Nonclassical antifolates, part 3: Synthesis, biological evaluation and molecular modeling study of some new 2-heteroarylthio-quinazolin-4-ones

Article abstract of DOI:10.1016/j.ejmech.2012.12.061

A new series of 2-heteroarylthio-6-substituted-quinazolin-4-one analogs was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 21, 25, and 39 proved to be active DHFR inhibitors with IC₅₀ range of 0.3-0.8 μM. Compounds 25, 28, 33, 35 and 36 showed broad spectrum antimicrobial activity comparable to the known antibiotic gentamicin. Compound 29 showed broad spectrum antitumor activity toward several tumor cell lines with GI values range of 25.8-41.2%. Molecular modeling studies concluded that recognition with key amino acid Arg38 and Lys31 are essential for binding and biological activities. Flexible alignment; electrostatic and hydrophobic mappings revealed that the obtained model could be useful for the development of new DHFR inhibitors.

Full text of DOI:10.1016/j.ejmech.2012.12.061

European Journal of Medicinal Chemistry 63 (2013) 33e45
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Nonclassical antifolates, part 3: Synthesis, biological evaluation and molecular
modeling study of some new 2-heteroarylthio-quinazolin-4-onesq
,
Fatmah A.M. Al-Omary a, Ghada S. Hassan a b, Shahenda M. El-Messery c, Mahmoud N. Nagi d,
,
El-Sayed E. Habib e, Hussein I. El-Subbagh f
*
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
b Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt
c Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt
d Department of Pharmacology, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
e Department of Pharmaceutics and Pharmaceutical Technology (Microbiology), College of Pharmacy, Taibah University, Almadinah Almunawwarah 344, Saudi Arabia
f Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future University, 12311 Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 2-heteroarylthio-6-substituted-quinazolin-4-one analogs was designed, synthesized, and
Received 8 September 2012
Received in revised form
24 December 2012
Accepted 26 December 2012
Available online 13 February 2013
evaluated for their in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 21, 25,
and 39 proved to be active DHFR inhibitors with IC50 range of 0.3e0.8 mM. Compounds 25, 28, 33, 35 and
36 showed broad spectrum antimicrobial activity comparable to the known antibiotic gentamicin.
Compound 29 showed broad spectrum antitumor activity toward several tumor cell lines with GI values
range of 25.8e41.2%. Molecular modeling studies concluded that recognition with key amino acid Arg38
and Lys31 are essential for binding and biological activities. Flexible alignment; electrostatic and hy-
drophobic mappings revealed that the obtained model could be useful for the development of new DHFR
inhibitors.
Keywords:
Synthesis
Quinazolin-4-ones
DHFR inhibition
Antimicrobial testing
Antitumor screening
Molecular modeling study
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
chemotherapeutic agents against bacterial and parasitic infections
as well as cancer [3].
Dihydrofolate reductase (DHFR) plays a crucial role in the bio-
synthesis of nucleic acids. It catalyzes the NADPH reduction of 7,8-
dihydrofolate to 5,6,7,8-tetrahydrofolate and intimately couples
with thymidylate synthase. Thymidylate synthase is the key
enzyme that catalyzes the reductive methylation of deoxyuridine
monophosphate (dUMP) to deoxythymidine monophosphate
(dTMP) utilizing N5,N10-methylene-tetrahydrofolate as a cofactor.
Hence, the inhibition of DHFR or thymidylate synthase activity
leads to cellular deficiency of tetrahydrofolate cofactors, that re-
sults in disruption of the biosynthesis of purines and pyrimidines,
and finally to cell death [1,2]. Thus, DHFR inhibition has long
been identified as an important target for the development of
DHFR inhibitors are broadly classified as either classical or non-
classical antifolates. The classical antifolates possess p-amino-
benzoylglutamate side-chain embedded in the molecule, whereas
non-classical inhibitors of DHFR does not possess the glutamate
moiety, but rather have a lipophilic side-chain. Literature citations
revealed numerous compounds which categorized under the non-
classical DHFR inhibitors such as: trimethoprim (TMP, 1), trime-
trexate (TMQ, 2) and piritrexim (PTX, 3), beside others belong to the
quinazoline heterocycle [4e12], (Chart 1).
Recently, a new series of 2,6-substituted-quinazolin-4-ones was
designed, synthesized, and evaluated for their in vitro DHFR inhi-
bition in our laboratories [13,14]. This study allowed the allocation
of compounds 4e8 as active DHFR inhibitors with IC50 values
around 0.4
mM (Chart 1). Compounds 6 and 8 characterized by
bearing 2-thioallylic hydrophobic
p-system. Molecular modeling
studies of this class of compounds revealed the importance of the
main pharmacophoric groups (the 4-carbonyl fragment, the basic
q
For parts 1 and 2 see Refs. [13,14].
* Corresponding author. Tel./fax: þ20 2 26186111.
nitrogen atom at N-1, and the hydrophobic
p-system regions) as
E-mail address: subbagh@yahoo.com (H.I. El-Subbagh).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.061

34
F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
OCH3
OCH3
OCH3
OCH3
NH2 CH3
NH2 CH3
NH2
N
OCH3
N
N
N
N
H
OCH3
H2N
N
N
H2N
OCH3
OCH3
H2N
N
PTX, 3
1
TMP,
TMQ, 2
CH3
N
CH3
N
CH3O
CH3O
O
N
O
Bn
Ph
N
N
CH3
CH3
N
S
S
4
5
O
Bn
O
Bn
N
O
N
H
N
N
S
Bn
S
N
N
N
N
N
CH3
S
7
8
6
Chart 1. Structures of literature antifolate lead compounds.
well as of their relative spatial distances. The substitution pattern
and spatial considerations of the -systems in regard to the qui-
activity will be the candidates for treating cancer and bacterial or
parasitic infections.
p
nazoline nucleus proved to be critical for DHFR inhibition [13,14].
In continuation to our previous efforts [13e27], a new series of
2-arylthio or heteroarylthio-quinazolin-4-one analogs, of the gen-
eral formula presented in Chart 2 was designed to possess 2-
2. Results and discussion
2.1. Chemistry
arylthio or heteroarylthio functions as hydrophobic
p-system re-
gions replacing the 2-thioalkyl and 2-thioallyl function of 4e8. In
addition, 6-chloro, 6-methyl, or 6,7-dimethoxy functions, repre-
senting electron donating and electron withdrawing substituents;
a phenyl or benzyl group at position 3 were introduced to the
quinazolin-4-one nucleus in resemblance to the lead compounds
4e8. Most of the functions designed to be accommodated on the
quinazoline ring such as thioether, aryl, heteroaryl groups are
known to contribute to DHFR inhibition activity [28,29]. The aim of
this study is to locate novel synthetic lead compound(s), and their
in vitro testing as DHFR inhibitor(s). In addition, these derivatives
were tested for their in vitro antimicrobial activity against a panel of
standard strains of Gram-positive and Gram-negative bacteria.
Furthermore, the new compounds were screened for their in vitro
antitumor activity using the NCI’s disease-oriented human cell
lines assay [30e33]. Compounds possessing DHFR inhibition
The synthetic strategy to obtain the targets of the general for-
mula presented in Chart 2 is depicted in Scheme 1. The starting
materials 2-mercapto-6-methyl-3-phenyl (or benzyl)-quinazolin-
4(3H)-ones (14, 15), 6-chloro-2-mercapto-3-phenyl (or benzyl)-
quinazolin-4(3H)-ones (16, 17) and 6,7-dimethoxy-2-mercapto-3-
phenyl (or benzyl)-quinazolin-4(3H)-ones (18, 19) were prepared
adopting reported procedures [24e26]. The 2-thioxo-function of
the starting materials 14e19 was then alkylated using variety of
halogenated aryl or heteroaryl derivatives.
