20 ml), ethyl cyanoacetate and/or ethyl acetoacetate (5 mmol) was added. The mix-
ture was heated at reꢀuxing temperature for 48 h, left to cool and poured into ice;
the crude solid product deposited was collected by ꢁltration and washed with water,
dried, and recrystallised to aꢂord 14 and 15, respectively.
Ethyl 3‑(2‑benzamido‑5‑iodophenyl)‑2‑cyano‑3‑oxopropanoate (14) Brown crys-
−
1
tals, mp 113–115 °C (ethanol), yield 90%. FT-IR (KBr, cm ): 3248 ν , 3057
NH
ν
aromatic, 2959 ν aliphatic, 2215 ν , 1757 ν ester, 1673 ν . MS (m/z
CH
CH
CN
CO
C=O
∔
∔
(
%)): 462 (M , not observed), 444 (M–H O] , 3.16), 395 (16.32), 372 (59.44), 305
2
(
13.20), 105 (3.84), 77 (8.64).
Ethyl 2‑(2‑benzamido‑5‑iodobenzoyl)‑3‑oxobutanoate (15) Black crystals, mp
−
1
1
2
07 °C (ethanol), yield 89%. FT-IR (KBr, cm ): 3248 ν , 3057 ν aromatic,
NH CH
1
985 ν aliphatic, 1756 ν ester, 1690 ν
ketone, 1669 ν
amide. HNMR
C=O
CH
CO
C=O
(
300 MHz, DMSO-d ): 11.48 (s, 1H, NH, D O exchangeable), 8.37–7.47 (m, 8H,
6
2
Ar–H), 4.35 (q, 2H, CH CH , J = 7.2 Hz), 3.44 ( , 1H, CH–), 2.69 (s, 3H, COCH ),
2
3
S
3
∔
1
.31 (t, 3H, CH CH , J = 7.2 Hz). MS (m/z (%)): 479 (M , not observed), 461 (M–
2 3
∔
H O] , 0.04), 395 (28.50), 349 (25.20), 305 (53.00), 105 (100.00), 77 (41.40), 63
2
(
38.00).
Reaction of 6‑iodo‑2‑phenylquinazolin‑4(3H)‑one (2) with α‑d‑glucose pentaacetate
and/or β‑d‑glucose pentaacetate; General procedure
A mixture of quinazolinone 2 (5 mmol, 1.75 g), α-d-glucose pentaacetate, and/or
β-d-glucose pentaacetate (5 mmol) in absolute ethanol (20 ml) was heated at reꢀux
temperature for 48 h and left to cool; the crude solid product deposited after cooling
was collected by ꢁltration, dried, and recrystallised to give 16 and 17, respectively.
(
2R,3R,4S,5R,6S)‑2‑Acetoxymethyl)‑6‑(6‑iodo‑4‑oxo‑2‑phenylquinazolin‑3(4H)‑yl)
tetrahydro‑2H‑pyran‑3,4,5‑triyltriacetate (16) Black crystals, mp 209–210 °C
1
3

M. A. E.-A. M. El-Hashash et al.
−
1
(
benzene), yield 60%. FT-IR (KBr, cm ): 3165 ν aromatic, 2922 ν aliphatic,
CH
CH
1
1
746 ν ester, 1676 ν
amide. HNMR (400 MHz, CDCl ): 8.65–7.35 (m, 8H,
CO
C=O 3
Ar–H), 6.33 (s, 1H, -N–CH–O–), 5.47–5.09 (m, 3H, 3CHOCO), 4.25 (d, 1H, O–
CH–CH –, J = 2.7 Hz), 4.10 (d, 1H, O–CH–CH –, J = 2.4 Hz), 3.73 (t, 1H, O–CH–
2
2
CH –, J = 5.1 Hz), 2.17–2.00 (m, 12H, 4COCH ). MS (m/z (%)): 349 (3.00), 331
2
3
(
39.00), 287 (4.00), 242 (21.30), 115 (100.00).
