Synthesis and biological evaluation of some amino- and sulfanyl-3H-quinazolin-4-one derivatives as potential anticancer agents

Article abstract of DOI:10.1007/s00706-015-1508-6

A series of 6-substituted quinazolinone derivatives were prepared by the reaction of 6-bromoquinazolinones with aryl or alkyl amines and thiols, in the presence of a Pd(OAc)₂/Xantphos system, under Buchwald-Hartwig-type reaction conditions. The 6-bromoquinazolinones were obtained in the three-components reaction of 5-bromoisatoic anhydride, triethyl orthoformate and an appropriate amine. Biological screening of the potential cytotoxicity of synthesized compounds on HT29 and HCT116 cell lines, as well as on the lymphocytes, showed that some derivatives of quinazolinone have significant anticancer activities. The detailed synthesis, spectroscopic data, and biological assays were reported.

Full text of DOI:10.1007/s00706-015-1508-6

Monatsh Chem
DOI 10.1007/s00706-015-1508-6
ORIGINAL PAPER
Synthesis and biological evaluation of some amino- and
sulfanyl-3H-quinazolin-4-one derivatives as potential
anticancer agents
Zbigniew Malinowski1 Emilia Fornal2 Monika Nowak1
•
•
•
Renata Kontek3 Gabriela Gajek3 Bartłomiej Borek1
•
•
Received: 13 March 2015 / Accepted: 1 June 2015
Ó Springer-Verlag Wien 2015
Abstract
A
series of 6-substituted quinazolinone
Graphical abstract
derivatives were prepared by the reaction of 6-bromo-
quinazolinones with aryl or alkyl amines and thiols, in the
presence of a Pd(OAc)2/Xantphos system, under Buch-
wald–Hartwig-type
reaction
conditions.
The
6-bromoquinazolinones were obtained in the three-com-
ponents reaction of 5-bromoisatoic anhydride, triethyl
orthoformate and an appropriate amine. Biological
screening of the potential cytotoxicity of synthesized
compounds on HT29 and HCT116 cell lines, as well as on
the lymphocytes, showed that some derivatives of quina-
zolinone have significant anticancer activities. The detailed
synthesis, spectroscopic data, and biological assays were
reported.
Keywords Quinazolinones Á
Buchwald–Hartwig reaction Á Palladium coupling Á
Anticancer activity Á Sulfides
Introduction
Quinazolinone and quinazoline derivatives are a group of
compounds, which arouse a great interest for medicinal
chemistry, due to their diverse biological and pharmaceutical
properties [1, 2]. Additionally, the quinazolinone skeleton is
an important structural moiety present in many naturally
occurring alkaloids [3], such as febrifugine [4, 5] isolated
from an Asian plant Dichroa febrifuga or aniquinazolines
A-D [6] and isolated from the culture of Aspergillus nidu-
lans, and also it is a significant building block in the synthesis
of various bioactive substances. Compounds incorporating
3H-quinazolin-4-one moiety were reported to possess anal-
gesic [7], anti-inflammatory [8, 9], antibacterial [10, 11],
anticonvulsant [12, 13], antifungal [14, 15], and particularly
antitumor activity [16–18]. Furthermore, it was demon-
strated that some of the quinazoline and quinazolinone
derivatives can act as inhibitors of tubulin polymerization
[19, 20] or as apoptosis inducers [21].
& Zbigniew Malinowski
zbigmal@uni.lodz.pl
1
Department of Organic Chemistry, Faculty of Chemistry,
University of Lodz, Tamka 12 Street, 91-403 Lodz, Poland
2
Laboratory of Separation and Spectroscopic Method
Applications, Center for Interdisciplinary Research, The John
Paul II Catholic University of Lublin, Krasnicka Avenue 102,
20-718 Lublin, Poland
3
Laboratory of Cytogenetics, Department of General Genetics,
Molecular Biology, and Plant Biotechnology, Faculty of
Biology and Environmental Protection, University of Lodz,
Banacha 12/16 Street, 90-237 Lodz, Poland
123

Z. Malinowski et al.
Recently, we have started research on the synthesis of
new derivatives of polyazanaphthalenes (quinazolinones,
phthalazinones), and their benzo-analogs, with potential
anticancer activity. In our previous paper [22], we descri-
bed a methodology for the synthesis of N-substituted
6-aminobenzo[h]quinazolinones involving the Pd-cat-
alyzed C–N bond formation reaction. The biological tests
of 3H-benzo[h]quinazolin-4-one derivatives showed that
compounds with the amino group in 6-position of lactam
skeleton were highly toxic against HT29 cells, while its
cytotoxicity against A549 cells and lymphocytes was sig-
nificantly lower.
corresponding bromolactams under palladium-catalyzed
Buchwald–Hartwig-type reactions.
Starting bromoquinazolinones 2a, 2b were obtained
using two approaches as shown in Scheme 1. Initially, the
synthesis of quinazolinones 2a, 2b was carried out
according to a strategy based on the Niementowski reaction
and involved the condensation of 1 with formamide [22–
26] and then alkylation of nitrogen atom in reaction with
appropriate benzyl or methyl halides under MW irradiation
conditions (Scheme 1, 1 ? 2).
