Synthesis, biological evaluation and molecular modeling study of some new methoxylated 2-benzylthio-quinazoline-4(3H)-ones as nonclassical antifolates

Article abstract of DOI:10.1016/j.bmcl.2016.08.022

A new series of 2,3,6-substituted-quinazolin-4-ones was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 28 and 61 proved to be active DHFR inhibitors with IC₅₀0.02 and

Full text of DOI:10.1016/j.bmcl.2016.08.022

Accepted Manuscript
Synthesis, biological evaluation and molecular modeling study of some new
methoxylated 2-benzylthio-quinazoline-4(3H)-ones as nonclassical antifolates
Shahenda M. El-Messery, Ghada S. Hassan, Mahmoud N. Nagi, El-Sayed E.
Habib, Sarah T. Al-Rashood, Hussein I. El-Subbagh
PII:
S0960-894X(16)30843-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.08.022
BMCL 24148
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 June 2016
1 August 2016
9 August 2016
Please cite this article as: El-Messery, S.M., Hassan, G.S., Nagi, M.N., Habib, E.E., Al-Rashood, S.T., El-Subbagh,
H.I., Synthesis, biological evaluation and molecular modeling study of some new methoxylated 2-benzylthio-
quinazoline-4(3H)-ones as nonclassical antifolates, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.08.022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, biological evaluation and molecular modeling study of some new
methoxylated 2-benzylthio-quinazoline-4(3H)-ones as nonclassical antifolates.
Shahenda M. El-Messery,a* Ghada S. Hassan,b Mahmoud N. Nagi,c El-Sayed E. Habib,d,e Sarah T.
Al-Rashood,f Hussein I. El-Subbagh.b,g*
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
P.O.Box 35516, Mansoura, Egypt.
bDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, P.O.Box 35516,
Mansoura, Egypt.
cDepartment of Pharmacology, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi
Arabia.
dDepartment of Pharmaceutics and pharmaceutical technology (Microbiology), College of
Pharmacy, Taibah University, Almadinah Almunawwarah, 344, Saudi Arabia
eDepartment of Microbiology, Faculty of Pharmacy, Mansoura University, P.O.Box 35516,
Mansoura, Egypt
fDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O.Box
2457, Riyadh 11451, Saudi Arabia.
gDepartment of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences & Pharmaceutical
Industries, Future University, 12311 Cairo, Egypt.
Abstract
A new series of 2,3,6-substituted-quinazolin-4-ones was designed, synthesized, and evaluated for
their in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 28 and 61 proved
to be active DHFR inhibitors with IC50 0.02 and 0.01 µM, respectively. Molecular modeling studies
concluded that recognition with the key amino acid Phe34 is essential for binding and hence DHFR
inhibition. Compounds 34, 56 and 66 showed broad spectrum antimicrobial activity comparable to
Gentamicin and Ciprofloxacin. Compounds 40 and 64 showed broad spectrum antitumor activity
toward several tumor cell lines and proved to be 10 fold more active than 5-FU, with GI50 MG-MID
values of 2.2 and 2.4 µM, respectively.
Keywords: Synthesis, quinazolin-4-ones, DHFR inhibition, antimicrobial testing, antitumor
screening, molecular modeling study.
Correspondence: Hussein I. El-Subbagh, Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura
University, Mansoura 35516, Egypt
E-mail: subbagh@yahoo.com.
Tel: +20-50-2247800, Fax: +20-50-2247496
Shahenda M. El-Messery Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt
E-mail: shahenda76@yahoo.com
Tel: +20-50-2247800, Fax +20-50-2247496
1

Dihydrofolate reductase (DHFR) enzyme has a crucial role in conjunction with thymidylate synthase
(TS) in the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine
monophosphate (dTMP) utilizing N5,N10-methylene-tetrahydrofolate as a cofactor. Inhibition of
DHFR or TS activity leads to “thymineless death” of the cell [1,2]. DHFR inhibition is an attractive
goal for the developing of chemotherapeutic agents against bacterial and parasitic infections as well
as cancer [3]. Previous studies conducted in our laboratories [4-22], especially what has been
reported about the DHFR inhibition [17-19], allowed the allocation of certain pharmacophoric
characteristics for the active quinazoline DHFR inhibitors. Bearing a basic nitrogen atom at N-1, a 4-
carbonyl function, and hydrophobic π-system regions and their relative spatial distances are the
major pharmacophoric requirements. The type of substitution, in addition to the spatial
considerations of the π-systems in regard to the quinazoline nucleus manipulated the DHFR
inhibition activity [17-19,22]. Compounds A-C (Figure 1) represent the active DHFR inhibitors
obtained as a result of implementing these pharmacophoric features with IC50 range of 0.3-1.0 µM.
Figure 1: Structures of some literature antifolate lead compounds.
In continuation to our previous efforts, a new series of 2,3,6-substituted-quinazolin-4-ones was
designed. The new analogues possess methoxylated 2-(arylmethylthio)- as hydrophobic π-system
regions replacing the 2-heteroaryl functions reported earlier [22], as an attempt to explore the scope
and limitations of activity of this class of compounds. In addition, 6-Chloro, 6-methyl, or 6,7-
dimethoxy functions, were introduced representing electron donating and electron withdrawing
substituents. Also, substituted phenyl or benzyl group were added to position 3- of the quinazolin-4-
one nucleus as a realization of the pharmacophoric requirements of this class of DHFR inhibitors.
Most of the function groups used in this design are known to contribute to DHFR inhibition activity
[23,24]. The aim of this study is to locate novel synthetic lead compound(s), and its in vitro testing as
DHFR inhibitors. As an application, the synthesized compounds were also tested against a panel of
2

