Synthesis and cytotoxicity of novel thioxo-quinazolino[3,4-a]quinazolinones

Article abstract of DOI:10.3906/kim-1512-80

Various thioxo-quinazolino[3,4-a]quinazolinones were prepared and evaluated for their cytotoxicity in MOLT-4 (lymphoblastic leukemia) and MCF-7 (breast adenocarcinoma) cell lines. Synthesis of the target compounds was started from isatoic anhydride. Successive reaction of isatoic anhydride with benzylamine and 2-nitrobenzaldehyde, reduction of the nitro group, and reaction with CS2 gave 12-benzyl-6-thioxo-6,7,11b,12-tetrahydro-13H-quinazolino[3,4-a]quinazolin-13-one. The latter compound reacted with various 2-chloro-N-substituted acetamides to afford the corresponding fused quinazolinone derivatives.

Full text of DOI:10.3906/kim-1512-80

Turk J Chem
Turkish Journal of Chemistry
(2017) 41: 125 – 134
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1512-80
Research Article
Synthesis and cytotoxicity of novel thioxo-quinazolino[3,4-a]quinazolinones
Negar MOHAMMADHOSSEINI1, Mina SAEEDI2,3, Shahram MORADI1,
Mohammad MAHDAVI5, Omidreza FIRUZI4, Alireza FOROUMADI5,
Abbas SHAFIEE5,∗
1Faculty of Chemistry, Tehran-North Branch, Islamic Azad University, Tehran, Iran
2Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
3Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran
4Medicinal and Natural Product Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
5Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran, Iran
Received: 26.12.2015
•
Accepted/Published Online: 05.04.2016
•
Final Version: 22.02.2017
Abstract:Various thioxo-quinazolino[3,4-a]quinazolinones were prepared and evaluated for their cytotoxicity in MOLT-4
(lymphoblastic leukemia) and MCF-7 (breast adenocarcinoma) cell lines. Synthesis of the target compounds was started
from isatoic anhydride. Successive reaction of isatoic anhydride with benzylamine and 2-nitrobenzaldehyde, reduction of
the nitro group, and reaction with CS2 gave 12-benzyl-6-thioxo-6,7,11b,12-tetrahydro-13H -quinazolino[3,4-a]quinazolin-
13-one. The latter compound reacted with various 2-chloro-N -substituted acetamides to afford the corresponding fused
quinazolinone derivatives.
Key words: Quinazolinones, cytotoxicity, thioxo-quinazolino[3,4-a]quinazoline, 2-nitrobenzaldehyde
1. Introduction
Quinazoline and its derivatives, a large and important class of heterocyclic compounds, have attracted lots
of attention due to their efficient and versatile biological activities1 such as antimicrobial,2 anticancer,3
anticholinesterase,4 and antihypertensive.5 Among the versatile and efficient medicinal properties of quina-
zolines, their anticancer activity has occupied significant attention since raltitrexed6 and thymitaq7 (Figure)
have been used in cancer chemotherapy. Hence, synthesis of quinazolinones has been the focus of attention and
various methods have been reported for their synthesis in the literature.8
Figure. The structure of raltitrexed and thymitaq.
∗Correspondence: shafieea@tums.ac.ir
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MOHAMMADHOSSEINI et al./Turk J Chem
Quinazolinones are usually prepared by the reaction of 2-aminobenzonitrile and 3-phenyl-acryloyl chlo-
ride followed by oxidative ring closure under basic conditions.9 Cyclization reaction of 2-aminobenzoic acids
and the corresponding modified protocols have been versatile and efficient procedures for the synthesis of
qinazolinones.10,11 Palladium-mediated N -heterocyclisation of 2-nitrophenyl ketones with formamide12 as well
aspalladium-mediated cyclocarbonylation of o-iodoanilines with heterocumulenes in the presence of CO (g) are
newly reported protocols in the field of quinazolinones.13
Recently, isatoic anhydride has been reported as a versatile starting material,14,15 and in this regard vari-
ous procedures have been developed by our research group leading to the synthesis of a wide spectrum of function-
alized quinazolinones. Obviously, 2-aminobenzamides obtained from reaction of isatoic anhydride and amines
are efficient bident nucleophiles to react with various electrophiles such as dimethyl acetylenedicarboxylate,16
aldehydes,17−19 Vilsmeier reagent,20 boronic acids,21 carbon disulfide and anthranilic acids,22 and N ,N ’-
dialkylcarbodiimides.23
In continuation of our research program for the synthesis of novel heterocycles24−27 using isatoic
anhydride,16−23 herein we report the synthesis of a wide range of novel thioxo-quinazolino[3,4-a]quinazolinones
10 to investigate their cytotoxicity (Scheme).
Scheme. Synthesis of novel thioxo-quinazolino[3,4-a]quinazolinones 10.