Compounds 14e19 were reacted with 5-bromothiazole-2-amine
(20), 2-chloronicotinic acid (27), ethyl 2-amino-5-bromothiazole-4-
carboxylate (34) or 2-chloro-4-nitrobenzoic acid (41) and potassium
carbonate in dimethylformamide to afford 2-(2-aminothiazol-5-
ylthio)-3-phenyl (or benzyl)-6-substituted (or 6,7-dimethoxy)-
quinazolin-4(3H)-ones (21e26), 2-(3-benzyl-6-substituted (or 6,7-
dimethoxy)-4-oxo-3,4-dihydro quinazoline-2-ylthio)nicotinic acid
derivatives (28e33), ethyl 2-amino-5-(3-phenyl (or benzyl)-6-
substituted (or 6,7-dimethoxy)-4-oxo-3,4-dihydroquinazolin-2-
ylthio)thiazole-4-carboxylate analogs (35e40) or 2-(3-phenyl (or
benzyl)-6-substituted (or 6,7-dimethoxy)-4-oxo-3,4-dihydroquin-
azolin-2-ylthio)-4-nitrobenzoic acid derivatives (42e47), respec-
tively, Scheme 1. Structure elucidation of the synthesized in-
termediates and final products was attained bythe aid of elementary
analyses, 1H & 13C NMR spectroscopy, and mass spectrometry.
O
(CH2)n
R1
R2
N
N
S
R3
n = 0, 1
R1 = CH3, Cl, OCH3
R2 = H, OCH3
2.2. Dihydrofolate reductase (DHFR) inhibition
COOH
NO2
COOH
The synthesized compounds (21e47) were evaluated as in-
hibitors of bovine liver DHFR using reported procedure [34]. Results
were reported as IC50 values (Tables 2 and 3). Compounds 21, 22, 25,
35, 38, and 39 proved to be the most active DHFR inhibitors with
EtOOC
N
N
R3
=
N
S
NH2
S
NH2
IC50 values range of 0.3e1.0
mM, while compounds 23, 24, 26, 36, 37,
Chart 2. Structures of the designed new DHFR inhibitors.
40, and 47 were considered of moderate activity with IC50 range of

F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
35
O
O
(CH2)n
(CH2)n
R1
R2
R1
R2
N
S
N
N
S
N
S
N
COOH
N
H2N
N
28-33
21-26
Br
S
20
NH2
COOH
DMF
K2CO3
DMF
K2CO3
N
Cl
27
NCS
(CH2)n
O
(CH2)n
R1
R2
R1
R2
COOH
N
C2H5OH, Et3N
+
N
H
S
NH2
9:
12:
R1=CH3, R2=H
n = 0
14-19
13: n = 1
10: R1=Cl, R2=H
11: R1=R2=OCH3
EtOOC
Br
N
DMF
K2CO3
DMF
NO2
K2CO3
S
34
NH2
Cl
COOH
41
O
N
O
(CH2)n
(CH2)n
R1
R2
R1
R2
N
S
N
N
S
S
COOH
COOEt
N
O2N
H2N
42-47
35-40
Scheme 1. Synthesis of the target compounds 21e47.
1.0e5.0
m
M, the rest of the tested compounds were considered
quinazoline analogs, namely, 2-aminothiazole (21e26), 2-nicotinic
acid (28e33), ethyl 2-aminothiazole-4-carboxylate (35e40) or 4-
nitrobenzoic acid derivatives (42e47). The 2-thioether function
affects the magnitude of DHFR inhibition. The order of activity is 2-
aminothiazole or ethyl 2-aminothiazole-4-carboxylate (IC50 0.3e
inactive with IC50 > 5 mM. Methotrexate (IC50 0.08 mM) was used as
a positive control.
2.2.1. Structure activity relationship (SAR)
In the present investigation, the type of 2-, 3- or 6-substituent
on the studied quinazolines manipulated the DHFR inhibition ac-
tivity. Four different groups of compounds were synthesized differ
in the type of the 2-thioether function attached to the used
4.0
12.0
m
m
M) > 2-nicotinic acid or 4-nitrobenzoic acid (IC50 7.0e
M). In the 6-methyl series, the presence of 3-phenyl and 2-
M; replacing of
the 3-phenyl by 3-benzyl or the introduction of 4-ethyl carboxylate
aminothiazole-5-thiol produced 21 with IC50 0.8
m

36
F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
Table 1
Physicochemical properties of the newly synthesized compounds 21e47.
O
N
O
N
O
N
O
N
(C H )
(CH )
(CH )
2 n
2 n
(CH )
2 n
2 n
R
R
R
R
R
R
R
1
2
1
2
1
2
1
N
S
N
N
S
N
S
S
S
R
S
2
COOH
COOH
N
COOEt
N
N
H N
2
H N
2
O N
2
21-26
28-33
35-40
42-47
Compd.
R1
R2
n
Yield%
M.p., ꢀC
Molecular formulasa
21
22
23
24
25
26
28
29
30
31
32
33
35
36
37
38
39
40
42
43
44
45
46
47
CH3
CH3
Cl
H
H
H
H
OCH3
OCH3
H
H
H
H
OCH3
OCH3
H
H
H
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
42
25
56
48
55
30
49
47
61
23
46
52
44
63
31
37
63
49
22
39
52
43
28
35
122e5
148e50
132e4
161e3
144e6
152e5
131e4
138e40
121e3
155e7
110e2
129e31
125e7
119e21
133e5
145e7
124e7
153e5
139e41
154e8
114e9
155e8
149e51
158e61
C18H14N4OS2
C19H16N4OS2
C17H11ClN4OS2
C18H13ClN4OS2
C19H16N4O3S2
C20H18N4O3S2
C21H15N3O3S
C22H17N3O3S
Cl
OCH3
OCH3
CH3
CH3
Cl
C
C
20H12ClN3O3S
21H14ClN3O3S
Cl
OCH3
OCH3
CH3
CH3
Cl
C22H17N3O5S
C23H19N3O5S
C21H18N4O3S2
C22H20N4O3S2
C20H15ClN4O3S2
C21H17ClN4O3S2
C22H20N4O5S2
C23H22N4O5S2
C22H15N3O5S
C23H17N3O5S
Cl
H
OCH3
OCH3
CH3
CH3
Cl
Cl
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OCH3
OCH3
C
C
21H12ClN3O5S
22H14ClN3O5S
C23H17N3O7S
C24H19N3O7S
a
Analyzed for C, H, N; results were within ꢁ0.4% of the theoretical values for the given formulas.
Table 2
DHFR inhibition (IC50
,
m
M), and antimicrobial activity results of compounds 21e26, 28e33.
O
O
N
(CH )
(CH )
2 n
2 n
R
R
R
R
1
2
1
2
N
S
N
S
N
S
COOH
N
N
H N
2
21-26
28-33
Compd.
R1
R2
n
DHFR inhibition (IC50
,
m
M)
S. aureus
B. subtilis
M. luteus
E. coli
P. aeruginosa
21
22
23
24
25
26
28
29
30
31
32
33
CH3
CH3
Cl
H
H
H
H
OCH3
OCH3
H
H
H
0
1
0
1
0
1
0
1
0
1
0
1
e
e
0.8
1.0
4.0
2.0
0.5
3.0
8.0
10.0
8.0
10.0
7.0
5.0
Nd
Nd
e
e
e
e
e
e
e
e
e
e
18 (8.0)
16
18 (8.0)
17
20 (4.0)
15
15
22 (4.0)
18
20 (4.0)
18
22 (4.0)
16
16
14
e
e
e
Cl
15
18 (4.0)
16
16
e
e
OCH3
OCH3
CH3
CH3
Cl
16 (4.0)
e
14
e
14
12
12
e
e
e
e
Cl
H
14
18
e
e
e
OCH3
OCH3
e
OCH3
OCH3
e
20 (4.0)
22 (4.0)
27 (2.0)
20 (2.0)
22 (4.0)
26 (2.0)
25 (2.0)
22 (2.0)
15
15
18 (2.0)
18 (2.0)
22 (2.0)
18 (4.0)
21 (0.5)
28 (1.0)
e
12
19 (1.0)
25 (2.0)
Gentamicin
Sulphacetamide
e
e
Inhibition zone (mm): (ꢂ) not active (8 mm), weak activity (8e12 mm), moderate activity (12e15 mm), strong activity (>15 mm). Solvent: DMSO (8 mm). MICs showed in
parentheses. Nd, not determined.

F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
37
Table 3
DHFR inhibition (IC50, mM), and antimicrobial activity results of compounds 35e40, 42e47.
O
N
O
N
(C H )
(CH )
2 n
R
R
2 n
R
R
1
2
1
2
N
S
N
S
S
COOH
COOEt
N
H N
2
O N
2
35-40
42-47
Compd.
