Non-classical antifolates. Part 2: Synthesis, biological evaluation, and molecular modeling study of some new 2,6-substituted-quinazolin-4-ones

Article abstract of DOI:10.1016/j.bmc.2010.03.019

A new series of 2,6-substituted-quinazolin-4-ones was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 22, 33-37, 39-43, and 45 proved to be active DHFR inhibitors with IC₅₀ range of 0.4-1.0 μM. Compound 18 showed broad-spectrum antimicrobial activity comparable to the known antibiotic gentamicin. Compounds 34 and 36 showed antitumor activity at GI₅₀ (MG-MID) concentrations of 11.2, and 24.2 μM, respectively. Molecular modeling study including flexible alignment; electrostatic, hydrophobic mappings; and pharmacophore prediction were performed. A main featured pharmacophore model was developed which justifies the importance of the main pharmacophoric groups as well as of their relative distances. The substitution pattern and spatial considerations of the π-systems in regard to the quinazoline nucleus proved critical for biological activity.

Full text of DOI:10.1016/j.bmc.2010.03.019

Bioorganic & Medicinal Chemistry 18 (2010) 2849–2863
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Non-classical antifolates. Part 2: Synthesis, biological evaluation, and
molecular modeling study of some new 2,6-substituted-quinazolin-4-ones q
Fatmah A. M. Al-Omary a, Laila A. Abou-zeid b, Mahmoud N. Nagi c, El-Sayed E. Habib d,
Alaa A.-M. Abdel-Aziz a, Adel S. El-Azab a, Sami G. Abdel-Hamide a, Mohamed A. Al-Omar a,
Abdulrahman M. Al-Obaid a, Hussein I. El-Subbagh a,
*
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
c Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
d Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, 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 2,6-substituted-quinazolin-4-ones was designed, synthesized, and evaluated for their
Received 30 January 2010
Revised 6 March 2010
Accepted 9 March 2010
Available online 12 March 2010
in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 22, 33–37, 39–43, and 45
proved to be active DHFR inhibitors with IC50 range of 0.4–1.0
trum antimicrobial activity comparable to the known antibiotic gentamicin. Compounds 34 and 36
showed antitumor activity at GI50 (MG-MID) concentrations of 11.2, and 24.2 M, respectively. Molecular
lM. Compound 18 showed broad-spec-
l
modeling study including flexible alignment; electrostatic, hydrophobic mappings; and pharmacophore
prediction were performed. A main featured pharmacophore model was developed which justifies the
importance of the main pharmacophoric groups as well as of their relative distances. The substitution
Keywords:
Synthesis
Quinazolin-4-ones
DHFR inhibition
Antimicrobial activity
Antitumor screening
Molecular modeling study
pattern and spatial considerations of the
for biological activity.
p
-systems in regard to the quinazoline nucleus proved critical
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
arylthio function,8,9 the antimalarial drug 5 with its 6-arylthio moi-
ety,10 the antileukemic activity of the dithiocarbamate 6, and the
carbodithioate 7,11 then The 6-acrylamide analogue 8,12 (Chart 1).
In continuation of our previous efforts,13–26 a new series of 2,6-
substituted-quinazolin-4-one analogues of the general formulae
A–C (Chart 2), was designed to possess 6-methyl, 6-nitro or 6-amino
functions, representing electron donating and electron withdrawing
substituents; a benzyl group at position 3-, based on what was con-
cluded from a previous study, that DHFR inhibition activity could be
obtained only, upon the existence of a benzyl group at N-3 of the qui-
nazoline ring, rather than 3-methyl or 3-phenyl functions.26 In addi-
tion, alkyl, allyl or cinnamyl thioether groups introduced at position
2- in resemblance to the lead compounds 4 and 5 (A). The 6-amino
function of A was used to introduce an allyl and cinnamyl amines,
or acrylamide and cinnamamide functions (B); these 6-amide com-
pounds resemble the active 6-acrylamide analogue 8. The secondary
amines of A were converted to their corresponding tertiary amines
(C) through their alkylation using methyl, allyl, or cinnamyl
functions, to block the NH-function to produce methotrexate type
analogues. Most of the functions designed to be accommodated on
the quinazoline ring such as thioether, alkyl, aryl, arylalkyl and nitro
groups are known to increase lipid solubility of polar compounds, a
character very much needed for the non-classical DHFR
Dihydrofolate reductase (DHFR) catalyzes the reduction of fo-
late or 7,8-dihydrofolate to tetrahydrofolate and intimately cou-
ples with thymidylate synthase (TS). TS is a crucial enzyme that
catalyzes the reductive methylation of deoxyuridine monophos-
phate (dUMP) to deoxythymidine monophosphate (dTMP) utiliz-
ing N5,N10-methylene-tetrahydrofolate as
a cofactor, which
functions as source of the methyl group as well as the reductant.