(
2R,3R,4S,5R,6R)‑2‑Acetoxymethyl)‑6‑(6‑iodo‑4‑oxo‑2‑phenylquinazolin‑3(4H)‑yl)
tetrahydro‑2H‑pyran‑3,4,5‑triyltriacetate (17) Brown crystals, mp 122–123 °C
−1
(
benzene), yield 75%. FT-IR (KBr, cm ): 3165 ν aromatic, 2974ν aliphatic,
CH
CH
1
1
751 ν ester, 1675 ν
amide. HNMR (400 MHz, CDCl ): 8.65–7.35 (m, 8H,
CO
C=O 3
Ar–H), 5.72 (s, 1H, –N–CH–O–), 5.27–5.10 (m, 3H, 3CHOCO), 4.30–4.25 (m,
2
4
H, O–CH–CH –), 4.12 (t, 1H, O–CH–CH –, J = 1.8 Hz), 2.17–2.01 (m, 12H,
2 2
COCH ). MS (m/z (%)): 448 (1.00), 434 (69.80), 361(100.00), 349 (33.10), 245
3
(
24.00), 204 (42.80).
3
‑Benzyl‑6‑iodo‑2‑phenylquinazolin‑4(3H)‑one (18) A mixture of quinazolinone 2
(
5 mmol, 1.75 g) and benzylchloride (5 mmol, 0.63 ml) in absolute ethanol (20 ml)
was heated at reꢀux temperature for 12 h and left to cool; the crude solid prod-
uct deposited after cooling was collected by ꢁltration, dried, and recrystallised from
−
1
benzene to give 18 as beige crystals: mp 227–228 °C, yield 70%. FT-IR (KBr, cm ):
1
3
061 ν aromatic, 2923 ν aliphatic, 1676 ν
amide. HNMR (400 MHz,
CH
CH
C=O
∔
CDCl ): 8.58–7.25 (m, 13H, Ar–H), 4.46 (s, 2H, –CH Ph). MS (m/z (%)): 438 (M ,
3
2
5
1
.15), 395 (100.00), 367 (84.34), 322 (40.54), 286 (30.47), 272 (96.50), 127 (68.34),
05 (18.38), 77 (46.92).
6
‑Iodo‑2‑phenylquinazoline‑4(3H)‑thione (19) A mixture of quinazolinone 2
(
5 mmol, 1.75 g) and phosphorus pentasulꢁde (5 mmol, 1.11 g) in absolute toluene
(
20 ml) was heated at reꢀux temperature for 24 h. The reaction mixture was ꢁltered
oꢂ while hot and concentrated. The crude solid product deposited after cooling was
collected by ꢁltration, dried, and recrystallised from DMF to give 19 as yellow crys-
−1
tals: mp 247–248 °C, yield 65%. FT-IR (KBr, cm ): 3239 ν , 3061 ν aromatic,
NH
CH
∔
1
200 ν . MS (m/z (%)): 364 (M , 100.00), 330 (89.13), 204 (27.76), 134 (13.36),
C=S
1
01 (16.37), 77 (22.03).
Ethyl 2‑(6‑iodo‑4‑oxo‑2‑phenylquinazolin‑3(4H)‑yl)acetate (20) A mixture of
quinazolinone 2 (10 mmol, 3.48 g), ethyl chloroacetate (10 mmol, 1 ml), and anhy-
drous potassium carbonate 5 g in dry acetone (50 ml) was heated in a water bath for
2
4 h. The excess solvent was evaporated and the reaction mixture was then poured
into water, and the crude solid product was collected by ꢁltration, dried, and recrys-
tallised from ethanol to give 20 as white crystals: mp 122–123 °C, yield 60%. FT-IR
−1
(
KBr, cm ): 3065 ν aromatic, 2978 ν aliphatic, 1754 ν ester, 1680 ν
.