2. Results and discussion
2.1. Chemistry
The synthesis of novel desired thioxo-quinazolino[3,4-a]quinazoline derivatives 10 is depicted in the Scheme.
Compounds 3, 5, 6, and 8 were prepared according to our previous report.19 Reaction of isatoic anhydride 1
and benzylamine 2 in water at ambient temperature for 4 h gave 2-amino-N -benzylbenzamide 3, which reacted
with 2-nitrobenzaldehyde 4 in refluxing EtOH in the presence of K2 CO3 followed by reduction of the nitro
group in the presence of Zn/NH4 Cl to give 2-(2-aminophenyl)-3-benzyl-2,3-dihydroquinazolin-4(1H)-one 6.
Next, reaction of the latter compound with an excess amount of carbon disulfide (CS2) 7 in refluxing EtOH in
the presence of KOH afforded 12-benzyl-6-thioxo-6,7,11b,12-tetrahydro-13H -quinazolino[3,4-a]quinazolin-13-
one 8. Finally, reaction of compound 8 and 2-chloro-N -substituted acetamides 9 (obtained from chloroacetyl
chloride and different amines in DMF at room temperature) in acetone at room temperature led to the formation
of the desired products 10 (Table).
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MOHAMMADHOSSEINI et al./Turk J Chem
Table. Synthesis and cytotoxicity of thioxo-quinazolino[3,4-a]quinazolinones 10.
MOLT-4
MCF-7 cells
Entry
R
Product 10
IC50 (µM) IC50 (µM)
14.9 ꢀ 0.8 21.8 ꢀ 1.9
1
2
3
4
5
6
7
8
Cyclohexyl
C6H5
10a
10b
10c
10d
10e
10f
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
2-MeO-C6H4
3-MeO-C6H4
4-MeO-C6H4
2,5-diMeO-C6H3
3,4-diMeO-C6H3
10g
3,4,5-triMeO-C6H2 10h
9
2-Cl-C6H4
3-Cl-C6H4
4-Cl-C6H4
2,5-diCl-C6H3
3,4-diCl-C6H3
2-Me-C6H4
3-Me-C6H4
4-Me-C6H4
Cisplatin
10i
10j
10k
10l
10m
10n
10o
10p
10
11
12
13
14
15
16
17
54.1 ꢀ 1.3 > 100
> 100
> 100
> 100
> 100
> 100
3.3 ꢀ 0.3
> 100
> 100
> 100
> 100
> 100
12.3 ꢀ 3.0
The structures of all products derived from 10 were elucidated by IR, 1 H NMR, and 13 C NMR
spectroscopy as well as chemical analysis. For example, in the 1 H NMR spectrum of compound 10a, cyclohexyl
protons were detected in the range of 1.09–3.54 ppm. Methylene protons were observed at 3.70 and 4.49 ppm.
Moreover, a CH singlet signal was distinguished at 6.29 ppm. Aromatic protons (H1 , H2 , H3 , H4 , H8 , H9 ,
H10 , H11 , and phenyl as depicted in the Scheme) were detected in the range of 6.79–8.27 ppm according to the
expected chemical shifts and coupling patterns. Furthermore, amide NH proton was observed at 10.30 ppm.
The 13 C NMR spectrum showed the expected 28 signals including 19 aromatic (between 115.8 and 168.0 ppm)
and 9 aliphatic signals (between 24.5 and 69.7 ppm).
2.2. Biological investigation
The effects of sixteen molecules derived from compound 10 on cell viability were evaluated in two cancer cell
lines, namely MOLT-4 (lymphoblastic leukemia) and MCF-7 (breast adenocarcinoma). Cisplatin was used as
a positive control (Table). Based on calculated IC50 values, most of compounds showed no cytotoxicity and,
among them, compounds 10a and 10k were found to be cytotoxic active derivatives. Compound 10a depicted
activity against MOLT-4 and MCF-7 with IC50 values = 14.9 and 21.8 µM, respectively, compared with cisplatin
(IC50 values = 3.3 and 12.3). It is obvious that the cyclohexyl group induced higher activity compared with the
other aryl group connected to acetamide moiety. Compound 10k showed cytotoxicity against MOLT-4 (IC50
=
54.1 µM) and no activity was observed against MCF-7. It is clear that introduction of methoxy, chlorine, and
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MOHAMMADHOSSEINI et al./Turk J Chem
methyl groups led to the elimination of cytotoxic activity and only the presence of chlorine at the 4-position of
aryl connected to acetamide moiety induced cytotoxicity against MOLT-4.
3. Experimental
3.1. Apparatus and chemicals
Melting points were determined on a Kofler hot stage apparatus and are uncorrected. 1 H and 13 C NMR spectra
were recorded on a Bruker 500-MHz instrument using TMS as an internal standard. IR spectra were acquired
on a Nicolet Magna 550-FT spectrometer. All reagents were obtained from Merck and Aldrich.