Alternatively, N-substituted lactams 2 were prepared
from corresponding isatoic anhydride (Scheme 1,
3 ? 6 ? 2). The isatoic anhydrides are valuable starting
substances for the synthesis of many important compounds
[27–31]. During the research on the preparation of isatin
derivatives, we observed that when commercially available
isatin (3) was treated with large excess of NBS (5 equiv-
alents) in MeCN at an ambient temperature with access to
air, 5-bromoisatoic anhydride (6) was formed as a main
product in 77 % yield. Surprisingly, 5,7-dibromoisatin (5)
In the present paper, we report results of further research
on the synthesis of novel derivatives of 3H-quinazolin-4-
ones with amines and thiols, containing various sub-
stituents which would affect the activity of the target
compounds. In addition, some of the newly synthesized
compounds were evaluated for anticancer activity against
human colon carcinoma cell lines HT29 and HCT116.
[32, 33] was not produced during this reaction. The 1H, 13
C
Results and discussion
Chemistry
NMR spectra of 6 confirmed the proposed structure and
were in accordance with those reported in the literature
[34–36]. On the other hand, the use of 1 equivalent of NBS
in the reaction with isatin, at the same reaction conditions,
resulted in the formation of 5-bromoisatin (4) in good
yield. In this case, the formation of anhydride 6 was not
The target 6-amino and 6-sulfanyl derivatives of N-methyl-
and N-benzylquinazolinones were synthesized from
Scheme 1
O
Bn
N
Br
H
NH2
7
iv
v
O
O
O
i
COOH
NH2
Br
Br
R1
Br
vi
N
O
ii
O
N
H
3
N
N
H
O
2a R1 = Me 2b R1 = Bn
,
1
6
iii
O
Br
O
N
H
X
4
X = H, 5 X = Br
123

Synthesis and biological evaluation of some amino- and sulfanyl-3H-quinazolin-4-one…
Scheme 2
Compound
R1
R2
R3
H
H
H
H
H
H
H
R4
Yield / %a
8a
8b
8c
8d
9a
9b
9c
Me
Me
Me
Me
Bn
Bn
Bn
2-(morpholin-4-yl)-CH2CH2-
4-EtO-C6H4-
50
62
46
20b
57
35
34
2-[3,4-(MeO)2-C6H3]-CH2CH2-
4-Me-3-NO2-C6H3-
Ph
4-EtO-C6H4-
2-[3,4-(MeO)2-C6H3]-CH2CH2-
9d
9e
Bn
Bn
(morpholin-4-yl)-
[4-(2-F-C6H4)piperazin-1-yl]-
89c
88c
10a
10b
11
Me
Me
Bn
n-Pr
Bn
75
78
80
i-Pr
aThe yield of isolated compound
bThe NMR yield
cCompounds 9d, 9e were synthesized according to the methods described in Ref. [14]
observed. In our view, the preparation of 5-bromoisatoic
anhydride 6 is a one-pot process involving in the first step
bromination of 3 by N-bromosuccinimide, followed by ring
expansion of bromoderivative 4, with the insertion of an
oxygen atom between the C-2/C-3 carbon atoms.
(320 W, 8 min) led mainly to the expected derivative 7,
which was separated in 60 % yield. Thereby, the prepara-
tion of amide 7 from 6 proved conclusively the structure of
anhydride 6. In next step, compound 7 was successfully
converted into the benzylquinazolinone 2b, in 76 %
yield, in the reaction with triethyl orthoformate, in the
presence of KAl(SO4)2Á12H2O [42, 43] (MW, 148 °C,
15 min). Based on these research results, 3-methyl
derivative 2a was synthesized in the one-pot three-com-
ponent reaction between 5-bromoisatoic anhydride, triethyl
orthoformate, and methylamine. Lactam 2a was achieved
in 60 % yield.
The mechanism of this transformation is not clear and
needs more studies. However, it seems that NBS as well as
oxygen from air can play important roles in the preparation
process of 5-bromoisatoic anhydride from isatin. In our
opinion, the presented methodology can be complementary
to existing methods for the synthesis of this type of com-
pounds [37–41].
Finally, 5-bromoisatoic anhydride was used for the
preparation of bromoquinazolinones 2a, 2b. Treatment of
anhydride 6 with benzylamine under MW conditions
The final aminoquinazolinones 8, 9 were successfully
synthesized via palladium-catalyzed Buchwald–Hartwig
cross-coupling reaction of N-methyl- and N-
123

Z. Malinowski et al.
benzylbromoquinazolinones 2a, 2b with aromatic and ali-
phatic amines, as depicted in Scheme 2.