R1
R2
n
DHFR inhibition (IC50
,
m
M)
S. aureus
B. subtilis
M. luteus
E. coli
P. aeruginosa
35
36
37
38
39
40
42
43
44
45
46
47
CH3
CH3
Cl
H
H
H
H
OCH3
OCH3
H
H
H
0
1
0
1
0
1
0
1
0
1
0
1
e
e
1.0
4.0
3.0
1.0
0.3
2.0
10.0
12.0
10.0
8.0
10.0
3.0
24 (2.0)
24 (2.0)
e
26 (2.0)
26 (2.0)
e
14
20 (4.0)
18 (4.0)
24 (2.0)
12
18 (4.0)
e
e
e
Cl
20 (4.0)
e
22 (2.0)
e
14
e
20 (4.0)
e
e
OCH3
OCH3
CH3
CH3
Cl
e
18
e
20
e
e
e
e
e
e
e
e
e
e
e
e
14
e
20 (4.0)
15
20 (4.0)
e
e
e
e
Cl
H
e
e
e
OCH3
OCH3
e
OCH3
OCH3
e
18 (8.0)
18
27 (2.0)
20 (2.0)
e
22 (2.0)
e
e
e
e
Gentamicin
Sulphacetamide
Nd
Nd
25 (2.0)
22 (2.0)
18 (2.0)
18 (2.0)
21 (0.5)
28 (1.0)
19 (1.0)
25 (2.0)
e
e
Inhibition zone (mm): (ꢂ) not active (8 mm), weak activity (8e12 mm), moderate activity (12e15 mm), strong activity (>15 mm). Solvent: DMSO (8 mm). MICs showed in
parentheses. Nd, not determined.
to the thiazole function of 21 produced the equipotent derivatives
22 and 35 (IC50 1.0 M); the introduction of 4-ethyl carboxylate to
the thiazole function of 22 (IC50 4.0 M) produced 36 with four fold
and Pseudomonas aeruginosa). The primary screen was carried out
using the agar disc-diffusion method [35,36]. The antibiotics gen-
m
m
tamicin (100
mg/disc), and the DHFR inhibitor sulphacetamide
decrease in DHFR inhibition activity. Replacing of the thiazole
moiety of 21 by either 2-nicotinic acid or 4-nitrobenzoic acid
functions yielded compounds 28, 29, 42, 43 with almost 10 fold
decrease in potency. In the 6-chloro series, the presence of 3-phenyl
(100 g/disc) were used as positive controls. The obtained results
m
revealed that the tested compounds expressed varying degrees of
activity against the tested microorganisms (Tables 2 and 3). Strong
activity (>15 mm inhibition zone) against the Gram-positive bac-
teria was observed for compounds 23e28, 32, 33, 35, 36, 38, 40, 46
and 47 against S. aureus; 23e33, 35, 36, 38, 40, 44, and 46 against B.
subtilis, and 25 and 36 against M. luteus. In case of Gram-negative
bacteria, strong activity was observed for compounds 25e27, 32,
33, 35, 36, 38 and 46 against E. coli. Compounds 25, 28, 33, 35, and
36 showed a remarkable broad-spectrum activity against both
Gram-positive and Gram-negative bacteria. Compounds 23, 29e31,
40, 44 appeared to be selective and active against Gram-positive
bacteria. The minimal inhibitory concentrations (MICs) for the most
active compounds was carried out using the micro-dilution sus-
ceptibility method as shown in Tables 2 and 3. The Gram-positive
bacteria B. subtilis and S. aureus are sensitive to majority of the
tested compounds. Comparing the potency of the active anti-
bacterial compounds and their DHFR inhibition revealed that
compounds 25, 35 and 38 might exert their activity through DHFR
inhibition.
and 2-aminothiazole-5-thiol produced 23 with IC50 4.0
mM;
replacing the 3-phenyl function by 3-benzyl or the introduction of
4-ethyl carboxylate to the thiazole moiety of 23 produced de-
rivatives 24 and 37 (IC50 2.0 and 3.0
duction of 4-ethyl carboxylate to the thiazole function of 24 (IC50
4.0 M) produced 38 with two fold increase in DHFR inhibition
mM, respectively); the intro-
m
activity. Replacing of the thiazole moiety of 23 by either 2-nicotinic
acid or 4-nitrobenzoic acid functions yielded compounds 30, 31, 44,
45 with almost two fold decrease in potency. In the 6,7-dimethoxy
series, the presence of 3-phenyl and 2-aminothiazole-5-thiol pro-
duced 25 with IC50 0.5
benzyl function produced 26 (IC50 3.0
in DHFR inhibition activity. The introduction of 4-ethyl carboxylate
to the thiazole moiety of 25 yielded 39 (IC50 0.3 M) the most
mM; replacing of the 3-phenyl of 25 by 3-
mM) with six fold decrease
m
potent DHFR inhibitor in this study. Replacing of the thiazole
moiety of 25 by either 2-nicotinic acid or 4-nitrobenzoic acid
functions yielded compounds 32, 33, 46, 47 with almost 6e20 fold
decrease in potency. In general, the type of substituent at positions
2-, 3-, and 6- of the quinazoline nucleus proved to manipulate and
contribute to the DHFR inhibition activity in the following order: 2-
thiazolethio- > 2-pyridinethio- > 2-phenylthio-; 3-phenyl- > 3-
benzyl-; and 6,7-dimethoxy- > 6-methyl- > 6-chloro-.
2.4. Antitumor screening
The synthesized compounds 21e47 were subjected to the Na-
tional Cancer Institute (NCI) in vitro disease-oriented human cells
screening panel assay for in vitro antitumor activity. A single dose
(10 mM) of the test compounds were used in the full NCI 60 cell lines
2.3. Antimicrobial activity
panel assay which includes nine tumor subpanels namely; Leuke-
mia, Non-small cell lung, Colon, CNS, Melanoma, Ovarian, Renal,
Prostate, and Breast cancer cells [30e33]. The data reported as
mean-graph of the percent growth of the treated cells, and pre-
sented as percentage growth inhibition (GI%) caused by the test
compounds. Only compound 29 showed broad spectrum potency
The synthesized compounds (21e47) were tested for their
in vitro antimicrobial activity against a panel of standard strains of
Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis and
Micrococcus luteus), and Gram-negative bacteria (Escherichia coli

38
F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
toward several tumor cell lines with GI values range of 25.8e41.2%.
Concerning activity toward individual cell lines, leukemia SR cell
line showed sensitivity toward compounds 21, 23, 31 and 33 with
GI values of 37.8, 29.1, 32.8 and 29.8%, respectively; also CAKI-1
renal cancer cell line toward 22, 24, 26, 29 and 31 with GI values
of 30.3, 25.6, 25.4, 41.2 and 30.3%, respectively; while PC-3 prostate
cancer cell line toward 24, 29 and 31 with GI values of 28.2, 32.8 and
25.2%, respectively (Table 4).
The aligned binding conformations of 39 revealed a clear pref-
erence for binding where the compound occupies the site quite
well as seen in Fig. 2. Compound 39 occupies a space deep in the
cavity and the ester tail extends toward the solvent showing
a strong hydrophobic interaction with the hydrophobic residues in
the DHFR active site. The presence of the 2-amino function at the
thiazole moiety of 39 as a hydrogen bond donor is important for
DHFR inhibition activity that is in contrary to the least active
compound 43 which lacks such association and functional group. In
addition, N1 and 4-carbonyl moieties of the quinazoline ring per-
formed hydrogen bonding with the backbone amino acids of the
active site. Compound 39 is involved also in van der Waals in-
teractions through its phenyl ring with Ala126 and Phe36. (Residue
was not shown in Fig. 2 for clarity.) Meanwhile, compound 43 did
not interact with any amino acid in the active site indicating that
the ligand in question projects out of the active site, this might be
attributed to either the ligand atoms and the remaining surface
area are outside of the binding pocket, or they are in an interior
region with difficult or practically no distinct binding.
3. Molecular dynamic study
The investigated quinazolines were subjected to molecular
modeling study to evaluate their recognition profile at DHFR
binding-pocket, in comparison to the dihydrofolate inhibitor 2,4-
diamino-6-[N-(20,50-dimethoxybenzyl)-N-methylamino]quinazo-
line (COQ). The tertiary complex of DHFR, NADPH and COQ was
used as a reference for modeling and docking [37e42]. At DHFR
pocket, COQ formed bifurcated H-bonding with the key residue
Glu32 and Ile10 [43e46], (Fig. 1). Molecular dynamic study indi-
cated that the tested quinazoline’s recognition with the key amino
acid Arg38 and Lys31 is essential for binding and biological activity.
