Synthesis of New Benzoxazinone, Quinazolinone, and Pyrazoloquinazolinone Derivatives and Evaluation of Their Cytotoxic Activity Against Human Breast Cancer Cells

Article abstract of DOI:10.1002/jhet.3158

Synthesis of 2-[3-(3,4-dichlorophenyl)-3-oxoprop-1-enyl]-4H-benzo[3,1]oxazin-4-one 2 as a precursor to synthesize quinazolinones 5, 7 pyrazolylquinazolinones, 4, 12, 13, 14, 16 benzothiazine thione 10, benzene derivative 3 pyrroloquinazolinone 6, benzoimidazoquinazolinone 8, and pyrazolotriazepinoquinazolinone 15. The structures of the newly synthesized compounds were established by their elemental analyses and spectral data. Most of the synthesized compounds exhibited high antitumor and cytotoxic activity against human breast cancer cells.

Full text of DOI:10.1002/jhet.3158

Month 2018
Synthesis of New Benzoxazinone, Quinazolinone, and
Pyrazoloquinazolinone Derivatives and Evaluation of Their Cytotoxic
Activity Against Human Breast Cancer Cells
a
Eman A. El-Bordany
and Rania S. Alib,c
*
aDepartment of Chemistry, Faculty of Science, Ain Shams University, Cairo 11566, Egypt
bFaculty of Industrial Education, Helwan University, Cairo 11795, Egypt
cDepartment of Chemistry, Faculty of Science, Taif University, Taif 21974, Saudi Arabia
*
E-mail: emanslime@yahoo.com
Received December 6, 2017
DOI 10.1002/jhet.3158
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
Synthesis of 2-[3-(3,4-dichlorophenyl)-3-oxoprop-1-enyl]-4H-benzo[3,1]oxazin-4-one 2 as a precursor to
synthesize quinazolinones 5, 7 pyrazolylquinazolinones, 4, 12, 13, 14, 16 benzothiazine thione 10, benzene
derivative 3 pyrroloquinazolinone 6, benzoimidazoquinazolinone 8, and pyrazolotriazepinoquinazolinone
15. The structures of the newly synthesized compounds were established by their elemental analyses and
spectral data. Most of the synthesized compounds exhibited high antitumor and cytotoxic activity against hu-
man breast cancer cells.
J. Heterocyclic Chem., 00, 00 (2018).
INTRODUCTION
reported that different synthesized pyrazolyl derivatives
showed anticancer activity because of their B-Raf
inhibitory activities [24–27]. Cancer is one of the most
dangerous, fast propagating diseases and causes death. This
encouraged us to synthesize some novel benzoxazinone,
and quinazolinone ring system attached to a pyrazole
moiety. Such skeleton appeared of highly promising
antitumor activity, to evaluate their in vitro anticancer
activities against breast cancer.
Compounds of benzoxazinone and quinazolinone
attracted much attention of chemists [1–8]. Benzoxazinone
derivatives have broad range of biological activities,
such as protein methyltransferases inhibitors [9],
antifungal [10,11], anticancer activity [12], treatment of
cardiovascular diseases [13–16], and antibacterial [17]. On
the other hand, quinazolin-4(3H)-one is a heterocyclic
alkaloid ring system frequently encountered in medicinal
chemistry [18]. These alkaloids have attracted focus by
synthetic and medicinal chemists. Quinazolin-4(3H)-one
structure analogous has been found to have different
biological properties including anticonvulsant, sedative,
RESULTS AND DISCUSSION
Chemistry. The synthesis of 2-[3-(3,4-dichlorophenyl)
analgesic,
tranquilizer,
antimicrobial,
anesthetic,
-3-oxoprop-1-en-1-yl]-4H-3,1-benzoxazin-4-one
2
was
antihypertensive, antiviral, anticancer, anti-inflammatory,
diuretic, and muscle relaxant [19–23]. Also, it has been
performed by heating 2-(4-(3,4-dichlorophenyl)-4-oxobut-
2-enamido)benzoic acid 1 [28] in freshly distilled acetic
© 2018 Wiley Periodicals, Inc.

E. A. El-Bordany and R. S. Ali
Vol 000
anhydride as shown in (Scheme 1). The structure of
compound 2 was confirmed by its spectral data, its
infrared (IR) spectrum reveals two strong absorption
bands at 1759 cmꢀ1 (C═O benzoxazine) and 1671 cmꢀ1
(C═O unsaturated ketone). Further support for the
(Scheme 2). The structures of compounds 3 and 4 are
substantiated from their IR spectra that exhibit absorption
1
bands corresponding to NH and C═O groups. H NMR
spectrum of compound 3 supports its structure as it
shows multiplet signals for –CH2─CH– group, aromatic
protons, and exchangeable signals for protons of NH and
NH2 groups. 1H NMR spectrum of compound 4 is in
accord with the proposed structure as it shows the signals
corresponding to ABX pattern of the –CH─CH2– moiety.