Table 1 The IC50 values obtained for HT29 and HCT116 cell lines
and normal human lymphocytes
Previously, we have revealed that Xantphos, Pd(OAc)2
and tBuOK used in the ratio: 15 mol %/15 mol %/1.5 mol,
in 1,4-dioxane as solvent, are an effective system for C–N
bond formation [22]. The employment of the above con-
ditions resulted in obtaining compounds 8a–8d and 9a–9c,
in moderate to good yields (34–60 %). In all cases, the
synthesis of derivatives 8, 9 was carried out in 1,4-dioxane,
under an argon and conventional heating at 100 °C, for
24 h. The achieved results, summarize in Scheme 2,
demonstrated that the yields obtained were dependent on
the choice of an applied amine and on the used starting
lactam. Generally, N-methyl derivatives 8 were obtained in
slightly better yields than N-benzyl derivatives 9. More-
over, the arylation of 2-(3,4-dimethoxyphenyl)ethanamine
with 2a or 2b gave corresponding products 8c or 9c in
lower yield, in comparison with the arylation of
4-ethoxyaniline—compounds 8b, 9b (Scheme 2). On the
other hand, unsubstituted aromatic amine, such as aniline,
afforded appropriate product 9a in higher yield than
4-ethoxyaniline (compound 9b).
Compounds
IC50/lM
HT29
HCT116
Lymphocytes
8b
29.70 3.24
33.40 5.07
59.10 3.89
50.90 4.89
29.10 3.33
45.90 4.15
74.00 5.24
120.67 4.62
90.00 6.17
46.00 3.91
28.67 4.40
55.33 4.58
184.67 4.23
178.00 5.98
120.67 6.87
196.67 6.61
71.33 3.71
143.33 3.84
9b
9d
9e
10a
10b
cytotoxicity was assessed by the means of an MTT test and
evaluated by IC50 values. Compounds were studied in the
concentration range from 5 to 350 lM. The obtained IC50
values are summarized in Table 1.
As can be seen, the most interesting results were obtained
for compound 9e, containing [4-(2-fluorophenyl)piperazin-
1-yl] moiety. Compound 9e showed significant activity
towards both HT29 (IC50 = 50.90 lM) and HCT116
(IC50 = 46.00 lM) cells lines. Simultaneously, 9e was the
least toxic for lymphocytes from among tested compounds
(IC50 = 196.67 lM). Thereby, compound 9e demonstrated
a carcinoma-specific cytotoxicity against human colon
cancer cells—a strong cytotoxicity activity against both
colon cancer cell lines. A similar correlation was observed
for the sulfanyl derivatives of 3-methylquinazolinone 10a,
10b. In particular, compound 10a, substituted by propyl-
sulfanyl group, exhibited the most potent cytotoxicity from
all tested quinazolinones: IC50 (HT29) = 29.10 lM and
IC50 (HCT116) = 28.67 lM. However, it had almost a
threefold lower IC50 value against normal cells than com-
pound 9e. In the case of N-methyl and N-benzyl lactams 8b,
9b, insertion of 4-ethoxyphenylamino group at 6-position of
quinazoline skeleton resulted in potent activity towards only
HT29 cell line, IC50 = 29.70 lM and IC50 = 33.40 lM,
respectively. Lactams 8b, 9b indicated significantly less
cytotoxicity against HCT116 cancer cells (IC50 = 74.00 -
lM and IC50 = 120.67 lM, respectively). On the other
hand, the IC50 values for the toxic effect on normal lym-
phocytes were comparable to the one obtained for 9e.
Additionally, we aimed to explore the possibility of
synthesis of novel quinazolinone derivatives, containing a
new C-S bond via palladium-catalyzed cross-coupling
reactions [44–47] of lactams 2 with thiols (Scheme 2). Aryl
sulfides are an important building block for many natural
products and biologically and pharmaceutically active
compounds [48–50]. Our initial studies showed that when
Xantphos and Pd(OAc)2 were used in the ratio 15 mol %/
15 mol %, to the reaction of 2a with 1-propanethiol, the
outcomes were poor. Alkylsulfanylquinazolinone 10a was
formed in trace amounts only. It was observed that increase
the amount of Xantphos/Pd(OAc)2 system gave a better
yield of 10a. The optimal conditions for the arylation of
alkylthiols were obtained by applying Xantphos/Pd(OAc)2
system in the ratio 30 mol %/30 mol % in the presence of
DIPEA and in 1,4-dioxane as solvent. The need for an
increase of ligand to palladium source ratio may result
from the deactivation of the palladium catalyst by thiols
[51]. Based on these results, we synthesized compounds
10a–10b and 11 in high yields (Scheme 2). The chemical
structures of compounds 8, 9 and 10, 11 were determined
1
by IR, H NMR, 13C NMR, and HRMS tests.