standard strains of Gram-positive and Gram-negative bacteria; and screened for their in-vitro
antitumor activity using the NCI’s disease-oriented human cell lines assay [25-28].
The title compounds were prepared according to the synthetic strategy described in Schemes 1 and 2.
The starting materials 6-methyl- (4-9), 6-chloro- (10-15), or 6,7-dimethoxy- (16-21) -3-(phenyl, 4-
methoxyphenyl, 2-methoxyphenyl, 2,4-dimethoxyphenyl, 4-chlorophenyl or 4-methylphenyl)-
quinazolin-4(3H)-one-2-thiones and the 6-methyl-, 6-chloro- or 6,7-dimethoxy-3-(benzyl)-
quinazolin-4(3H)-one-2-thiones (58-60) were prepared adopting reported procedures [17-19,22]. The
2-thioxo- function of the starting materials 4-21 was then alkylated using 1-(chloromethyl)-4-
methoxybenzene and potassium carbonate in dimethylformamide to afford the quinazolin-4(3H)-
ones derivatives; 2-[(4-methoxybenzyl)-thio]-3-phenyl-6-methyl- (22-27), 2-[(4-methoxybenzyl)-
thio]-3-phenyl-6-chloro- (28-33) and 2-[(4-methoxybenzyl)-thio]-3-phenyl-6,7-dimethoxy- (34-39).
Compounds 4-21 were also reacted with 1-(chloromethyl)-3,4,5-trimethoxybenzene and potassium
carbonate in dimethylformamide to afford 2-[(3,4,5-trimethoxybenzyl)-thio]-3-phenyl-6-methyl- (40-
45), 2-[(3,4,5-trimethoxybenzyl)-thio]-3-phenyl-6-chloro- (46-51) and 2-[(3,4,5-tri-methoxybenzyl)-
thio]-3-phenyl-6,7-dimethoxy- (52-57) -quinazolin-4(3H)-ones. The 2-thioxo-function of the starting
materials 58-60 was then alkylated using 1-(chloromethyl)-4-methoxybenzene and potassium
carbonate in dimethylformamide to afford 2-[(4-methoxy-benzyl)-thio]-3-benzyl-6-methyl- (61), 2-
[(4-methoxy-benzyl)-thio]-3-benzyl-6-chloro- (62) and 2-[(4-methoxy-benzyl)-thio]-3-benzyl-6,7-
dimethoxy- (63) quinazolin-4(3H)-one. Compounds 58-60 were also reacted with 1-(chloromethyl)-
3,4,5-trimethoxybenzene and potassium carbonate in dimethylformamide to afford 2-[(3,4,5-
trimethoxy-benzyl)-thio]-3-benzyl-6-methyl- (64), 2-[(3,4,5-trimethoxy-benzyl)-thio]-3-benzyl-6-
chloro- (65) and 2-[(3,4,5-trimethoxy-benzyl)-thio]-3-benzyl-6,7-dimethoxy- (66) quinazolin-4(3H)-
one. Structure elucidation of the synthesized products was attained by the aid of elementary analyses,
1H & 13C NMR spectroscopy, and mass spectrometry. The obtained data were in agreement with the
desired structures. 1H NMR spectrum of 22 showed three singlet absorptions at δ 1.42, 2.96, and 5.34
ppm attributed to the methyl‚ methoxy and methylene protons, respectively; in addition to the
aromatic proton absorption which appeared in their expected range. Signals which observed at δ 37.5
and 172.6 in the 13C NMR spectrum of 40 were correlated to methyl and carbonyl carbons. Further
evidence for the formation of synthesized structures was attained by recording the mass spectra. The
mass spectrum of compound 45 showed a molecular ion peak at m/z 462, which is in conformity
with its molecular formula.
3

Scheme 1: Synthesis of the target compounds 22-57
Scheme 2: Synthesis of the target compounds 61-66
4