The magnetic nonequivalence of the protons Ha and Hb
of the methylene group shows an AB system associated
with these protons, also constitutes a geminal pairs with
coupling constant (J = 17.1, 16.95 Hz), and afforded the
eight lines of AB part of an ABX system. The fact that
the protons of methelene group behaves as an AB part of
an ABX system can be attributed either to the attachment
of this group to an asymmetric center or its presence in a
rigid cyclic system. The mass spectra of compounds 3
1
assigned structure is gained from its H NMR spectrum
that shows two doublet signals due to two olefinic
protons at δ = 7.72, 8.10 ppm with coupling constant
J = 17.6 Hz in accord with its existence in the E-
configuration.
Several interesting reactions were performed depending
on the functionality of benzoxazinone derivative 2 with
various nucleophiles to yield important novel derivatives
with promising biological activities.
Stirring a solution of benzoxazinone 2 in n-butanol with
hydrazine hydrate at room temperature afforded the
hydrazide derivative 3. However, upon heating the same
mixture, it yielded the quinazolinone derivative
4
Scheme 1. Synthesis of 2-[3-(3,4-dichlorophenyl)-3-oxoprop-1-en-1-yl]-4H-3,1-benzoxazin-4-one.
Scheme 2. Reactions of 2-(3-(3,4-dichlorophenyl)-3-oxoprop-1enyl)-4H-benzo[d][1,3]oxazin-4-one 2 with various active nitrogen nucleophiles.
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Month 2018 Synthesis of New Benzoxazinone, Quinazolinone, and Pyrazoloquinazolinone Derivatives
and Evaluation of Their Cytotoxic Activity Against Human Breast Cancer Cells
and 4 do not show the signals for molecular ion peaks; this
may be due to their ease decomposition in the ionization
chamber, but they show some important peaks (cf.
Experimental). However, heating compound 3 in boiling
n-butanol for 3 h yielded compound 4.
afforded the Z-configuration isomer imidazoquinazolinone
8 via hetero ring opening of oxazinone nucleus followed
by cyclization (Scheme 2). The IR spectrum of compound
8 shows bands characteristic for C═O and C═N groups.
Further evidence is gained from its 1H NMR; the Z-
configuration has been assigned to 8 based on the
appearance of two doublet signals for the olefinic protons
at δ 8.42, 9.64 ppm with coupling constant J = 9.9 Hz.
The reaction of benzoxazinone derivative 2 with sulfur-
containing compounds such as thiophenol and phosphorus
pentasulfide yielded another interesting bioactive
phenylthiobenzoxazinone and benzothiazine thione
derivatives 9 and 10, respectively (Scheme 3). The
structures of compounds 9 and 10 were confirmed from
their analytical as well as spectral data. Thus, their IR
spectra exhibit bands characteristic for C═O and C═N
groups as well as an absorption band for C═S group in
case of compound 10. The 1H NMR spectrum of
compound 9 shows the multiplet signals of ABX pattern
of –CH─CH2– moiety instead of signals of the vinyl
protons, which supports the addition of thiophenol to the
β-carbon of the enone system. However, the appearance
of two doublet signals in the 1H NMR spectrum of
compound 10 at δ 6.42, 7.11 ppm with J = 13 suggests
its existence in the E-configuration. Mass spectra of
compound 10 shows the molecular ion peak at m/z = 378
(Scheme 3).
Treatment of benzoxazinone derivative 2 with formamide
yielded quinazolinone derivative 5 (Scheme 2). The
structure of 5 is evidenced from its analytical as well as
spectral data. Thus, IR spectrum of 5 shows absorption
bands characteristic for NH, NH2, and C═O groups.
1
Further support is gained from its H NMR spectrum that
shows multiplet signals for protons of –CH2─CH– group
as well as signals for aromatic, NH, and NH2 protons. The
downfield value for the signal of NH2 protons is in accord
with the existence of compound 5 as the chelated form
shown (Scheme 2). Refluxing ethyl glycinate
hydrochloride with benzoxazinone 2 in dry pyridine
yielded quinazolinone 6 (Scheme 2). The IR spectrum of 6
exhibits absorption bands characteristic to NH and C═O
groups. Furthermore, the 1H NMR of 6 supports its
structure as it shows signals for alkyl and aryl protons as
well as an exchangeable broad singlet signal in the
downfield region for NH proton. The appearance of
signals corresponding to protons of two ethyl groups as
well as an exchangeable broad singlet signal in the
downfield region for NH proton is a good evidence for the
addition of another mole of ethyl glycinate to α,β-
unsaturated enone system. In addition, the higher δ value
for the signal of NH proton and the lower absorption
frequency of ester group suggest the chelation shown.
Heating of compound 2 in n-butanol with piperidine gives
the benzoic acid derivative 1 [28]. It seems that piperidine
acts as a base that catalyzes the ring opening of
benzoxazinone. Refluxing of benzoxazinone derivative 2
with ethanolamine in n-butanol gave the quinazolinone
derivative 7 (Scheme 2). The IR spectrum of compound 7
shows absorption bands due to OH, CH2, C═O, and C═N
The aim in this part of work was to attach new groups at
position-3 of pyrazolylquinazolinone 4 on the hope of
getting new biologically active pharmacophores.