Conclusion
Anticancer activity
In summary, we described a simple method for preparation
of amino and sulfanyl derivatives of N-methyl- and N-
benzylquinazolinones (compounds 8, 9 and 10, 11), in
satisfactory yields. The applied strategy for the C–N and
C–S bond formation was based on the Buchwald–Hartwig-
type reaction between N-substituted bromoquinazolinones
2a, 2b and appropriate amines or thiols, in the presence of a
The anticancer activities of amino and sulfanyl derivatives
8b, 9b, 10a, 10b and additionally compounds 9d, 9e [22]
(Scheme 2) were evaluated using two human colorectal
carcinoma cell lines: HT29 and HCT116 (Table 1). The
cytotoxic effect of quinazolinones 8, 9, 10 was also
investigated on the normal human lymphocytes. The
123

Synthesis and biological evaluation of some amino- and sulfanyl-3H-quinazolin-4-one…
Pd(OAc)2/Xantphos system. The results of biological tests
showed that these types of compounds could have potential
applications in pharmacology and design of new anticancer
agents.
purification. 3H-Quinazolin-4-one [22–25], 6-bromo-3H-
quinazolin-4-one [22–25], 5,7-dibromo-1H-indole-
2,3-dione (5) [52], 3-benzyl-6-(morpholin-4-yl)-3H-quina-
zolin-4-one (9d) [22], and 3-benzyl-6-[4-(2-fluorophenyl)
piperazin-1-yl]-3H-quinazolin-4-one (9e) [22] were pre-
pared according to known procedures.
Experimental
5-Bromo-1H-indole-2,3-dione (4)
The reaction was carried out in a round-bottom flask fitted
with a magnetic stirrer bar and a rubber septum with a
needle as vent. To a solution of 1.00 g isatin (3, 6.8 mmol)
Melting points were determined on a Boetius hot-stage
apparatus. 1H, 13C, and 19F NMR spectra were recorded on a
Bruker Avance III spectrometer at 600 MHz, 150 MHz, and
565 MHz, respectively. The residual CDCl3 or DMSO-d6
signal was used for reference (CDCl3 at 7.26 ppm or DMSO-
in 95 cm3 acetonitrile,
a solution of 1.21 g NBS
(6.8 mmol) in 15 cm3 acetonitrile was added at tempera-
ture 0 °C. Next reaction was continued for 4 days at
ambient temperature. The separated solid was collected by
filtration and washed with water (2 9 10 cm3). Then the
crude product was purified by crystallization from ethanol
to give pure 5-bromoisatin as an orange solid in 54 %
yield. M.p.: 248–252 °C (Ref. [53] 250–254 °C).
1
d6 at 2.54 ppm for H NMR and CDCl3 at 77.0 ppm or
DMSO-d6 at 39.0 ppm for 13C NMR). 19F NMR spectra were
1
1
1
obtained without H-decoupling. 2D homonuclear H H
COSY spectra and heteronuclear 1H 13C COSY spectra
(HSQC and HMBC) were used to assign the proton and
carbon signals. IR spectra were recorded on a Nexus FT-IR
spectrometer. Microwave reactions were performed in a
Synthos 3000 microwave reactor from Anton Paar. LC-
HRMS analyses were carried out using a liquid chro-
matograph (Agilent Technologies series 1200) coupled to a
tandem mass spectrometer (Agilent Technologies 6538
UHD Accurate Mass Q-TOF LC/MS) equipped with a
HPLC-chip cube allowing nanoelectrospray ionization of
analytes. The instrument is housed in the Laboratory of
Separation and Spectroscopic Method Applications, Center
for Interdisciplinary Research, KUL, Lublin, Poland.
Instrument control and data acquisition were performed
using an Agilent Technologies Mass Hunter Acquisition
module (version B.04). High-resolution mass spectra were
acquired in the positive ion scan mode at m/z = 100 - 1000.
The capillary potential was set at -1750 V and the frag-
mentor was set at 100 V. Mobile phase consisted of water–
acetonitrile—0.1 % formic acid. A large capacity chip
(0.160 mm3, 150 mm C18) was used. Internal mass cali-
bration was enabled, using two reference mass ions
(121.0509 and 922.0098). The mass accuracy for MS scans
was \1 ppm. The analytical thin-layer chromatography
tests (TLC) were carried out on Supelco silica gel plates
(supported on aluminum, layer thickness 200 lm) and the
spots were visualized using UV lamp. The flash column
chromatography purifications were performed on Fluka sil-
ica gel (Silica gel 60, 0.035–0.070 mm). All reactions with
organopalladium compounds were performed under an
argon atmosphere using standard Schlenk technique.
6-Bromo-2H-3,1-benzoxazine-2,4(1H)-dione (6)
The reaction was carried out in a round-bottom flask fitted
with a magnetic stirrer bar and a rubber septum with a
needle as vent. To a solution of 1.00 g isatin (3, 6.8 mmol)
in 95 cm3 acetonitrile, a solution of 6.05 g NBS (34 mmol)
in 40 cm3 acetonitrile was added at temperature 0 °C. Next
reaction was continued for 22 days at ambient temperature,
with access to air. The separated solid was collected by
filtration and washed with acetonitrile (2 9 10 cm3). The
pure product was obtained as a yellow solid in 77 % yield.
M.p.: 245–247 °C (Ref. [35] 230–232 °C, Ref. [36]
286–288 °C).