This is the exclusive de novo sources of dTMP, hence inhibition
of DHFR or TS activity in the absence of salvage, leads to ‘thymine-
less death’.1,2 Thus, DHFR inhibition has long been an attractive
goal for the development of chemotherapeutic agents against bac-
terial and parasitic infections as well as cancer.3 Upon reviewing
the literature, it was noticed that in addition to the non-classical
DHFR inhibitors: Trimethoprim (TMP, 1),4 trimetrexate (TMQ,
2),5,6 and piritrexim (PTX, 3),5,7 some other interesting inhibitors,
most of them belong to the quinazoline heterocycle were also lo-
cated; such as the anti-colorectal cancer thymitaq (4) with its 5-
q
For part 1 see Ref. 26.
* Corresponding author. Tel.: +966 1 4677349; fax: +966 1 4676220.
E-mail address: subbagh@yahoo.com (H.I. El-Subbagh).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.019

2850
F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
OCH3
OCH3
NH2 CH3
NH2 CH3
OCH3
OCH3
NH2
N
OCH3
N
N
H
N
N
OCH3
H2N
OCH3
OCH3
H2N
N
H2N
N
N
1
TMQ, 2
3
PTX,
TMP,
N
H
N
O
S
SCH3
N
NH2
CH3
HN
H2N
S
Ar
N
N
N
H
S
N
N
NH2
6
4
5
S
N
O
N
S
HN
Br
H
N
HN
H3C
N
O
Br
N
7
8
Chart 1.
3:1. The 6-nitro function of 12 was subjected to metal reduction
using Fe/HCl. Chromatographic separation of the obtained products
allowed the identification of the reported 6-amino analogue 1726
along with 6-amino-3-benzyl-quinazolin-4(3H)-one (18) resulted
from the reductive elimination of 2-CH3S- function. The 2-thioxo
function of 9 and 10 was then alkylated using allyl or cinnamyl bro-
mide to give the thioether derivatives 19, 20, 22, and 23. Chromato-
graphic purification of the crude products obtained upon treating 10
with allyl bromide, allowed the separation of the target compound
20, along with the disulfide analogue 21 resulted from the oxidation
of the 2-thiol function. Oxidation of thiol into disulfide is well docu-
mented.29 The 6-nitro function of both 20 and 23 was subjected to
metal reduction using Fe/HCl to produce the corresponding 6-amino
analogues 24 and 25, respectively (Scheme 1).
O
R1
Bn
N
R2
N
S
A
R1 = CH3, NO2, NH2, allyl-NH-, cinnamyl-NH-
2 = CH3, Et, Bn, allyl, cinnamyl
R
R2
N
O
O
N
H
N
R1
Bn
S
R1
Bn
S
N
N
O
R2
R3
N
Variety of amines and mercaptanes undergo Michael-type addi-
B
C
tions to
a,b-unsaturated functions to produce the b-amino or b-
mercapto derivatives.30–34 Upon treating 10 with acryloyl chloride
in NEt3/CH2Cl2, a material was isolated and identified to be the b-
mercapto analogue, ethyl 3-(3-benzyl-6-nitro-4-oxo-3,4-dihydro-
quinazolin-2-ylthio)-propanoate (26). The only possible source of
ethyl group to generate the ester 26 is the NEt3 exist in the reaction
medium. This was proven by repeating the reaction without using
such base, where the free acids 3-(3-benzyl-6-methyl or nitro-4-
oxo-3,4-dihydroquinazolin-2-ylthio)-propanoic acid (27 and 28),
were produced instead. The same procedure was adopted to pre-
pare 3-benzyl-1-cinnamoyl-6-(methyl or nitro)-2-thioxo-2,3-dihy-
dro-quinazolin-4(1H)-one (29 and 30). Spectral analysis of 29 and
30 revealed that acylation with cinnamoyl chloride occurred at N-1
of the quinazoline ring instead of the Michael addition of –SH
R
1 = H, Ph
R
1 = H, Ph
R2 = H, CH3, allyl, cinnamyl
R2 = CH3, allyl, cinnamyl
R
3 = CH3, allyl, cinnamyl
Chart 2.
