Synthesis, antitumor testing and molecular modeling study of some new 6-substituted amido, azo or thioureido-quinazolin-4(3H)-ones

Article abstract of DOI:10.1016/j.bioorg.2019.102923

A new series of 6-substituted amido, azo or thioureido-quinazolin-4(3H)-one was synthesized and tested for their in-vitro antitumor activity. Compounds 21, 53 and 60 showed broad spectrum antitumor activity with average IC₅₀ values of 6.7, 7.6

Full text of DOI:10.1016/j.bioorg.2019.102923

BioorganicChemistry88(2019)102923
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Bioorganic Chemistry
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Synthesis, antitumor testing and molecular modeling study of some new 6-
substituted amido, azo or thioureido-quinazolin-4(3H)-ones
⁎
Mohamed A. Sabrya, , Heba A. Ewidab, Ghada S. Hassana, Mariam A. Ghalya,
⁎
Hussein I. El-Subbagha,
a Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, P.O. Box 35516, Mansoura, Egypt
b Department of Pharmacology and Biochemistry, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future University in Egypt, 12311 Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of 6-substituted amido, azo or thioureido-quinazolin-4(3H)-one was synthesized and tested for their
in-vitro antitumor activity. Compounds 21, 53 and 60 showed broad spectrum antitumor activity with average
IC50 values of 6.7, 7.6 and 9.1 μM, respectively compared with methotrexate (1, IC50 19.26 μM). As an attempt to
reveal the mechanism of the antitumor potency, cell cycle analysis and DHFR inhibition were performed.
Compounds 59 and 61 induced their cytotoxicity in Hela (IC50 10.6 μM) and HCT-116 (IC50 15.5 μM) cell lines,
respectively through Pre-G1 apoptosis, inhibiting cell growth at G2-M phase. Compounds 29, 33, 59 and 61
showed DHFR inhibitory potency at IC50 0.2, 0.2, 0.3 and 0.3 μM, respectively. The active DHFR inhibitors
showed high affinity binding toward the amino acid residues Thr56, Ser59 and Ser118. The active compounds
obeyed Lipinski’s rule of five and could be used as template model for further optimization.
Synthesis
Substituted-quinazolin-4(3H)-one
Antitumor testing
Cell viability assay
DHFR inhibition
Molecular modeling study
1. Introduction
in the DHFR active site [20,21].
Compounds containing thioether (7–9), amide (10), azo- (11) and
Quinazolines as anticancer agents have been in the focus since the
discovery of raltitrexed (1) and thymitaq (2) as thymidylate synthetase
(TS) inhibitors [1–3]. 6-Arylamino-7-chloro-quinazoline-5,8-dione deri-
vatives (3) showed antineoplastic potency against human A549 lung, and
SNU-638 stomach cancer cell lines [4–6]. Quinazolines inhibit EGFR
(epidermal growth factor receptor) tyrosine kinase overexpression
through the inhibition of EGFR autophosphorylation and EGF-stimulated
signal transduction [7–9]. Quinazolines also exert their antitumor ac-
tivity through inhibition of the DNA repair enzyme system [10–14]. The
pteridine containing lead methotrexate (MTX, 4), the quinazoline ske-
leton of trimetrexate (5) and its pyrido[2,3-d]pyrimidine bioisoster pir-
itrexim (6) are dihydrofolate reductase (DHFR) inhibitors exhibited an
essential role in clinical medicine as antitumor agents [15–17], Fig. 1.
The activity of DHFR is linked to TS which catalyzes the reductive
methylation of dUMP to dTMP [18]. The inhibition of DHFR prevents
the growth of cancer cells and depletes the cell from thymine causing
thymineless death. Accordingly, DHFR inhibition becomes the target
for new antitumor drugs [19]. This revealed the importance of quina-
zoline nucleus concerning its binding affinity toward DHFR. Different
hydrophobic and hydrophilic interactions for quinazolines were ob-
served with Phe31, phe34, Thr56, Ser59 and Lys55 amino acid residues
thioureido- (12) functional groups known to enhance antitumor ac-
tivity, Fig. 2 [22–26]. Combining the inherited antitumor and DHFR
inhibition activity of quinazolines and the antitumor potency enhancer
functional groups in one structure is anticipated to produce compounds
with more antitumor and/or DHFR inhibition activities [20,21,27–30].
The designed hybrids and their structure features (A, B, and C), fitted
with electron donating or withdrawing functional groups, illustrated in
Fig. 2, were synthesized and evaluated for their antitumor activity. As
an attempt to reveal the antitumor potency mode of action, cell cycle
analysis was performed and DHFR inhibition activity was evaluated.
Moreover, Conformational analysis, molecular docking, flexible align-
ment, surface mapping, ADMET-study and compliance to the Lipinski's
rule of five were investigated. The present study, aim to synthesis new
quinazoline analogues, hoping to reach potentiated antitumor with
DHFR inhibitory activity.
2. Results and discussion
2.1. Chemistry
The designed compounds A, B and C were synthesized adopting the
⁎ Corresponding authors.
E-mail addresses: mohamedas1992@yahoo.com (M.A. Sabry), subbagh@yahoo.com (H.I. El-Subbagh).
https://doi.org/10.1016/j.bioorg.2019.102923
Received 28 January 2019; Received in revised form 1 April 2019; Accepted 10 April 2019
Availableonline10April2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Fig. 1. Structures of some lead compounds.
Fig. 2. Combining structure features of quinazoline, thioether, amide (A), azo (B) and thioureido (C) moieties.
routes outlined in Schemes 1–4. The starting materials 2-Mercapto-3-(4-
substituted-phenyl)-6-substituted-quinazolin-4(3H)-ones (16, 17) were
obtained by reacting 5-nitro-anthranilic acid (13) and 4-substituted-
phenyl isothiocyanates (14 and 15) using reported reaction conditions
[21,29–31]. The 2-mercapto- functions of 16 and 17 was methylated
using methyl iodide to produce 18 and 19. Refluxing the 6-nitro-qui-
nazolines 18 and 19 with stannous chloride in acetone gave the 6-
amino analogues 20 and 21, (Scheme 1, Table 1).
The 6-amino function of 20 and 21 was utilized to synthesize the
amide derivatives 25–30, 32, 33 and 37–42 using variety of acid
2

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Scheme 1. Synthesis of the target compounds 16–21.
chlorides (Scheme 2), the azo derivatives 45–48, 50, 51, 53 and 54
using various phenolic coupling reagents (Scheme 3), and the thiourea
analogues 56–61 upon treating with 4-substituted phenyl iso-
thiocyanate derivatives (Scheme 4). Structure elucidation of the syn-
thesized compounds was attained by the use of elemental analyses, in
conjunction with MS, 1H and 13C NMR spectra.
2.2. Biological evaluation
2.2.1. Antitumor screening
The synthesized compounds 16–21, 25–30, 32, 33, 37–42, 45–48,
50, 51, 53, 54 and 56–61 were evaluated for their antitumor activity
via standard MTT assay [32–34], using four human tumor cell lines
namely; HCT-116 colorectal carcinoma, MCF-7 breast, PC-3 prostate
Scheme 2. Synthesis of the target compounds 25–30, 32, 33 and 37–42.
3

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Scheme 3. Synthesis of the target compounds 45–48, 50, 51, 53 and 54.
and Hela epithelioid carcinoma. Methotrexate (1) was used as a stan-
dard anticancer drug (IC50 19.26 μM). Cytotoxicity was expressed as the
concentration that caused 50% loss of the cell monolayer (IC50),
Table 1. The tested compounds exhibited cytotoxic antitumor potency
of various magnitudes. Compounds 21, 53 and 60 showed remarkable
broad spectrum cytotoxic potency and considered to be the most active
members in this study with average IC50 values of 6.7, 7.6 and 9.1 μM,
respectively. Compounds 17, 20, 54, 57, 59 and 61 showed strong
activities with average IC50 values of 10.5, 13.7, 17.1, 11.8, 18.9,
19.9 μM, respectively. Meanwhile, some of the tested compounds
showed selective potency toward certain tumor cell lines, such as 37,
viability assay was performed [36–38] using mutant Hela cell line and
59 (cytotoxicity IC50 value of 10.62 μM). The flow cytometry experi-
ment results for cell cycle analysis indicated that 59 increased the
percentage of apoptotic cells at Pre-G1 phase from 1.91% to 26.59%;
increased the percentage of cells at G2/M phase from 6.3% to 44.01%;
decreased the percentage of cells in the S phase from 35.09% to 26.44%
and decreased the percentage of cells in G0/G1 phase from 58.61% to
29.55% causing cell cycle arrest (Fig. 3). Cell viability assay was also
performed for 61 using the mutant HCT-116 cell line which exhibited
strong cytotoxicity with IC50 values of 15.49 μM. The flow cytometry
experiment results for cell cycle analysis indicated that 61 increased the
percentage of apoptotic cells in Pre-G1 phase from 1.66% to 18.41%;
increased the percentage of cells in G2/M phase from 8.84% to 31.77%;
decreased the percentage of cells in the S phase from 39.27% to 29.52%
and decreased the percentage of cells in G0/G1 phase from 51.89% to
38.71% causing cell cycle arrest (Fig. 4).
51, 56 and 58 against Hela epithelioid carcinoma (15.33
1.6,
1.8 μM, respectively); 46 against
2.0 μM); 45, 47, 57 and 61 against both
1.5, 18.82 1.7, 8.77 0.9,
24.46
2.0, 12.88
1.5, 20.49
colorectal HCT-116 (24.10
colorectal HCT-116 (16.14
15.49
1.4 μM, respectively) and Hela epithelioid carcinoma
(28.82
2.1, 18.75
1.6, 7.84
0.9, 12.42
1.3 μM, respec-
tively); 59 against breast MCF-7, prostate PC3 and Hela epithelioid
2.2.2. Dihydrofolate reductase (DHFR) inhibition
carcinoma (18.07
1.5, 18.94
0.6, 5.48
1.6, 10.62
1.0 μM, respectively);
As an attempt to reveal the antitumor potency mode of action,
DHFR inhibition activity was evaluated. The synthesized compounds
16–21, 25–30, 32, 33, 37–42, 45–48, 50, 51, 53, 54 and 56–61 were
subjected to Dihydrofolate reductase inhibition evaluation assay using
reported procedure, bovine liver DHFR enzyme was used in the test due
60 against colorectal HCT-116, breast MCF-7 and Hela cells
(8.01
0.8, 8.70
0.7 μM, respectively).
Anticancer drugs usually induce cytotoxicity via apoptosis through
activation of signaling pathways leading to G2/M arrest [35]. MTT cell
Scheme 4. Synthesis of the target compounds 56–61.
4