3.2. Preparation of fused quinazolinone derivatives 10
Compounds 3, 5, 6, and 8 were prepared according to our previous report.19
A mixture of 12-benzyl-6-thioxo-6,7,11b,12-tetrahydro-13H -quinazolino[3,4-a]quinazolin-13-one 8 (1 mmol),
2-chloro-N -substituted acetamide 9 (1 mmol), and potassium iodide (1 mmol) in acetone (8 mL) was heated at
room temperature for 5–48 h. After completion of the reaction, water (8 mL) was added to the reaction mixture,
and the precipitates were filtered off and recrystallized from EtOH /H2 O (90/10) to give the corresponding
product 10.
3.2.1. N-Cyclohexyl-2-((13-oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)
thio)acetamide (10a)
Yield: 32%, mp 173–175 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 1.09–1.22 (m, 2H, Cyclohexyl), 1.28–1.39 (m,
2H, Cyclohexyl), 1.53–1.60 (m, 2H, Cyclohexyl), 1.62–1.69 (m, 2H, Cyclohexyl), 1.85–1.97 (m, 2H, Cyclohexyl),
3.48–3.54 (m, 1H, Cyclohexyl), 3.70 (s, 2H, CH2), 4.49 (s, 2H, SCH2), 6.29 (s, 1H, CH), 6.79 (d, J = 7.5 Hz,
1H, H4), 6.90 (d, J = 7.5 Hz, 1H, H8), 7.13–7.21 (m, 5H, Ph), 7.45 (t, J = 7.5 Hz, 1H, H9), 7.53–7.63 (m, 4H,
H2 , H3 , H10 , H11), 8.27 (d, J = 7.5 Hz, 1H, H1), 10.30 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 24.5,
25.5, 32.6, 32.7, 34.8, 44.8, 48.1, 48.2, 69.7, 115.8, 123.3, 124.7, 125.2, 126.7, 126.9, 127.0, 128.2, 128.4, 128.6,
129.5, 131.2, 132.1, 135.1, 137.2, 139.5, 156.4, 162.8, 168.0; IR (cm−1) υ: 3478, 1664, 1551; Anal. Calcd for
C30 H30 N4 O2 S: C, 70.56; H, 5.92; N, 10.97; Found: C, 70.79; H, 6.10; N, 10.69.
3.2.2. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-pheny-
lacetamide (10b)
Yield: 25%, mp 172–174 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.96 (d, J = 18.5 Hz, 1H, CH2), 4.07 (d, J =
18.5 Hz, 1H, CH2), 4.17 (d, J = 20.0 Hz, 1H, CH2), 4.50 (d, J = 20.0 Hz, 1H, CH2), 6.57 (s, 1H, CH), 6.71
(d, J = 9.0 Hz, 1H, H4), 6.77 (t,J = 9.0 Hz, 1H, H2), 6.97 (d, J = 9.0 Hz, 1H, H8), 7.00–7.11 (m, 4H, Ph),
7.13 (t, J = 9.0 Hz, 1H, H9), 7.19 (t, J = 9.0 Hz, 1H, H10), 7.29–7.38 (m, 3H, Ph), 7.56–7.63 (m, 3H, Ph),
7.66–7.75 (m, 2H, H3 , H11), 8.07 (d, J = 9.0 Hz, 1H, H1), 10.29 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ:
37.0, 44.5, 68.9, 117.0, 119.6, 123.9, 125.1, 125.6, 126.3, 126.8, 127.0, 128.4, 128.9, 129.2, 129.3, 130.0, 131.2,
132.7, 137.9, 139.4, 140.1, 141.4, 155.2, 162.6, 164.0, 166.3; IR (cm−1) υ: 3396, 1655, 1543; Anal. Calcd for
C30 H24 N4 O2 S: C, 71.41; H, 4.79; N, 11.10; Found: C, 71.22; H, 4.58; N, 11.23.
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MOHAMMADHOSSEINI et al./Turk J Chem
3.2.3. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(2-
methoxyphenyl)acetamide (10c)
Yield: 79%, mp 179–181 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.63 (s, 3H, OCH3), 3.97 (d, J = 19.0 Hz, 1H,
CH2), 4.01 (d, J = 19.0 Hz, 1H, CH2), 4.18 (d, J = 20.0 Hz, 1H, CH2), 4.48 (d, J = 20.0 Hz, 1H, CH2),
6.60 (s, 1H, CH), 6.74–6.79 (m, 2H, H4 , H3 ’), 6.91 (t, J = 9.5 Hz, 1H, H2), 6.97–6.77 (m, 5H, Ph), 7.10 (d,
J = 9.2 Hz, 1H, H8), 7.14–7.22 (m, 2H, H9 , H11), 7.41 (t, J = 9.2 Hz, 1H, H10), 7.57–7.74 (m, 4H, H3 , H4 ’,
H5 ’, H6 ’), 8.08 (d, J = 9.5 Hz, 1H, H1), 9.55 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 36.1, 44.5, 55.9,
69.0, 111.4, 113.3, 117.0, 120.8, 120.9, 122.1, 125.4, 125.6, 126.3, 126.8, 127.0, 127.7, 128.3, 128.7, 128.9, 129.2,
131.2, 132.7, 137.9, 139.9, 141.3, 149.2, 155.4, 162.6, 166.8; IR (cm−1) υ: 3251, 1684, 1657; Anal. Calcd for
C31 H26 N4 O3 S: C, 69.64; H, 4.90; N, 10.48; Found: C, 69.36; H, 5.06; N, 10.73.