inhibition.27,28 The aim of this study is to locate novel synthetic lead
compound(s), and their in vitro testing as DHFR inhibitor(s). Com-
pounds possessing such activity will be candidates for treating can-
cer, bacterial and parasitic infections.
2. Chemistry
group to the
a,b-unsaturated function, as evidenced by the exis-
The synthetic strategy to obtain the targets A–C (Chart 2) is
depicted in Schemes 1–3. The starting materials 3-benzyl-2-
mercapto-6-methyl-quinazolin-4(3H)-one (9) and 3-benzyl-2-mer-
capto-6-nitro-quinazolin-4(3H)-one (10) were prepared adopting
reported procedures.24–26 The 2-thioxo- function of 9 and 10 was
then alkylated using variety of alkyl halides to give the thioether
derivatives 11–16. A close examination of the NMR spectrum of 3-
benzyl-2-ethylthio-6-nitro-quinazolin-4(3H)-one revealed its exis-
tence in two inseparable positional isomers; (S-Et:N-Et) in ratio of
tence of the C@S absorptions at d 173.5, 174.5 ppm, respectively.
This might be attributed to the steric hindrance caused by the phe-
nyl ring of the cinnamoyl moiety (Scheme 2).
The synthesized 6-amino-quinazolines 17, 24, and 25 were then
acylated using acryloyl chloride or cinnamoyl chloride to afford the
target acrylamides (31–33), and cinnamamides (34–36), respec-
tively, in reasonable yields. Meanwhile, the 6-amino function of
17 and 24 were alkylated using allyl bromide. Chromatographic

F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
2851
O
N
O
N
O
R1
Bn
H2N
Bn
H2N
Bn
S
N
N
N
Fe, HCl
EtOH
CH3
+
S
N
R2
17
18
11:
R
1 = R2 = CH3
12:
R1 = NO2, R2 = CH3
13: R1 = CH3, R2 = Et
14: R1 = NO2, R2 = Et
15: R1 = CH3, R2 = Bn
16:
R1 = NO2, R2 = Bn
R2X
O
O
O2N
O
N
N
R
Bn
R
Bn
S
Br
N
S
N
O
N
N
S
+
N
N
H
S
NO2
. HCl
9: R = CH3
10:
19: R = CH3
20:
21
R = NO2
R = NO2
Br
Fe, HCl,
EtOH
O
N
O
N
H2N
Bn
R
Bn
Fe, HCl,
EtOH
N
N
S
R
S
24: R = H
22: R = CH3
23:
25: R = Ph
R = NO2
Scheme 1.
O
N
R
Bn
N
SH
Cl
Cl
Ph
9: R = CH3
10: R = NO2
O
2
O
2
3
O
O
N
R
Bn
O2N
Bn
S
Cl
N
N
O
Toluene
O
N
S
OEt
O
26
O
N
R
Bn
S
N
O
29: R = CH3
30: R = NO2
OH
27: R = CH3
28: R = NO2
Scheme 2.