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Table 1
In vitro antitumor assay and DHFR inhibition results (IC50 μM) of 16–21, 25–30, 32, 33, 37–42, 45–48, 50, 51, 53, 54, and 56–61.
Compound R1
R2
R3
–
In vitro Cytotoxicity (IC50, µM)
Average cytotoxicity (IC50
µM)
,
DHFR inhibition (IC50 μM),
n = 3
HCT-116
9.25
MCF-7
PC3
Hela
MTX (1)
16
17
18
19
20
21
25
26
27
28
29
30
32
33
37
38
39
40
41
42
45
46
47
48
50
51
53
–
–
0.7
25.32
5.1 > 100
1.2 9.97
1.6 28.78
> 100
1.8 13.69
> 100
1.1 19.3
0.08
15.0
12.9
18.6
10.8
9.6
0.005
1.98
0.97
1.78
1.28
CH3
CH3O
CH3
H
NO2 92.90
NO2 11.84
NO2 39.34
NO2 63.09
NH2 10.88
NH2 7.66
98.2
10.5
H
0.8
13.02
3.5 57.76
5.0 89.66
1.3 18.21
1.0 7.25
3.6 40.21
5.5 97.34
1.5 9.30
0.8
CH3
3.2 47.72
4.5 83.47
1.0 16.32
2.9 46.3
5.6 83.4
CH3O CH3
CH3
CH3
0.9
0.4
13.7
6.7
0.87
CH3O CH3
0.6
5.73
0.4
9.45
0.8
3.87
4.3 71.16
4.5 81.33
4.9 76.77
5.0 84.36
4.0 64.47
5.3 94.91
3.9 51.70
4.4 63.31
2.8 15.33
5.1 86.52
3.5 51.38
> 100
10.9
18.7
15.0
45.6
32.9
0.2
0.93
1.45
1.78
2.36
2.94
0.003
2.98
1.06
0.001
2.84
2.56
3.78
CH3
CH3
CH3
4-(CH3)C6H4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
51.03
59.48
48.95
53.73
48.18
54.93
42.93
44.46
20.56
67.32
42.20
83.46
83.77
72.01
16.14
24.10
18.82
27.02
37.01
34.47
6.58
3.9 67.83
4.3 72.47
3.8 71.72
4.0 75.53
3.7 64.31
4.1 83.25
3.2 57.20
3.5 60.89
1.9 27.65
4.4 80.20
3.3 54.94
4.7 92.59
4.9 > 100
4.5 88.81
1.5 43.93
2.0 44.84
1.7 31.15
2.2 36.02
3.0 46.19
2.8 40.27
4.5 68.07
4.8 76.40
4.6 76.76
4.8 82.94
4.4 62.79
4.9 89.24
3.8 61.41
3.9 71.26
1.8 36.20
4.9 87.52
3.7 53.78
5.5 > 100
> 100
4.1 64.5
4.7 72.4
4.3 68.6
4.9 74.1
3.9 59.9
5.1 80.6
3.3 53.3
3.6 60.0
1.6 24.9
5.0 80.4
3.1 50.6
94.0
4-(CH3O)C6H4
4-FC6H4
CH3O 4-(CH3)C6H4
CH3O 4-(CH3O)C6H4
CH3O 4-FC6H4
48.9
20.9
0.2
CH3
2-thienyl
CH3O 2-thienyl
CH3
CH3
CH3
CH3
36.8
38.8
40.7
> 100
> 100
> 100
10.3
13.0
15.3
> 100
23.6
1.3
CH3CH2
CH3(CH)2
CH3O CH3
CH3O CH3CH2
CH3O CH3(CH)2
> 100
95.9
5.3 97.03
2.7 48.88
2.9 52.68
2.1 35.28
2.3 45.48
3.3 47.98
2.6 41.90
5.7 > 100
3.1 28.82
3.4 30.15
2.5 18.75
3.1 23.04
3.3 34.05
2.9 24.46
0.9 4.89
89.5
CH3
4-(HO)C6H4
2.1 34.4
2.3 37.9
1.6 26.0
1.9 32.9
2.4 41.3
2.0 35.3
1.25
1.26
0.98
CH3O 4-(HO)C6H4
CH3
4-(HO)-3-(CH3)C6H3
CH3O 4-(HO)-3-(CH3)C6H3
CH3
2-(HO)-5-(CH3)C6H3
2.12
0.002
4.58
CH3O 2-(HO)-5-(CH3)C6H3
CH3
2-(HO)-1-
0.4
8.39
1.2 20.28
2.5 26.19
0.6
10.60
0.5
7.6
1.1 17.1
1.5 24.4
57.3
naphthalenyl
54
CH3O 2-(HO)-1-
–
13.94
1.7 22.87
1.8 11.49
49.6
2.79
naphthalenyl
56
57
58
59
60
61
CH3
CH3
CH3
CH3
–
–
–
–
–
–
32.03
8.77
1.8 26.39
1.1 16.59
2.3 33.26
1.5 18.94
1.9 12.88
1.3 7.84
2.4 20.49
1.6 10.62
1.2 5.48
2.1 12.42
2.6
5.9
0.9
0.3
43.6
0.3
0.87
CH3O
Cl
0.9
14.09
2.7 32.50
2.4 18.07
0.9
11.8
1.8 29.7
1.0 18.9
0.25
32.66
28.16
8.01
0.007
0.003
3.58
CH3O CH3
CH3O CH3O
CH3O Cl
0.8
8.70
0.6
14.36
0.7
9.1
15.49
1.4 23.12
1.8 28.65
1.3 19.9
0.001
to its structure similarity to that of human DHFR [39–41]. Results were
reported as IC50 values (Table 1). Methotrexate (1) was used as re-
ference drug (IC50 0.08 μM). Compounds 29, 33, 59 and 61 proved to
be the most active DHFR inhibitors in this study with IC50 values of 0.2,
0.2, 0.3 and 0.3 μM, respectively. Compounds 51 and 56–58 showed
moderate activity with IC50 1.3, 2.6, 5.9 and 0.9 μM, respectively.
Compounds 16–21, 25, 26 and 45–47 proved to be of slight activity
with IC50 15.0, 12.9, 18.6, 10.8, 9.6, 10.9, 18.7, 15.0, 10.3, 13.0 and
15.3 μM, respectively. The rest of the tested compounds were inactive
especially compounds 40–42 and 48 with IC50 more than 100 μM.
collection of phenols gave the corresponding diazenyl analogues with
marginal antitumor activity except 6-[(2-Hydroxy-naphthalen-1-yl)
diazenyl]-2-(methyl-thio)-3-(4-methyl-phenyl)-quinazolin-4(3H)-one
(53) and 6-[(2-Hydroxy-naph-thalen-1-yl)diazenyl]-3-(4-methoxy-
phenyl)-2-(methyl-thio)quina-zolin-4(3H)-one (54) which showed re-
markable antitumor potency with average IC50 values of 7.6 and
17.1 μM, respectively. The 6-amino functions of 20 and 21 were further
utilized to produce the thiourea analogues 1-(4-Methoxy-phenyl)-3-(2-
(methyl-thio)-4-oxo-3-(4-methyl-phenyl)-3,4-di-hydro-quinazolin-6-yl)
thiourea (57), 1-(3-(4-Methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-di-
hydro-quinazolin-6-yl)-3-(4-methyl-phenyl)thiourea (59) and 1-(4-Me-
thoxy-phenyl)-3-(3-(4-methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-di-
hydro-quinazolin-6-yl)-thiourea (60) which showed remarkable anti-
tumor activity with average IC50 values of 11.8, 18.9 and 9.1 μM, re-
spectively. In regard to DHFR inhibition activity, the starting materials 2-
mercapto-3-(4-substituted-phenyl)-6-nitro-quinazolin-4(3H)-ones (16
and 17) showed marginal DHFR inhibition activity with IC50 values of
15.0 and 12.9 μM, respectively. Methylation of the 2-mercapto func-
tions produced 18 and 19 (IC50 values of 18.6 and 10.8 μM, respec-
tively), or reduction of the 6-nitro groups gave the 6-amino derivatives
20 and 21 (IC50 values of 9.6 and 10.9 μM, respectively) without sen-
sible enhancement of the DHFR inhibition activity. Acylation of the 6-
amino functions of 20 and 21 with a collection of substituted aromatic,
3. Structure activity relationship
Two main series of compounds were investigated, namely: 3-(4-
methyl-phenyl)- and 3-(4-methoxy-phenyl)-quinazolin-4(3H)-one for
their antitumor activity and DHFR inhibition. In regard to antitumor
activity, the starting material 17 showed remarkable antitumor potency
with average IC50 values of 10.5 μM. Reduction of the 6-nitro groups
gave the 6-amino derivatives 20 and 21 (average IC50 values of 13.7
and 6.7 μM, respectively) with enhanced antitumor potency. Acylation
of the 6-amino functions with a collection of acid chlorides produced
the corresponding amides which proved to be devoid of antitumor
potency. Diazotization of the 6-amino functions of 20 and 21 with a
5

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Fig. 3. Cell cycle analysis and apoptosis effect in Hela cell line treated with 59 against control Hela cell line (upper panel), which evaluated by flow cytometry (lower
panel).
heterocyclic or aliphatic acid chlorides produced the corresponding
amides with marginal DHFR inhibition activity except compounds 4-
methoxy-N-(3-(4-methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-dihydro-
quinazolin-6-yl)benzamide (29) and N-(3-(4-methoxy-phenyl)-2-(me-
thyl-thio)-4-oxo-3,4-dihydro-quinazolin-6-yl)thiophene-2-carboxamide
(33) which showed remarkable potency with IC50 values of 0.2 μM
each. Aromatic and heterocyclic amides at position 6- of the quinazo-
line nucleus contributed to the antifolate potency more than aliphatic
amides. Meanwhile, the combination of 6-(aromatic or heterocyclic)-
amides and 3-(4-methoxy-phenyl)- favor the activity rather than the
combination of 6-(aliphatic)-amides and 3-(4-methyl-phenyl)-quinazo-
line. Diazotization of the 6-amino functions of 20 and 21 with variety of
phenols gave the corresponding diazenyl analogues with marginal
DHFR inhibition activity except 6-[(2-Hydroxy-5-methyl-phenyl)dia-
zenyl]-3-(4-methoxy-phenyl)-2-(methyl-thio)quinazolin-4(3H)-one
(51) which showed moderate DHFR inhibition potency with IC50 values
of 1.3 μM. The 6-amino functions of 20 and 21 were further utilized to
produce the thiourea analogues 56–61 with moderate inhibition ac-
tivity except 1-(3-(4-Methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-di-
hydro-quinazolin-6-yl)-3-(4-methyl-phenyl)-thiourea (59) and 1-(4-
Chloro-phenyl)-3-(3-(4-methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-di-
hydro-quinazolin-6-yl)-thiourea (61) which expressed considerable
DHFR inhibition potency with IC50 values of 0.3 μM each. Generally,
the 3-(4-methoxy-phenyl)- series favor the DHFR inhibition activity
rather than 3-(4-methyl-phenyl)-quinazolin-4(3H)-one.
understanding about the molecular structure behavior of the most ac-
tive DHFR inhibtor 29 and the least active 48, in comparison to
methotrexate (MTX, 1). Conformational analysis was performed using
MMFF94 force-field (RMS gradient of 0.01 kcal/Å mol) implemented in
MOE 2009.10 [44,45]. The lowest energy conformers for 1, 29 and 48
were shown in Fig. 5. Molecular docking was performed for the binding
pocket of human dihydrofolate reductase binding domain which is in
tertiary complex with dihydronicotinamide adeninedinucleotide phos-
phate (NADPH) and 1, (code ID 1DLS) to predict the binding affinity of
the newly synthesized compounds as hDHFR inhibitors [46].
Fig. 6 illustrates the 2D binding mode of 1, 29 and 48. The obtained
docking data showed that the binding of 1 to hDHFR is expressed as a
complex interaction where the receptor site undergo a kind of iso-
merizational change leading to the tight binding with hydrogen
bonding of the central amide carbonyl function as hydrogen bond ac-
ceptor (HBA) to Thr56 and Ser59.
MTX (1) binds also through hydrogen and ionic bonding with Lys55,
arene-arene interaction with Phe34 and Phe31 amino acid residues
(Fig. 6a). The most active 29 showed high affinity binding toward the
amino acid residues Thr56, Ser59 and Ser118 as hydrogen bonding
acceptor (Fig. 6b); which is more or less the same fashion and type of
binding as the reference 1 do. This could be the reason for the re-
markable activity of 29 with IC50 0.2
0.003 μM. On the other hand,
48 (IC50 > 100 μM) did not bind with those amino acid residues and
the S-methyl and part of the terminal phenyl groups were exposed out
the enzyme pocket explaining its poor activity (Fig. 6c). Further mod-
eling studies and docking comparisons between structurally related
compounds were done to uncover the reason behind the unique activity
of 29 and the modest potency of the other closely related analogs.
4. Molecular modeling simulation study
Molecular modeling methods [42,43] were used to get a better
6