Table 1: Physicochemical properties and DHFR inhibition (IC50, µM) of the synthesized
compounds 22-57 and 61-66
DHFR
inhibition
Molecular
Formulaea
Yield
%
M.p.
oC
Compound
R
R1
R2
(IC50, µM)
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
61
62
63
64
65
66
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
Cl
H
58
67
58
62
73
46
56
65
75
78
62
69
55
53
58
67
79
50
67
69
55
42
59
69
54
59
56
58
50
65
70
68
63
65
77
76
49
58
58
76
72
72
170-172 C23H20N2O2S
189-191 C24H22N2O3S
123-125 C24H22N2O3S
155-157 C25H24N2O4S
148-150 C23H19ClN2O2S
132-134 C24H22N2O2S
115-117 C22H17ClN2O2S
190-192 C23H19ClN2O3S
137-139 C23H19ClN2O3S
129-131 C24H21ClN2O4S
177-179 C22H16Cl2N2O2S
202-204 C23H19ClN2O2S
178-180 C24H22N2O4S
211-213 C25H24N2O5S
244-246 C25H24N2O5S
138-140 C26H26N2O6S
166-168 C24H21ClN2O4S
199-201 C25H24N2O4S
152-154 C25H24N2O4S
140-142 C26H26N2O5S
214-216 C26H26N2O5S
115-117 C27H28N2O6S
252-254 C25H23ClN2O4S
166-168 C26H26N2O4S
138-140 C24H21ClN2O4S
0.1
1.2
0.9
0.7
0.1
1.0
0.02
0.1
0.8
1.0
3.0
4.0
4.0
0.8
0.6
0.3
6.0
2.0
7.0
10.0
1.0
3.0
0.9
1.5
0.8
3.0
0.1
4.0
2.0
3.0
0.9
1.2
0.8
3.0
0.9
1.2
0.01
1.1
0.8
1.0
4.0
2.0
4-CH3O
2-CH3O
2,4-(CH3O)2
4-Cl
4-CH3
H
Cl
Cl
Cl
Cl
4-CH3O
2-CH3O
2,4-(CH3O)2
4-Cl
4-CH3
H
Cl
CH3O CH3O
CH3O CH3O 4-CH3O
CH3O CH3O 2-CH3O
CH3O CH3O 2,4-(CH3O)2
CH3O CH3O 4-Cl
CH3O CH3O 4-CH3
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
H
4-CH3O
2-CH3O
2,4-(CH3O)2
4-Cl
4-CH3
H
4-CH3O
2-CH3O
2,4-(CH3O)2
4-Cl
4-CH3
H
94-96
C25H23ClN2O5S
188-190 C25H23ClN2O5S
167-169 C26H25ClN2O6S
188-190 C24H20Cl2N2O4S
191-193 C25H23ClN2O4S
155-157 C26H26N2O6S
195-195 C27H28N2O7S
232-234 C27H28N2O7S
211-213 C28H30N2O8S
215-217 C26H25ClN2O6S
170-172 C27H28N2O6S
110-112 C24H22N2O2S
149-151 C23H19ClN2O2S
189-191 C25H24N2O4S
128-130 C26H26N2O4S
125-127 C25H23ClN2O4S
176-178 C27H28N2O6S
Cl
CH3O CH3O
CH3O CH3O 4-CH3O
CH3O CH3O 2-CH3O
CH3O CH3O 2,4-(CH3O)2
CH3O CH3O 4-Cl
CH3O CH3O 4-CH3
H
H
CH3
Cl
-
-
-
-
-
-
CH3O CH3O
H
H
CH3
Cl
CH3O CH3O
aAnalysed for C,H,N,; results were within 0.4 % of the theoretical values for the given formulae.
5

The synthesized compounds (22-57 and 61-66) were evaluated as inhibitors of bovine liver DHFR
using reported procedure [29]. Results were reported as IC50 values (Table 1). Compounds 28, and
61 proved to be the most active DHFR inhibitors with IC50 values of 0.02 and 0.01 µM, respectively;
while compounds 22, 24-27, 29-31, 35-37, 42, 44, 46, 48, 52, 54, 56, 63 and 64 considered of
moderate activity with IC50 range of 0.1-1.0 µM; compounds 23, 39, 45, 50, 53, 57, 62 and 66
showed weak activity with IC50 range of 1.1-2.0 µM. Methotrexate (IC50 0.008 µM) was used as a
positive control. Compounds 22-57 and 61-66 were further tested for their in vitro antimicrobial
activity against a panel of standard strains of the Gram-positive bacteria (Staphylococcus aureus and
Bacillus subtilis), the Gram-negative bacteria (Escherichia coli and Pseudomonas aeuroginosa), and
the yeast-like pathogenic fungus Candida albicans. The primary screen was carried out using the
agar disc-diffusion method using Müller-Hinton agar medium [30,31]P.
Table 2: Antimicrobial activity results of the tested compounds.
Compound
S. aureus B. subtilis
E. coli
P. aeuroginosa C. albicans
22
28
18 (4.0)
14
22 (2.0)
16
16 (8.0)
-
12
-
-
-
31
34
38
46
52
56
15 (8.0)
24 (2.0)
20 (4.0)
17 (8.0)
15 (8.0)
22 (4.0)
20 (4.0)
26.5 (2.0) 25 (2.0)
35 (0.5)
Nd
18 (4.0)
26 (2.0)
18 (4.0)
20 (4.0)
19 (8.0)
26 (2.0)
22 (4.0)
-
-
-
-
-
-
12
-
-
-
-
-
Nd
Nd
21
14 (8.0)
15 (8.0)
16 (8.0)
18 (8.0)
18 (8.0)
16 (8.0)
20.8 (0.5)
38 (0.25)
Nd
16 (8.0)
12
-
19 (1.0)
36 (1.0)
Nd
66
Gentamicin
Ciprofloxacin 32 (0.5)
Clotrimazole Nd
Inhibition Zone (mm): (-) Not active (8 mm), Weak activity (8-12 mm), Moderate
activity (12-15 mm), Strong activity (> 15 mm). Solvent: DMSO (8 mm). MICs showed
in parentheses. Nd, not determined.
The results of the preliminary antimicrobial testing of the synthesized compounds are shown in Table
2. The majority of the synthesized compounds showed varying degrees of inhibition against the
tested microorganisms. Compounds 22, 28, 31, 34, 38, 46, 52, 56 and 66 proved moderate active
against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. The inhibitory
activity against the tested Gram-negative bacteria Pseudomonas aeuroginosa was lower than the
other tested microorganisms. The minimal inhibitory concentration (MIC) for the most active
compounds 22, 31, 34, 38, 46, 52, 56 and 66 was carried out using the micro-dilution susceptibility
method in Müller-Hinton Broth; as shown in Table 2. It could be concluded that the Gram-positive
bacteria Bacillus subtilis and to a lesser extent Staphylococcus aureus beside the Gram-negative
bacteria Escherichia coli are sensitive to the tested compounds. In the present study, compounds 34,
6