3.2.4. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(3-
methoxyphenyl)acetamide (10d)
Yield: 70%, mp 178–180 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.71 (s, 3H, OCH3), 3.96 (d, J = 15.0 Hz, 1H,
CH2), 4.06 (d, J = 15.0 Hz, 1H, CH2), 4.18 (d, J = 16.2 Hz, 1H, CH2), 4.51 (d, J = 16.2 Hz, 1H, CH2), 6.56
′
′
(s, 1H, CH), 6.64 (d, J = 7.0 Hz, 1H, H4 ), 6.76–6.78 (m, 2H, H8 , H4), 6.88 (d, J = 7.0 Hz, 1H, H6 ), 7.03 (d,
′
J = 7.2 Hz, 1H, H11), 7.06–7.15 (m, 4H, Ph), 7.16–7.24 (m, 2H, H2 , Ph), 7.03 (s, 1H, H2 ), 7.35 (t, J = 7.2 Hz,
′
1H, H3), 7.59 (t, J = 7.2 Hz, 1H, H10), 7.65–7.75 (m, 2H, H9 , H5 ), 8.08 (d, J = 7.2 Hz, 1H, H1), 10.28 (s,
1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 37.0, 44.5, 55.4, 68.9, 104.1, 105.4, 109.3, 111.9, 117.0, 123.9, 125.1,
125.6, 126.4, 128.4, 128.6, 128.9, 129.3, 130.0, 131.2, 132.7, 137.9, 140.1, 140.6, 140.7, 141.4, 155.2, 156.0, 162.6,
166.3; IR (cm−1) υ: 3507, 1699, 1650; Anal. Calcd for C31 H26 N4 O3 S: C, 69.64; H, 4.90; N, 10.48; Found: C,
69.88; H, 5.13; N, 10.59.
3.2.5. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(4-
methoxyphenyl)acetamide (10e)
Yield: 96%, mp 150–152 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.71 (s, 3H, OCH3), 3.93 (d, J = 15.0 Hz, 1H,
CH2), 4.04 (d, J = 15.0 Hz, 1H, CH2), 4.18 (d, J = 16.2 Hz, 1H, CH2), 4.51 (d, J = 16.2 Hz, 1H, CH2), 6.56
′
(s, 1H, CH), 6.77–6.78 (m, 2H, H4 , H11), 6.89 (d, J = 8.8 Hz, 2H, H3 , H5 ), 7.01 (t, J = 7.6 Hz, 1H, Ph),
′
′
7.03–7.22 (m, 5H, H2 , Ph), 7.35 (t, J = 7.6 Hz, 1H, H10), 7.49 (d, J = 8.8 Hz, 2H, H2 , H6 ), 7.61 (t, J = 7.6
′
Hz, 1H, H3), 7.70 (d, J = 7.6 Hz, 1H, H8), 7.71–7.75 (t, J = 7.6 Hz, 1H, H9), 8.07 (d, J = 7.6 Hz, 1H, H1),
10.16 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 36.9, 44.5, 55.6, 68.9, 114.2, 114.3, 117.0, 120.9, 121.2,
123.9, 124.0, 125.1, 125.6, 126.4, 126.8, 127.0, 128.4, 128.9, 131.2, 132.6, 132.7, 137.9, 140.1, 141.5, 155.8, 162.6,
165.7; IR (cm−1) υ: 3359, 1672, 1643; Anal. Calcd for C31 H26 N4 O3 S: C, 69.64; H, 4.90; N, 10.48; Found: C,
69.48; H, 5.11; N, 10.77.