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Fig. 4. Cell cycle analysis and apoptosis effect in HCT-116 cell line treated with 61 against control HCT-116 cell line (upper panel), which evaluated by flow
cytometry (lower panel).
Fig. 5. The lowest energy conformer of (a) MTX 1, (b) the most active 29 (c) and the least active 48 with balls and cylinders.
7

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Fig. 6. 2D binding mode and residues involved in the recognition of (a) MTX (1, IC50 0.08 μM), (b) the most active compound 29 (IC50 0.2
least active compound 48 (IC50 > 100 μM) docked and minimized in the hDHFR binding pocket.
0.003 μM), and (c) the
Comparison of 26 (IC50 15.0
1.78 μM) and 29 (IC50
Flexible Alignment computational procedures were performed using
MOE/MMFF94 flexible alignment tool [48–50]. 100 conformers of each
compound were generated and minimized with a distance-dependent
dielectric model. Fig. 8a showed that the most active compounds 29,
33, 59 and 61 are perfectly aligned especially at the quinazoline
moiety. Fig. 8b showed that the least active compounds 40–42 and 48
are aligned well especially at the quinazoline moiety. Fig. 8c showed
that the least active compounds are completely deviated from the most
active counterparts.
0.2
0.003 μM) was performed to predict the reason of the efficacy
difference. The structure difference was the 4-methoxy-phenyl group in
29 instead of 4-methyl-phenyl group in 26. This difference led to the
change of orientation and the binding poses inside the pocket.
Compound 26 lacked the essential binding toward the amino acid re-
sidues Thr56 and Ser59 to which 29 bonded, in addition to the arene-
arene interaction with Phe31. The same analogy was adopted to com-
pare 32 (IC50 20.9
1.06 μM) and 33 (IC50 0.2
0.001 μM).
Comparison of the equipotent compounds 29 and 33 (IC50 0.2 μM);
revealed that the structure difference lies in the 4-methoxy-phenyl-
carboxamide group in 29 and 2-thienyl-carboxamide group in 33. This
structure difference did not affect the orientation and the binding poses
inside the enzyme pocket. The same could be applied for 47
Surface mapping comparison of 29, 48 and MTX (1) was performed
to detect their surface properties and the similarity in the position of
hydrophilic and hydrophobic sites that bind to the active sites (Fig. 9),
[51]. As noticed in Fig. 9a and b, it is clear that there is similarity in the
positions of the hydrophilic site at the two sides of the structure and in
the presence of hydrophobic aromatic ring in the center of the mole-
cule, in addition to the obvious similarity between the pteridine ring in
MTX (1) and quinazoline ring in the newly synthesized compounds.
Lipinski’s rule of five is a rule of thumb to determine the drug
likeness or investigating the properties that would make a chemical
compound with a certain pharmacological or biological activity likely
orally active drug in humans [52–54]. As a part of the molecular
modeling study, the compliance of the most active newly synthesized
compounds 29, 33, 59 and 61 to the Lipinski rule of five was checked as
illustrated in Table 2. It can be observed that all of the active
(15.38
(1.3
0.98 μM) and 48 (> 100 μM); 50 (23.6
0.002 μM); 48 (> 100 μM) and 51 (1.3
2.12 μM) and 51
0.002 μM).
Additional ligands docking and alignment inside hDHFR binding
pocket was performed [47]. The binding pocket surface map was cal-
culated showing MTX (1) occupying the whole space lying into the
groove pocket (Fig. 7a). Compound 29 pocket alignments showed a
similar pattern filling the area of the active site with its bulkiness,
forming favorable binding contacts (Fig. 7b). On the other hand, the
structure of 48 was exposed out the surface wall of the active site ex-
plaining its poor DHFR inhibitory activity (Fig. 7c).
8

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Fig. 7. The aligned conformations of (a) MTX (1, space filled yellow, IC50 0.08 μM), (b) the most active 29 occupying the DHFR binding pocket (space filled orange,
IC50 0.2 0.003 μM) and (c) the most inactive 48 (space filled cyan, IC50 > 100 μM) exposing out of the DHFR binding pocket surface map.
compounds obey Lipinski’s rule of five.
toward the amino acid residues Thr56, Ser59 and Ser118 as hydrogen
bond acceptor. Compounds 29, 33, 59 and 61 obeyed Lipinski’s rule of
five and have comparable ADMET data with that of MTX (1).
In the present study, it seemed that compound 21, 59 and 61
probably exerts its antitumor potency (average IC50 values of 6.7, 18.9
and 19.9 μM, respectively) through DHFR inhibition (IC50 value of 10.9,
0.3 and 0.3 μM, respectively). The cell cycle analysis gave evidence that
compounds 59 and 61 exerted their antitumor activity probably
through dual modes, apoptotic behavior and DHFR inhibition. The
other active antitumor exerted their potency with some other me-
chanism(s). The active compounds and their biological results as well as
molecular modeling studies could be considered as a template for future
investigation and development.
The pharmacokinetics ADMET property limitations [55–57] for the
active compounds were calculated. The ADMET data were predicted
and showed in Table 3. Based on the predicted values of the most active
compounds 29, 33, 59 and 61, the new compounds have comparable
ADMET data with that of 1. The active compounds are capable of pe-
netrating blood brain barrier (BBB) and can be absorbed by intestine.
Their estimated log S values are −4, which indicate low water solu-
bility. All active compounds are expected to be non-substrate/non-in-
hibitors of CYP 2D6 enzyme which suggests low possibility of drug-drug
interaction occurrence. Furthermore, toxicity tests data showed that all
of the compounds are non-toxic nor-mutagenic in Ames test and all are
non-carcinogenic.
5. Conclusion
6. Experimental
Two main series of compounds were investigated, namely: 3-(4-
methyl-phenyl)- and 3-(4-methoxy-phenyl)-quinazolin-4(3H)-ones.
Compounds 21, 53 and 60 showed broad spectrum remarkable cyto-
toxic potency and considered to be the most active members in this
study with average IC50 values of 6.7, 7.6 and 9.1 μM, (Fig. 10). MTT
cell viability assay was performed using the mutant Hela, the mutant
HCT-116 cell lines against compound 59 and 61, respectively. Both
compounds induced their cytotoxicity via Pre-G1 apoptosis and in-
hibited cell growth at G2-M. Compounds 29, 33, 59 and 61 proved to
be the most active DHFR inhibitors in this study with IC50 values of 0.2,
0.2, 0.3 and 0.3 μM, respectively.
All melting points (°C) were measured by Stuart melting point ap-
paratus SMP30 and they are uncorrected. 1H, 13C NMR, DEPT-135, 2D
COSY and 2D HSQC spectra were recorded on Bruker Avance III HD FT-
high resolution- 1H NMR (400 MHz), 13C NMR (100 MHz) at Faculty of
Pharmacy-Mansoura University; chemical shifts are expressed in δ ppm
with reference to TMS. Mass spectra was carried out on Direct Inlet part
to mass analyzer in Thermo Scientific GCMS model ISQ at the Regional
Center for Mycology and Biotechnology (RCMB), Al-Azhar University,
Nasr City, Cairo. Reaction times were determined using TLC on silica
gel plates 60F245 E. Merk, using chloroform/methanol 9:1 or chloro-
form alone as eluting system and the spots were visualized by UV
(366–245 nm). Starting materials and reagents were purchased from
The most active DHFR inhibitor 29 showed high affinity binding
9

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Fig. 8. (a) Flexible alignment of the most active compounds 29 (orange), 33 (yellow), 59 (red) and 61 (brown), (b) Flexible alignment of the least active compounds
40 (green), 41 (blue), 42 (violet) and 48 (cyan), (c) Flexible alignment of the most active compounds 29 (orange) and 33 (yellow) against the least active compounds
42 (violet) and 48 (cyan).
Fig. 9. (a) Surface map for MTX (1) in pocket side. (b) Surface map for the most active compound 29 in pocket side. (c) Surface map for the least active compound 48
in pocket side. Pink: hydrophilic, blue: mild polar, green: hydrophobic.
10