56 and 66 proved to be the most active broad spectrum antimicrobial agents against the used strains.
Compounds 56 (IC50 0.9 µM) and 66 (IC50 2.0µM) might exert their antimicrobial activity through
DHFR inhibition.
Compounds 22-57 and 61-66 were also subjected to the National Cancer Institute (NCI) in vitro
antitumor activity. A single arbitrary dose (10 µM) of the test compounds were used in the full NCI
60 cell lines panel assay which includes nine tumor subpanels. The most active members of this
study, compounds 40, 41, 43, 48, and 64-66 proved lethal to numerous cancer cell lines and passed
the primary anticancer assay (Table 3). Consequently, those active compounds were carried over and
tested against tumor cell lines at a 5-log dose range [25-28]. Three response parameters, GI50, TGI,
and LC50 were monitored for each cell line, using the known drug 5-Fluorouracil (5-FU) as a positive
control. Compounds 40, 41, 43, 48, and 64-66 proved to be 2-10 fold more active than 5-FU, with
GI50 MG-MID values of 2.2, 10.7, 4.3, 3.8, 2.4, 3.5 and 2.6 µM, respectively; detailed results are
shown in Table 4. Compounds 64 (IC50 1.0 µM) may exert its activity through DHFR inhibition.
Three groups of compounds synthesized differ in the type of the substituent at position 6- of the used
quinazolines, namely, 6-methyl- (22-27, 40-45, 61 and 64), 6-chloro- (28-33, 46-51, 62 and 65), or
6,7-dimethoxy- (34-39, 52-57, 63 and 66) derivatives. The 2-thioether function affects the
magnitude of DHFR inhibition. In the 3-phenyl series, the 2-(4-methoxybenzyl)thio- function with
order of activity 6-methyl- > 6-chloro- > 6,7-dimethoxy- (22-39) contributed to the DHFR inhibition
activity more than 2-(3,4,5-trimethoxybenzyl)thio- group with order of activity 6-chloro- > 6,7-
dimethoxy- > 6-methyl- (40-57). In the 3-benzyl series, the 2-(4-methoxy-benzyl)thio- function (61-
63) contributed to the DHFR inhibition activity more than 2-(3,4,5-trimethoxybenzyl)thio- group
(64-66). In both groups, the order of activity was 6-methyl- > 6,7-dimethoxy- > 6-chloro-. In the 6-
methyl series, the combination of 3-phenyl- and 2-(4-methoxy-benzyl)thio- produced 22 with IC50
0.1 µM; the introduction of 4-methoxy- or 4-methyl- groups to the 3-phenyl- function of 22
produced compounds with a pronounced decrease in activity; while the introduction of 4-chlorine
atom preserved the DHFR inhibition potency of 22. Replacing of the 2-(4-methoxy-benzyl)thio-
moiety of 22 by 2-(3,4,5-trimethoxybenzyl)thio- function produced 40-45 with a remarkable
decrease in potency. Replacing the 3-phenyl- of 22 by 3-benzyl- group produced one of the most
active members of this study, compound 61 (IC50 0.01 µM) with 10 fold increase in activity. In the 6-
chloro series, the presence of 3-phenyl- and 2-(4-methoxy-benzyl)thio- combination produced 28
with IC50 0.02 µM. The 6,7-dimethoxy series, showed the least active members of this investigation.
7