C=O
CH
CH
CO
1
HNMR (300 MHz, DMSO-d ): 8.52–7.52 (m, 8H, Ar–H), 5.28 (s, 2H, CH ), 4.20
6
2
(
q, 2H, CH CH , J = 7.2 Hz), 1.19 (t, 3H, CH CH , J = 7.2 Hz). MS (m/z (%)):
2 3 2 3
1
3

Synthesis and anticancer activity of novel quinazolinone…
∔
4
34 (M , 3.58), 361 (71.27), 331 (21.56), 205 (24.46), 204 (33.52), 104 (15.42), 77
(
18.09).
Ethyl 2‑(6‑iodo‑2‑phenyl‑4‑thioxoquinazolin‑3(4H)‑yl)acetate (21) Method A A
solution of compound 20 (5 mmol, 2.2 g) and phosphorus pentasulꢁde (5 mmol,
1
.1 g) in dry toluene (20 ml) was heated at reꢀux temperature for 1 h, ꢁltered while
hot, and left to cool: the solid product was collected by ꢁltration, dried, and recrys-
tallised from dioxane to give 21 as red crystals: mp 120–122 °C, yield 40%. FT-IR
−
1
(
KBr, cm ): 3062 ν aromatic, 2965 ν aliphatic, 1731 ν ester, 1203 ν
.
C=S
CH
CH
CO
1
HNMR (300 MHz, CDCl ): 8.96–7.36 (m, 8H, Ar–H), 4.70 (s, 2H, CH ), 4.29 (q,
3
2
2
H, CH CH , J = 6.9 Hz), 1.31 (t, 3H, CH CH , J = 6.9 Hz). MS (m/z (%)): 450
2 3 2 3
∔
(
M , 9.11), 377 (36.99), 360 (19.92), 331 (40.18), 250 (100.00), 204 (69.11), 177
14.36), 104 (14.52), 77 (12.89).
(
Method B A mixture of quinazolinthione 19 (10 mmol, 3.48 g), ethyl chloro-
acetate (10 mmol, 1 ml), and anhydrous potassium carbonate 5 g in dry acetone
(
50 ml) was heated in a water bath for 24 h. The excess solvent was evaporated and
the reaction mixture was poured into water; the crude solid product was collects by
ꢁ
ltration, dried, and recrystallised from dioxane to give 21 as red crystals: mp 122–
1
23 °C, yield 60%.
2
‑(6‑Iodo‑4‑oxo‑2‑phenylquinazolin‑3(4H)‑yl)acetohydrazide (22) A mixture of
compound 20 (5 mmol 2.2 g) and hydrazine hydrate (5 mmol, 0.25 ml) in abso-
lute ethanol (30 ml) was heated at reꢀux temperature for 10 h and left to cool: the
crude solid product deposited after cooling was collected by ꢁltration, dried, and
recrystallised from ethanol to give 22 as yellow crystals: mp > 300 °C, yield 75%.
−1
FT-IR (KBr, cm ): 3400, 3307 ν
, 3078 ν aromatic, 2989 ν aliphatic,
CH CH
NH2,NH
1
1
8
670 ν . HNMR (300 MHz, DMSO-d ): 9.40 (s, 1H, NH, D O exchangeable),
C=O
6
2
.39–7.47 (m, 8H, Ar–H), 4.96 (s, 2H, CH –CO), 4.29 (s, 2H, NH , D O exchange-
2
2
2
∔
able). MS (m/z (%)): 420 (M , 0.98), 348 (22.58), 313 (100.00), 285 (55.21), 256
(
20.70), 245 (27.04), 228 (12.58), 158 (11.62), 95 (15.52), 55 (11.16).
3
‑(2‑(3,5‑Dimethyl‑1H‑pyrazol‑1‑yl)‑2‑oxoethyl)‑6‑iodo‑2‑phenylquinazo‑
lin‑4(3H)‑one (23) A mixture of acetohydrazide derivative 22 (2.5 mmol, 1 g) and
acetyl acetone (2.5 mmol, 0.25 ml) in ethanol (20 ml) was heated at reꢀux tem-
perature for 7 h, left to cool, and the crude solid product deposited was collected by
ꢁ
ltration, dried, and recrystallised from ethanol to give 23 as yellow crystals: mp
−1
1
55–157 °C, yield 50%. FT-IR (KBr, cm ): 3096 ν aromatic, 2984 ν aliphatic,
CH CH
1
1
674 ν . HNMR (300 MHz, DMSO-d ): 9.21–7.52 (m, 8H, Ar–H), 6.20 ( , 1H,
C=O
6
S
pyrazolo-H), 4.80 (s, 2H, CH –C=O), 2.73 (s, 3H, CH ), 2.41 (s, 3H, CH ). MS
2
3
3
∔
(
(
m/z (%)): 484 (M , 0.81), 453 (6.41), 331 (34.42), 312 (12.46), 258 (21.18), 204
20.03), 136 (100.00).