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Table 2
6.1.2. General procedure for the synthesis of 3-(4-substituted-phenyl)-2-
Calculated parameters of Lipinski's rule of five for compounds 29, 33, 59 and
61.
(methyl-thio)-6-nitro-quinazolin-4(3H)-ones (18 and 19)
A
mixture of the 2-mercapto-quinazoline analog (16 or 17,
Compound
Parameter
nVsg
0.005 mol), methyl iodide (3 ml), and anhydrous potassium carbonate
(1 g) in acetone (50 ml) was stirred at room temperature for 8 h. The
excess carbonate was filtered off, washed with acetone, the filtrate was
then evaporated under vacuum, and the obtained residue was dried,
and recrystallized from ethanol to give 18 and 19.
Log Pa
TPSAb
MWc
nHBAd
nHBDe
nRBf
29
33
59
61
4.97
5.02
5.82
6.17
80.23
71.00
98.05
98.05
447.5
5
4
4
4
1
1
2
2
6
5
7
7
0
1
1
1
423.52
462.60
483.02
6.1.2.1. 2-(Methyl-thio)-6-nitro-3-(4-methyl-phenyl)quinazolin-4(3H)-one
(18). White crystals (1.31 g, 80%), m.p. 200–2 °C. 1H NMR (CDCl3): δ
2.40 (s, 3H, CH3Ph), 2.48 (s, 3H, SCH3), 7.13 (d, 2H, J = 8.0 Hz , ArH),
7.31 (d, 2H, J = 8.0 Hz, ArH), 7.65 (d, 1H, J = 8.8 Hz, ArH), 8.43–8.46
(dd, 1H, J = 2.4, 8.8 Hz, ArH), 9.02 (d, 1H, J = 2.4 Hz, ArH), 13C NMR:
δ 15.8, 21.5, 119.8, 124.2, 127.7, 128.5, 128.8, 130.7, 132.5, 140.9,
144.7, 151.6, 160.6, 163.4. MS (m/z, %) for C16H13N3O3S: (M+ 327.0,
100).
a
Calculated lipophilicity.
Total polar surface area.
Molecular weight.
b
c
d
e
f
Number of hydrogen bond acceptor.
Number of hydrogen bond donor.
Number of rotatable bonds.
g
Number of violation from Lipinski's rule of five.
Sigma-AldrichCo., U.S.A., El-Gomhoria and El-Nasr Pharmaceutical and
Chemical Co., Egypt. Dihydrofolate reductase (DHFR) inhibition ac-
tivity experiments were performed at Pharmacology and Biochemistry
Department, Faculty of Pharmacy; Future University in Egypt. In vitro
antitumor activity was performed in the Department of
Pharmacognosy, Faculty of Pharmacy, Mansoura University. Cell Cycle
analysis was performed at the confirmatory diagnostic unit, VACSERA,
Egypt.
6.1.2.2. 3-(4-Methoxy-phenyl)-2-(methyl-thio)-6-nitroquinazolin-4(3H)-
one (19). White crystals (1.32 g, 77%), m.p. 230–2 °C. 1H NMR
(CDCl3): δ 2.58 (s, 3H, SCH3), 3.91 (s, 3H, OCH3), 7.09 (d, 2H,
J = 8.8 Hz
, ArH), 7.25 (d, 2H, J = 8.8 Hz, ArH), 7.74 (d, 1H,
J = 8.8 Hz, ArH), 8.52–8.55 (dd, 1H, J = 2.4, 8.8 Hz, ArH), 9.12 (d,
1H, J = 2.4 Hz, ArH), 13C NMR: δ 15.9, 55.6, 115.2, 119.7, 124.2,
127.5, 127.7, 128.8, 130, 144.7, 151.6, 160.8, 160.9, 163.8. MS (m/z,
%) for C16H13N3O4S: (M+ 343.0, 100).
6.1.3. General procedure for the synthesis of 6-amino-3-(4-substituted-
6.1. Chemistry
phenyl)-2-(methyl-thio)quinazolin-4(3H)-ones (20 and 21)
A
mixture of the 6 nitro-quinazoline derivative (18 or 19,
6.1.1. General procedure for the synthesis of 2-mercapto-3-(4-substituted-
phenyl)-6-nitro-quinazolin-4(3H)-ones (16 and 17)
0.005 mol) and SnCl2·2H2O (0.025 mol, 5.64 g), in acetone (50 ml), was
heated under reflux for 2 h. the reaction mixture was allowed to cool
then poured into ice. The pH was rendered slightly basic (pH 8) by
addition of 5% aqueous sodium bicarbonate and the resulting basic
mixture was stirred at room temperature for 0.5 hr. The separated solid
was filtered, washed with water, and dried. The dried solid was then
extracted with acetone, the extract was then evaporated under vacuum,
and the obtained residue was dried, and recrystallized from ethanol to
give 20 and 21.
A mixture of 5-nitroanthranilic acid (13, 0.91 g, 0.005 mol), the
appropriate substituted phenylisothiocyanate derivative (14 or 15,
0.005 mol), and TEA (2 ml) in ethanol (50 ml) was heated under reflux
for 4 h. The reaction mixture was then stirred at room temperature for
12 h. The separated yellow solid was filtered, washed with methanol,
dried and recrystallized from ethanol to give 16 and 17.
6.1.1.1. 2-Mercapto-6-nitro-3-(4-methyl-phenyl)quinazolin-4(3H)-one
(16). Yellowish white crystals (0.93 g, 60%), m.p. 270–2 °C. 1H NMR
(DMSO‑d6): δ 3.38 (s, 3H, CH3Ph), 7.17 (d, 2H, J = 8.4 Hz, ArH), 7.30
(d, 2H, J = 8.4 Hz, ArH), 7.56 (d, 1H, J = 8.8 Hz , ArH), 8.56–8.59 (dd,
1H, J = 2.4, 8.8 Hz, ArH), 8.60 (d, 1H, J = 2.4 Hz, ArH), 13.46 (s, 1H,
SH). 13C NMR: δ 21.3, 116.9, 117.6, 124.0, 128.9, 130.1, 130.6, 136.7,
138.3, 143.2, 144.0, 159.3, 177.7. MS (m/z, %) for C15H11N3O3S: (M+
313.0, 76.55), 312 (100).
6.1.3.1. 6-Amino-2-(methyl-thio)-3-(4-methyl-phenyl)quinazolin-4(3H)-
one (20). Greyish white crystals (0.89 g, 60%), m.p. 213–5 °C. 1H NMR
(CDCl3): δ 2.47 (s, 3H, CH3Ph), 2.51 (s, 3H, SCH3), 3.93 (s, 2H, NH2),
7.11–7.14 (dd, 1H, J = 2.8, 8.4 Hz , ArH), 7.21 (d, 2H, J = 8.0 Hz,
ArH), 7.36 (d, 2H, J = 8.0 Hz, ArH), 7.46 (d, 1H, J = 2.8 Hz, ArH), 7.5
(d, 1H, J = 8.4 Hz, ArH), 13C NMR: δ 15.4, 21.4, 109.7, 120.7, 123.3,
127.4, 128.9, 130.3, 133.6, 140.0, 141.2, 144.6, 154.0, 161.9. MS (m/z,
%) for C16H15N3OS: (M+ 297.9, 73.80), 89 (100).
6.1.1.2. 2-Mercapto-3-(4-methoxy-phenyl)-6-nitroquinazolin-4(3H)-one
(17). Yellowish white crystals (1 g, 65%), m.p. 287–90 °C. 1H NMR
(DMSO‑d6): δ 3.82 (s, 3H, OCH3), 7.04 (d, 2H, J = 8.8 Hz, ArH), 7.21
(d, 2H, J = 8.8 Hz, ArH), 7.57 (d, 1H, J = 8.8 Hz , ArH), 8.57–8.60 (dd,
1H, J = 2.4, 8.8 Hz, ArH), 8.61 (d, 1H, J = 2.4 Hz, ArH), 13.46 (s, 1H,
SH), 13C NMR: δ 55.8, 114.7, 117.0, 117.6, 124.0, 130.2, 130.6, 131.9,
143.3, 144.0, 159.4, 159.5, 177.9. MS (m/z, %) for C15H11N3O4S: (M+
329.0, 70.02), 114 (100).
6.1.3.2. 6-Amino-3-(4-methoxy-phenyl)-2-(methyl-thio)quinazolin-4(3H)-
one (21). Greyish white crystals (0.98 g, 63%), m.p. 191–3 °C. 1H NMR
(CDCl3): δ 2.51 (s, 3H, SCH3), 3.89 (s, 3H, OCH3), 3.94 (s, 2H, NH2),
7.05 (d, 2H, J = 8.0 Hz, ArH), 7.1–7.13 (dd, 1H, J = 2.8, 8.4 Hz , ArH),
7.24 (d, 2H, J = 8.0 Hz, ArH), 7.45 (d, 1H, J = 2.8 Hz, ArH), 7.49 (d,
1H, J = 8.8 Hz, ArH), 13C NMR: δ 15.4, 55.5, 109.7, 114.8, 120.7,
123.3, 127.4, 128.7, 130.3, 141.2, 144.6, 154.3, 160.5, 162.1. MS (m/z,
Table 3
Predicted ADMET data MTX (1) versus the most active compounds 29, 33, 59 and 61.
Compound
BBB
HIA
AQ sol log S
CYP 2D6 substrate/inhibition
AMES toxicity
carcinogenicity
MTX (1)
29
0.9930
0.9770
0.9763
0.9778
0.9772
0.9088
0.8492
0.7401
0.9491
0.9474
−3
−4
−4
−4
−4
Non-substrate/noninhibitor
Non-substrate/noninhibitor
Non-substrate/noninhibitor
Non-substrate/noninhibitor
Non-substrate/noninhibitor
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Nontoxic
Noncarcinogenic
Noncarcinogenic
Noncarcinogenic
Noncarcinogenic
Noncarcinogenic
33
59
61
11