3.2.6. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(2,5-
dimethoxyphenyl)acetamide (10f)
Yield: 41%, mp 187–188 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.58 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.97
(d, J = 14.8 Hz, 1H, CH2), 4.10 (d, J = 14.8 Hz, 1H, CH2), 4.19 (d, J = 16.4 Hz, 1H, CH2), 4.45 (d, J = 16.4
′
Hz, 1H, CH2), 6.60 (d, J = 2.2 Hz, 1H, H6 ), 6.62 (s, 1H, CH), 6.76 (d, J = 8.8 Hz, 1H, H4), 6.90 (d, J = 8.8
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MOHAMMADHOSSEINI et al./Turk J Chem
′
Hz, 1H, H8), 6.98–7.05 (m, 3H, H2 , H9 , H3 ), 7.09 (d, J = 8.8 Hz, 1H, H11), 7.14–7.22 (m, 3H, Ph), 7.41 (td,
′
J = 2.4, 8.8 Hz, 1H, H10), 7.60 (t, J = 8.8 Hz, 1H, H3), 7.65–7.74 (m, 3H, Ph, H4 ), 8.07 (d, J = 8.8 Hz, 1H,
H1), 9.06 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 36.2, 44.5, 55.8, 56.4, 69.5, 106.2, 107.5, 108.3, 112.0,
117.0, 124.1, 125.4, 125.6, 126.3, 126.8, 127.0, 128.4, 128.7, 128.9, 130.8, 131.2, 132.8, 137.9, 139.9, 141.2, 143.2,
153.4, 155.4, 162.7, 167.0; IR (cm−1) υ: 3257, 1686, 1655; Anal. Calcd for C32 H28 N4 O4 S: C, 68.07; H, 5.00;
N, 9.92; Found: C, 68.25; H, 4.83; N, 10.03.
3.2.7. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(3,4-
dimethoxyphenyl)acetamide (10g)
Yield: 54%, mp 192–193 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.52 (d, J = 14.5 Hz, 1H, CH2), 3.80 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 3.96 (d, J = 14.5 Hz, 1H, CH2), 4.32 (d, J = 15.7 Hz, 1H, CH2), 4.70 (d, J =
′
′
15.7 Hz, 1H, CH2), 6.46 (s, 1H, CH), 6.74–6.78 (m, 3H, H2 , H5 , H4), 6.87 (d, J = 6.7 Hz, 1H, H6 ), 7.00
′
(d, J = 7.1 Hz, 1H, H8), 7.04–7.07 (m, 3H, H2 , H3 , H9), 7.18–7.28 (m, 3H, Ph), 7.45 (td, J = 1.0, 7.1 Hz,
1H, H10), 7.52–7.60 (m, 3H, Ph, H11), 8.26 (d, J = 7.1 Hz, 1H, H1), 10.04 (s, 1H, NH); 13 C NMR (125 MHz,
CDCl3) δ: 35.6, 44.7, 55.8, 56.1, 69.5, 104.3, 111.2, 111.4, 115.9, 123.1, 124.5, 125.5, 126.5, 126.8, 126.9, 128.1,
128.4, 128.8, 129.5, 131.2, 132.1, 137.1, 139.3, 140.6, 145.5, 146.1, 149.0, 157.1, 162.7, 166.8; IR (cm−1) υ:
3253, 1662, 1606; Anal. Calcd for C32 H28 N4 O4 S: C, 68.07; H, 5.00; N, 9.92; Found: C, 68.32; H, 4.82; N,
9.70.
3.2.8. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(3,4,5-
trimethoxyphenyl)acetamide (10h)
Yield: 20%, mp 200–202 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.49 (d, J = 13.8 Hz, 1H, CH2), 3.77 (s, 3H,
OCH3), 3.79 (s, 6H, 2 × OCH3), 3.94 (d, J = 13.8 Hz, 1H, CH2), 4.32 (d, J = 15.7 Hz, 1H, CH2), 4.69 (d,
J = 15.7 Hz, 1H, CH2), 6.30 (s, 1H, CH), 6.73–6.77 (m, 4H, H2 , H6 , H2 , H11), 7.01 (d, J = 7.4 Hz, 1H, H8),
′
′
7.05–7.08 (m, 3H, H4 , H9 , H10), 7.18–7.26 (m, 2H, Ph), 7.44 (t,J = 7.4 Hz, 1H, H3), 7.52–7.61 (m, 3H, Ph),
7.26 (d, J = 7.4 Hz, 1H, H1), 10.24 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 35.6, 44.7, 55.9, 60.9, 69.5,
96.9, 113.2, 116.0, 123.0, 123.10, 125.5, 126.4, 126.8, 126.9, 127.1, 128.9, 129.5, 131.0, 131.3, 132.2, 134.4, 137.0,
139.3, 140.7, 153.2, 157.2, 162.6, 167.0; IR (cm−1) υ: 3434, 1689, 1662; Anal. Calcd for C33 H30 N4 O5 S: C,
66.65; H, 5.08; N, 9.42; Found: C, 67.42; H, 5.40; N, 9.23.