2852
F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
O
N
O
H
N
H
Bn
Ph
N
Bn
N
N
O
R
O
R
N
S
S
34:
35:
31:
32:
R = CH3
R = CH3
R = CH2-CH=CH2
R = CH2-CH=CH2
36: R = CH2-CH=CH-Ph
33: R = CH2-CH=CH-Ph
Cl
O
Cl
Ph
O
O
O
N
O
H
N
Br
Bn
N
Bn
H2N
Bn
S
N
N
N
R
+
R
R
N
S
N
S
17: R = CH3
37:
39:
38:
40:
R = CH3
R = CH2-CH=CH2
R = CH3
24: R = CH2-CH=CH2
R = CH2-CH=CH2
25: R = CH2-CH=CH-Ph
HCHO,
NaCNBH4
Br
Ph
Ph
O
O
N
CH3
N
O
N
H
Ph
N
Bn
Ph
N
Bn
Bn
S
N
+
N
N
R
R
S
CH3
N
S
42: R = CH3
44: R = CH2-CH=CH2
43:
45:
R = CH3
41
R = CH2-CH=CH2
Scheme 3.
purification of the obtained products allowed the separation of 6-
allylamino-3-benzyl-2-methylthio-quinazolin-4(3H)-one (37)
uate compounds proved to be bovine DHFR inhibitors. Under the
assay conditions, no significant difference (P >0.05) was observed
between bovine DHFR and hDHFR, using methotrexate (IC50
along with the 6-diallyl amino 38, also 6-allylamino-2-allylthio-
3-benzylquinazolin-4(3H)-one (39) in addition to the 6-diallyl
amino 40. The secondary amine 37 was subjected to reductive
methylation using formaldehyde and NaCNBH3 to produce the N-
methylated tertiary amine, 6-allyl-(methyl)-amino-3-benzyl-2-
methylthio-quinazolin-4(3H)-one (41), in good yield. Similarly,
17 and 24 was alkylated using cinnamyl bromide. Careful
chromatographic purification of the obtained products allowed
the separation of 3-benzyl-6-cinnamylamino-2-methylthio-qui-
nazolin-4(3H)-one (42) along with the 6-dicinnamyl-amino
analogue 43, also 2-allylthio-3-benzyl-6-cinnamylamino-quinazo-
lin-4(3H)-one (44) in addition to the 6-dicinnamyl-amino deriva-
tive 45 (Scheme 3).
0.08 lM) as a positive control.
3.1.1. Structure–activity relationship (SAR)
The type of substituent at positions 2- and 6- of the studied
quinazolines manipulated the DHFR inhibition activity. The 2-thio-
ether function affects the magnitude of DHFR inhibition. The order
of activity is CH2@CH–CH2–S– or Ph–CH@CH–CH2–S– (0.5–
5.0
presence of 6-CH3 group (0.5–2.0
than the 6-nitro (3.0–5.0 M) or the 6-amino functions (8.0
In the 6-methyl series, the existence of 2-cinnamylthio-function
(22, IC50 0.5 M) favors the activity rather than 2-allylthio- (19,
IC50 2.0 M). The combination of 6-methyl- and 2-cinnamylthio-
(22, IC50 0.5 M) favors the activity rather than the combination
of 6-nitro- and 2-cinnamylthio- (23, IC50 5.0 M). In the 6-acrylam-
l
M) > CH3–S–, CH3CH2–S–, and Bn–S– (10.0–35.0
M) favors the activity rather
M).