Therefore, when compounds 3 and 4 were submitted to
react with O-chlorobenzaldehyde, our expectation is the
formation of the same product. Actually, different
products are obtained. Compound 3 reacted with two
moles of aldehydes producing compound 11; however,
compound 4 reacted with one mole of aldehydes to
afford the quinazolinone derivative 12 (Scheme 4). The
structures of compounds 11 and 12 are substantiated from
their micro analytical and spectral data. The IR spectra
shows absorptions due to NH and C═O groups. Further
1
groups. While the H NMR supports the structure as it
exhibits two triplets signals for protons of –CH2─CH2–
group as well as exchangeable broad singlet signal for OH
proton. Furthermore, when compound 2 was allowed to
react with O-phenylinediamine in boiling butanol, it
1
support for their structures is gained from their HNMR
spectra where the devoid of any signals for protons of the
Scheme 3. Reactions of 2-(3-(3,4-dichlorophenyl)-3-oxoprop-1enyl)-4H-benzo[d][1,3]oxazin-4-one 2 with sulfur containing compounds.
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E. A. El-Bordany and R. S. Ali
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Scheme 4. Utility of 3-amino-2-(3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)quinazolin-4(3H)-one (4) as nitrogen nucliophile.
two NH2 groups and appearance of three broad singlet
signals for protons of three NH groups is a good
evidence for the incorporation of two moles of aldehyde,
and this supports structure 11. In addition, 1HNMR
spectrum supports structure 12 as it is also devoid of any
signals for protons of the NH2 group and exhibits one
broad singlet signal for NH proton, which is in accord
with the condensation with one mole of aldehyde. The
EIM spectrum of compound 11 does not show the
molecular ion peak, probably because of its ease
decomposition in the ionization chamber; however, it
exhibits the molecular ion peak of compound 12 beside
Further evidence is gained from their 1H NMR
spectra where they show signals due to NH, alkyl, and
aromatic protons. A good evidence for the suggested
structures comes from the disappearance of the signal
corresponding to protons of NH2 group.
When compound
4
is allowed to react with
chloroacetonitrile or acetyl chloride, it affords compounds
15 and 16 (Scheme 4). The IR spectrum of compound 15
exhibits characteristic bands of NH, C═O, and C═N
groups and devoid of any band corresponding to CN
group. A good evidence for the structure of compound 15
1
is obtained from its H NMR spectrum as it shows an
some abundant peaks. Treatment of
4
with O-
exchangeable broad singlet signals for protons of NH2
group at δ 5.16 ppm in addition to broad singlet signal
for proton of NH group at δ 14.11 ppm. The higher δ
value for the signal of NH proton and the lower
absorption frequency of C═O group in azepine nucleus
methylbenzaldehyde and/or propionaldehyde in boiling n-
butanol afforded quinazolinone derevatives 13 and 14,
respectively (Scheme 4). The IR spectra of 13 and 14
exhibit bands due to NH, C═O, and C═N groups.
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Month 2018 Synthesis of New Benzoxazinone, Quinazolinone, and Pyrazoloquinazolinone Derivatives
and Evaluation of Their Cytotoxic Activity Against Human Breast Cancer Cells
suggest the chelation shown in Scheme 4. The structure of
compound 16 is in a good agreement with its analytical and
spectral data (cf. Experimental).
(IC50 = 71.73 μg/mL), and compound 11 had the lowest
cytotoxicity effect (IC50 = 102.35 μg/mL). No significant
difference recorded between compounds 13
(IC50 = 105.4 μg/mL) and 16 (IC50 = 106.03 μg/mL).
As depicted from the biological assessment results, the
synthesized compounds exhibited broad spectrum and
higher antitumor activity. The substitution at derivatives
13 and 16 was not suitable for cytotoxic properties
against used breast cell line. The reduction of activity
observed in some prepared compounds may be attributed
to its low solubility of the main medium used in NR
assay. The cytotoxic activity of prepared derivatives in
different experiments was mainly related to its
intermediate metabolites because they are capable of
inducing a frequency of damages in the DNA and
apoptosis in human breast cancer cells.
ACUTE TOXICITY AND ANTITUMOR ACTIVITY
Cytotoxicity effects.
Cytotoxicity assay experiments
were conducted to determine the effects of the newly
synthesized hybrid molecules on the cell viability of
human breast carcinoma cells by using the neutral red
(NR) assay method as previously described. The
cytotoxicity test provides a quantitative estimation of a
number of viable cells in a culture, where it is based on
the ability of viable cells to incorporate and bind the
supravital dye neutral red in the lysosomes [29]. The
analysis of the cancer cell viability showed that all the
tested complexes are cytotoxic to target carcinoma cells
(Table 1). The cytotoxic effect is a dependent factor, as
an increase of cell line viability is related to decrease of
the tested compounds' concentrations.