Table 3: Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10
µM concentration of compounds 37, 40, 41, 43, 46-48, 54, 64-66
% Growth Inhibition (GI %)a
Subpanel tumor cell lines
37
40
41
43
46
47
48
54
64
65
66
Leukemia
HL-60(TB)
K-562
-
87.8
89.6
78.2
45.1
79.7
68.9
83.1
44.0
-
L
-
-
L
L
83.0
86.2
69.2
46.0
82.0
85.4
87.2
70.6
31.3
76.9
88.1
89.8
83.3
45.3
93.8
12.9
20.6
36.3
40.6
85.6
88.7
L
61.8
30.3
-
28.6
27.6
33.8
-
87.3
84.7
82.6
84.0
72.7
53.2
28.8
76.2
MOLT-4
RPMI-8226
SR
77.0
92.3
54.4
Non-small cell lung cancer
A549/ATCC
HOP-62
19.2
-
60.7
62.2
32.1
78.0
52.6
79.8
91.7
49.0
34.8
-
30.1
19.7
26.2
60.7
81.1
52.6
77.3
82.2
72.0
95.4
-
26.1
28.0
11.1
21.0
14.3
-
20.2
-
68.9
48.2
57.1
63.9
44.9
89.2
-
12.0
-
60.0
60.0
43.4
78.7
53.8
82.3
L
57.8
62.3
28.7
74.9
52.5
79.5
L
63.2
58.6
37.4
79.6
54.6
77.7
L
HOP-92
NCI-H23
24.5
21.7
19.4
-
42.9
20.2
-
34.2
20.1
14.9
16.6
-
NCI-322M
NCI-H460
NCI-H522
10.3
16.7
31.4
61.6
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
-
L
67.3
24.3
65.1
66.4
78.1
64.7
50.7
L
15.5
20.0
38.2
47.5
33.0
50.2
20.3
-
72.0
43.2
78.9
72.2
92.3
83.6
83.2
-
L
L
92.6
59.2
85.4
79.9
89.4
84.6
66.6
-
50.3
73.4
69.9
85.7
74.7
70.3
54.5
81.9
76.4
88.8
81.9
27.9
-
-
57.0
74.9
70.8
89.5
74.7
74.2
50.0
79.2
75.1
88.3
76.4
71.4
37.2
19.4
12.0
21.9
-
33.3
19.3
17.0
18.7
-
12.0
24.6
33.4
45.9
23.6
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-75
U251
13.7
10.4
52.6
67.2
96.5
L
19.4
48.6
22.3
55.6
30.9
55.9
93.7
80.0
70.4
75.2
22.3
15.8
21.1
24.3
11.4
15.2
16.7
12.7
-
58.4
68.2
35.5
L
30.1
26.2
10.1
37.2
13.2
48.5
69.0
L
59.1
71.1
94.2
L
62.2
80.3
L
-
-
-
L
67.0
L
71.0
61.5
-
74.5
67.2
Melanoma
LOX IMVI
MALME-3M
M14
16.6
17.4
-
64.0
57.6
82.8
L
37.0
40.4
46.2
L
68.4
74.5
88.8
L
20.7
23.5
-
18.0
-
66.8
67.1
84.1
L
22.7
37.0
23.0
100
-
60.2
70.0
64.4
L
60.7
61.2
64.7
98.2
78.6
56.3
74.4
53.5
84.8
62.3
78.1
85.2
L
-
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
38.0
-
75.6
22.5
-
12.4
-
85.3
47.9
80.0
49.8
79.2
35.3
26.0
45.2
22.7
43.3
-
-
99.1
48.0
82.2
52.6
78.6
L
-
59.2
82.4
-
-
50.7
72.3
-
21.1
-
60.8
L
19.5
-
18.8
10.3
35.9
15.1
11.2
33.9
-
27.5
36.7
76.6
19.8
75.6
65.7
Ovarian cancer
IGORV1
14.0
-
66.8
L
38.0
40.1
32.8
19.4
15.8
54.2
26.3
64.3
L
27.5
22.2
18.3
15.4
15.7
24.5
-
16.3
-
52.3
99.5
37.0
46.7
44.7
79.8
40.6
16.9
51.8
16.9
11.0
15.4
37.1
-
75.7
L
64.7
L
60.8
93.8
55.8
46.3
-
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
16.8
-
42.8
53.6
47.2
82.2
86.4
42.6
56.8
54.5
98.6
54.7
28.3
10.5
13.7
10.7
-
46.9
54.7
64.9
87.3
76.1
48.0
53.3
47.1
86.1
75.7
10.8
20.5
-
L
70.2
Renal cancer
786-0
A498
-
62.4
84.0
55.3
72.4
85.8
63.0
75.9
15.9
62.0
33.3
45.8
47.2
17.8
52.8
70.4
L
10.7
18.3
21.1
26.1
31.1
24.0
52.2
-
49.0
95.0
55.8
57.4
38.5
29.7
57.4
-
53.2
93.0
53.3
71.3
L
56.6
76.0
59.7
73.3
86.9
61.5
72.9
63.3
L
41.4
-
49.1
16.4
33.0
25.9
18.3
52.1
74.6
11.5
57.1
22.0
-
ACHN
55.1
86.7
80.4
54.8
81.8
60.8
74.3
95.5
58.8
65.3
CAKI-1
RXF 393
SN12C
UO-31
31.3
24.5
-
60.0
68.0
44.6
63.7
Prostate cancer
PC-3
44.4
51.0
25.0
91.3
26.8
37.7
80.4
38.9
54.1
44.9
51.2
Breast cancer
MCF7
16.9
24.6
13.3
-
80.1
62.1
71.8
80.4
71.8
L
75.2
40.3
22.3
28.6
32.2
53.2
84.6
55.2
93.3
67.9
66.9
83.0
23.1
37.0
-
15.6
36.8
13.9
-
84.4
44.3
60.4
60.5
56.1
84.4
23.0
22.6
39.2
12.5
11.4
23.0
82.0
55.6
79.3
65.2
65.9
L
85.7
61.2
69.5
78.3
66.1
L
89.9
64.0
73.3
83.2
64.1
L
MDA-MB-231/ATCC
HS 578T
BT-549
-
T-47D
MDA-MB-468
24.4
25.7
29.1
42.7
23.9
13.4
a -, GI ˂ 10%; nt, not tested; L, compound proved lethal to the cancer cell line.
8