Preparation of N-substituted 6-bromo-3H-quinazolin-4-
ones 2a, 2b
Reactions were carried out in an Anton Paar Synthos 3000
Microwave reactor.
Method A
3H-Quinazolin-4-ones 2a, 2b were prepared according to
the already reported procedure from appropriate 6-bromo-
3H-quinazolin-4-one [22]. Products 2a and 2b were puri-
fied by flash chromatography.
Method B
A
mixture of 0.2 g 5-bromoisatoic anhydride (6,
0.826 mmol), benzylamine (0.826 mmol), and 6 cm3 THF
was charged to PTFE tube, sealed in ceramic case and placed
in the rotor and then the reaction mixture was irradiated for
8 min at constant power 320 W (constant power mode) and
then cooled to 35 °C. Next, 20 cm3 water was added to the
reaction mixture and stirring was continued at ambient
1,4-Dioxane was distilled from sodium benzophenone
ketyl prior to use. Commercially available solvents and
reagents, DMF, NBS, anthranilic acid, formamide, benzyl
bromide, methyl iodide, isatin, and 4,5-bis(diphenylphos-
phino)-9,9-dimethylxanthene (Xantphos), were purchased
from Sigma-Aldrich and were used without further
123

Z. Malinowski et al.
charged with quinazolinone 2a or 2b (0.19 mmol), 5 cm3
freshly distilled dioxane, Pd(OAc)2 (15 mol %), XantPhos
(15 mol %), KOt-Bu (0.28 mmol), and the appropriate
amine (0.57 mmol). The whole mixture was stirred and
heated in an oil bath at 100 °C for 24 h. After this time, the
reaction mixture was cooled to an ambient temperature and
diluted with 5 cm3 chloroform. The solid was removed by
filtration and washed with 5 cm3 chloroform. The filtrate was
concentrated to dryness and the residue was purified by flash
chromatography to give pure product.
temperature for 15 min. The reaction mixture was extracted
with chloroform (3 9 15 cm3) and the organic layers were
combined together and dried over MgSO4. Evaporation of
solvent gave a crude 2-amino-N-benzyl-5-bromobenzamide
(7), which was purified by flash column chromatography.
Next,
a
mixture of 0.10 g bromobenzamide
7
(0.3277 mmol), 6 cm3 triethyl orthoformate, and 0.012 g
KAl(SO4)2Á12H2O (0.0250 mmol) was charged to PTFE
tube, sealed in ceramic case and placed in the rotor. The
reaction mixture was heated to 148 °C, held for 15 min at
148 °C (constant temperature mode) and then cooled to
35 °C. After cooling, 20 cm3 water was added to the reaction
mixture and stirring was continued at ambient temperature
for 15 min. Next, the reaction mixture was extracted with
chloroform (3 9 15 cm3) and the organic layers were
combined together and dried over MgSO4. Evaporation of
solvent gave a product, 2b, which was purified by flash
column chromatography.
The 6-sulfanylquinazolinones 10, 11 were prepared
according to procedure described above, using quinazoli-
none 2a or 2b (0.19 mmol), 5 cm3 freshly distilled dioxane
as solvent and Pd(OAc)2 (30 mol %), XantPhos
(30 mol %), DIPEA (0.38 mmol), and the appropriate
thiol (0.57 mmol).
3-Methyl-6-[[2-(morpholin-4-yl)ethyl]amino]-3H-
quinazolin-4-one (8a, C15H20N4O2)
Gum; yield: 50 %, Rf = 0.2 (MeCN/MeOH/AcOEt 1/0.5/
2-Amino-N-benzyl-5-bromobenzamide (7)
White solid, yield: 60 % (Method B), Rf (PE/AcOEt;
1/1) = 0.72; m.p.: 144–146 °C (Ref. [56, 57] 140–141 °C).
1
1); IR (KBr): v = 1728, 1659 cm-1; H NMR (600 MHz,
CDCl3): d = 7.84 (s, 1H), 7.53 (d, 1H, J = 8.8 Hz, 8 ArH),
7.33 (d, 1H, J = 2.8 Hz, 5 ArH), 7.08 (dd, 1H, J = 8.8,
2.8 Hz, 7 ArH), 4.69 (s, 1H, NH), 3.77-3.73 (m, 4H, MOR),
3.57 (s, 1H, Me) 3.28 (t, 2H, J = 5.8 Hz, CH2), 2.69–2.67
(m, 2H, CH2), 2.49–2.46 (m, 4H, MOR) ppm; 13C NMR
(150 MHz, CDCl3): d = 161.8, 147.8, 143.1, 140.7, 128.7,
123.3, 122.1, 104.8, 67.1, 57.0, 53.5, 40.0, 34.1 ppm.