EXPERIMENTAL
Chemistry.
Melting points of the synthesized
IC50 values. The IC50 value means a 50% cell viability
maximum inhibition concentration, and the more effective
compound as anti-cell line viability has a lower IC50 value.
The results for compounds 3–16 indicated that compound
10 possessed the highest cytotoxicity effect
(IC50 = 37.21 μg/mL) followed by 6 (IC50 = 45.31 μg/
mL), 9 (IC50 = 45.83 μg/mL), 8 (IC50 = 61.78 μg/mL),
and 4 (IC50 value = 71.73 μg/mL), 7 (IC50 = 130.94 μg/
mL) of moderate activities, and finally, compound (3) had
the lowest cytotoxicity effect (IC50 value = 255.31 μg/
mL). The IC50 values for pyrazolylquinazolinone
derivatives 4 and 11–16 showed that compound 14 has
the highest cytotoxicity effect (IC50 = 60.93 μg/mL)
compounds were determined in open-glass capillaries on
Stuart-SMP20 melting point apparatus (Bibby Scientific
Limited, Staffordshire, UK) and are uncorrected.
Elemental analyses (C, H, and N) were conducted using
the Perkin-Elmer 2400 Analyzer, series II (Perkin Elmer
Co., Shelton, UK); their results were found to be in good
agreement ( 0.3%) with the calculated values. The IR
spectra were recorded on a Perkin Elmer Alpha platinum-
1
ATR spectrometer (Perkin Elmer Co.), and the H NMR
was measured on a Bruker WPSY 300 MHz spectrometer
(Bruker, MA, USA) in dimethyl sulfoxide (DMSO-d6)
using trimethylsilane as an internal standard. The
1H NMR chemical shifts were reported as parts per
million (ppm) downfield from trimethylsilane. The
splitting patterns are designated as follows: s, singlet; d,
doublet; and m, multiplet. Mass spectra were recorded on
Varian MAT 311 at 70 ev. All of the microanalyses and
spectral analyses were performed at the Microanalytical
Center, Faculty of Science, Cairo University, Egypt. All bi-
ological experiments were carried out in Biotechnology
Laboratories, Cairo University Research Park (CURP),
followed by 15 (IC50
= 61.39 μg/mL) and 12
(IC50 = 71.63 μg/mL), as compared with compound 4
Table 1
Cytotoxicity (IC50) of the tested compounds
Compounds
MCF-7
5-FU
3
4
5
6
7
8
9
10
11
12
13
14
15
16
38.4 0.21
255.4 0.78
71.73 0.21
104.42 2.49
45.31 1.58
130.96 2.65
61.78 0.92
45.83 0.76
37.21 2.34
102.35 2.59
71.63 3.27
105.4 4.32
60.93 1.45
61.39 3.24
106.03 1.12
Egypt.
(E) 2-[3-(3,4-Dichlorophenyl)-3-oxoprop-1-en-1-yl]-4H-3,1-
benzoxazin-4-one (2).
Heating of 1 gm of 2-[4-(3,4-
dichlorophenyl)-4-oxobut-2-enoyl]aminobenzoic acid
1
[28] with 2 mL freshly distilled acetic anhydride for 1 h
on a water bath, leave to cool. The solid product
formed was filtered off to afford the benzoxazinone 2
(69%) as orange crystal (Dioxane); mp 202–203°C; IR
(KBr) (vmax, cmꢀ1): 3062 (ArH), 1759, 1671 (C═O)
1
cmꢀ1. H NMR (DMSO-d6, 300 MHz) δH (ppm): 7.16–
8.35 (m, 7H, Ar─H), 7.72 (d, 1H, CH═CH─CO,
J = 17.6 Hz), 8.10 (d, 1H, CH═CH─CO, J = 17.6 Hz).
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E. A. El-Bordany and R. S. Ali
Vol 000
Anal. Calcd for C17H9Cl2NO3 (346.16): C, 58.98; H, 2.62;
Calcd for C17H13Cl2N3O2 (362.21): C, 56.37; H, 3.62; N,
N, 4.05. Found: C, 58.88; H, 2.59; N, 4.09.
11.60. Found: C, 56.72; H, 3.83; N, 11.96.
4-(3,4-Dichlorophenyl)-N-(2-(hydrazinecarbonyl)phenyl)-2-
Ethyl-3-(3,4-dichlorophenyl)-5-(3-(2-ethoxy-2-oxoethyl)-4-
oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrrole-2-carboxylate
(6). A mixture of benzoxazinone 2 (0.35 g, 0.001 mol)
and ethyl glycinate hydrochloride (0.28 g, 0.002 mol)
was heated under reflux in dry pyridine (10 mL) for 4 h.
The reaction mixture was poured into crushed ice
containing concentrated hydrochloric acid (2 mL).
hydrazinyl-4-oxobutanamide
(3).
A
mixture
of
benzoxazinone 2 (0.35 g, 0.001 mol) and hydrazine
hydrate (0.5 mL) was stirred in n-butanol (10 mL) 2 h.