The amino acid Lys31 is not one of the key residues of the recog-
nition of the parent ligand COQ but it plays a crucial role in the
recognition of the tested compounds. Fig. 1 showed the binding
mode and residues involved in the recognition of the most active
To probe similarity between the 3D structures of the most active
compounds 25 and 39, flexible alignment was employed. Our initial
approach was to employ MOE/MMFF94 flexible alignment to
automatically generate superposition of the compounds under
investigation with minimal user bias [47]. 200 conformers of each
compound were generated and minimized with
a distance-
compound 39 (IC50 0.3
m
M), and the most inactive compound 43
dependant dielectric model. A low energy set of 100 was selected
for further analysis. The top scoring alignment of both the least
(IC50 12.0
mM) docked and minimized in the DHFR binding pocket.
Table 4
Percentage growth inhibition (GI%) of in vitro subpanel tumor cell lines at 10 mM concentration of compounds 21e33.
Subpanel tumor cell linesa
21
22
23
24
25
26
28
29
30
31
32
33
Leukemia
CCRF-CEM
HL-60(TB)
MOLT-4
RPMI-8226
SR
e
e
e
13.7
e
e
22.8
e
e
16.5
e
e
e
e
e
e
17.8
e
e
e
e
e
e
e
e
e
e
e
e
13.2
13.8
12.5
29.8
e
e
e
16.8
18.9
e
e
e
e
15.3
25.8
14.4
16.7
16.3
32.8
e
16.8
e
e
12.5
14.8
15.9
e
10.4
e
37.8
29.1
Non-small cell lung cancer
A549/ATCC
EKVX
NCI-H226
NCI-H23
NCI-H522
CNS cancer
SF-295
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
25.9
19.9
e
12.9
25.7
10.9
14.0
11.9
17.1
e
12.7
e
16.1
10.4
e
26.6
21.2
e
e
25.4
18.4
e
e
23.1
14.0
e
12.4
e
15.1
e
12.0
e
e
13.8
e
16.0
e
19.8
24.4
e
13.1
11.4
17.3
e
e
e
17.3
e
e
e
e
16.6
e
e
e
e
e
e
e
e
14.2
14.2
30.0
e
e
e
15.7
e
e
e
e
e
e
e
SNB-19
SNB-75
e
20.4
10.7
13.3
18.4
Melanoma
LOX IMVI
MALME-3M
MDA-MB-435
SK-MEL-5
UACC-62
Ovarian cancer
IGORV1
OVCAR-4
NCI/ADR-RES
Renal cancer
A498
e
e
e
e
e
10.4
12.3
e
11.2
e
14.9
10.8
e
e
e
e
e
e
10.8
e
e
e
e
e
e
13.8
e
e
e
e
e
e
13.6
e
e
e
e
e
e
e
e
e
e
21.8
14.4
30.4
e
e
e
e
e
12.9
e
e
e
12.2
e
14.4
16.7
11.6
14.3
e
21.8
e
e
e
e
12.0
13.8
e
13.0
e
e
e
e
e
e
e
16.3
16.2
12.7
e
e
e
14.7
20.6
e
e
e
e
e
14.3
e
e
e
e
e
e
e
11.2
25.6
e
e
e
e
11.4
41.2
21.5
11.2
20.6
e
e
e
e
CAKI-1
RXF 393
SN12C
UO-31
20.9
e
30.3
e
24.1
e
17.8
e
25.4
e
13.1
e
e
30.3
e
17.7
e
15.9
e
e
e
e
e
e
e
e
e
e
e
e
e
20.7
29.4
e
14.8
22.2
17.4
12.1
20.5
20.3
12.0
14.6
Prostate cancer
PC-3
10.1
22.6
10.2
28.2
12.0
22.4
11.7
32.8
e
25.2
e
e
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
e
10.8
15.2
e
19.0
21.5
e
e
e
e
e
e
e
e
16.6
30.5
12.5
e
e
e
e
e
e
e
13.5
e
18.1
e
11.2
e
18.6
e
19.1
e
10.4
e
24.7
e
e
T-47D
e
e
19.1
10.4
e
e
15.0
14.1
e
a
e, GI < 10%; nt, not tested.

F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
39
Fig. 1. The binding mode and residues involved in the recognition for COQ, the most active compound 39 (IC50 0.3
mM) and the most inactive compound 43 (IC50 12.0
m
M) docked
and minimized in the DHFR binding pocket.
strain energy and the best alignment score is shown in Fig. 3. The
most active compounds 25 and 39 align completely (Fig. 3a), also
the least active compounds 29, 42, and 43 align perfectly (Fig. 3b);
while compounds 39, 42 and 43 showed a dramatic deviation from
alignment (Fig. 3c). Examination of the flexible alignment results of
the active versus the inactive DHFR inhibitors in this study revealed
that the inactive compounds 42 and 43 were aligned in a distinct
different pattern from that of the most active compound 39 (Fig. 3c).
Flexible alignment projected the importance of the 2-aminothiazole
moiety of 39 as a five membered ring bearing basic center for ac-
tivity; and the pyridine-2-carboxylic acid (28e33) or 4-nitrobenzoic
acid (42e47) functions as a six membered ring bearing acidic center
for the diminished potency. This evidence is supported by the DHFR
inhibition data displayed in Tables 2 and 3.
points such as N-1 of the quinazoline nucleus, the thiazole ring, the
3-phenyl fragment, 2-S-linkage directly attached to quinazoline
moiety, 2-amino group at the thiazole function and the
p-system
represented by aryl group of the quinazoline core, align fairly well
with almost complete superposition of the quinazoline rings
ꢀ
(0.4 A). This superposition suggests the possible existence of a re-
gion on the receptor suited for specific recognition of such features.
This hypothesis is supported by biological results which strongly
suggest that these seven chosen mark points could be used as
a pharmacophore model for further optimization.
As an attempt to investigate the reasons behind the diminished
DHFR inhibition activity of 29, 42 and 43, electrostatic mapping was
carried out for the lowest energy conformer of compound 43, to
examine the similarity and dissimilarity in the electronic and
electrostatic binding characteristics of the molecule surface and the
conformational properties, in comparison to the most active com-
pound 39. Fig. 5 showed some common features of 39, which are
negative charged hydrogen bond acceptoredonor region located on
the 4-carbonyl and N-1; a non-polar area located on the aryl moiety
attached to the quinazoline core distributed on both sides of the
aryl parts; and hydrophilic region located on 4-carbonyl, N-1 of the
quinazoline core. Such results indicated the structure and hence
biological similarity among those active analogs. On the contrary,
the low activity of the other compounds, represented by 43 may be
attributed to the difference in electrostatic mapping in which the
hydrogen bond area was mainly located on the carboxylic group.
Similarly, hydrophobic surface mapping study of the most active
compound 39 (Fig. 6a) pointed out the hydrophobic region which is
responsible for the interaction with the key amino acid residues
inside the enzyme active pocket. On the other hand, the hydro-
phobic distributions of the least active compound 43 (Fig. 6b) lack
such hydrophobic moieties and hence the required lipophilicity,
instead a hydrophilic region was located on the carboxylic acid
fragment. The obtained charge distribution, electrostatic, hydro-
phobic mapping and conformation of the active agents suggest
a distinct different interaction of the active and inactive molecules
with the potential protein-binding site.
Atomic-level details of the structure of pharmaceutically rele-
vant receptors are not available; in such cases, 3D superposition of
putative ligands can be used to deduce specific structural re-
quirements for biological activity [47,48]. Structure superposition
of the most active compounds 21, 25 and 39 (Fig. 4) with mark
Fig. 2. The aligned conformation of compound 39 (IC50 0.3 mM) in the DHFR binding
pocket.