3.2.9. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(2-
chlorophenyl)acetamide (10i)
Yield: 23%, mp 154–156 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.88 (d, J = 14.4 Hz, 1H, CH2), 3.96 (d, J = 14.4
Hz, 1H, CH2), 3.38 (d, J = 15.8 Hz, 1H, CH2), 4.55 (d, J = 15.8 Hz, 1H, CH2), 6.27 (s, 1H, CH), 6.86–6.98
′
(m, 3H, H2 , H4 , H11), 7.01–7.14 (m, 5H, Ph), 7.25–7.33 (m, 3H, H4 , H5 , H8), 7.42 (t, J = 8.3 Hz, 1H, H10),
′
7.52–7.62 (m, 3H, H3 , H3 , H9), 8.28 (m, 2H, H1 , H6 ), 9.49 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ:
35.6, 44.8, 69.7, 113.1, 116., 122.5, 123.5, 124.5, 124.6, 125.2, 126.6, 126.9, 127.5, 128.1, 128.3, 128.4, 129.0,
129.4, 130.9, 132.0, 134.9, 137.2, 138.2, 139.4, 141.0, 155.6, 162.8, 167.5; IR (cm−1) υ: 3450, 1691, 1650; Anal.
Calcd for C30 H23 ClN4 O2 S: C, 66.85; H, 4.30; N, 10.39. Found: C, 66.56; H, 4.09; N, 10.58.
′
′
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MOHAMMADHOSSEINI et al./Turk J Chem
3.2.10. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(3-
chlorophenyl)acetamide (10j)
Yield: 17%, mp 199–201 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.53 (d, J = 14.2 Hz, 1H, CH2), 3.90 (d, J = 14.2
Hz, 1H, CH2), 4.32 (d, J = 17.7 Hz, 1H, CH2), 4.72 (d, J = 17.7 Hz, 1H, CH2), 6.30 (s, 1H, CH), 6.75–6.77
′
(m, 2H, H4 , H11), 7.00–7.10 (m, 3H, H2 , H9 , H4 ), 7.18-7.28 (m, 5H, H3, H8 , H10 , H5 , H6 ), 7.48–7.62 (m,
′
′
6H, H2’, Ph), 7.27 (d, J = 7.5 Hz, 1H, H1), 10.24 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 35.7, 44.7,
69.6, 116.0, 117.5, 119.7, 123.2, 124.5, 125.6, 126.6, 126.9, 127.0, 128.2, 128.5, 128.8, 129.6, 129.9, 130.9, 131.4,
132.2, 134.6, 139.4, 139.7, 140.8, 149.6, 157.1, 162.7, 167.4; IR (cm−1) υ: 3322, 1683, 1661; Anal. Calcd for
C30 H23 ClN4 O2 S: C, 66.85; H, 4.30; N, 10.39. Found: C, 66.62; H, 4.58; N, 10.21.
3.2.11. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(4-
chlorophenyl)acetamide (10k)
Yield: 22%, mp 212–214 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.53 (d, J = 14.2 Hz, 1H, CH2), 3.91 (d, J = 14.2
Hz, 1H, CH2), 4.31 (d, J = 14.2 Hz, 1H, CH2), 4.72 (d, J = 14.2 Hz, 1H, CH2), 6.30 (s, 1H, CH), 6.73–6.77
′
(m, 2H, H2 , H4), 7.02–7.08 (m, 5H, Ph), 7.16 (d, J = 7.5 Hz, 1H, H8), 7.24 (d, J = 8.7 Hz, 2H, H3 , H5 ),
′
′
7.38 (d, J = 8.7 Hz, 2H, H2 , H6 ), 7.49 (t, J = 7.5 Hz, 1H, H10), 7.52–7.62 (m, 3H, H3 , H9 , H11), 8.26 (d,
′
J = 7.6 Hz, 1H, H1), 10.19 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 35.7, 44.7, 69.6, 113.3, 116.0, 116.6,
120.7, 123.1, 124.4, 125.5, 126.5, 126.8, 128.1, 128.4, 128.8, 128.9, 129.1, 129.5, 131.4, 132.2, 136.8, 139.3, 140.8,
157.1, 162.7, 167.3; IR (cm−1) υ: 3242, 1662, 1556; Anal. Calcd for C30 H23 ClN4 O2 S: C, 66.85; H, 4.30; N,