Method C: one-pot procedure
A
mixture of 0.2 g 5-bromoisatoic anhydride (6,
0.826 mmol), MeNH2—solution in THF (c = 0.11 g/cm3,
0.826 mmol), 6 cm3 triethyl orthoformate, and 0.03 g
KAl(SO4)2Á12H2O (0.0632 mmol) was charged to PTFE
tube, sealed in ceramic case and placed in the rotor. The
reaction was heated to 148 °C, held for 15 min at 148 °C
(constant temperature mode). After cooling to 35 °C, pro-
duct 2a was separated as described above—method B.
6-[(4-Ethoxyphenyl)amino]-3-methyl-3H-quinazolin-4-one
(8b, C17H17N3O2)
Light brown solid; yield: 62 %, Rf = 0.56 (MeCN/AcOEt
1/1); m.p.: 174–176 °C; IR (KBr): v = 1647 cm-1
;
1H
NMR (600 MHz, CDCl3): d = 7.86 (s, 1H), 7.67 (d, 1H,
J = 2.7 Hz, 5 ArH), 7.56 (d, 1H, J = 8.8 Hz, 8 ArH),
7.28–7.27 (m, 1H, 7 ArH), 7.14–7.11 (m, 2H, ArH),
6.90–6.88 (m, 2H, ArH), 5.78 (s, 1H, NH), 4.04 (q, 2H,
J = 7.0 Hz, CH2), 3.56 (s, 3H, Me), 1.42 (t, 3H,
J = 7.0 Hz, Me) ppm; 13C NMR (150 MHz, CDCl3):
d = 161.6, 155.8, 145.3, 143.8, 141.7, 134.4, 128.8, 123.6,
123.3, 122.9, 115.8, 108.6, 64.0, 34.1, 15.1 ppm.
6-Bromo-3-methyl-3H-quinazolin-4-one (2a)
White solid, yield: 55 % (method A), 60 % (method C);
Rf = 0.32 (DCM/MeOH 15/0.5); m.p.: 116–119 °C (Ref.
1
[54] 338–340 °C as HBr salt); IR (KBr): 1675 cm-1; H
NMR (600 MHz, CDCl3): d = 8.44 (d, 1H, J = 2.3 Hz, 5
ArH), 8.04 (s, 1H), 7.82 (dd, 1H, J = 8.7, 2.3 Hz, 7 ArH),
7.57 (d, 1H, J = 8.7 Hz, 8 ArH), 3.59 (s, 3H, Me) ppm;
13C NMR (150 MHz, CDCl3): d = 160.5, 147.3, 147.2,
137.5, 129.5, 129.3, 123.6, 121.1, 34.3 ppm.
6-[[2-(3,4-Dimethoxyphenyl)ethyl]amino]-3-methyl-3H-
quinazolin-4-one (8c, C19H21N3O3)
3-Benzyl-6-bromo-3H-quinazolin-4-one (2b)
White solid, yield: 61 % (method A), 76 % (method B);
Rf = 0.48 (Hex/AcOEt 1/1); m.p.: 127–129 °C (Ref. [55]
124–126 °C).
Yellow solid; yield: 46 %, Rf = 0.4 (AcOEt/acetone 1/1);
m.p.: 48–51 °C; IR (KBr): v = 1660 cm-1; 1H NMR (600,
MHz, CDCl3): d = 7.83 (s, 1H), 7.51 (d, 1H, J = 8.8 Hz,
ArH), 7.36 (d, 1H, J = 2.8 Hz, ArH), 6.99 (dd, 1H,
J = 8.8, 2.8 Hz, ArH), 6.82 (d, 1H, J = 8.1 Hz, ArH),
6.76 (dd, 1H, J = 8.1, 1.9 Hz, ArH), 6.73 (d, 1H,
J = 1.9 Hz, ArH), 4.05 (brs, 1H, NH), 3.86 (s, 6H,
2 9 OMe), 3.56 (s, 3H, Me), 3.49 (t, 2H, J = 6.8 Hz,
CH2), 2.90 (t, 2H, J = 6.8 Hz, CH2) ppm; 13C NMR
(150 MHz, CDCl3): d = 161.7, 149.3, 148.0, 147.4, 143.1,
General procedure for the palladium-catalyzed C–N and
C–S bond formations: synthesis of 6-aminoquinazolinones
8, 9, and 6-sulfanylquinazolinones 10, 11
The synthesis of 6-aminoquinazolinones 8, 9. The reaction
was carried out under an argon atmosphere in an oven-dried
resealable Schlenk flask. A resealable Schlenk flask was
123

Synthesis and biological evaluation of some amino- and sulfanyl-3H-quinazolin-4-one…
140.6, 131.6, 128.7, 123.4, 122.1, 120.9, 112.2, 111.7,
105.0, 56.1, 56.1, 45.1, 34.8, 34.1 ppm.