40
F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
Fig. 3. (a) Flexible alignment of the most active compounds 25 (blue), and 39 (red). (b) Flexible alignment of the least active compounds 29 (yellow), 42 (violet), and 43 (cyan). (c)
Flexible alignment of the most active compound 39 (pink) against the least active compounds 42 (yellow) and 43 (cyan). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
4. Conclusion
positions 2-, 3- and 6- of the studied quinazolin-4-ones manipulate
the DHFR inhibition activity. In general, the 2-thiazolethio-sub-
stituent on the quinazoline nucleus contributed to the DHFR inhi-
bition activity more than the 2-phenylthio- or the 2-pyridinethio-
moieties; also 3-phenyl-, and 6,7-dimethoxy substituent favor the
activity rather than 3-benzyl-, 6-methyl or 6-chloro functions.
Compounds 25, 28, 33, 35, and 36 showed a remarkable broad-
spectrum activity against both Gram-positive and Gram-negative
bacteria. Compounds 25, 35 and 38 might exert their activity
through DHFR inhibition. Meanwhile, compound 29 showed broad
spectrum potency toward several tumor cell lines with GI values
range of 25.8e41.2%. Molecular modeling study was performed and
concluded that recognition with key amino acid Arg38 and Lys31 is
essential for binding and biological activities of the investigated
quinazolines. Flexible alignment; electrostatic and hydrophobic
mappings were also performed. This model justifies the importance
of the main pharmacophoric groups (the 4-carbonyl fragment, the
Compounds 21, 22, 25, 35, 38, and 39 proved to be the most
active DHFR inhibitors with IC50 range of 0.3e1.0
activity relationship studies revealed that, the type of substituent at
mM. Structure
basic nitrogen atom at N-1, and the hydrophobic
p-system regions).
The substitution pattern and spatial considerations of the
p-sys-
tems in regard to the quinazoline nucleus proved to be critical for
biological activity. Therefore, the obtained model could be useful
for the development of new DHFR inhibitors.
5. Experimental part
Melting points (ꢀC) were determined on Mettler FP80 melting
point apparatus and are uncorrected. Microanalyses were per-
formed on a PerkineElmer 240 elemental analyzer at the Central
Research Laboratory, College of Pharmacy, King Saud University. All
of the new compounds were analyzed for C, H and N and agreed
Fig. 4. Electrostatic and hydrophobic map of the lowest energy conformers for the
most active compound 39 and the least active compound 43, maps are color coded: red
for a hydrogen bond and a hydrophilic region, and green for a hydrophobic region. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
41
Fig. 5. Electrostatic maps of the lowest energy conformers for the most active compound 39 and the least active compound 43. Red for a hydrogen bond and a hydrophilic region,
and green for a hydrophobic region. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
with the proposed structures within ꢁ0.4% of the theoretical
values. 1H, 13C NMR spectra were recorded on a Bruker 500 MHz FT
spectrometer (the Central Research Laboratory, College of Phar-
anhydrous potassium carbonate (1.5 g, 0.01 mol) in DMF (10 ml)
was heated under reflux for 14 h. Solvent was then removed under
reduced pressure. The obtained residue was dissolved in CH2Cl2,
then washed the organic layer several times with water, and finally
dried. The obtained solid was recrystallized from ethanol to obtain
macy, King Saud University); chemical shifts are expressed in d ppm
with reference to TMS. Mass spectral (MS) data were obtained on
a Perkin Elmer, Clarus 600 GC/MS and Joel JMS-AX 500 mass
spectrometers. Thin layer chromatography was performed on
precoated (0.25 mm) silica gel GF254 plates (E. Merck, Germany),
compounds were detected with 254 nm UV lamp. Silica gel (60e
230 mesh) was employed for routine column chromatography
separations. DHFR inhibition activity experiments were performed
at Pharmacology Department, College of Pharmacy, King Saud
University. Bovine liver DHFR enzyme, methotrexate (MIX) was
used in the assay (Sigma Chemical Co, USA). The in vitro anti-
microbial testing was performed at Department of Microbiology,
Faculty of Pharmacy, Mansoura University, Egypt. The agar disc-
diffusion method and a panel of standard strains (S. aureus IFO
3060, B. subtilis IFO 3007, M. luteus IFO 3232, E. coli IFO 3301, and P.
aeruginosa IFO 3448) were employed. In vitro antitumor activity
was conducted at the NCI, Bethesda, MD. All modeling experiments
were conducted with Hyperchem 8.0.5 package from Hypercube
running on a PC computer. The docking of the candidates into DHFR
pocket was performed with MOE software. Starting coordinate of
DHFR enzyme in tertiary complex with reduced-nicotinamide
adenine dinucleotide phosphate (NADPH) and COQ, code ID 1LY4,
was obtained from the Protein Data Bank. Compounds 14e19 were
previously reported [24e26].
compounds 21e24, (Table 1). 21: 1H NMR (DMSO-d6)
d 2.26 (s, 3H,
CH3), 3.77 (brs, 2H, NH2), 7.00 (d, 1H, J ¼ 8 Hz, AreH), 7.14 (d, 2H,
J ¼ 8.5 Hz, AreH), 7.39 (t,1H, J ¼ 7.5 Hz, AreH), 7.46 (t, 2H, J ¼ 7.5 Hz,
AreH), 7.53 (d, 1H, J ¼ 7 Hz, AreH), 7.79 (s, 1H, AreH), 7.96 (s, 1H,
thiazoleeH). 13C NMR
d 20.2, 115.2, 115.7, 116.0, 127.0, 128.8, 129.0,
133.9,135.8,136.6,137.6,139.4,150.1,159.8,162.2,175.5. MS m/z (%):
367 (2.5, Mþ). 22: 1H NMR (DMSO-d6)
d
2.34 (s, 3H, CH3), 3.45 (brs,
2H, NH2), 5.09 (s 2H, benzyliceH), 7.11 (d,1H, J ¼ 8.5 Hz, AreH), 7.23
(d, 2H, J ¼ 4.5 Hz, AreH), 7.31 (t, 1H, J ¼ 4 Hz, AreH), 7.50 (t, 2H,
J ¼ 7 Hz, AreH), 7.61 (d, 1H, J ¼ 7.5 Hz, AreH), 7.77 (s, 1H, AreH),
7.99 (s, 1H, thiazoleeH). 13C NMR
d 20.4, 43.1, 113.5, 115.2, 115.7,
126.7, 127.4, 128.2, 131.9, 134.2, 136.2, 136.7, 137.3, 137.4, 150.1, 161.9,
175.0. MS m/z (%): 380 (1.2, Mþ). 23: 1H NMR (DMSO-d6)
d
7.28 (t,
2H, J ¼ 8 Hz, AreH), 7.33 (t, 1H, J ¼ 7 Hz, AreH), 7.42 (d, 2H,
J ¼ 7.5 Hz, AreH), 7.49 (d, 2H, J ¼ 2.5 Hz, AreH), 7.83 (d, 1H,
J ¼ 2.5 Hz, AreH), 7.89 (s, 1H, thiazoleeH), 13.15 (s, 2H, NH2). 13
C
NMR d 115.8,117.7,117.9,126.4,128.1,128.9,134.9,135.5,138.4,139.1,
149.9, 158.8, 161.2, 176.1, 206.3. MS m/z (%): 386 (24.3, Mþ). 24: 1
NMR (DMSO-d6)
H
d
5.10 (s, 2H, benzyliceH), 7.19 (d, 1H, J ¼ 7.5 Hz,
AreH), 7.26 (t, 2H, J ¼ 8 Hz, AreH), 7.34 (t,1H, J ¼ 7.5 Hz, AreH), 7.48
(d, 2H, J ¼ 8.5 Hz, AreH), 7.59 (d, 1H, J ¼ 7 Hz, AreH), 8.63 (s, 1H,
thiazoleeH), 8.72 (s, 1H, AreH). 13.53 (s, 2H, NH2). 13C NMR
d 113.9,
116.7, 117.3, 123.7, 127.5, 128.3, 135.9, 136.8, 142.0, 143.0, 143.1, 144.3,
149.9, 158.6, 160.9, 176.5. MS m/z (%): 400 (1.6, Mþ).