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
Fig. 10. Structures of the most active antitumors 21 and 53, and the most active DHFR inhibitors 29, 33.
%) for C16H15N3O2S: (M+ 313.1, 81.95), 90 (100).
157.4, 161.2, 163.5, 165.0. MS (m/z, %) for C23H18FN3O2S: (M+ 419.0,
100).
6.1.4. General procedure for the synthesis of 4-substituted-N-(3-(4-
substituted-phenyl)-2-(methyl-thio)-4-oxo-3,4-dihydro-quinazolin-6-yl)
benzamides (25–30)
6.1.4.4. N-(3-(4-Methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-dihydro-
quinazolin-6-yl)-4-methyl-benzamide (28). Yellowish-white powder
(1.88 g, 87%), m.p. 270–3 °C. 1H NMR (DMSO-d6): δ 2.40 (s, 3H,
CH3Ph), 2.47 (s, 3H, SCH3), 3.85(s, 3H, OCH3), 7.10 (d, 2H, J = 7.6 Hz,
ArH), 7.35–7.37 (m, 4H, ArH), 7.63 (d, 1H, J = 8.4 Hz, ArH), 7.92 (d,
2H, J = 6.8 Hz, ArH), 8.25 (d, 1H, J = 8.4 Hz, ArH), 8.57 (s, 1H, ArH),
10.5 (s, 1H, NH), 13C NMR: δ 15.5, 21.5, 55.9, 115.1, 116.8, 120.0,
126.9, 127.9, 128.2, 129.0, 129.5, 131.1, 132.1, 137.5, 142.4, 144.0,
157.7, 160.5, 161.4, 165.9. MS (m/z, %) for C24H21N3O3S: (M+ 431.1,
31.21), 119 (100).
A mixture of 6-amino-quinazoline derivative (20 or 21, 0.005 mol)
and the appropriate substituted benzoyl chloride (22–24, 0.007 mol) in
pyridine (10 ml) was stirred at room temperature for 2 h. The reaction
mixture was poured into ice and the formed precipitate was filtered,
washed with water, dried and recrystallized from ethanol to give
25–30.
6.1.4.1. 4-Methyl-N-(2-(methyl-thio)-4-oxo-3-(4-methyl-phenyl)-3,4-
dihydro-quinazolin-6-yl)benzamide (25). Off white powder (1.79 g,
86%), m.p. 280–3 °C. 1H NMR (DMSO‑d6): δ 2.40 (s, 3H, CH3Ph),
2.42 (s, 3H, CH3Ph), 2.48(s, 3H, SCH3), 7.31–7.37 (m, 6H, ArH), 7.64
(d, 1H, J = 8.4, ArH), 7.93 (d, 2H, J = 7.6 Hz, ArH), 8.26 (d, 1H,
J = 8.4 Hz, ArH), 8.57 (s, 1H, ArH), 10.5 (s, 1H, NH), 13C NMR: δ 15.5,
21.3, 21.5, 116.8, 120.0, 126.9, 127.9, 128.2, 129.5, 129.6, 130.4,
132.1, 133.9, 137.5, 139.9, 142.3, 144.0, 157.2, 161.2, 165.9. MS (m/z,
%) for C24H21N3O2S: (M+ 415.1, 100).
6.1.4.5. 4-Methoxy-N-(3-(4-methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-
dihydro-quinazolin-6-yl)benzamide (29). Greyish white powder (1.97 g,
88%), m.p. 290–3 °C. 1H NMR (CDCl3): δ 2.54 (s, 3H, SCH3), 3.86 (s,
3H, OCH3), 3.90 (s, 3H, OCH3), 6.98–7.03 (m, 4H, ArH), 7.22 (d, 2H,
J = 8.8 Hz, ArH), 7.68 (d, 1H, J = 8.8 Hz, ArH), 7.88 (d, 2H,
J = 8.8 Hz, ArH), 8.08 (d, 1H, J = 2.4 Hz, ArH), 8.27 (s, 1H, NH),
8.51–8.54 (dd, 1H, J = 2.4, 8.8 Hz, ArH), 13C NMR: δ 15.6, 55.49,
55.50, 114.0, 114.9, 116.9, 119.9, 126.6, 127.2, 127.9, 128.3, 129.1,
130.2, 136.3, 144.6, 157.5, 160.6, 161.9, 162.6, 165.2. MS (m/z, %) for
6.1.4.2. 4-Methoxy-N-(2-(methyl-thio)-4-oxo-3-(4-methyl-phenyl)-3,4-
dihydro-quinazolin-6-yl)benzamide (26). Greyish white powder (1.81 g,
84%), m.p. 281–3 °C. 1H NMR (DMSO‑d6): δ 2.41 (s, 3H, CH3Ph), 2.47
(s, 3H, SCH3), 3.85(s, 3H, OCH3), 7.09 (d, 2H, J = 8.0 Hz, ArH),
7.31–7.38 (m, 4H, ArH), 7.63 (d, 1H, J = 8.4 Hz, ArH), 8.01 (d, 2H,
J = 8.4 Hz, ArH), 8.25 (d, 1H, J = 8.4 Hz, ArH), 8.55 (s, 1H, ArH),
10.45 (s, 1H, NH), 13C NMR: δ 15.5, 21.3, 55.9, 114.2, 116.7, 120.0,
126.9, 127.0, 127.9, 129.6, 130.2, 130.4, 133.9, 137.6, 139.9, 143.9,
157.2, 161.2, 162.5, 165.5. MS (m/z, %) for C24H21N3O3S: (M+ 431.2,
100).
C
24H21N3O4S: (M+ 447.1, 94.93), 135 (100).
6.1.4.6. 4-Fluoro-N-(3-(4-methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-
dihydro-quinazolin-6-yl)benzamide (30). White powder (1.79 g, 82%),
m.p. 305–8 °C. 1H NMR (DMSO‑d6): δ 2.47 (s, 3H, SCH3), 3.84 (s, 3H,
OCH3), 7.10 (d, 2H, J = 8.8 Hz, ArH), 7.34–7.41 (m, 4H, ArH), 7.64 (d,
1H, J = 8.8, ArH), 8.06–8.10 (m, 2H, ArH), 8.21–8.23 (dd, 1H, J = 2,
8.8 Hz, ArH), 8.54 (d, 1H, J = 2.0 Hz, ArH), 10.61 (s, 1H, NH), 13C
NMR: δ 15.5, 55.9, 115.1, 115.8, 116.0, 117.0, 120.0, 127.0, 127.9,
128.9, 130.9, 131.0, 131.1, 131.39, 131.42, 137.2, 144.2, 157.8, 160.5,
161.4, 163.4, 165.1. MS (m/z, %) for C23H18FN3O3S: (M+ 435, 100).
6.1.4.3. 4-Fluoro-N-[2-(methyl-thio)-4-oxo-3-(4-methyl-phenyl)-3,4-
dihydro-quinazolin-6-yl]benzamide (27). White powder (1.7 g, 81%),
m.p. 275–8 °C. 1H NMR (DMSO‑d6): δ 2.41 (s, 3H, CH3Ph), 2.47 (s,
3H, SCH3), 7.32 (d, 2H, J = 8.4 Hz, ArH), 7.36–7.42 (m, 4H, ArH), 7.65
(d, 1H, J = 8.8, ArH), 8.07–8.11 (m, 2H, ArH), 8.23–8.26 (dd, 1H,
J = 2.4, 8.8 Hz, ArH), 8.55 (d, 1H, J = 2.4 Hz, ArH), 10.61 (s, 1H, NH),
13C NMR: δ 15.5, 21.3, 115.8, 116.0, 116.9, 120.0, 124.4, 127.0, 127.9,
129.6, 130.4, 130.9, 131.0, 131.4, 133.9, 137.3, 140.0, 144.1, 150.1,
6.1.5. General procedure for the synthesis of N-(3-(4-substituted-phenyl)-2-
(methyl-thio)-4-oxo-3,4-dihydro-quinazolin-6-yl)thiophene-2-
carboxamides (32 and 33)
A mixture of 6-amino-quinazoline derivative (20 or 21, 0.005 mol)
and thiophene-2-carbonyl chloride (31, 0.733 g, 0.007 mol) in pyridine
(10 ml) was stirred at room temperature for 2 h. The reaction mixture
12