lM). The
l
l
l
l
l
l
3. Results and discussion
l
ide series (31–33), the order of activity is determined by the type of
the 2-thioether function. Compounds bearing 2-cinnamylthio-
function is 10-fold more active than the 2-allylthio, and 50-fold
more active than the 2-methylthio-. In the 6-cinnamamide series
(34–36), and in contrast to the 6-acrylamide series, the type of
the 2-thioether moieties does not affect the magnitude of activity;
all are in the remarkable side with IC50 values of 0.6–0.9 lV. The
6-cinnamamide moiety proved to contribute to activity more than
the 6-acrylamide function. Another piece of evidence proving the
3.1. Dihydrofolate reductase (DHFR) inhibition
The synthesized compounds (9–45) were evaluated as inhibi-
tors of bovine liver and human DHFR using reported procedure,35
results were reported as IC50 values (Tables 1 and 2). Compounds
22, 33–37, 39–43, and 45 proved to be the most active inhibitors
of bovine liver DHFR with IC50 range of 0.4–1.0 lM. The analogy
and similarity among bovine liver DHFR and hDHFR is almost
75%.35 In this study, hDHFR enzyme was used to pursue and reval-

F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
2853
Table 1
DHFR inhibition (IC50, lM), and antimicrobial activity results of compounds 9–25
O
N
O
N
O
N
O2N
N
R1
Bn
R2
H2N
Bn
N
N
S
N
O
S
N
NO2
. HCl
21
9-17, 19, 20, 22-25
18
Compd
R1
R2
DHFR inhibition (IC50
,
lM)
Inhibition zone (mm); MICs (lg/ml)
bDHFR
hDHFR
S. aureus
B. subtilis
M. luteus
E. coli
P. aeuroginosa
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
CH3
NO2
CH3
NO2
CH3
NO2
CH3
NO2
NH2
—
CH3
NO2
—
CH3
NO2
NH2
NH2
SH
SH
S-CH3
S-CH3
S-Et
S-Et
S-Bn
S-Bn
S-CH3
30.0
15.0
20.0
30.0
35.0
10.0
10.0
30.0
10.0
40.0
2.0
3.0
10.0
0.5
5.0
8.0
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
4.0
2.0
Nd
1.0
3.0
5.0
6.0
—
18 (4.0)
18 (8.0)
—
—
—
—
—
—
14 (Nd)
16 (16.0)
—
—
—
—
—
—
17 (4.0)
—
—
—
15 (Nd)
—
—
—
20 (4.0)
—
—
—
—
—
—
—
24 (2.0)
—
—
—
—
—
—
—
—
—
20 (8.0)
—
—
—
22 (4.0)
—
—
—
15 (Nd)
—
—
—
—
—
—
—
—
—
—
28 (2.0)
—
—
—
—
—
—
—
20 (4.0)
S-CH2–CH@CH2
S-CH2–CH@CH2
—
S-CH2–CH@CH–Ph
S-CH2–CH@CH–Ph
S-CH2–CH@CH2
S-CH2–CH@CH–Ph
27 (2.0)
—
—
—
—
—
—
—
—
—
21 (8.0)
—
—
17 (16.0)
—
—
25
8.0
25 (2.0)
22 (2.0)
Gentamicin
Sulfacetamide
18 (2.0)
18 (2.0)
21 (0.5)
28 (1.0)
19 (1.0)
25 (2.0)
20 (2.0)
(—) Not active (8 mm), weak activity (8–12 mm), moderate activity (12–15 mm), strong activity (>15 mm). Solvent: DMSO (8 mm). MICs (lg/ml) showed in parentheses. Nd,
not determined.
role of the 2-thioether function in determining the magnitude of
activity appears in the 6-allylamino and the 6-cinnamylamino
derivatives (37–40). In the 6-allylamino series, the effect of the 2-
thioether group proved to be of minimal influence as presented
by the almost equal potency of 37 and 39 (IC50 0.6 and 0.8 lM,
respectively). Replacing 6-allylamino by 6-diallylamino moiety en-
the Gram-positive bacteria was observed for compounds 27, 30,
35, 44 against S. aureus; 9, 10, 27, 35, 43 against B. subtilis; and
10, 35, 44 against M. luteus. In case of Gram-negative bacteria,
strong activity was noticed for compounds 9, 26, 35, 37, 42–45
against E. coli; and compounds 9, 14, 23, 26, 30 against Pseudomo-
nas aeuroginosa. Selectivity was noticed among the tested com-
pounds. Compounds 14 and 23 possessed activity solely confined
toward the used Gram-negative bacteria, while 10, 27 and 28
showed activity solely confined toward the used Gram-positive
bacteria. Compounds 18, 30, 35, 44 and 45 showed a remarkable
broad-spectrum activity against both Gram-positive and Gram-
negative bacteria. Compound 18 proved to be the most active
broad-spectrum antibiotic in this study with potency comparable
to the known antibiotic gentamicin. There is no antifungal activity
observed for all of the tested compounds. The minimal inhibitory
concentrations (MICs) for the most active compounds was carried
out using the micro-dilution susceptibility method as shown in Ta-
bles 1 and 2. The Gram-positive bacteria B. subtilis and the Gram-
negative bacteria E. coli are sensitive to majority of the tested com-
pounds. Comparing the potency of the active antibacterials and
their DHFR inhibition revealed that compounds 23, 35, and 43–
45 might exert their activity through DHFR inhibition.