The solid that was separated out was filtered off and
dried to afford 3 (70%); as white crystals (Ethanol); mp
>300°C; IR (KBr) (vmax, cmꢀ1): 3322, 3276, 3207 (NH,
NH2), 3068 (ArH), 1690, 1678 (C═O), 1635 (C═N)
The solid product was filtered off to afford
6
1
cmꢀ1. H NMR (DMSO-d6, 300 MHz) δH (ppm): 3.10
(63%) as yellow crystals (Toluene); mp 162–164°C; IR
(KBr) (vmax, cmꢀ1): 3242 (NH), 3069 (ArH), 2986, 1732,
1682 (C═O), 1609 (C═N). 1H NMR (DMSO-d6,
300 MHz) δH (ppm): 0.86 (t, 3H, CH2CH3), 1.23 (t, 3H,
CH2CH3), 3.53 (q, 2H, CH2CH3), 3.67 (q, 2H, CH2CH3),
4.44 (s, 2H, CH2), 7.15–8.68 (m, 8H, 7Ar─H + CH
pyrrol), 11.97 (s, 1H, NH, exchangable). MS, m/z (%):
514 (M+, 0), 458 (48), 291 (26), 201(26). Anal. Calcd for
C25H21Cl2N3O5 (514.36): C, 58.38; H, 4.12; N, 8.17.
(m, 1H, CH2─CH, ABX system), 3.42 (br.s, 2H, NH2,
exchangeable), 3.62 (m, 1H, CH2─CH, ABX system),
4.42 (m, 1H, CH2─CH, ABX system), 4.65 (br.s, 2H,
NH2, exchangeable), 7.12–7.90 (m, 7H, Ar─H), 8.50 (s,
1H, CH─NH─NH2, exchangeable), 10.00 (s, 1H,
CO─NH─NH2,
exchangeable),
11.75
(s,
1H,
Ph─NH─CO, exchangeable). MS, m/z (%): 409 (M+, 0),
357 (31), 344 (26), 215 (10), 173 (17), 119 (100), 115
(17), 103 (12), 98 (14), 90 (23), 89 (17). Anal. Calcd for
C17H17Cl2N5O3 (410.26): C, 49.77; H, 4.18; N, 17.07.
Found: C, 58.45; H, 4.19; N, 8.25.
2-[3-(3,4-Dichlorophenyl)-3-oxoprop-1-en-1-yl]-3-(2-
hydroxyethyl)quinazolin-4(3H)-one (7).
A mixture of
Found: C, 50.12; H, 4.43; N, 17.35.
3-Amino-2-(3-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-
5-yl)quinazolin-4(3H)-one (4). A mixture of benzoxazinone
2 (0.35 g, 0.001 mol) and hydrazine hydrate (0.5 mL)
was heated 4 h under reflux in n-butanol (10 mL). The
solid that was separated out was filtered off and dried to
benzoxazinone 2 (0.35 g, 0.001 mol) in n-butanol
(10 mL) and ethanolamine (0.06 g, 0.001 mol) was
refluxed for 4 h. The reaction mixture was concentrated,
and the solid product was separated out to afford 7
(69%) as orange crystals (Toluene); mp 173–175°C; IR
(KBr) (vmax, cmꢀ1): 3377 (OH), 3099 (ArH), 2937
(olifenic-H), 1690, 1654 (C═O). 1H NMR (DMSO-d6,
300 MHz) δH (ppm): 3.56 (s, 1H, OH, exchangeable),
4.01 (t, 2H, N─CH2─CH2), 4.34 (t, 2H, CH2─CH2─OH),
7.18–7.95 (m, 9H, 7Ar─H, and 2 olefinic protons). MS,
m/z (%): 389 (M+, 2), 369 (5), 347 (11), 217 (100), 173
(58), 172 (36), 145 (15), 75 (10). Anal. Calcd for
C19H14Cl2N2O3 (389.23): C, 58.63; H, 3.63; N, 7.20.
afford
4 (64%) as white crystals (Dioxane); mp
244–246°C; IR (KBr) (vmax, cmꢀ1): 3331, 3279, 3183
(NH, NH2), 3072 (ArH), 1668 (C═O). 1H NMR
(DMSO-d6, 300 MHz) δH (ppm): 3.38 (dd, 1H, Ha,
Jab = 17.1 Hz, Jac = 12 Hz), 3.77 (dd, 1H, Hb,
Jab = 16.95 Hz, Jbc = 6.3 Hz), 5.45 (dd, 1H, Hc,
Jca = 11.7 Hz, Jcb = 6.3 Hz), 5.61 (br.s, 2H, NH2,
exchangeable), 7.49–8.15 (m, 8H, 7Ar─H
+ NH,
exchangeable). MS, m/z (%): 373 (M+, 0), 357 (31),
344 (27), 215 (11), 173 (17), 119 (100), 115 (17), 103
(13), 98 (14), 90 (23), 89 (17). Anal. Calcd for
C17H13Cl2N5O (374.23): C, 54.56; H, 3.50; N, 18.71.