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
was poured into ice and the formed precipitate was filtered, washed
MS (m/z, %) for C20H21N3O2S: (M+ 367.0, 98.91), 182 (1 0 0).
with water, dried and recrystallized from ethanol to give 32 and 33.
6.1.6.4. N-(3-(4-Methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-dihydro-
quinazolin-6-yl)acetamide (40). Off-white powder (1.44 g, 81%), m.p.
310–2 °C. 1H NMR (DMSO‑d6): δ 2.09 (s, 3H, COCH3), 2.45 (s, 3H,
SCH3), 3.84 (s, 3H, OCH3), 7.09 (d, 2H, J = 8.8 Hz, ArH), 7.35 (d, 2H,
J = 8.8 Hz, ArH), 7.58 (d, 1H, J = 8.8 Hz, ArH), 7.97–8 (dd, 1H,
J = 2.4, 8.8 Hz, ArH), 8.36 (d, 1H, J = 2.4 Hz, ArH), 10.29 (s, 1H,
NH), , 13C NMR: δ 15.5, 24.5, 55.9, 115.0, 115.3, 120.1, 126.7, 127.1,
128.9, 131.1, 137.5, 143.7, 157.4, 160.4, 161.3, 169.0. MS (m/z, %) for
6.1.5.1. N-(2-(Methyl-thio)-4-oxo-3-(4-methyl-phenyl)-3,4-dihydro-
quinazolin-6-yl)thiophene-2-carboxamide (32). Off white powder
(1.81 g, 89%), m.p. 225–8 °C. 1H NMR (DMSO‑d6): δ 2.41 (s, 3H,
CH3Ph), 2.47 (s, 3H, SCH3), 7.24–7.26 (dd, 1H, J = 3.6, 4.0 Hz, ArH),
7.31–7.38 (m, 4H, ArH), 7.65 (d, 1H, J = 8.8 Hz, ArH), 7.9 (d, 1H,
J = 4.8 Hz, ArH), 8.09 (d, 1H, J = 3.2 Hz, ArH), 8.23–8.26 (dd, 1H,
J = 2.4, 8.8 Hz, ArH), 8.49 (d, 1H, J = 2.4 Hz, ArH), 10.57 (s, 1H, NH),
13C NMR: δ 15.6, 21.3, 116.8, 120.1, 124.4, 127.1, 127.8, 128.7, 129.6,
130.4, 132.8, 133.9, 137.0, 140.1, 144.1, 150.1, 157.4, 160.5, 161.2.
MS (m/z, %) for C21H17N3O2S2: (M+ 407.2, 100).
C
18H17N3O3S: (M+ 355.1, 100).
6.1.6.5. N-(3-(4-Methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-dihydro-
quinazolin-6-yl)propion-amide (41). Yellowish-white powder (1.44 g,
78%), m.p. 275–8 °C. 1H NMR (DMSO‑d6): δ 1.10 (t, 3H, J = 7.6 Hz,
CH3), 2.36 (q, 2H, J = 7.6 Hz, COCH2), 2.45 (s, 3H, SCH3), 3.84 (s, 3H,
OCH3), 7.09 (d, 2H, J = 8.4 Hz, ArH), 7.35 (d, 2H, J = 8.4 Hz, ArH),
7.58 (d, 1H, J = 8.8 Hz, ArH), 7.99–8.02 (dd, 1H, J = 2.0, 8.8 Hz, ArH),
8.38 (d, 1H, J = 2.0 Hz, ArH), 10.21 (s, 1H, NH), 13C NMR: δ 10.0,
15.5, 30.0, 55.9, 115.0, 115.4, 120.1, 126.7, 127.1, 129.0, 131.1,
137.6, 143.6, 157.3, 160.4, 161.3, 172.7. MS (m/z, %) for
6.1.5.2. N-(3-(4-Methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-dihydro-
quinazolin-6-yl)thiophene-2-carboxamide (33). Yellowish-white powder
(1.9 g, 90%), m.p. 190–3 °C. 1H NMR (DMSO-d6): δ 2.47 (s, 3H, SCH3),
3.85 (s, 3H, OCH3), 7.10 (d, 2H, J = 8.8 Hz, ArH), 7.24–7.27 (dd, 1H,
J = 3.6, 4.0 Hz, ArH), 7.37 (d, 2H, J = 8.8 Hz, ArH), 7.64 (d, 1H,
J = 8.8 Hz, ArH), 7.89–7.91 (dd, 1H, J = 1.2, 4.8 Hz, ArH), 8.08–8.09
(dd, 1H, J = 1.2, 4.0 Hz, ArH), 8.22–8.25 (dd, 1H, J = 2.4, 8.8 Hz,
ArH), 8.48 (d, 1H, J = 2.4 Hz, ArH), 10.56 (s, 1H, NH), 13C NMR: δ
15.5, 55.9, 115.1, 116.8, 120.1, 125.5, 127.1, 127.7, 128.7, 128.9,
129.9, 131.1, 132.8, 137.0, 140.1, 144.1, 157.8, 160.5, 161.3. MS (m/z,
%) for C21H17N3O3S2: (M+ 423.0, 100).
C
19H19N3O3S: (M+ 369.1, 100).
6.1.6.6. N-(3-(4-Methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-dihydro-
quinazolin-6-yl)butyr-amide (42). Greyish-white powder (1.53 g, 80%),
m.p. 292–5 °C. 1H NMR (CDCl3): δ 0.98 (t, 3H, J = 7.6 Hz, CH3),
1.68–1.77 (m, 2H, CH2), 2.25 (t, 2H, J = 7.6 Hz, COCH2), 2.51 (s, 3H,
SCH3), 3.87 (s, 3H, OCH3), 7.04 (d, 2H, J = 8.8 Hz, ArH), 7.22 (d, 2H,
J = 8.8 Hz, ArH), 7.61 (d, 1H, J = 9.2 Hz, ArH), 8.00 (d, 1H,
J = 2.8 Hz, ArH), 8.44–8.46 (dd, 1H, J = 2.8, 9.2 Hz, ArH), 8.59 (s,
1H, NH), 13C NMR: δ 13.8, 15.6, 19.0, 39.2, 55.5, 114.9, 116.1, 119.8,
127.0, 127.5, 128.4, 130.2, 136.7, 144.3, 157.1, 160.6, 162.0, 172.0.
MS (m/z, %) for C20H21N3O3S: (M+ 384.0, 100).
6.1.6. General procedure for the synthesis of N-(3-(4-substituted-phenyl)-2-
(methyl-thio)-4-oxo-3,4-dihydro-quinazolin-6-yl)- derivatives of acetic,
propionic or butyric acid amides (37–42)
A mixture of 6-amino-quinazoline derivative (20 or 21, 0.005 mol),
the appropriate acid chloride (34–36, 0.007 mol) and TEA (0.5 ml) in
methylene chloride (20 ml) was stirred at room temperature for 2 h.
Then the reaction mixture was evaporated under vacuum and the se-
parated solid was washed with water, dried and recrystallized from
ethanol to give 37–42.
6.1.7. General procedure for the synthesis of 6-[(4-hydroxy-3-substituted-
phenyl)diazenyl]-3-(4-substituted-phenyl)-2-(methyl-thio)quinazolin-
4(3H)-ones (45–48)
6.1.6.1. N-(2-(Methyl-thio)-4-oxo-3-(4-methyl-phenyl)-3,4-dihydro-
quinazolin-6-yl)acetamide (37). Greyish-white powder (1.31 g, 77%),
m.p. 250–2 °C. 1H NMR (CDCl3): δ 1.84 (s, 3H, COCH3), 2.47 (s, 3H,
CH3Ph), 2.53 (s, 3H, SCH3), 7.22 (d, 2H, J = 8.0, ArH), 7.37 (d, 2H,
J = 8.0 Hz, ArH), 7.65 (d, 1H, J = 8.8 Hz, ArH), 8.03 (d, 1H,
J = 2.4 Hz, ArH), 8.42 (s, 1H, NH), 8.52–8.55 (dd, 1H, J = 2.4,
8.8 Hz, ArH). 13C NMR: δ 15.6, 21.4, 24.0, 115.9, 119.7, 127.2,
127.6, 128.9, 130.5, 133.4, 136.7, 140.4, 144.4, 156.8, 162.0, 169.1.
MS (m/z, %) for C18H17N3O2S: (M+ 339.1, 100).
A stirred solution of 6-amino-quinazoline derivative (20 or 21,
0.005 mol) in 4.0 ml of conc. HCl was cooled in an ice-bath to 0–5 °C
and diazotized with a solution of 0.4 g NaNO2 (0.005 mol) in 2 ml H2O.
The cold diazonium solution was added slowly to a well stirred solution
of phenol or o-cresol (43 or 44, 0.005 mol) in 2.5 M NaOH (10 ml) at
0–5 °C. The reaction mixture was stirred for another 2 h. The crude
product was filtered, washed with cold dil. HCl, dried and recrystallized
from ethanol to give 45–48.
6.1.6.2. N-(2-(Methyl-thio)-4-oxo-3-(4-methyl-phenyl)-3,4-dihydro-
quinazolin-6-yl)propion-amide (38). Yellowish-white powder (1.33 g,
75%), m.p. 240–2 °C. 1H NMR (CDCl3): δ 1.18 (t, 3H, J = 7.2 Hz,
CH3), 2.16 (q, 2H, J = 7.6 Hz, COCH2), 2.48 (s, 3H, CH3Ph), 2.53 (s,
3H, SCH3), 7.22 (d, 2H, J = 8.0 Hz, ArH), 7.38 (d, 2H, J = 8.0 Hz,
ArH), 7.65 (d, 1H, J = 9.2 Hz, ArH), 8.00 (d, 1H, J = 2.8 Hz, ArH), 8.07
(s, 1H, NH), 8.5–8.53 (dd, 1H, J = 2.8, 9.2 Hz, ArH), 13C NMR: δ 9.6,
15.6, 21.5, 30.2, 115.9, 119.8, 127.2, 127.6, 128.9, 130.4, 133.4,
136.5, 140.3, 144.4, 156.9, 161.9, 172.5. MS (m/z, %) for
6.1.7.1. 6-[(4-Hydroxy-phenyl)diazenyl]-2-(methyl-thio)-3-(4-methyl-
phenyl)quinazolin-4(3H)-one (45). Dark yellow crystals (1.39 g, 69%),
m.p. 275–8 °C. 1H NMR (DMSO‑d6): δ 2.42 (s, 3H, CH3Ph), 2.51 (s, 3H,
SCH3), 6.97 (d, 2H, J = 8.4 Hz, ArH), 7.35–7.38 (m, 4H, ArH), 7.76 (d,
1H, J = 8.4 Hz, ArH), 7.86 (d, 2H, J = 8.4 Hz, ArH), 8.26 (d, 1H,
J = 8.4 Hz, ArH), 8.44 (s, 1H, ArH), 10.43 (s, 1H, OH). 13C NMR: δ
15.7, 20.4, 56.0, 115.2, 118.6, 120.6, 121.9, 123.3, 127.7, 127.9,
128.7, 129.1, 131, 135.4, 138.7, 149.2, 150.5, 153.4, 160.6, 161.2,
161.3. MS (m/z, %) for C26H20N4O2S: (M+ 402.1, 100).
C
19H19N3O2S: (M+ 353.0, 28.79), 68 (100).
6.1.7.2. 6-[(4-Hydroxy-phenyl)diazenyl]-3-(4-methoxy-phenyl)-2-
6.1.6.3. N-(2-(Methyl-thio)-4-oxo-3-(4-methyl-phenyl)-3,4-dihydro-
quinazolin-6-yl)butyramide (39). Off-white powder (1.52 g, 83%), m.p.
214–6 °C. 1H NMR (CDCl3): δ 0.98 (t, 3H, J = 7.2 Hz, CH3), 1.66–1.73
(m, 2H, CH2), 2.13 (t, 2H, J = 7.2 Hz, COCH2), 2.48 (s, 3H, CH3Ph),
2.53 (s, 3H, SCH3), 7.22 (d, 2H, J = 8.0 Hz, ArH), 7.37 (d, 2H,
J = 8.0 Hz, ArH), 7.64 (d, 1H, J = 8.8 Hz, ArH), 8.01 (d, 1H,
J = 2.4 Hz, ArH), 8.08 (s, 1H, NH), 8.47–8.5 (dd, 1H, J = 2.4, 8.8 Hz,
ArH), 13C NMR: δ 13.7, 15.6, 18.9, 21.4, 39.0, 116.1, 119.8, 127.2,
127.7, 128.8, 130.4, 133.4, 136.4, 140.3, 144.4, 156.9, 161.9, 171.8.
(methyl-thio)quinazolin-4(3H)-one (46). Dark orange crystals (1.29 g,
62%), m.p. 276–8 °C. 1H NMR (DMSO‑d6): δ 2.51 (s, 3H, SCH3), 3.85 (s,
3H, OCH3), 6.97 (d, 2H, J = 8.8 Hz, ArH), 7.12 (d, 2H, J = 8.8 Hz,
ArH), 7.41 (d, 2H, J = 8.8 Hz, ArH), 7.75 (d, 1H, J = 8.8 Hz, ArH), 7.86
(d, 2H, J = 8.8 Hz, ArH), 8.24–8.27 (dd, 1H, J = 2.0, 8.8 Hz, ArH), 8.44
(d, 1H, J = 2.0 Hz, ArH), 10.45 (s, 1H, OH). 13C NMR: δ 15.7, 56.0,
115.1, 116.5, 120.5, 122.0, 125.6, 127.6, 127.9, 128.7, 131.0, 145.7,
149.1, 149.5, 160.6, 160.9, 161.4, 161.8. MS (m/z, %) for
C
26H20N4O3S: (M+ 418.1, 100).
13