Table 4: Median growth (GI50) and total growth (TGI) inhibitory concentrations, (µM) of
in vitro subpanel tumor cell lines
Subpanel tumor cell linesa
Compound
40
MG-MIDb
Activity
I
II
III
IV
V
VI
VII VIII IX
GI50
TGI
LC50
1.8
c
3.0
1.3
5.4
3.9
4.0
87.0 86.3 60.9 82.2 84.6 76.9
4.2
3.4
b
2.3
84.3
c
2.2
6.2
c
c
c
c
c
c
c
c
c
41
GI50
TGI
LC50
3.4
90.2
c
34.5 18.2 30.9 19.7 46.9 39.7 52.8 5.3
10.7
89.1
c
c
c
c
c
c
c
89.4
c
b
c
88.1
c
c
c
91.4
c
43
GI50
TGI
LC50
2.5
17.7 4.4
9.6
4.2
56.2 96.5 87.4 84.6 77.4 86.7 92.5
5.1
15.4 3.7
4.1
84.9
c
4.3
66.1
95.5
c
c
c
c
c
c
89.9
c
c
48
GI50
TGI
LC50
2.4
86.5
c
5.8
c
c
3.1
c
c
7.9
4.2
6.7
63.1 91.7 87.9 93.5
10.6 5.9
3.5
84.8
c
3.8
83.2
c
c
c
c
c
c
c
64
GI50
TGI
LC50
2.4
4.0
1.6
3.5
3.2
3.3
4.3
2.9
2.8
85.2 65.9 76.1 46.7 58.4 57.6 61.4 60.2 47.5
2.4
40.7
93.3
c
98.2 87.1 81.9
c
92.7 97.9
c
c
65
GI50
TGI
LC50
3.0
5.5 3.1 4.2
4.4
5.2 4.4
87.9 66.5 86.3 49.8 55.2 75.6 52.3
4.0
c
c
3.3
59.1
c
3.5
47.9
93.3
c
97.6 87.5 81.8 98.9
c
96.5
66
GI50
TGI
LC50
1.9
c
c
3.6 2.2 3.4 2.6
6.4
4.7
63.1 87.8 24.5 45.9 62.7 49.1
4.0
c
c
2.5
47.4
99.3
2.6
38.9
87.1
94.5
c
80.6 78.7
c
85.7
5-FU
GI50
TGI
LC50
15.1
c
c
c
c
c
8.4
c
72.1 70.6 61.4 45.6 22.7 76.4
22.6
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
aI, leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian
c
cancer; VII, renal cancer; VIII, prostate cancer; IX, breast cancer. bFull panel mean-graph midpoint (µM),
Compounds showed values > 100 µM.
The pattern of structure-activity variation in the 6-chloro- and 6,7-dimethoxy- derivatives is more or
less similar to what has been noticed in the 6-methyl- series. 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. The antitumor activity was confined to the 2-(3,4,5-trimethoxy-benzyl)thio- rather
than the 2-(4-methoxybenzyl)thio- analogues. In the 3-phenyl series, the 6-methyl- derivative 40
proved to be the most active member with GI50 value of 2.2 µM. The introduction of 2- or 4-
methoxy- group to the 3-phenyl function of 40 decreased the antitumor potency as shown in 41 and
43 with GI50 values of 10.7 and 4.3 µM, respectively; replacing the 6-methyl- by 6-chloro- regained
some of the potency (48, GI50 3.8 µM). In the 3-benzyl series, the 6-methyl derivative 64 exhibited
GI50 value of 2.4 µM. Replacing the 6-methyl- of 64 by 6-chloro- moiety produced 65 with a
decreased potency (GI50 3.5 µM); while the introduction of 6,7-dimethoxy- yielded 66 (GI50 2.6 µM)
9

and helped to regain some of the activity of 64 .
The inhibitory activity of the new synthesized against DHFR was experimentally determined.
Compounds 28 and 61 proved to be the active members in the present study (IC50 0.02 and 0.01 µM,
respectively) compared to the positive control MTX (IC50, 0.008 µM). Molecular modeling study
was necessary to obtain the binding mode in addition to get a consistent, more precise picture of the
biologically active molecules at the atomic level and furthermore, to clarify the reasons behind
diminished activity of the inactive candidates. A comparative modeling study of the active DHFR
inhibitor 61 (IC50, 0.01 µM) and the least active compound 41 (IC50, 10 µM), against MTX was
performed. The tertiary complex of human dihydrofolate reductase (hDHFR) crystal structure (pdb
ID: 1DLS obtained from the protein data bank), NADPH and MTX were used as reference for
modeling and docking [32]. Conformational search of initial structures of the selected molecules
were constructed using MOE. The MM (calculations in vacuo, bond dipole option for and RMSD
gradient of 0.01 kcal/mol) energy minima were determined by a semi-empirical method AM1 (as
implemented). The energy-minimized geometry was used for the docking calculation and the various
2D descriptors using molecular modeling software MOE 2009.10 [33]. An analysis of the respective
docking pose of active DHFR inhibitor compound 61 shows high affinity binding energy of -13.1387
Kcal/mol (even higher than MTX -12.9876 Kcal/mol) with Phe34 amino acid via arene-arene
interaction (Figure 2) while the inactive compound 41 shows no binding with any amino acid residue
in DHFR binding pocket (Figure 3) with less binding affinity energy of -11.3412 Kcal/mol. Each
compound has its own unique feature of binding profile which could explain their different patterns
in DHFR inhibitory activity regardless of their similarity in chemical structure.
Figure 2: 3D binding mode and residues involved in the recognition for the
most active compound 61 (IC50 0.01 µM).
10

Figure 3: 2D binding mode and residues involved in the recognition and the
least active compound 41 (IC50 10.0 µM) docked and minimized in the DHFR
binding pocket.
Figure 4: (a) Flexible alignment of the most active compounds and 61 (red), IC50 0.01
µM and 28 (cyan), IC50 0.02 µM. (b) Flexible alignment of the most active compound 61
(red), IC50 0.01 µM and the least active compound 41 (yellow), IC50 10.0 µM.
Figure 5: (a) Surface map for the most active compound 61 (orange) in pocket side. (b)
Surface map for the least active compound 41 (cyan) in pocket side. Pink: hydrophilic,
blue: mild polar, green: hydrophobic.
Flexible alignment technique [34] was performed using MOE/MMFF94 to automatically produce
superposition of the compounds under investigation with minimal user bias. 200 conformers of each
11