Found: C, 58.54; H, 3.54; N, 7.14.
(Z)-3-(Benzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-1-(3,4-
dichlorophenyl)prop-2-en-1-one (8).
A
solution of
benzoxazinone
2
(0.35 g, 0.001 mol) and O-
phenylenediamine (0.11 g. 0.001 mol) in n-butanol
(10 mL) was heated under reflux for 3 h. The solid was
separated out on cooling and filtered off to afford 8
(62%) as brownish red crystals (Toluene); mp 153–
157°C; IR (KBr) (vmax, cmꢀ1): 3064 (ArH), 1686, 1609
Found: C, 54.43; H, 3.25; N, 18.54.
2-(1-Amino-3-(3,4-dichlorophenyl)-3-oxopropyl)quinazolin-
4(3H)-one (5). A solution of benzoxazinone 2 (0.35 g,
0.001 mol) in formamide (5 mL) was refluxed for 2 h.
The reaction mixture was poured onto ice-cold water, and
the solid product formed was filtered off to afford
quinazolinone 5 (74%) as orange crystals (toluene/
ethanol); mp 148–149°C; IR (KBr) (vmax, cmꢀ1): 3218,
3285, 3300 (NH, NH2), 3072 (ArH), 1690, 1668 (C═O),
1
(C═N). H NMR (DMSO-d6, 300 MHz) δH (ppm): 7.62–
8.79 (m, 11H, Ar─H), 8.42 (d, 1H, CH═CH─CO,
J = 9.4 Hz), 9.63 (d, 1H, CH═CH─CO, J = 9.9 Hz). MS,
m/z (%): 418 (M+, 0), 246 (100), 219 (30). Anal. Calcd
for C23H13Cl2N3O (418.28): C, 66.05; H, 3.13; N, 10.05.
1
1611(C═N). H NMR (DMSO-d6, 300 MHz) δH (ppm):
3.00 (m, 1H, CH─CH2, ABX system), 3.55 (m, 1H,
CH─CH2, ABX system), 4.96 (m, 1H, CH─CH2, ABX
system), 7.27–8.91 (m, 9H, 7Ar─H, 2H, NH2,
exchangeable), 12.22 (s, 1H, NH, exchangeable). MS m/z
(%): 362 (M+, 43), 187 (36), 175 (43), 174 (43), 137
(36), 136 (36), 106 (21), 85 (64), 64 (100), 54 (71). Anal.
Found: C, 66.10; H, 3.19; N, 10.12.
2-(3-(3,4-Dichlorophenyl)-3-oxo-1-(phenylthio)propyl)-4H-
benzo[d][3,1]oxazin-4-one
(9).
A
solution
of
benzoxazinone 2 (0.35 g, 0.001 mol) in dry benzene
(10 mL) and thiophenol (0.11 g, 0.001 mol) was
refluxed for 3 h, concentrated, and cooled; the solid
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Month 2018 Synthesis of New Benzoxazinone, Quinazolinone, and Pyrazoloquinazolinone Derivatives
and Evaluation of Their Cytotoxic Activity Against Human Breast Cancer Cells
product was filtered off, washed by petroleum ether to
afford 9 (54%) as yellow crystals (Petroleum ether 60–
80); mp 182–184°C; IR (KBr) (vmax, cmꢀ1): 3068
(ArH), 1758, 1679 (C═O), 1607 (C═N). 1H NMR
(DMSO-d6, 300 MHz) δH (ppm): 3.60 (m, 1H,
CH─CH2), 3.83 (m, 1H, CH─CH2), 4.37 (m, 1H,
CH─CH2), 7.12–8.42 (m, 12H, Ar─H). Anal. Calcd for
C23H15Cl2NO3S (456.34): C, 60.54; H, 3.31; N, 3.07.
Jac = 11.1 Hz), 4.24 (dd, 1H, Hb, Jab = 17.1 Hz,
Jbc = 8.7 Hz), 5.44 (dd, 1H, Hc, Jca = 10.9 Hz,
Jcb = 8.5 Hz), 5.61 (br.s, 1H, NH, exchangeable), 7.48–
8.17 (m, 12H, 11Ar─H + CH═N). MS, m/z (%): 495
(M+, 4), 397 (5), 357 (45), 282 (4), 213 (16), 138 (30),
119 (100). Anal. Calcd for C24H16Cl3N5O (496.78): C,
58.03; H, 3.25; N, 14.10. Found: C, 57.74; H, 3.11; N,
13.87.
2-(3-(3,4-Dichlorophenyl)-1H-pyrazol-5-yl)-3-((2-
Found: C, 60.51; H, 3.26; N, 3.05.
E-1-(3,4-Dichlorophenyl)-3-(4-thioxo-4H-3,1-benzothiazin-
2-yl)prop-2-en-1-one (10). A mixture of benzoxazinone 1
methylbenzylidene)amino)quinazolin-4(3H)-one (13).