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BioorganicChemistry88(2019)102923
6.1.7.3. 6-[(4-Hydroxy-3-methyl-phenyl)diazenyl]-2-(methyl-thio)-3-(4-
methyl-phenyl)quina-zolin-4(3H)-one (47). Orange crystals (1.19 g,
57%), m.p. 212–5 °C. 1H NMR (DMSO‑d6): δ 2.23 (s, 3H, CH3Ph),
2.42 (s, 3H, CH3Ph), 2.52 (s, 3H, SCH3), 6.98 (d, 1H, J = 8.8 Hz, ArH),
7.36–7.40 (m, 4H, ArH), 7.70–7.73 (dd, 1H, J = 2.4, 8.4 Hz, ArH),
7.75–7.78 (m, 2H, ArH), 8.24–8.27 (dd, 1H, J = 2.4, 8.8 Hz, ArH), 8.44
(d, 1H, J = 2.4 Hz, ArH), 10.38 (s, 1H, OH). 13C NMR: δ 15.6, 16.5,
21.3, 115.5, 120.5, 121.8, 124.1, 125.3, 125.7, 127.6, 127.9, 129.5,
130.5, 133.8, 140.1, 145.5, 149, 149.7, 160, 160.4, 161.2. MS (m/z, %)
for C23H20N4O2S: (M+ 416.1, 100).
at 0–5 °C. The reaction mixture was stirred for another 2 h. The crude
product was filtered, washed with cold dil. HCl, dried and recrystallized
from ethanol to give 53 and 54.
6.1.9.1. 6-[(2-Hydroxy-naphthalen-1-yl)diazenyl]-2-(methyl-thio)-3-(4-
methyl-phenyl)quina-zolin-4(3H)-one (53). Dark red crystals (1.2 g,
53%), m.p. 286–9 °C.. 1H NMR (CDCl3): δ 2.50 (s, 3H, CH3Ph), 2.58
(s, 3H, SCH3), 6.94 (d, 1H, J = 9.2 Hz, ArH), 7.26 (d, 2H, J = 8.4 Hz,
ArH), 7.4 (d, 2H, J = 8.0, ArH), 7.45 (d, 1H, J = 6.8 Hz, ArH), 7.59 (t,
1H, J = 6.8 Hz, ArH), 7.65 (d, 1H, J = 7.6 Hz, ArH), 7.77 (d, 2H,
J = 9.2 Hz, ArH), 8.21–8.24 (dd, 1H, J = 2.4, 9.2 Hz, ArH), 8.55 (d, 1H,
6.1.7.4. 6-[(4-Hydroxy-3-methyl-phenyl)diazenyl]-3-(4-methoxy-phenyl)-
2-(methyl-thio)quina-zolin-4(3H)-one (48). Brown crystals (1.17 g,
54%), m.p. 265–8 °C. 1H NMR (DMSO‑d6): δ 2.23 (s, 3H, CH3Ph),
2.51 (s, 3H, SCH3), 3.86 (s, 3H, OCH3), 6.99 (d, 1H, J = 8.8 Hz, ArH),
7.12 (d, 2H, J = 8.8 Hz, ArH), 7.41 (d, 2H, J = 8.8 Hz, ArH), 7.70–7.73
(dd, 1H, J = 2.0, 8.8 Hz, ArH), 7.75 (d, 1H, J = 8.8 Hz, ArH), 7.76 (s,
1H, ArH), 8.23–8.26 (dd, 1H, J = 2.0, 8.8 Hz, ArH), 8.43 (d, 1H,
J = 2.0 Hz, ArH), 10.4 (s, 1H, OH). 13C NMR: δ 15.7, 16.5, 56.0, 115.1,
115.5, 120.6, 121.8, 124.0, 125.3, 125.6, 127.6, 127.9, 128.7, 131.0,
145.5, 149.0, 149.6, 160.0, 160.6, 160.8, 161.4. MS (m/z, %) for
J = 2.4 Hz, ArH), 8.65 (d, 1H, J = 8.0 Hz, ArH), 16.15 (s, 1H, OH). 13
C
NMR: δ 15.6, 21.5, 117.7, 120.7, 122.0, 124.1, 124.6, 125.9, 128.0,
128.2, 128.6, 128.7, 129.0, 130.4, 130.5, 133.1, 133.4, 139.9, 140.4,
143.1, 147.3, 158.8, 161.6, 169.8. MS (m/z, %) for C22H18N4O3S: (M+
452.1, 100).
6.1.9.2. 6-[(2-Hydroxy-naphthalen-1-yl)diazenyl]-3-(4-methoxy-phenyl)-
2-(methyl-thio)quina-zolin-4(3H)-one (54). Dark red crystals (1.29 g,
55%), m.p. 309–12 °C. 1H NMR (CDCl3): δ 2.58 (s, 3H, SCH3), 3.92
(s, 3H, OCH3), 6.95 (d, 1H, J = 9.2 Hz, ArH), 7.10 (d, 2H, J = 8.8 Hz,
ArH), 7.29 (d, 2H, J = 8.8, ArH), 7.44 (t, 1H, J = 7.6 Hz, ArH), 7.59 (t,
1H, J = 7.2 Hz, ArH), 7.66 (d, 1H, J = 8.0 Hz, ArH), 7.76–7.79 (m, 2H,
ArH), 8.21–8.24 (dd, 1H, J = 2.4, 9.2 Hz, ArH), 8.55 (d, 1H, J = 2.4 Hz,
ArH), 8.66 (d, 1H, J = 8.0 Hz, ArH), 16.15 (s, 1H, OH). 13C NMR: δ
15.7, 55.6, 115.0, 117.7, 120.7, 122.0, 124.1, 124.6, 125.9, 127.9,
128.17, 128.22, 128.7, 129.0, 130.2, 130.4, 133.4, 139.9, 143.1, 147.1,
159.2, 160.7, 161.7, 169.9. MS (m/z, %) for C23H20N4O3S: (M+ 468.1,
100).
C
23H20N4O3S: (M+ 432.1, 100).
6.1.8. General procedure for the synthesis of 6-[(2-hydroxy-5-methyl-
phenyl)diazenyl)]-3-(4-substituted-phenyl)-2-(methyl-thio)quinazolin-
4(3H)-ones (50 and 51)
A stirred solution of 6-amino-quinazoline derivative (20 or 21,
0.005 mol) in 4.0 ml of conc. HCl was cooled in an ice-bath to 0–5 °C
and diazotized with the solution of 0.4 g NaNO2 (0.005 mol) in 2 ml
H2O. The cold diazonium solution was added slowly to a well stirred
solution of p-cresol (49, 0.54 g, 0.005 mol) in 2.5 M NaOH (10 ml) at
0–5 °C. The reaction mixture was stirred for another 2 h. The crude
product was filtered, washed with cold dil. HCl, dried and recrystallized
from ethanol to give 50 and 51.
6.1.10. General procedure for the synthesis of 1-(4-substituted-phenyl)-3-
(3-(4-substituted-phenyl)-2-(methyl-thio)-4-oxo-3,4-dihydro-quinazolin-6-
yl)thioureas (56–61)
A
solution of the 6-aminoquinazoline derivative (20 or 21,
0.005 mol), and the appropriate substituted phenylisothiocyanate de-
rivative (14, 15 or 55, 0.005 mol) in pyridine (10 ml) was stirred at
room temperature for 6 h. Then the reaction mixture was poured into
cold dil. HCl. The formed precipitate was filtered, washed with water,
dried and recrystallized from ethanol to give (56–61).
6.1.8.1. 6-[(2-Hydroxy-5-methyl-phenyl)diazenyl]-2-(methyl-thio)-3-(4-
methyl-phenyl)quina-zolin-4(3H)-one (50). Dark brown crystals (1.02 g,
49%), m.p. 208–10 °C. 1H NMR (DMSO‑d6): δ 2.30 (s, 3H, CH3Ph), 2.43
(s, 3H, CH3Ph), 2.53 (s, 3H, SCH3), 6.99 (d, 1H, J = 8.4 Hz, ArH),
7.25–7.28 (dd, 1H, J = 2.0, 8.4 Hz, ArH), 7.36–7.41 (m, 4H, ArH), 7.60
(d, 1H, J = 2.0 Hz, ArH), 7.77 (d, 1H, J = 8.8 Hz, ArH), 8.45–8.48 (dd,
1H, J = 2.4, 8.8 Hz, ArH), 8.6 (d, 1H, J = 2.4 Hz, ArH), 10.8 (s, 1H,
OH). 13C NMR: δ 15.7, 20.4, 21.3, 118.6, 120.6, 121.9, 123.2, 127.7,
127.9, 129.1, 129.5, 130.5, 133.7, 135.2, 138.7, 140.1, 149.2, 149.5,
153.4, 160.8, 161.2. MS (m/z, %) for C22H18N4O2S: (M+ 416.1, 100).
6.1.10.1. 1-(2-(Methyl-thio)-4-oxo-3-(4-methyl-phenyl)-3,4-dihydro-
quinazolin-6-yl)-3-(4-methyl-phenyl)thiourea
(56). Yellowish-white
crystals (1.83 g, 82%), m.p. 108–10 °C. 1H NMR (CDCl3): δ 2.40 (s,
3H, CH3Ph), 2.42 (s, 3H, CH3Ph), 2.53 (s, 3H, SCH3), 7.18 (d, 2H,
J = 8.4 Hz, ArH), 7.23–7.28 (m, 4H, ArH), 7.33 (d, 2H, J = 8.0 Hz,
ArH), 7.66 (d, 1H, J = 8.4 Hz, ArH), 8.0 (d, 1H, J = 2.0 Hz, ArH), 8.05
(s, 1H, NH), 8.08 (s, 1H, NH), 8.18–8.21 (dd, 1H, J = 2.0, 8.4 Hz, ArH).
13C NMR: δ 15.6, 21.1, 21.4, 119.9, 121.7, 125.6, 125.9, 127.0, 128.7,
130.1, 130.4, 130.5, 132.5, 133.1, 135.8, 140.4, 146.1, 158.3, 161.6,
180.5. MS (m/z, %) for C24H22N4OS2: (M+ 446.0, 23.62), 444 (100).
6.1.8.2. 6-[(2-Hydroxy-5-methyl-phenyl)diazenyl]-3-(4-methoxy-phenyl)-
2-(methyl-thio)quina-zolin-4(3H)-one (51). Dark orange crystals (1.1 g,
51%), m.p. 229–32 °C. 1H NMR (DMSO‑d6): δ 2.30 (s, 3H, CH3Ph), 2.52
(s, 3H, SCH3), 3.86 (s, 3H, OCH3), 6.99 (d, 1H, J = 8.4 Hz, ArH), 7.12
(d, 2H, J = 8.8 Hz, ArH), 7.25–7.28 (dd, 1H, J = 2.0, 8.4 Hz, ArH), 7.42
(d, 2H, J = 8.8 Hz, ArH), 7.60 (s, 1H, ArH), 7.77 (d, 1H, J = 8.8 Hz,
ArH), 8.45–8.47 (dd, 1H, J = 2.4, 8.8 Hz, ArH), 8.60 (d, 1H, J = 2.0 Hz,
ArH), 10.8 (s, 1H, OH). 13C NMR: δ 15.7, 20.4, 56.0, 115.2, 118.6,
120.6, 121.9, 123.3, 127.7, 127.9, 128.7, 129.1, 131.0, 135.4, 138.7,
149.2, 150.5, 153.4, 160.6, 161.2, 161.3. MS (m/z, %) for
6.1.10.2. 1-(4-Methoxy-phenyl)-3-(2-(methyl-thio)-4-oxo-3-(4-methyl-
phenyl)-3,4-dihydro-quinazolin-6-yl)thiourea (57). Off-white crystals
(1.97 g, 85%), m.p. 164–6 °C. 1H NMR (CDCl3):
δ 2.46 (s, 3H,
CH3Ph), 2.54 (s, 3H, SCH3), 3.86 (s, 3H, OCH3), 6.99 (d, 2H,
J = 8.8 Hz, ArH), 7.19 (d, 2H, J = 8.4 Hz, ArH), 7.29–7.36 (m, 4H,
ArH), 7.67 (d, 1H, J = 8.0 Hz, ArH), 7.81 (s, 1H, NH), 7.82 (s, 1H, NH),
7.99 (s, 1H, ArH), 8.15 (d, 1H, J = 8.0 Hz, ArH). 13C NMR: δ 15.6, 21.4,
55.6, 115.3, 120.0, 122.0, 124.0, 128.0, 128.1, 128.7, 130.5, 132.4,
132.5, 133.1, 135.6, 140.4, 146.3, 158.5, 161.5, 180.9. MS (m/z, %) for
C
23H20N4O2S: (M+ 432, 100).
6.1.9. General procedure for the synthesis of 6-[(2-hydroxy-naphthalen-1-
yl)diazenyl]-3-(4-substituted-phenyl)-2-(methyl-thio)quinazolin-4(3H)-one
(53 and 54)
C
24H22N4O2S2: (M+ 462.0, 2.87), 428 (100).
A stirred solution of 6-amino-quinazoline derivative (20 or 21,
0.005 mol) in 4.0 ml of conc. HCl was cooled in an ice-bath to 0–5 °C
and diazotized with the solution of 0.4 g NaNO2 (0.005 mol) in 2 ml
H2O. The cold diazonium solution was added slowly to a stirred solu-
tion of naphthalen-2-ol (52, 0.72 g, 0.005 mol) in 2.5 M NaOH (10 ml)
6.1.10.3. 1-(4-Chloro-phenyl)-3-(2-(methyl-thio)-4-oxo-3-(4-methyl-
phenyl)-3,4-dihydro-quinazolin-6-yl)thiourea (58). Pale yellow crystals
(2.07 g, 89%), m.p. 129–32 °C. 1H NMR (CDCl3): δ 2.32 (s, 3H, CH3Ph),
2.53 (s, 3H, SCH3), 7.17 (d, 2H, J = 8.4 Hz, ArH), 7.23–7.37 (m, 6H,
14