compound were generated then minimized by a distance-dielectric dependent model. To investigate
the similarity between the 3D structures of the most active compounds 61 (IC50, 0.01 µM) and 28
(IC50, 0.02 µM) as selected examples, flexible alignment was employed where it was clear that both
compounds have shown good alignment profile (Figure 4a). On the contrary, Figure 4b obviously
indicated a different alignment profiles for compounds 61 (IC50, 0.01 µM) and 41 (IC50, 10.0 µM).
These findings are consistent with the obtained DHFR inhibition experimental data. To study the
reasons for the decreased DHFR inhibition potency of 41 hydrophobic surface mapping studies were
conducted. Compound 61 showed more intense greener hydrophobic distributions which could be
responsible for the interaction with amino acid residues inside the enzyme active pocket (Figure 5a).
On the other hand, the hydrophobic regions of the least active compound 41 seemed to be more near
to the mild polar blue side (Figure 5b), hence the required lipophilicity for the effective binding to
DHFR could be decreased. The performed molecular modeling studies with proposed binding mode
analysis for 61 revealed some features which could embrace the importance of hydrophobicity and
aromatic π-systems as crucial criteria required for interaction with the active site.
In Conclusion, compounds 28 and 61 proved to be the most active DHFR inhibitors with IC50 0.02
and 0.01 µM, respectively. Structure activity relationship revealed that, the type of substituent at
positions 2-, 3- and 6- of the studied quinazolin-4-ones manipulate the DHFR inhibition activity. The
order of activity in the 2-(4-methoxybenzyl)thio- derivatives was 6-methyl- > 6-chloro- > 6,7-
dimethoxy-; while in case of the 2-(3,4,5-trimethoxybenzyl)thio- was 6-chloro- > 6,7-dimethoxy- >
6-methyl-. Compounds 34, 56 and 66 showed remarkable broad spectrum antimicrobial activity
against both Gram-positive and Gram-negative bacteria comparable to the known antibiotics
Gentamicin and Ciprofloxacin. Compounds 56 (IC50 0.9 µM) and 66 (IC50 2.0µM) may exert their
activity through DHFR inhibition. Meanwhile, Compounds 40 and 64 showed broad spectrum
antitumor activity toward several tumor cell lines and proved to be 10 fold more active than 5-FU,
with GI50 MG-MID 2.2 and 2.4 µM, respectively. Compound 64 (IC50 1.0 µM) might exert its
activity through DHFR inhibition. Molecular modeling studies concluded that recognition with key
amino acid Phe34 is essential for binding and DHFR inhibition. The binding mode for the most
active compound 61 at the active site of hDHFR was consistent with the experimental data. Analysis
of this binding mode emphasis the common features highlighting the importance of hydrophobicity
and aromatic π-systems in binding to the DHFR active site. Figure 6 showed the structures of the
most active DHFR inhibitors 28 and 61; the broad spectrum antibacterial 34; in addition to the
antitumor agents 40, 64.
12

Figure 6: Structures of the most active DHFR inhibitors 28, 61; the broad spectrum
antibacterial 34; and the antitumor agents 40, 64.
Comparing the obtained results to that for the leads A-C (Figure 1, IC50 range of 0.3-1.0 µM), clearly
shows that the introduction of methoxylated 2-(arylmethyl-thio)- as hydrophobic π-system regions to
the used quinazolines (compounds 61 and 28, IC50 0.01 and 0.02 µM, respectively) confirmed the
feasiblity of the reported pharmacophoric features mentioned in the introduction and hence
contributed with positive impact to the DHFR inhibition activity. The methoxylated 2-(arylmethyl-
thio)-function containing compounds proved to be 30-100 fold more active than the 2-allylthio-, 2-
thiazolylthio- or 2-thiadiazolylthio- groups shown in A-C. The obtained model could be useful for
further development of new DHFR inhibitors.
Acknowledgements
Thanks are due to the NCI, Bethesda, MD, for performing the antitumor testing of the synthesized
compounds
References and Notes
1.
2.
3.
4.
Masur, H. J. Infect. Dis. 1990, 161, 858−864.
Berman, E.M.; Werbel, L.M. J. Med. Chem. 1991, 34, 479-485.
Borst, P.; Quellette, M. Annu. Rev. Microbiol. 1995, 49, 427-460.
Hassan, G.S.; El-Messery, S.M.; Al-Omary, F.A.M.; El-Subbagh, H.I. Bioorg. & Med. Chem.
lett. 2012, 22, 6318–6323.
5.
El-Messery, S.M.; Hassan, G.S.; Al-Omary, F.A.M.; El-Subbagh, H.I. Eur. J. Med. Chem.
2012, 54, 615-625.
13