6-(Benzylsulfanyl)-3-methyl-3H-quinazolin-4-one
(10b, C16H14N2OS)
Light yellow solid; yield: 78 %, Rf = 0.64 (AcOEt/MeCN
1/1); m.p.: 108–111 °C; IR (KBr): v = 1674 cm-1
3-Benzyl-6-(phenylamino)-3H-quinazolin-4-one
(9a, C21H17N3O)
Beige solid; yield: 57 %, Rf = 0.28 (AcOEt/PE 1/1); m.p.:
;
1H
NMR (600 MHz, CDCl3): d = 8.22 (d, 1H, J = 2.0 Hz, 5
ArH), 7.98 (s, 1H), 7.62 (dd, 1H, J = 8.5, 2.2 Hz, 7 ArH),
7.57 (d, 1H, J = 8.5 Hz, 8 ArH), 7.36–7.33 (m, 2H, PhH),
7.30–7.28 (m, 2H, PhH), 7.25–7.23 (m, 1H, PhH), 4.23 (s,
2H, CH2), 3.58 (s, 3H, Me) ppm; 13C NMR (150 MHz,
CDCl3): d = 161.1, 147.2, 146.7, 146.5, 137.5, 136.9,
136.7, 135.2, 129.0, 128.8, 128.0, 127.6, 125.6, 122.5,
38.7, 34.2 ppm.
172–174 °C; IR (KBr): v = 1660 cm-1
;
1H NMR
(600 MHz, CDCl3): d = 7.97 (s, 1H), 7.88 (d, 1H,
J = 2.7 Hz, 5 ArH), 7.61 (d, 1H, J = 8.8 Hz, 8 ArH),
7.42 (dd, 1H, J = 8.8, 2.7 Hz, 7 ArH), 7.36–7.29 (m, 7H,
PhH), 7.17–7.16 (m, 2H, PhH), 7.04–7.02 (m, 1H, PhH),
5.98 (brs, 1H, NH). 5.18 (s, 2H, CH2) ppm; 13C NMR
(150 MHz, CDCl3): d = 161.0, 143.8, 143.4, 142.0, 141.7,
136.0, 129.7, 129.2, 128.9, 128.4, 128.1, 124.4, 123.5,
122.8, 119.5, 110.9, 49.8 ppm.
3-Benzyl-6-(propan-2-ylsulfanyl)-3H-quinazolin-4-one
(11, C18H18N2OS)
Light yellow solid; yield: 80 %, Rf = 0.60 (AcOEt/DCM/
PE 1/9/3); m.p.: 59–61 °C; IR (KBr): v = 1674 cm-1; H
NMR (600 MHz, CDCl3): d = 8.27 (d, 1H, J = 2.1 Hz, 5
ArH), 8.07 (s, 1H), 7.70 (dd, 1H, J = 8.5, 2.1 Hz, 7 ArH),
7.61 (d, 1H, J = 8.5 Hz, 8 ArH), 7.36–7.30 (m, 5H, PhH),
5.19 (s, 2H, CH2), 3.56–3.51 (m, 1H, CH), 1.34 (d, 6H,
J = 6.7 Hz, 2 9 Me) ppm; 13C NMR (150 MHz, CDCl3):
d = 160.7, 146.5, 146.1, 136.9, 136.6, 135.8, 134.6, 129.2,
128.5, 128.2, 128.0, 127.8, 124.5, 122.7, 49.9, 38.1, 31.0,
23.2 ppm.
3-Benzyl-6-[(4-ethoxyphenyl)amino]-3H-quinazolin-4-one
(9b, C23H21N3O2)
Light yellow solid; yield: 35 %, Rf = 0.32 (AcOEt/PE
1
1
10/1); m.p.: 199–200 °C; IR (KBr): v = 1663 cm-1; H
NMR (600 MHz, CDCl3): d = 7.94 (s, 1H), 7.68 (d, 1H,
J = 2.9 Hz, 5 ArH), 7.57 (d, 1H, J = 8.8 Hz, 8 ArH),
7.34–7.27 (m, 6H, ArH), 7.13–7.12 (m, 2H, ArH), 6.90–6.88
(m, 2H, ArH), 5.78 (brs, 1H, NH), 5.17 (s, 2H, CH2), 4.03 (q,
2H, J = 6.9 Hz, CH2), 1.42 (t, 3H, J = 6.9 Hz, Me) ppm;
13C NMR (150 MHz, CDCl3): d = 161.0, 155.9, 145.5,
143.3, 136.1, 134.2, 129.1, 134.2, 129.1, 128.4, 128.1, 123.8,
123.5, 123.0, 115.8, 108.8, 64.0, 49.8, 15.0 ppm.
Cells cultures
3-Benzyl-6-[[2-(3,4-dimethoxyphenyl)ethyl]amino]-3H-
quinazolin-4-one (9c, C25H25N3O3)
The experiments were performed with the use of HCT116
(colorectal carcinoma) and HT29 (colorectal adenocarci-
noma) cancer cells (human colon cancer cells) derived
from the American Type Culture Collection (ATCC; CCL-
247, HTB-38) and human lymphocytes obtained from the
Blood Donation Centre (Lodz, Poland).