5.1. Chemistry
5.1.2. 2-(2-Aminothiazol-5-ylthio)-6,7-dimethoxy-3-phenyl or
benzyl-quinazolin-4(3H)-ones (25, 26)
5.1.1. 2-(2-Aminothiazol-5-ylthio)-3-phenyl or benzyl-6-
substituted-quinazolin-4(3H)-ones (21e24)
A mixture of 2-amino-5-bromothiazole (20, 1.79 g, 0.01 mol),
the appropriate 2-thioxo-quinazoline analogs (18 or 19, 0.01 mol),
A mixture of 2-amino-5-bromothiazole (20, 1.79 g, 0.01 mol),
the appropriate 2-thioxo-quinazoline analogs (14e17, 0.01 mol),
Fig. 6. (a) Surface map for the most active compound 39 in pocket side. (b) Surface map for the least active compound 43. Pink hydrogen bond, blue mild polar, green hydrophobic.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

42
F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
anhydrous potassium carbonate (1.5 g, 0.01 mol) in DMF (10 ml)
was heated under reflux for 14 h. Solvent was then removed
under reduced pressure and continued as mentioned under
5.1.5. Ethyl 2-amino-5-[(3-phenyl or benzyl)-6-substituted-4-oxo-
3,4-dihydroquinazolin-2-ylthio)]thiazole-4-carboxylate
A mixture of 2-amino-5-bromothiazole-4-carboxylate (34, 2.22 g,
0.01 mol), the appropriate 2-thioxo-quinazoline analogs (14e17,
0.01 mol), anhydrous potassium carbonate (1.5 g, 0.01 mol) in DMF
(10 ml) was heated under reflux for 14e16 h. Solvent was then
removed under reduced pressure and continued as mentioned
compounds 21e24 (Table 1). 25: 1H NMR (DMSO-d6)
d 2.89 (brs,
2H, NH2), 4.35 (s, 6H, OCH3), 7.31 (d, 2H, J ¼ 8 Hz, AreH), 7.42 (t,
1H, J ¼ 7.5 Hz, AreH), 7.51 (t, 2H, J ¼ 8 Hz, AreH), 7.96 (s, 1H,
thiazoleeH), 8.52 (s, 2H, AreH). 13C NMR
d 55.2, 55.9, 114.6, 116.5,
117.1, 123.5, 128.4, 129.1, 135.1, 138.8, 141.9, 142.8, 143.5, 144.6,
under compounds 21e24 (Table 1). 35: 1H NMR (DMSO-d6)
d 1.12
149.9, 161.1, 177.1. MS m/z (%): 412 (3.0, Mþ). 26: 1H NMR (DMSO-
(s, 3H, CH3), 1.29 (t, 3H, J ¼ 13.5 Hz, CH2CH3), 3.22e3.25 (q, 2H,
CH2CH3), 4.28 (s, 2H, NH2), 7.35 (d, 2H, J ¼ 8 Hz, AreH), 7.44 (s, 1H,
AreH), 7.62 (t, 3H, J ¼ 7.5 Hz, AreH), 7.99 (d, 2H, J ¼ 8.5 Hz, AreH). MS
d6)
H), 6.74 (s, 2H, AreH), 7.04e7.39 (m, 5H, AreH), 7.96 (s, 1H,
thiazoleeH). 13C NMR
34.1, 46.5, 55.7, 56, 105.6, 105.9, 106.9,
d 3.42 (brs, 2H, NH2), 3.85 (s, 6H, OCH3), 5.19 (s, 2H, benzylice
d
m/z (%): 438 (2.1, Mþ). 36: 1H NMR (DMSO-d6)
d 1.15 (s, 3H, CH3),1.32
111.4, 126.7, 127.3, 127.5, 128.1, 135.9, 143.3, 155.0, 155.5, 161.4,
(t, 3H, J ¼ 14 Hz, CH2CH3), 3.20e3.24 (q, 2H, CH2CH3), 5.49 (s, 2H,
benzyliceH), 7.24 (s,1H, AreH), 7.39 (d, 2H, J ¼ 8.5 Hz, AreH), 7.58 (t,
3H, J ¼ 8 Hz, AreH), 8.22 (d, 2H, J ¼ 7.5 Hz, AreH), 13.15 (s, 2H, NH2).
177.2. MS m/z (%): 426 (5.1, Mþ).
5.1.3. 2-(3-Phenyl or benzyl-6-substituted-4-oxo-3,4-dihydro
quinazoline-2-ylthio)nicotinic acid derivatives (28e31)
MS m/z (%): 452 (1.2, Mþ). 37: 1H NMR (DMSO-d6)
d 1.27 (t, 3H,
J ¼ 14.5 Hz, CH2CH3), 3.12e3.14 (q, 2H, CH2CH3), 7.28 (d, 2H, J ¼ 7.5 Hz,
AreH), 7.57 (t, 2H, J ¼ 8 Hz, AreH), 7.75 (d, 2H, J ¼ 2 Hz, AreH), 7.85
(d,1H, J ¼ 2 Hz, AreH), 8.14 (d,1H, J ¼ 2 Hz, AreH),13.16 (s, 2H, NH2).
A mixture of 2-chloronicotinic acid (27, 1.57 g, 0.01 mol), the
appropriate 2-thioxo-quinazoline analogs (14e17, 0.01 mol),
anhydrous potassium carbonate (1.5 g, 0.01 mol) in DMF (10 ml)
was heated under reflux for 24 h. Solvent was then removed under
reduced pressure and continued as mentioned under compounds
13C NMR
d 117.4, 117.9, 125.4, 126.4, 127.4, 128.8, 129.3, 129.5, 129.6,
131.7, 134.7, 135.1, 135.5, 138.4, 139.1, 146.5, 158.9, 176.1. MS m/z (%):
459 (0.9, Mþ). 38: 1H NMR (DMSO-d6)
d
1.33 (t, 3H, J ¼ 14.5 Hz,
21e24 (Table 1). 28: 1H NMR (DMSO-d6)
d
2.85 (s, 3H, CH3), 7.15 (d,
CH2CH3), 3.23e3.24 (q, 2H, CH2CH3), 5.21 (s, 2H, NH2), 5.65 (s, 2H,
benzyliceH), 7.24 (t, 1H, J ¼ 7 Hz, AreH), 7.30e7.34 (m, 4H, AreH),
7.45 (d, 1H, J ¼ 9 Hz, AreH), 7.83 (d, 1H, J ¼ 11 Hz, AreH), 7.91 (s,
1H, J ¼ 8 Hz, AreH), 7.26 (d, 2H, J ¼ 7.5 Hz, AreH), 7.31 (d, 1H,
J ¼ 7 Hz, AreH), 7.36 (d, 1H, J ¼ 8 Hz, AreH), 7.41 (d, 1H, J ¼ 7.5 Hz,
AreH), 7.48 (t, 2H, J ¼ 7.5 Hz, AreH), 7.52 (d, 1H, J ¼ 7 Hz, AreH),
7.62 (d, 1H, J ¼ 7 Hz, AreH), 7.76 (s, 1H, AreH), 13.0 (s, 1H, COOH).
1H, AreH).13C NMR
d 48.8,116.8,118.0,126.3,126.7,127.0,127.1,127.5,
128.2,128.4,130.4,131.0, 135.6,136.3,138.0, 141.5,158.5,160.8,175.5.
13C NMR
d
20.4, 115.7, 116.1, 126.7, 128.1, 128.7, 128.9, 129.0, 133.9,
MS m/z (%): 473 (2.5, Mþ).
136.6, 137.6, 137.6, 139.4, 141.2, 143.2, 144.5, 159.8, 161.3, 175.5. MS
m/z (%): 389 (0.2, Mþ). 29: 1H NMR (DMSO-d6)
d
2.37 (s, 3H, CH3),
5.1.6. Ethyl 2-amino-5-[(3-phenyl or benzyl)-6-substituted-4-oxo-
3,4-dihydroquinazolin-2-ylthio]thiazole-4-carboxylate
5.68 (s, 2H, benzyliceH), 7.11 (s, 1H, AreH), 7.24 (d, 2H, J ¼ 6 Hz,
AreH), 7.31e7.35 (m, 5H, AreH), 7.50 (d, 1H, J ¼ 7.5 Hz, AreH), 7.60
(d, 1H, J ¼ 7.5 Hz, AreH), 7.76 (t, 1H, J ¼ 8.5 Hz, AreH), 13.01 (s, 1H,
A
mixture of 2-amino-5-bromothiazole-4-carboxylate (34,
2.22 g, 0.01 mol), the appropriate 2-thioxo-quinazoline analogs (18
or 19, 0.01 mol), anhydrous potassium carbonate (1.5 g, 0.01 mol) in
DMF (10 ml) was heated under reflux for 14e16 h. Solvent was then
removed under reduced pressure and continued as mentioned
COOH). 13C NMR
d 20.4, 62.9, 115.3, 115.7, 126.7, 126.9, 127.1, 127.4,
128.2, 128.3, 129.4, 129.9, 131.5, 134.2, 136.6, 136.7, 137.1, 156.4,
162.5, 175.0. MS m/z (%): 403 (1.0, Mþ). 30: 1H NMR (DMSO-d6)
d
7.28 (d, 2H, J ¼ 7 Hz, AreH), 7.34 (d, 2H, J ¼ Hz, AreH), 7.43 (d, 2H,
under compounds 21e24 (Table 1). 39: 1H NMR (DMSO-d6)
d 1.54
J ¼ 7.5 Hz, AreH), 7.49 (t, 3H, J ¼ 10 Hz, AreH), 7.84 (d, 1H,
J ¼ 2.5 Hz, AreH), 7.89 (d, 1H, J ¼ 2 Hz, AreH), 13.15 (s, 1H, COOH).