A
mixture of quinazolinon 4 (0.37 g, 0.001 mol) and 2-
methylbenzaldehyde (0.129 g, 0.001 mol) was heated
under reflux in n-butanol (10 mL) for 6 h. The solid that
was separated after cooling was filtered off to afford
quinazolinone 13 (65%) as yellow crystals (n-butanol);
mp >300°C; IR (KBr) (vmax, cmꢀ1): 3219 (NH), 3057
(0.35 g, 0.001 mol) and phosphorus pentasulfide (0.22 g,
0.001 mol) was heated under reflux in dry benzene
(10 mL) for 6 h. The filtrate was concentrated. The solid
product was filtered off and washed by petroleum ether
80–100 to afford benzothiazin 10 (58%) as brownish red
crystals (Petroleum ether 80–100); mp 138–140°C; (KBr)
(vmax, cmꢀ1): 3071 (ArH), 1684 (C═O), 1606 (C═N),
1
(ArH), 1677 (C═O). H NMR (DMSO-d6, 300 MHz) δH
(ppm): 2.65 (s, 3H, CH3), 6.21 (s, 1H, CH═C, pyrazole),
7.25–8.21 (m, 13H, 11Ar─H + 1H, CH═N, +NH
exchangeable). Anal. Calcd for C25H17Cl2N5O (474.35):
C, 63.30; H, 3.61; N, 14.76. Found: C, 63.58; H, 3.32;
1
1170 (C═S). H NMR (DMSO-d6, 300 MHz) δH (ppm):
6.42 (d, 1H, CH═CH─CO, J = 13.1 Hz), 7.11 (d, 1H,
CH═CH─CO, J = 13 Hz), 7.70–8.09 (m, 7H, Ar─H).
MS, m/z (%): 378 (M+, 14), 344 (33), 227 (16), 173 (52),
172 (66), 145 (58), 119 (91), 90 (100). Anal. Calcd for
C17H9Cl2NOS2 (378.29): C, 53.98; H, 2.40; N, 3.70.
N, 14.52.
2-(3-(3,4-Dichlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-
3(propylideneamino)quinazolin-4(3H)-one (14). A mixture
of 4 (0.37 g, 0.001 mol) and propionaldehyde (0.06 g,
0.001 mol) was heated under reflux in n-butanol (10 mL)
for 3 h; the reaction mixture was left to cool. The
solid that was separated out was filtered off to afford 14
(55%) as white crystals (n-butanol); mp 220–222°C; IR
(KBr) (vmax, cmꢀ1): 3224 (NH), 3068 (ArH), 2929
Found: C, 53.95; H, 2.43; N, 3.72.
N-(2-(2-(2-Chlorobenzylidene)hydrazine-1-carbonyl)
phenyl)-2-(2-2chlorobenzylidene)hydrazinyl)-4-(3,4-
dichlorophenyl)-4-oxobutanamide (11).
A mixture of 3
(0.41 g, 0.001 mol) and 2-chlorobenzaldehyde (0.14 g,
0.001 mol) was heated 3 h under reflux in n-butanol
(10 mL); white precipitate was formed after cooling. The
solid that was separated out was filtered off to afford 11
(30%) as pale yellow crystals (Benzene); mp 244–247°C;
IR (KBr) (vmax, cmꢀ1): 3196 (NH), 3068 (ArH), 2901
1
(alkyl-H), 1661 (C═O), 1605 (C═N). H NMR (DMSO-
d6, 300 MHz) δH (ppm): 1.08 (t, 3H, CH3), 1.78 (q, 2H,
CH2), 3.47 (m, 1H, CH─CH2), 4.14 (m, 1H, CH─CH2),
5.61 (m, 1H, CHCH2), 5.79 (s, 1H, CH═N), 7.21–8.21
(m, 8H,7H, Ar─H + NH exchangeable). Anal. Calcd for
C20H17Cl2N5O (444): C, 57.98; H, 4.14; N, 16.90.
1
(alkyl-H), 1712 (C═O), 1599 (C═N). H NMR (DMSO-
d6, 300 MHz) δH (ppm): 3.27 (m, 1H, CH2─CH), 3.44
(m, 1H, CH2─CH), 4.80 (m, 1H, CH─CH2), 6.48 (s, 1H,
CH─NHN═CH, exchangeable), 6.87–8.027 (m, 17H,
15Ar-H + 2CH═N), 11.65 (br.s, 1H, CO─NH─N═CH,
Found: C, 57.63; H, 3.75; N, 16.52.
5-Amino-2-(3,4-dichlorophenyl)-1,14b-dihydro-
7H,9Hpyrazolo[10,50:5,6][1,2,5]triazepino[7,1-b]quinazolin-9-
one (15).