Yellow solid; yield: 34 %, Rf = 0.42 (PE/DCM/MeCN
1/1/0.5); m.p.: 137–139 °C; IR (KBr): v = 1664 cm-1; 1H
NMR (600 MHz, CDCl3): d = 7.82 (s, 1H), 7.43 (d, 1H,
J = 8.8 Hz, 8 ArH), 7.30 (d, 1H, J = 2.4 Hz, 5 ArH),
7.26–7.16 (m, 6H, PhH, NH), 6.91 (dd, 1H, J = 8.8,
2.4 Hz, 7 ArH), 6.74 (d, 1H, J = 8.1 Hz, PhH), 6.69–6.63
(m, 2H, PhH), 5.10 (s, 2H, CH2), 3.78 (s, 6H, 2 9 OMe),
3.40 (t, 2H, J = 6.8 Hz, CH2), 2.82 (t, 2H, J = 6.8 Hz,
CH2) ppm; 13C NMR (150 MHz, CDCl3): d = 161.1,
149.2, 147.9, 147.4, 142.5, 140.2, 136.1, 131.4, 128.9,
128.6, 128.1, 127.9, 123.4, 122.1, 120.7, 112.1, 111.6,
105.2, 56.0, 55.9, 49.5, 44.9, 34.7 ppm.
HCT116 cells were cultured in RPMI 1640 medium
(CytoGen) supplemented with 10 % FBS (Foetal Bovine
Serum, CytoGen) and penicillin/streptomycin solution
(1 %).
RPMI 1640 medium (CytoGen) was used for HT29 cells
containing FBS (10 %), penicillin/streptomycin solution
(1 %), and MEM non-essential amino acids solution (1 %).
Human lymphocytes were cultured in RPMI 1640 medium
complemented with inactivated FBS (15 %), penicillin/
streptomycin (1 %), and mitogen PHA (1 %, phyto-
hemagglutinin; CytoGen). Cells were cultured at 37 °C in a
5 % CO2 humidified atmosphere. All solvents and reagents
were obtained from Sigma-Aldrich.
3-Methyl-6-(propylsulfanyl)-3H-quinazolin-4-one
(10a, C12H14N2OS)
Yellow solid; yield: 75 %, Rf = 0.64 (acetone/DCM 1/1);
m.p.: 62–64 °C; IR (KBr): v = 1667 cm-1
;
1H NMR
(600 MHz, DMSO-d6): d = 8.31 (s, 1H), 7.95 (d, 1H,
J = 2.2 Hz, 5 ArH), 7.73 (dd, 1H, J = 8.6, 2.2 Hz, 7
ArH), 7.60 (d, 1H, J = 8.6 Hz, 8 ArH), 3.49 (s, 3H, Me),
3.03 (t, 2H, J = 7.2 Hz, CH2), 1.66–1.60 (m, 2H, CH2),
0.99 (t, 3H, J = 7.3 Hz, Me) ppm; 13C NMR (150 MHz,
DMSO-d6): d = 197.7, 185.5, 183.6, 173.5, 171.5, 165.5,
160.8, 159.5, 71.6, 71.2, 59.3, 50.7, 37.7 ppm.
Inhibition growth assay
Cancer cells and human lymphocytes were grown for 24 h on
96-well plates at a density of 6–8 9 103 cells/well and
8 9 105 cells/well, respectively. Then the cells were treated
123

Z. Malinowski et al.
10. Tiwari AK, Singh VK, Bajpai A, Shukla G, Singh S, Mishra AK
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with the tested compounds for 72 h. After the treatment,
MTT dye dissolved in PBS was added to each plate well for
4 h. Purple crystals formed in cancer cells after the reduction
of MTT were dissolved by DMSO (100 mm3/well) after
removing of the RPMI 1640 medium. In the case of lym-
phocytes, it was done by adding 100 mm3 of 20 % DMF and
50 % SDS mixture to each well for 24 h. Absorbance at
595 nm was measured with a spectrophotometer Pow-
erWave XS (BioTek Instruments, Inc.).
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The cell survival effect was expressed as the IC50 value
which is the concentration of the compound required to
reduce cell survival to 50 % as compared to the negative
control. The experiments were done in triplicate. All the
results were presented as the mean SD.
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MTT assay
MTT assay is a quantitative colorimetric method to
determine cell proliferation after the treatment with the
tested compounds. It is widely used to estimate the cyto-
toxic effect of chemicals on different types of cells. The
assay is based on the reduction of the yellow, water-soluble
´´
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tetrazolium
MTT
[3-(4,5-dimethylthiazolyl-2)-2,5-
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of living (not dead) cells which results in the formation of
an insoluble purple formazan product. Formazan crystals
are solubilized with organic solvent. The amount of for-
mazan is measured spectrophotometrically and it is directly
proportional to the number of living cells.
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Acknowledgements The authors gratefully acknowledge the use of
the mass spectrometry services and facilities of the Center for Inter-
disciplinary Research of The John Paul II Catholic University of
Lublin, Lublin, Poland, funded by POPW.01.03.00-06-003/09-00.
The authors are grateful to the University of Lodz for a partial
financial support.
33. Matesic L, Locke JM, Bremner JB, Pyne SG, Skropeta D, Ranson
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