10.39. Found: C, 66.97; H, 4.48; N, 10.11.
3.2.12. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(2,5-
dichlorophenyl)acetamide (10l)
Yield: 23%, mp 197–199 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.84 (d, J = 14.4 Hz, 1H, CH2), 3.97 (d, J =
14.4 Hz, 1H, CH2), 4.33 (d, 1H, CH2 ,J = 15.8 Hz), 4.58 (d, J = 15.8 Hz, 1H, CH2), 6.27 (s, 1H, CH), 6.79–6.84
′
(m, 3H, H2 , H4 , H11), 7.00 (d, J = 8.0 Hz, 1H, H8), 7.07–7.14 (m, 4H, Ph), 7.20 (d, J = 8.1 Hz, 1H, H3 ),
′
7.26 (d, J = 8.1 Hz, 1H, H4 ), 7.42 (t, J = 8.0 Hz, 1H, H10), 7.51–7.56 (m, 2H, Ph, H9), 7.59 (t, J = 7.5 Hz,
1H, H3), 8.27 (d, J = 7.5 Hz, 1H, H1), 8.41 (d, J = 2.1 Hz, 1H, H6 ), 9.57 (s, 1H, NH); 13 C NMR (125 MHz,
′
CDCl3) δ: 35.6, 44.6, 69.7, 114.0, 116.1, 121.3, 121.1, 122.1, 124.4, 124.6, 125.2, 126.6, 126.8, 126.9, 128.1,
128.4, 128.4, 129.5, 129.6, 130.9, 132.1, 135.9, 137.2, 139.3, 141.0, 155.6, 162.7, 167.6; IR (cm−1) υ: 3233, 1687,
1660; Anal. Calcd for C30 H22 Cl2 N4 O2 S: C, 62.83; H, 3.87; N, 9.77. Found: C, 62.74; H, 3.69; N, 10.14.
3.2.13. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(3,4-
dichlorophenyl)acetamide (10m)
Yield: 20%, mp 185–187 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 3.54 (d, J = 14.2 Hz, 1H, CH2), 3.87 (d, J = 14.2
Hz, 1H, CH2), 4.31 (d, J = 15.7 Hz, 1H, CH2), 4.74 (d, J = 15.7 Hz, 1H, CH2), 6.31 (s, 1H, CH), 6.67–6.75
′
(m, 2H, H2 , H4), 7.02–7.09 (m, 3H, H9 , H10 , H11), 7.15 (d, J = 7.8 Hz, 1H, H8), 7.21–7.26 (m, 2H, H3 , H5 ),
′
′
7.31 (d, J = 7.8 Hz, 1H, H6 ), 7.48–7.68 (m, 5H, Ph), 7.64 (d, J = 2.4 Hz, 1H H2 ), 8.26 (dd, J = 1.5, 7.8 Hz,
1H, H1), 10.37 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 35.7, 44.7, 69.6, 116.0, 118.2, 118.7, 121.2, 123.0,
124.4, 125.7, 126.5, 126.9, 127.0, 128.2, 128.3, 128.5, 128.9, 130.4, 131.4, 132.2, 132.7, 137.1, 137.7, 139.3, 140.7,
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MOHAMMADHOSSEINI et al./Turk J Chem
157.2, 162.7, 167.4; IR (cm−1) υ: 3306, 1685, 1661; Anal. Calcd for C30 H22 Cl2 N4 O2 S: C, 62.83; H, 3.87; N,
9.77. Found: C, 62.60; H, 3.69; N, 9.90.
3.2.14. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(2-
methylphenyl)acetamide (10n)
Yield: 18%, mp 210–212 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 2.08 (s, 3H, CH3), 3.85 (d, J = 14.4 Hz, 1H,
CH2), 3.89 (d, J = 14.4 Hz, 1H, CH2), 4.47–4.49 (m, 2H, CH2), 6.28 (s, 1H, CH), 6.72–6.80 (m, 2H, H2 , H4),
′
6.90 (t, J = 8.0 Hz, 1H, H9), 7.0–7.05 (m, 4H, Ph), 7.13–7.18 (m, 4H, Ph, H8 , H11 , H4 ), 7.21 (t, J = 8.0 Hz,
′
1H, H10), 7.41 (t, J = 8.0 Hz, 1H, H3), 7.56–7.64 (m, 3H, H3 , H5 , H6 ), 7.77 (d, J = 8.0 Hz, 1H, H1), 9.02
′
′
(s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 18.0, 35.4, 44.7, 69.7, 113.4, 116.2, 123.4, 124.7, 125.1, 125.3,
126.5, 126.6, 126.9, 127.2, 127.6, 128.2, 128.4, 128.6, 129.5, 130.3, 131.1, 132.1, 135.8, 137.1, 139.3, 140.9, 156.6,
162.8, 167.5; IR (cm−1) υ: 3252, 1690, 1670; Anal. Calcd for C31 H26 N4 O2 S: C, 71.79; H, 5.05; N, 10.80.
Found: C, 71.61; H, 5.26; N, 10.63.