hanced the activity of the 2-allylthio-analogue 39 by twofold (40,
IC50 0.4
logue 37 by fivefold (38, IC50 3.0
the concept is reversed. Compound 42 bearing 2-methylthio- func-
tion (IC50 0.5 M) proved to be more active than the 2-allylthio-
derivative 44 by 10-fold (IC50 5.0 M). Replacing the 6-cinnamyla-
mino function by 6-dicinnamylamino moiety gave compounds 43
lM) and suppressed the potency of the 2-methylthio- ana-
lM). In the 6-cinnamylamino series,
l
l
and 45 with almost equal potency (IC50 0.6 and 0.5 lM,
respectively).
3.2. Antimicrobial activity
The synthesized compounds (9–45) were tested for their
in vitro antimicrobial activity against a panel of standard strains
of Gram-positive bacteria, Gram-negative bacteria, and yeast-like
pathogenic fungus. The primary screen was carried out using the
agar disc-diffusion method.36,37 Gentamicin (100
l
g/disc), sulfa-
cetamide (100
lg/disc) and clotrimazole (100
l
g/disc) were used
3.3. Antitumor screening
as positive controls. The obtained results revealed that the tested
compounds expressed varying degrees of activity against the
tested microorganisms (Tables 1 and 2). Remarkable activity
(>20 mm inhibition zone) against the Gram-positive bacteria was
observed for compounds 10, 18, 28, 45 against Staphylococcus aur-
eus; 18, 28, 30, 44, 45 against Bacillus subtilis, and 18 against Micro-
coccus luteus. In case of Gram-negative bacteria, remarkable
activity was observed for compounds 14, 18, 23, and 30 against
Escherichia coli. Strong activity (>15 mm inhibition zone) against
The active DHFR inhibitors (22, 30, 34–37 and 42) were sub-
jected to the NCI’s in vitro anticancer screen. Compounds 34 and
36 passed the primary anticancer assay at an arbitrary concentra-
tion of 100
ried over and tested against a panel of 60 different tumor cell
lines at a 5-log dose range.38–41 Three response parameters, GI50
lM. Consequently, those active compounds were car-
,
TGI, and LC50 were calculated for each cell line, using 5-fluorouracil
(5-FU) as a positive control. The tested quinazolinone analogues

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F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
Table 2
DHFR inhibition (IC50, lM), and antimicrobial activity results of compounds 26–45
O
O
N
O
N
H
N
R1
Bn
S
R1
Bn R2
S
Bn
N
O
N
N
O
R1
N
OR2
S
O
29-30
31-36
26-28
R2
N
R2
N
O
O
N
Bn Ph
Bn
N
N
R1
S
R1
N
S
37-41
42-45
Compd
R1
R2
DHFR inhibition (IC50
,
l
M)
Inhibition zone (mm); MICs (lg/ml)
bDHFR hDHFR
S. aureus
B. subtilis
M. luteus
E. coli
P. aeuroginosa
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
NO2
CH3
NO2
CH3
NO2
CH3
Et
H
H
—
—
H
H
H
Ph
Ph
Ph
H
25.0
50.0
10.0
5.0
10.0
50.0
10.0
1.0
0.6
0.7
0.9
0.6
3.0
0.8
0.4
1.0
0.5
0.6
5.0
0.5
Nd
Nd
Nd
3.0
Nd
Nd
Nd
2.0
1.0
0.5
0.5
0.8
1.0
1.0
0.9
2.0
2.0
0.8
3.0
0.7
—
14 (Nd)
16 (4.0)
24 (2.0)
—
20 (4.0)
—
—
—
—
—
16 (Nd)
—
—
—
20 (4.0)
—
—
—
16 (Nd)
—
—
—
18 (4.0)
20 (4.0)
—
18 (8.0)
—
14 (Nd)
—
—
14 (Nd)
—
—
—
18 (8.0)
—
—
—
—
—
CH2–CH@CH2
CH2–CH@CH2–Ph
CH3
CH2–CH@CH2
CH2–CH@CH2–Ph
CH3
—
15 (Nd)
—
16 (8.0)
—
—
18 (8.0)
18 (8.0)
18 (8.0)
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
17 (Nd)
—
—
—
—
15 (Nd)
16 (16.0)
18 (8.0)
16 (16.0)
—
—
—
—
—
—
—
—
—
CH3
CH2–CH@CH2
H
CH2–CH@CH2
CH3
CH2–CH@CH2
CH2–CH@CH2
CH3
CH3
CH3
H
CH2–CH@CH–Ph
H
CH2–CH@CH–Ph
18 (8.0)
20 (4.0)
20 (4.0)
CH2–CH@CH2
CH2–CH@CH2
19 (8.0)
22 (4.0)
16 (16.0)
—
(—) Not active (8 mm), weak activity (8–12 mm), moderate activity (12–15 mm), strong activity (>15 mm). Solvent: DMSO (8 mm). MICs (lg/ml) showed in parentheses. Nd,
not determined.