A mixture of 4 (0.37 g, 0.001 mol) and
exchangeable),
12.22
(br.s,
1H,
Ph─NH─CO,
exchangeable). MS, m/z (%): 655 (M+, 0), 637 (2), 499
(1) 357 (2), 138 (87). Anal. Calcd for C31H23Cl4N5O3
(655.36): C, 56.81; H, 3.54; N, 10.69. Found: C, 57.04;
chloroacetonitrile (0.11 g, 0.001 mol) was heated under
reflux in dimethylformamide (10 mL) for 6 h. The
white solid that formed on hot was separated out and
filtered off to afford 15 (45%) as white crystals
(Dioxane); mp >300°C; IR (KBr) (vmax, cmꢀ1): 3335,
3280, 3198 (NH, NH2), 3068 (ArH), 2918 (alkyl-H),
1672 (C═O), 1610 (C═N). 1H NMR (DMSO-d6,
300 MHz) δH (ppm): 3.38 (m, 1H, CH2─CH pyrazol),
3.75 (m, 1H, CH2─CH pyrazol), 5.37 (m, 1H, CH2─CH
pyrazole), 5.61 (s, 2H, NH2, exchangeable), 6.99 (s, 1H,
CH═C, triazepine ring), 7.50–8.17 (m, 7H, Ar─H),
14.11 (br.s. 1H, NH, exchangeable). MS, m/z (%): 413
(M+, 0), 358(27). Anal. Calcd for C19H14Cl2N6O
(413.26): C, 55.22; H, 3.41; N, 20.34. Found: C, 54.98;
H, 3.68; N, 20.22.
H, 3.93; N, 10.77.
3-((2-Chlorobenzylidene)amino)-2-(3-(3,4-dichlorophenyl)-
4,5-dihydro-1H-pyrazol-5-yl)quinazolin-4(3H)-one (12).
A
mixture of
4
(0.37 g, 0.001 mol) and 2-
chlorobenzaldehyde (0.14 g, 0.001 mol) was heated 6 h
under reflux in n-butanol (10 mL); white precipitate was
formed after cooling. The solid that was separated out
was filtered off to afford quinazolinone 12 (78%)
as yellow crystals (n-butanol); mp 158–161°C; IR (KBr)
(vmax, cmꢀ1): 3253 (NH), 3068 (ArH), 2901 (alkyl-H),
1683 (C═O), 1599 (C═N). 1H NMR (DMSO-d6,
300 MHz) δH (ppm): 3.51 (dd, 1H, Ha, Jab = 17.25 Hz,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

E. A. El-Bordany and R. S. Ali
Vol 000
N-(2-(3-(3,4-Dichlorophenyl)-1H-pyrazol-5-yl)-4-
oxoquinazolin-3(4H)-yl)acetamide (16).
Absorbance of treated sample
%Cell death ¼ 100 ꢀ
ꢁ100:
A mixture of
Absorbance of untreated sample
quinazolinone 4 (0.37 g, 0.001 mol) and acetyl chloride
(0.103 g, 0.001 mol) was refluxed in n-butanol (10 mL).
A white precipitate was formed after half an hour, filtered
on hot to give 16 (55%) as white crystals (DMF); mp
>300°C; IR (KBr) (vmax, cmꢀ1): 3340, 3229 (NH), 3050
(ArH), 2975 (alkyl-H), 1676, 1669 (C═O), 1628 (C═N).
1H NMR (DMSO-d6, 300 MHz) δH (ppm): 2.49 (s, 3H,
CH3), 6.98 (s, 1H, CH═C pyrazol), 7.34 (br.s, 1H,
NH─CO, exchangeable), 7.55–8.34 (m, 7H, Ar─H), 8.52
(br.s, 1H, NH pyrazol, exchangeable). Anal. Calcd for
C19H13Cl2N5O2 (414.25): C, 55.09; H, 3.16; N, 16.91.
Found: C, 55.39; H, 3.42; N, 16.65.
On the other hand, the IC50 of the tested and control
compounds were calculated using XLfit5 software (IDBS)
and expressed in μg/mL at 95% confidence intervals. The
data were based on three independent experiments.
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CYTOTOXICITY ASSESSMENT
Reagent preparation and MCF-7 cell cultivation. The
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1640 supplemented with 100 μg/mL streptomycin,
100 μg/mL penicillin, and 10% (w/v) heat-inactivated
fetal bovine serum in a humidified 5% (v/v) CO2
atmosphere at 37°C.
Method of cytotoxicity assessment. A total of 30,000
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quadruplets for each treatment of the synthesized
derivatives 3–16. The cells were washed with pre-
warmed phosphate-buffered saline (PBS) and 125 μL of
media with appropriate concentration of each
synthesized compound. After the treatments, cells were
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reagent was added in each cell. The plates were
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incubation, plates were taken out, gently washed three
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shaker at 60 rpm for 30 min followed by keeping the
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reader, and their absorbance was measured in each well
at 540 nm. Each value is based on means of the
absorbance values acquired from quadruplet of
treatments and normalized to the mean value of
untreated control and expressed as % cell death with the
formula:
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018 Synthesis of New Benzoxazinone, Quinazolinone, and Pyrazoloquinazolinone Derivatives
and Evaluation of Their Cytotoxic Activity Against Human Breast Cancer Cells
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