N’‑(4‑Chlorobenzylidene)‑2‑(6‑iodo‑4‑oxo‑2‑phenylquinazolin‑3(4H)‑yl)aceto‑
hydrazide (24) A mixture of acetohydrazide derivative 22 (2.5 mmol, 1 g) and
p-chlorobenzaaldehyde (2.5 mmol, 0.35 g) in ethanol (20 ml) was heated at reꢀux
1
3

M. A. E.-A. M. El-Hashash et al.
temperature for 12 h, left to cool, and the crude solid product deposited after cool-
ing was collected by ꢁltration, dried, and recrystallised from dioxane to give 24 as
−
1
1
yellow crystals: mp 240–245 °C, yield 66%. FT-IR (KBr, cm ): 3210 ν , 3056
NH
ν
aromatic, 2977 ν aliphatic, 1708, 1665 ν , 1619 ν . HNMR (300 MHz,
CH
CH
C=O
C=N
DMSO-d ): 11.92 (s, 1H, NH, D O-exchangeable), 9.20 (s, 1H, CH=N–), 8.54–7.52
6
2
∔
(
m, 12H, Ar–H), 4.32 (s, 2H, COCH ). MS (m/z (%)): 542 (M , 3.07), 433 (14.13),
2
3
55 (13.19), 139 (19.11), 116 (19.17), 70 (100.00).
Cytotoxicity and antitumor evaluation
Materials and methods
Hepatocellular carcinoma (HEPG-2) and mammary gland breast cancer (MCF-7) cell
lines were obtained from ATCC via a holding company for biological products and
vaccines (VACSERA), Cairo, Egypt.
Doxorubicin was used as a standard anticancer drug for comparison.
Procedure MTT assay
The cell lines mentioned above were used to determine the inhibitory eꢂects of the
tested compounds on cell growth using the MTT assay. This colorimetric assay is based
on the conversion of the yellow tetrazolium bromide (MTT) to a purple formazan
derivative by mitochondrial succinate dehydrogenase in viable cells. Cell lines were
cultured in RPMI-1640 medium with 10% fetal bovine serum. Antibiotics added were
1
00 units/ml penicillin and 100 µg/ml streptomycin at 37 °C in a 5% CO incubator.
2
4
The cell lines were seeded in a 96-well plate at a density of 1.0 × 10 cells/well at 37 °C
for 48 h under 5% CO . After incubation the cells were treated with diꢂerent concentra-
2
tions of compounds and incubated for 24 h. After 24 h of drug treatment, 20 µl of MTT
solution at 5 mg/ml was added and incubated for 4 h. DMSO (100 µl) was added into
each well to dissolve the formed purple formazan. The colorimetric assay was meas-
ured and recorded at an absorbance of 570 nm using a plate reader (EXL 800, USA).
The relative cell viability in percentage was calculated as: (A570 of treated samples/
A570 of untreated sample) × 100 [26, 27].
Calculation of the IC50 for each compound
Cytotoxicity was estimated as the concentration that caused approximately 50% loss
of the monolayer. The assay was used to examine the newly synthesized compounds.
Doxorubicin was used as a standard anticancer drug for comparison. To calculate IC ,
50
you would need a series of dose-response data (e.g., drug concentrations x1, x2, xn and
growth inhibition y1, y2, yn). The values of y are in the range of 0–1.
1
3

Synthesis and anticancer activity of novel quinazolinone…
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