3.2.15. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(3-
methylphenyl)acetamide (10o)
Yield: 24%, mp 191–193 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 2.60 (s, 3H, CH3), 3.50 (d, J = 14.1 Hz, 1H,
CH2), 3.95 (d, J = 14.1 Hz, 1H, CH2), 4.34 (d, J = 15.7 Hz, 1H, CH2), 4.69 (d, J = 15.7 Hz, 1H, CH2), 6.29
′
(s, 1H, CH), 6.75–6.78 (m, 2H, H2 , H4), 6.89 (d, J = 7.0 Hz, 1H, H4 ), 6.99 (m, 2H, H8 , H10), 7.03–7.13 (m,
′
3H, H9 , H11 , Ph), 7.14–7.23 (m, 4H, Ph), 7.33 (s, 1H, H2 ), 7.48 (t, J = 7.4 Hz, 1H, H3), 7.54 (t, J = 7.0 Hz,
1H, H5 ), 7.58 (d, J = 7.0 Hz, 1H, H6 ), 8.27 (d, J = 7.4 Hz, 1H, H1), 9.96 (s, 1H, NH); 13 C NMR (125 MHz,
CDCl3) δ: 21.5, 35.7, 44.7, 69.6, 113.4, 116.0, 116.6, 120.3, 123.4, 124.5, 124.8, 125.4, 126.3, 126.6, 126.9, 127.0,
127.1, 128.2, 128.7, 129.5, 131.3, 137.2, 138.1, 138.8, 139.5, 140.9, 156.9, 162.7, 167.2; IR (cm−1): 3252, 1697,
1661; Anal. Calcd for C31 H26 N4 O2 S: C, 71.79; H, 5.05; N, 10.80. Found: C, 71.58; H, 5.29; N, 10.66.
′
′
3.2.16. 2-((13-Oxo-12-benzyl-11b,12-dihydro-13H-quinazolino[3,4-a]quinazolin-6-yl)thio)-N-(4-
methylphenyl)acetamide (10p)
Yield: 24%, mp 185–187 ◦ C. 1 H NMR (500 MHz, CDCl3) δ: 2.30 (s, 3H, CH3), 3.50 (d, J = 14.1 Hz, 1H,
CH2), 3.95 (d, J = 14.1 Hz, 1H, CH2), 4.33 (d, J = 15.7 Hz, 1H, CH2), 4.70 (d, J = 15.7 Hz, 1H, CH2), 6.29
(s, 1H, CH), 6.99–7.0 (m, 2H, H4 , H11), 7.05–7.12 (m, 5H, Ph), 7.19–7.21 (m, 2H, H2 , H9), 7.33 (d, J = 8.2
′
Hz, 2H, H3 , H5 ), 7.45–7.48 (m, 2H, H8, H10), 7.52–7.62 (m, 3H, H3 , H2, H6 ), 8.26 (d, J = 7.6 Hz, 1H,
′
′
H1), 9.93 (s, 1H, NH); 13 C NMR (125 MHz, CDCl3) δ: 20.8, 35.7, 44.7, 69.6, 116.0, 116.7, 119.6, 123.3, 124.5,
125.6, 126.3, 126.8, 127.0, 127.5, 128.2, 128.3, 129.4, 129.5, 131.3, 132.1, 135.6, 137.2, 139.4, 140.9, 156.9, 162.7,
167.1; IR (cm−1) υ: 3515, 1683, 1656; Anal. Calcd for C31 H26 N4 O2 S: C, 71.79; H, 5.05; N, 10.80. Found: C,
71.465; H, 5.31; N, 10.41.
3.3. Cytotoxicity assay
Reagents and chemicals. Fetal bovine serum (FBS), phosphate buffered saline (PBS), RPMI 1640, and
trypsin were purchased from Biosera (Ringmer, UK). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) and penicillin/streptomycin were obtained from Sigma-Aldrich (St Louis, MO, USA) and Invit-
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rogen (San Diego, CA, USA), respectively. Cisplatin was obtained from EBEWE Pharma (Unterach, Austria)
and dimethyl sulfoxide was purchased from Merck (Darmstadt, Germany).
Cell lines and cell culture. MOLT-4 (human lymphoblastic leukemia) and MCF-7 (human breast
adenocarcinoma) cells were obtained from the National Cell Bank of Iran, Pasteur Institute, Tehran, Iran.
Cells were kept at 37 ◦ C in humidified air containing 5% CO2 and were grown in suspension (MOLT-4) or
monolayer cultures (MCF-7). They were maintained in RPMI 1640 supplemented with 10% FBS, and 100
units/mL penicillin-G and 100 µg/mL streptomycin.
The in vitro cytotoxicity of compounds 10 was determined against two MOLT-4 and MCF-7 cell lines.
The inhibitory effect of the synthesized compounds on cancer cells’ growth was assessed by the MTT reduction
assay.28 The synthesized compounds were dissolved in dimethyl sulfoxide, and diluted in growth medium at
least 400 times. Maximum concentration of DMSO in the wells was 0.5%. Cells were seeded in 96-well plates
(5000 cells/well) and incubated at 37 ◦ C for 24 h. Different concentrations of test compounds in the range of
10–100 µM were added and, after 72 h of further incubation at 37 ◦ C, 0.5 mg/mL 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) dissolved in growth medium without phenol red was added to the
wells. After 4 h, formazan crystals were solubilized in 200 µL of dimethyl sulfoxide and the optical density was
measured at 570 nm, with background correction at 655 nm, using a microplate reader.
Acknowledgments
This work was supported by Tehran University of Medical Sciences and Iran National Science Foundation
(INSF).
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