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BioorganicChemistry88(2019)102923
ArH), 7.67 (d, 1H, J = 8.8 Hz, ArH), 8.03 (d, 1H, J = 2.4 Hz, ArH),
8.32–8.34 (dd, 1H, J = 2.4, 8.8 Hz, ArH), 8.38 (s, 1H, NH), 8.95 (s, 1H,
NH). 13C NMR: δ 15.7, 21.3, 119.4, 120.7, 126.5, 126.7, 127.0, 128.4,
129.0, 129.6, 130.6, 132.8, 133.1, 136.4, 140.7, 146.0, 158.0, 162.4,
180.7. MS (m/z, %) for C23H19ClN4OS2: (M + 2 467.1, 33.50), (M+
465.0, 12.31), 89 (100).
6.2.1.2. Cytotoxicity MTT assay for compounds 59 and 61. Tumor cell
Lines were acquired from American Type Culture Collection, cells were
cultured using Dulbecco's Modified Eagle Medium (DMEM)
(Invitrogen/Life Technologies) augmented with 10% fetal bovine
serum (Hyclone), 10 µg/ml of insulin (Sigma), and 1% penicillin-
streptomycin. All of the other reagents and chemicals were obtained
from Sigma chemical company. The cells (Hela and HCT-116) were
subcultured into a 96-well plate with 1 × 104 cells per well in medium,
at 37 °C, 5% CO2 and 95% air atmosphere, before being treated with or
without differenet concentrations of test compounds 59 and 61, each in
triplicate for 24 h. At the end of the incubation, the cells were harvested
and washed with phosphate buffered saline (PBS). 20 µl of MTT was
added to each well and incubated for 2 h before 200 µl DMSO was
added. The absorbance was measured on an ELISA reader (Multiskan
EX, Labsystems) at a test wavelength of 570 nm [36].
6.1.10.4. 1-(3-(4-Methoxy-phenyl)-2-(methyl-thio)-4-oxo-3,4-dihydro-
quinazolin-6-yl)-3-(4-methyl-phenyl)thiourea (59). Off-white crystals
(1.85 g, 80%), m.p. 136–8 °C. 1H NMR (CDCl3):
δ 2.40 (s, 3H,
CH3Ph), 2.53 (s, 3H, SCH3), 3.86 (s, 3H, OCH3), 7.03 (d, 2H,
J = 8.8 Hz, ArH), 7.21–7.26 (m, 6H, ArH), 7.66 (d, 1H, J = 8.4 Hz,
ArH), 7.91 (s, 1H, NH), 7.95 (s, 1H, NH), 7.99 (d, 1H, J = 2.4 Hz, ArH),
8.16–8.19 (dd, 1H, J = 2.4, 8.4 Hz, ArH), 13C NMR: δ 15.6, 21.1, 55.5,
115.0, 119.9, 121.8, 125.5, 125.9, 127.0, 128.1, 130.1, 130.6, 132.4,
135.7, 137.9, 146.1, 158.8, 160.6, 161.7, 180.5. MS (m/z, %) for
C
24H22N4O2S2: (M+ 462.0, 12.98), 460 (100).
6.2.1.3. Flow cytometry analysis of DNA content for cell cycle and
apoptosis. Hela and HCT-116 cell lines (each 2 × 105 cells/well) each
in 12-well plates were incubated with different concentrations of test
compounds 59 (for Hela cell line) and 61 (for HCT-116 cell line) for
various time-periods before the cells were harvested. The cells were
fixed gently in 70% ethanol (in PBS) in ice overnight and were then
suspended in PBS containing 40 µg/ml PI, 0.1 mg/ml RNase A (Sigma,
USA) and 0.1% triton x-100. After 30 min at 37 °C in the dark, the cells
were analyzed by flow cytometry (Becton Dickinson, San Jose, CA,
USA) equipped with an argon laser at 488 nm. Then cell cycle and
apoptosis were determined and examined [58–60].
6.1.10.5. 1-(4-Methoxy-phenyl)-3-(3-(4-methoxy-phenyl)-2-(methyl-
thio)-4-oxo-3,4-dihydro-quinazolin-6-yl)thiourea (60). Yellowish white
crystals (1.96 g, 82%), m.p. 168–70 °C. 1H NMR (CDCl3): δ 2.53 (s,
3H, SCH3), 3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.99 (d, 2H,
J = 8.8 Hz, ArH), 7.03 (d, 2H, J = 8.8 Hz, ArH), 7.21 (d, 2H,
J = 8.8 Hz, ArH), 7.29 (d, 2H, J = 8.8 Hz, ArH), 7.66 (d, 1H,
J = 8.8 Hz, ArH), 7.84 (s, 1H, NH), 7.85 (s, 1H, NH), 7.99 (d, 1H,
J = 2.0 Hz, ArH), 8.16–8.19 (dd, 1H, J = 2.0, 8.8 Hz, ArH), 13C NMR: δ
15.7, 55.5, 55.6, 115.0, 115.2, 119.9, 121.8, 127.0, 128.0, 128.1,
130.0, 130.1, 132.4, 135.7, 146.1, 158.8, 159.2, 160.6, 161.7, 180.9.
MS (m/z, %) for C24H22N4O3S2: (M+ 462.0, 12.98), 460 (100).
6.2.2. Dihydrofolate reductase (DHFR) inhibition assay
The assay mixture contained 50 µM Tris–HCl buffer (pH 7.4), 50 µM
NADPH, 10 µl DMSO or the same volume of DMSO solution containing
the test compounds to a final concentration of 10-11–10-5 M, and 10 µl
of bovine liver dihydrofolate reductase (DHFR), in a final volume of
1.0 ml [61]. After addition of the enzyme, the mixture was incubated at
room temperature for 2.0 min, and the reaction was started by adding
5 µl of dihydrofolic acid, the difference in absorbance (ΔOD/min) was
measured by the spectrophotometer at 340 nm and 22 °C, kinetic pro-
gram (reading repeated every 15 s for 2.5 min). Results are reported as
% inhibition of enzymatic activity (Table 1) calculated by using the
following equation:
6.1.10.6. 1-(4-Chloro-phenyl)-3-(3-(4-methoxy-phenyl)-2-(methyl-thio)-
4-oxo-3,4-dihydro-quinazolin-6-yl)thiourea (61). Pale yellow crystals
(2.13 g, 88%), m.p. 143–6 °C. 1H NMR (CDCl3): δ 2.53 (s, 3H, SCH3),
3.77 (s, 3H, OCH3), 6.92 (d, 2H, J = 8.8 Hz, ArH), 7.20 (d, 2H,
J = 8.8 Hz, ArH), 7.25 (d, 2H, J = 8.4 Hz, ArH), 7.34 (d, 2H,
J = 8.4 Hz, ArH), 7.68 (d, 1H, J = 8.8 Hz, ArH), 8.03 (d, 1H,
J = 2.4 Hz, ArH), 8.24 (s, 1H, NH), 8.31 (d, 1H, J = 8.8 Hz, ArH),
8.75 (s, 1H, NH), 13C NMR: δ 15.7, 55.4, 115.0, 119.4, 120.8, 126.8,
127.1, 127.7, 129.1, 129.8, 130.0, 132.0, 133.1, 136.2, 146.1, 158.6,
160.7, 162.5, 180.7. MS (m/z, %) for C23H19ClN4O2S2: (M + 2 483.1,
13.47), (M+ 481.0, 6.75), 480 (100).
Fractional activity of enzyme = (Sample OD/min blank OD/min)
6.2. Biological evaluation
× d/12.3 × V × mgP/ml
where
6.2.1. Antitumor screening
6.2.1.1. In vitro antitumor evaluation. Four human tumor cell lines
namely; mammary gland breast cancer (MCF-7), colorectal carcinoma
(HCT-116), human prostate cancer (PC-3) and epitheliod Carcinoma
(Hela) were used in the antitumor activity evaluation assay. MTT assay
was used to determine the inhibitory effects of compounds on cell
growth of the cell lines mentioned above. This colorimetric assay is
based on mitochondrial succinate dehydrogenase conversion of the
yellow tetrazolium bromide (MTT) to a purple formazan derivative by
in viable cells. Cell lines were cultured in RPMI-1640 medium with 10%
fetal bovine serum. Antibiotics added were 100 units/ml penicillin and
100 µg/ml streptomycin at 37 °C in a 5% CO2 incubator. The cell lines
were seeded in a 96-well plate at a density of 1.0x104 cells/well at 37 °C
for 48 h under 5% CO2. After incubation the cells were treated with
different concentration of tested compounds and incubated for 72 h.
After 72 h of drug treatment, 20 µl of MTT solution at 5 mg/ml was
added and incubated for 4 h. After that, 100 µl dimethyl sulfoxide
(DMSO) were added into each well to dissolve the purple formazan
formed. The colorimetric assay is measured and recorded at 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) X 100 (Table 1), [32–34].
ΔOD/min: the spectrophotometer readings.
12.3: extinction coefficient for the DHFR reaction at 340 nm.
V: Enzyme volume used in the assay in ml.
d: the enzyme sample dilution factor.
mgP/ml: the original sample enzyme concentration before dilution.
6.3. Molecular modeling study
The molecular modeling calculations were done using “molecular
operating environment (MOE) version 2009.11” Chemical Computing
Group Inc. software. The most stable conformer of newly synthesized
‘global-minima’ analogs was docked into the binding pocket of dihydro-
folate reductase enzyme-binding domain which is in tertiary complex
with dihydro-nicotinamide-adenine-dinucleotide phosphate (NADPH)
and methotrexate (MTX), code ID 1DLS. It was obtained from the re-
search collaboratory for structural bioinformatics Protein Data Bank.
The hydrogens were added, then enzyme structure subjected to a re-
finement protocol by addition of the missed bonds and protein potential
fixation where the constraints on the enzyme were gradually removed
and minimized until the RMSD gradient was 0.01 kcal/mol Å. The
15

M.A. Sabry, et al.
BioorganicChemistry88(2019)102923
active site of the enzyme was detected using a radius of 10.0 Å around
MTX. The three-dimensional structures of some selected substituted
quinazoline derivatives, which represent most and least active dihydro-
folate reductase inhibitors, in their neutral forms, were built using the
builder interface of the MOE software. Conformational analysis of the
selected compounds was performed using MMFF94 force field with root
mean square (RMS) gradient of 0.01 kcal/mol Å. The flexible alignment
of selected compounds were done using the flexible alignment tool of
the program adjusting the energy cut off to 15 kcal/mol, and RMSD
tolerance to 0.5. Molecular surfaces were obtained from the surfaces
and maps tool of the MOE program. In the molecular surface maps, the
pink colored regions have potential hydrogen bonding ability, blue
regions are mild polar, and green ones are the hydrophobic regions
[45,62].
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16