(t, 3H, J ¼ 14 Hz, CH2CH3), 3.15 (s, 6H, OCH3), 3.52e3.59 (q, 2H,
CH2CH3), 7.41 (s, 2H, AreH), 7.49 (t,1H, J ¼ 7.5 Hz, AreH), 7.52 (t, 2H,
J ¼ 7 Hz, AreH), 7.84 (d, 2H, J ¼ 8.5 Hz, AreH), 12.89 (s, 2H, NH2). MS
13C NMR
d 117.4, 117.7, 117.9, 126.2, 126.4, 128.1, 128.7, 128.8, 128.9,
134.9, 135.4, 138.4, 138.7, 139.1, 149.9, 158.8, 161.2, 176.1. MS m/z
m/z (%): 484 (12, Mþ). 40: 1H NMR (DMSO-d6)
d 1.42 (t, 3H,
(%): 410 (2.2, Mþ). 31: 1H NMR (DMSO-d6)
d
5.66 (s, 2H, benzylice
J ¼ 13.5 Hz, CH2CH3), 3.24 (s, 6H, OCH3), 3.45e3.50 (q, 2H, CH2CH3),
5.52 (s, 2H, benzyliceH), 7.41 (t, 3H, J ¼ 8 Hz, AreH), 7.50 (s, 2H, Are
H), 7.67 (d, 2H, J ¼ 7.5 Hz, AreH), 11.32 (s, 2H, NH2). MS m/z (%): 498
(25, Mþ).
H), 7.24 (t, 2H, J ¼ 14 Hz, AreH), 7.25e7.28 (m, 4H, AreH), 7.44 (d,
2H, J ¼ 8.5 Hz, AreH), 7.81 (d, 1H, J ¼ 2.5 Hz, AreH), 7.83 (d, 1H,
J ¼ 2 Hz, AreH), 7.91 (s, 1H, AreH), 13.19 (s, 1H, COOH). 13C NMR
d
116.5, 116.8, 117.5, 117.8, 118.0, 119.3, 120.1, 126.3, 126.9, 127.1,
127.5, 128.2, 128.3, 128.4, 135.6, 136.3, 138.0, 158.5, 175.5. MS m/z
5.1.7. 2-(3-Phenyl or benzyl-6-substituted-4-oxo-3,4-
dihydroquinazolin-2-ylthio)-4-nitrobenzoic acid derivatives (42e
45)
(%): 423 (9.7, Mþ).
5.1.4. 2-(6,7-Dimethoxy-3-phenyl or benzyl-4-oxo-3,4-dihydro
quinazoline-2-ylthio)nicotinic acid derivatives (32, 33)
A mixture of 2-chloro-4-nitrobenzoic acid (41, 2.01 g, 0.01 mol),
the appropriate 2-thioxo-quinazoline analogs (14e17, 0.01 mol),
anhydrous potassium carbonate (1.5 g, 0.01 mol) in DMF (10 ml) was
heated under reflux for 24e36 h. Solvent was then removed under
reduced pressure and continued as mentioned under compounds
A mixture of 2-chloronicotinic acid (27, 1.57 g, 0.01 mol), the
appropriate 2-thioxo-quinazoline analogs (18 or 19, 0.01 mol),
anhydrous potassium carbonate (1.5 g, 0.01 mol) in DMF (10 ml)
was heated under reflux for 24 h. Solvent was then removed
under reduced pressure and continued as mentioned under
21e24 (Table 1). 42: 1H NMR (DMSO-d6)
d 2.38 (s, 3H, CH3), 7.14 (d,
1H, J ¼ 8 Hz, AreH), 7.26 (d, 2H, J ¼ 7 Hz, AreH), 7.31 (d, 1H, J ¼ 7 Hz,
AreH), 7.39 (d, 1H, J ¼ 8.5 Hz, AreH), 7.42 (t, 1H, J ¼ 7.5 Hz, AreH),
7.48 (t, 2H, J ¼ 7 Hz, AreH), 7.54 (d, 1H, J ¼ 7 Hz, AreH), 7.63 (d, 1H,
compounds 21e24 (Table 1). 32: 1H NMR (DMSO-d6)
d 3.84 (s,
6H, CH3O), 6.72 (s, 1H, AreH), 6.93 (s, 1H, AreH), 7.25 (t, 1H,
J ¼ 8 Hz, AreH), 7.30e7.32 (m, 7H, AreH), 11.28 (s, 1H, COOH). MS
J ¼ 7.5 Hz, AreH), 7.76 (s, 1H, J ¼ 8 Hz, AreH), 13.0 (s, 1H, COOH). 13
C
m/z (%): 435 (0.5, Mþ). 33: 1H NMR (DMSO-d6)
d
3.83 (s, 6H,
NMR d 20.2, 62.9, 115.2, 115.7, 116.0, 126.7, 128.0, 128.7, 128.9, 129.0,
CH3O), 5.67 (s, 2H, benzyliceH), 6.71 (s, 1H, AreH), 6.97 (s, 3H,
130.2,131.0,133.9,136.6,137.6,139.4,140.6,159.8,161.2,175.5. MS m/
AreH), 7.23e7.31 (m, 6H, AreH), 12.89 (s, 1H, COOH). 13C NMR
z (%): 433 (3.1, Mþ). 43: 1H NMR (DMSO-d6)
d
2.34 (s, 3H, CH3), 5.68
d
48.5, 55.8, 56, 97.8, 106.8, 108, 126.9, 127.1, 127.4, 128.2, 128.3,
(s, 2H, benzyliceH), 7.12 (d, 1H, J ¼ 8 Hz, AreH), 7.13 (s, 2H, AreH),
7.31e7.34 (m, 4H, AreH), 7.35 (d, 1H, J ¼ 8.5 Hz, AreH), 7.51 (d, 1H,
J ¼ 7 Hz, AreH), 7.61 (d, 1H, J ¼ 7 Hz, AreH), 7.76 (d, 1H, J ¼ 9.5 Hz,
129.5, 129.9, 130.5, 131, 135.1, 136.8, 146.8, 155.4, 158.9, 174.4. MS
m/z (%): 449 (5.7, Mþ).

F.A.M. Al-Omary et al. / European Journal of Medicinal Chemistry 63 (2013) 33e45
43
AreH), 13.03 (s, 1H, COOH). 13C NMR
d
20.2, 48.6, 62.9, 113.5, 115.2,
the antibacterial antibiotic gentamicin, sulphacetamide (100
mg/
115.7, 126.7, 126.9, 127.1, 127.4, 128.2, 128.3, 131.9, 134.2, 136.2, 136.6,
disc) were carefully placed on the agar cultures plates that had been
previously inoculated separately with the microorganisms. The
plates were incubated at 37 ꢀC, and the diameter of the growth
inhibition zones were measured after 24 h. The minimal inhibitory
concentrations (MIC) for the test compounds against the same
microorganisms used in the primary screening were carried out
using the microdilution susceptibility method in Müller-Hinton
Broth [36]. Test compounds, gentamicin, and Sulphacetamide were
136.7, 137.1, 137.4, 159.4, 175. MS m/z (%): 447 (8.5, Mþ). 44: 1H NMR
(DMSO-d6)
d
7.26 (s,1H, AreH), 7.29 (d, 2H, J ¼ 7.5 Hz, AreH), 7.34 (d,
2H, J ¼ 7 Hz, AreH), 7.43 (d, 2H, J ¼ 8 Hz, AreH), 7.48e7.49 (m, 1H,
AreH), 7.75 (dd, 1H, J ¼ 2.5 Hz, 2 Hz, AreH), 7.83 (dd, 1H, J ¼ 2.5 Hz,
2 Hz, AreH), 7.88 (t,1H, J ¼ 4 Hz, AreH),13.16 (s,1H, COOH).13C NMR
d
115.8,117.4,117.9,119.0,126.4,128.1,128.7,128.8,128.9,134.9,135.4,
135.5, 138.4, 138.7, 139.0, 149.9, 158.8, 161.2, 176.1. MS m/z (%): 454
(7.1, Mþ). 45: 1H NMR (DMSO-d6)
d
5.56 (s, 2H, benzyliceH), 7.25 (d,
dissolved in dimethylsulphoxide at concentration of 64
two fold dilutions of the solution were prepared (64 till 0.5
m
g/ml. The
g/ml).