6.
Abdel-Aziz, A.A.-M.; El-Azab, A.S.; El-Subbagh, H.I.; Al-Obaid, A.M.; Alanazi, A.M.; Al-
Omar, M.A. Bioorg. & Med. Chem. lett. 2012, 22, 2008-2014.
7.
8.
Taher, A.T.; Georgey, H.H.; El-Subbagh, H.I. Eur. J. Med. Chem. 2012, 47, 445-451.
Al-Omary, F.A.M.; Hassan, G.S.; El-Messery, S.M.; El-Subbagh, H.I. Eur. J. Med. Chem.
2012, 47, 65-72.
9.
El-Subbagh, H.I.; Abadi, A.H.; Al-Khamees, H.A. Arch. Pharm. Pharm. Med. Chem. 1997,
330, 277−282.
10. El-Subbagh, H.I.; Abadi, A.H.; El-Khawad, I.E.; Rashood, K.A. Arch. Pharm. Pharm. Med.
Chem. 1999, 332, 19−22.
11. El-Subbagh, H.I.; Abu-Zaid, S.M.; Mahran, M.A.; Badria, F.A.; Al-Obaid, A.M. J. Med.
Chem. 2000, 43, 2915-2921.
12. Al-Madi, S.H.; Al-Obaid, A.M.; El-Subbagh, H.I. Anticancer Drugs 2001, 12 835-841.
13. Abdel Hamid, S.G.; El-Obaid, H.A.; Al-Rashood, K.A.; Khalil, A.A.; El-Subbagh, H.I. Sci.
Pharm. 2001, 69, 351−366.
14. Khalil, A.A.; Abdel Hamide, S.G.; Al-Obaid, A.M.; El−Subbagh, H.I. Arch. Pharm. Pharm.
Med. Chem. 2003, 336, 95−103.
15. Abdel Hamid, S.G.; El-Obaid, H.A.; Al-Majed, A.A.; El-Kashef, H.A.; El-Subbagh, H.I. Med.
Chem. Res. 2001, 10, 378−389.
16. Al-Omar, M.A.; Abdel Hamide, S.G.; Al-Khamees, H.A.; El-Subbagh, H.I. Saudi Pharm. J.
2004, 12, 63-71.
17. Al-Rashood, S.T.; Aboldahab, I.A.; Abouzeid, L.A.; Abdel-Aziz, A.A-M.; Nagi, M.N.;
Abdulhamide, S.G.;Youssef, K.M.; Al-Obaid, A.M.; El-Subbagh, H I. Bioorg. & Med. Chem.
2006, 14, 8608-8621.
18. Al-Omary, F. A. M.; Abou-zeid, L.A.; Nagi, M.N.; Habib, E. E.; Abdel-Aziz, A. A.-M.; El-
Azab, A.S.; Abdel-Hamide, S. G.; Al-Omar, M. A.; Al-Obaid, A. M.; El-Subbagh, H. I.
Bioorg. & Med. Chem. 2010, 18, 2849-2863.
19. Al-Omary, F.A.M.; Hassan, G.S; El-Messery, S.M.; Nagi, M.N.; Habib, E.E.; El-Subbagh,
H.I. Eur. J. Med. Chem. 2013, 63, 33-45.
20. Hassan, G.S.; El-Messery, S.M.; Al-Omary, F.A.M.; Al-Rashood, S.T.; Shabayek, M.I.;
Abulfadl, Y.S.; Habib, E.E.; El-Hallouty, S.M.; Fayad, W.; Mohamed, K.M.; El-Menshawi,
B.S.; El-Subbagh H.I. Eur. J. Med. Chem. 2013, 66, 135-145.
21. El-Subbagh, H.I.; Hassan, G.S.; El-Messery, S.M.; Al-Omary, F.A.M.; Al-Rashood, S.T.;
Abulfadl, Y.S.; Shabayek, M.I. Eur. J. Med. Chem. 2014, 74, 234-245.
22. Al-Rashood, S.T.; Hassan, G.S.; El-Messery, S.M.; Nagi, M.N.; Habib, E.E.; Al-Omary,
F.A.M.; El-Subbagh, H.I. Bioorg. & Med. Chem. Lett. 2014, 24, 4557–4567.
23. Foye, W.O.; Lemke, T.L.; Williams, D.A. Principles of Medicinal Chemistry 4th Ed., Williams
and Wilkins: Media, PA, 2005 442-456.
24. Nordberg, M.G.; Kolmodin, K.; Aqvist, J.; Queener, S.F.; Hallberg, P. Eur. J. Pharm. Sci.
2004, 22, 43–53.
25. Grever, M.R.; Schepartz, S.A.; Chabner, B.A. Semin. Oncol. 1992, 19, 622−638.
26. Monks, A.; Scudiero, D.; Skehan, P. J. Natl. Cancer. Inst. 1991, 83 757−766.
27. Boyd, M.R.; Paull, K.D. Drug Rev. Res. 1995, 34, 91−109.
14

28. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D. Warren, J.R. ;
Bokesch, H.; Kenney, S.; Boyd, M.R. J. Natl. Cancer Inst. 1990, 82, 1107−1112.
29. Pignatello, R.; Sapmpinato, G.; Sorrenti, V.; Vicari, L.; Di-Giacomo, C.; Vanella, A.; Puglisi,
G. Pharm. Pharmacol. Commun. 1999, 5, 299−504.
30. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial
susceptibility testing, CLSI approved standards M100-S15., Wayne, PA, 2008.
31. Murray, P. R.; Baron, E. J.; Pfaller, M. A.; Tenover, F. C.; Yolken, R. H. In Manual of
Clinical Microbiology; Wood, G. L.; Washington, J. A., Eds. Am. Soc. Microbiol.: Washington
D.C.1995
32. Lewis, W.S.; Cody, V.; Galitsky, N.; Luft, J.R.; Pangborn, W.; Chunduru, S.K.;
Spencer, H.T.; Appleman, J.R.; Blakley, R.L. J. Biol. Chem. 1995, 270, 5057-5064.
33. Molecular Operating Environment (MOE), 2009.10; Chemical Computing Group Inc.
34. Cody, V.; Schwalbe, C.H. Crystallography Reviews 2006, 12, 301–333.
35. Profeta, S.; Allinger. N. L. J. Am. Chem. Soc. 1985, 107, 1907-1918.
36. Allinger. N. L. J. Am. Chem. Soc. 1977, 99, 8127-8134.
37. Labute, P.; Williams, C.; Feher, M.; Sourial, E.; Schmidt, J.M. J. Med. Chem. 2001, 44, 1483–
1490.
38. Kearsley, S.; Smith, G.M. Tetrahedron Comput. Methodol. 1990, 3, 615–633.
15

Graphical Abstract
Synthesis, biological evaluation and molecular modeling study of some new methoxylated 2-benzylthio-
quinazoline-4(3H)-ones as nonclassical antifolates.
Shahenda M. El-Messery,a* Ghada S. Hassan,b Mahmoud N. Nagi,c El-Sayed E. Habib,d,e Sarah T. Al-Rashood,f Hussein
I. El-Subbagh.b,g*
The DHFR inhibitors 28, 61; the broad spectrum antibacterial 34; and the antitumor agents 40, 64.
39.
16