2H, J ¼ 6.5 Hz, AreH), 7.29 (t, 2H, J ¼ 7.5 Hz, AreH), 7.33 (d, 3H,
J ¼ 7 Hz, AreH), 7.44 (d,1H, J ¼ 9 Hz, AreH), 7.82 (d,1H, J ¼ 2 Hz, Are
m
The microorganism suspensions at 106 CFU/ml concentrations
(colony forming unit/ml) were inoculated to the corresponding
wells. The plates were incubated at 37 ꢀC for 24 h. The MIC values
were determined as the lowest concentration that completely
inhibited visible growth of the microorganism as detected by un-
aided eye.
H), 7.89 (d, 2H, J ¼ 2 Hz, AreH), 13.50 (s, 1H, COOH). 13C NMR
d 115.6,
115.9, 116.2, 116.8, 117.3, 117.8, 118.6, 119.4, 123.6, 124.2, 127.1, 127.2,
128.2, 130.1, 135.9, 143.0, 143.1, 158.6, 176.5, 204.3. MS m/z (%): 467
(3.3, Mþ).
5.1.8. 2-(3-Phenyl or benzyl-6,7-dimethoxy-4-oxo-3,4-
dihydroquinazolin-2-ylthio)-4-nitrobenzoic acid derivatives (46,
47)
5.4. Antitumor screening
A mixture of 2-chloro-4-nitrobenzoic acid (41, 2.01 g, 0.01 mol),
the appropriate 2-thioxo-quinazoline analogs (18 or 19, 0.01 mol),
anhydrous potassium carbonate (1.5 g, 0.01 mol) in DMF (10 ml)
was heated under reflux for 24e36 h. Solvent was then removed
under reduced pressure and continued as mentioned under
Under a sterile condition, cell lines were grown in RPMI 1640
media (Gibco, NY, USA) supplemented with 10% fetal bovine serum
(Biocell, CA, USA), 5 ꢃ 105 cell/ml was used to test the growth in-
hibition activity of the synthesized compounds. The concentrations
of the compounds ranging from 0.01 to 100 mM were prepared in
phosphate buffer saline. Each compound was initially solubilized in
dimethyl sulfoxide (DMSO), however, each final dilution contained
less than 1% DMSO. Solutions of different concentrations (0.2 ml)
were pipetted into separate well of a microtiter tray in duplicate.
Cell culture (1.8 ml) containing a cell population of 6 ꢃ 104 cells/ml
was pipetted into each well. Controls, containing only phosphate
buffer saline and DMSO at identical dilutions, were also prepared in
the same manner. These cultures were incubated in a humidified
incubator at 37 ꢀC. The incubator was supplied with 5% CO2 at-
mosphere. After 48 h, cells in each well were diluted 10 times with
saline and counted by using a coulter counter. The counts were
corrected for the dilution [30e33].
compounds 21e24 (Table 1). 46: 1H NMR (DMSO-d6)
d 2.89 (s,
6H, OCH3), 7.31 (d, 2H, J ¼ 7 Hz, AreH), 7.42 (t, 1H, J ¼ 7.5 Hz, AreH),
7.49 (t, 2H, J ¼ 8 Hz, AreH), 7.51 (d, 2H, J ¼ 8 Hz, AreH), 7.60 (d, 1H,
J ¼ 8.5 Hz, AreH), 8.52 (d, 1H, J ¼ 8 Hz, AreH), 8.66 (s, 1H, AreH),
13.48 (s, 1H, COOH). MS m/z (%): 479 (5.2, Mþ). 47: 1H NMR
(DMSO-d6) d 3.87 (s, 6H, OCH3), 5.53 (s, 2H, benzyliceH), 6.74 (s, 1H,
AreH), 7.09 (s, 1H, AreH), 7.30e7.34 (m, 1H, AreH), 7.39 (d, 2H,
J ¼ 8 Hz, AreH), 7.42 (d, 2H, J ¼ 7.5 Hz, AreH), 7.48 (d, 2H, J ¼ 8.5 Hz,
AreH), 7.56 (s, 1H, AreH), 11.36 (s, 1H, COOH). 13C NMR
d 55.7, 56,
97.6, 105.6, 106.1, 107.1, 107.7, 116.5, 116.8, 116.9, 117.8, 118.0, 118.5,
118.9, 119.4, 121.3, 127.9, 128.7, 129.1, 129.4, 129.7, 206.4. MS m/z (%):
493 (12.1, Mþ).
5.5. Docking and molecular modeling study
5.2. Dihydrofolate reductase (DHFR) inhibition assay
The three-dimensional structures the quinazoline derivatives,
which presented best and worst biological profiles, in their neutral
forms were constructed using the MOE of Chemical Computing
Group Inc software. Lowest energy conformer of each new analog
‘global-minima’ was docked into the DHFR enzyme-binding
domain. For each of the quinazoline analogs, energy minimiza-
tions (EM) were performed using 1000 steps of steepest descent,
followed by conjugate gradient minimization to a RMS energy
The assay mixture contained 50
mM TriseHCl buffer (pH 7.4),
50
m
M NADPH, 20 L DMSO or the same volume of DMSO solution
m
containing the test compounds to a final concentration of 10ꢂ11
e
10ꢂ5 M, and 0.02 units of bovine liver DHFR, in a final volume of
2.0 ml. After addition of the enzyme, the mixture was incubated
at room temperature for 2.0 min, and the reaction was initiated
by adding 25
mM FH2, the change in absorbance (DA/min) was
measured at 340 nm. The activity under these conditions was
linear for 10 min [34] Results are reported as % inhibition of
enzymatic activity calculated using the following formula:
ꢀ
gradient of 0.01 kcal/mol A. The active site of the enzyme was
ꢀ
defined using a radius of 10.0 A around COQ. These selected frames
ꢀ
were minimized until RMS deviation values of 0.01 kcal/mol A were
ꢀ
ꢁ
reached for the active-site residues. Energy of binding was calcu-
lated as the difference between the energy of the complex and
individual energies of the enzyme and ligand [43e46].
D
A=mintest
% Inhibition ¼ 1 ꢂ
ꢃ 100
D
A=minDMSO
The % inhibition values were plotted versus drug concentration
(log scale). The 50% inhibitory concentration (IC50) of each com-
pound was obtained using the Graph Pad Prism program, version 3
(San Diego, CA).
5.5.1. Conformational investigation
Initial structures for the investigated molecules were con-
structed using the HYPERCHEM program version 8.0.5. The MM
(calculations in vacuo, bond dipole option for electrostatics, Pola-
keRibiere algorithm, and RMS gradient of 0.01 kcal/mol) con-
formational searching in torsional space was performed using the
‘multiconformer method’ [49,50]. Energy minima for investigated
compounds were determined by a semi-empirical method AM156
(as implemented in HyperChem 8.0.5). The conformations thus
obtained were confirmed as minima by vibrational analysis. Atom-
5.3. Determination of in vitro antimicrobial activity
The primary screen was carried out using the agar disc-diffusion
method [35] using Müller-Hinton agar medium. Sterile filter paper
discs (8 mm diameter) were moistened with the compound solu-
tion in dimethylsulphoxide of specific concentration 200 mg/disc,

44
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Acknowledgments
This research project was supported by a grant from the
“Research Center of the Center for Female Scientific and Medical
Colleges”, Deanship of Scientific Research, King Saud University.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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