showed a distinctive potential pattern of selectivity as well as a
broad-spectrum antitumor activity. With regard to sensitivity
against individual cell lines, compound 34 showed GI50 effective-
ness against breast MDA-MB-468 cancer cell line at concentration
of 11.2, and 24.2
lM, and TGI (MG-MID) values of 86.2 and
81.9 M, respectively; against nine tumor subpanel cell lines
l
(Table 3). Compound 34 is almost twofold more active than the po-
sitive control 5-FU. When a full panel GI50 mean-graph (MG-MID)
divided by a particular subpanel GI50 mean-graph, certain ratio will
be obtained which help to predict the selectivity of this compound
toward this cell lines subpanel. Ratios of 3–6 are considered mod-
of 2.2
lM. Compound 36 showed GI50 activity against colon HT29,
and ovarian OVCAR-4 cell lines at concentration of 0.6 and 0.4
lM,
respectively. Compounds 34 and 36 showed GI50 (MG-MID) values
Table 3
Median growth (GI50) and total growth (TGI) inhibitory concentrations, (
l
M) of in vitro subpanel tumor cell lines
Compd
Subpanel tumor cell linesa
MG-MIDb
Activity
I
II
III
IV
V
VI
VII
VIII
IX
34
GI50
TGI
51.6
1.7
80.9
34.5
1.9
68.4
2.4
26.9
1.1
2.9
1.6
3.0
11.2
86.2
c
c
c
c
c
c
c
c
c
c
36
GI50
TGI
2.2
76.4
1.9
3.0
37.0
2.2
86.9
3.3
2.6
68.0
2.5
68.6
24.2
81.9
c
c
c
5-FU
GI50
TGI
15.1
8.4
72.1
70.6
61.4
45.6
22.7
76.4
22.6
c
c
c
c
c
c
c
c
c
c
a
b
c
I, leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII, renal cancer; VIII, prostate cancer; IX, breast cancer.
GI50 full panel mean-graph midpoint ( M).
Compounds showed values >100 M. Median lethal concentration (LC50
100 M.
l
l
, lM) of in vitro subpanel tumor cell lines and LC50 full panel mean-graph midpoint (lM) are >
l

F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
2855
erately selective and those with ratios of 6 or more are taken as
selective.41 Compound 34 proved to be selective toward non-small
cell lung, ovarian and prostate cancer cell lines with selectivity ra-
tio of 6.6, 10.2 and 7.0, respectively; while 36 proved to be selec-
tive toward non-small cell lung, colon, CNS, melanoma, ovarian,
renal and breast cancer cell lines with selectivity ratio of 11.0,
12.7, 8.1, 11.0, 7.3, 9.3 and 9.7, respectively.
trifurcated H-bonds with the ‘catalytic triad’ residues Val8,
Tyr121 and Val115 (Fig. 1).42–46 Molecular dynamic study indicated
that the tested quinazoline’s recognition with the key amino acid
Glu30 and Ser59 is essential for binding and biological activity.
The amino acid Ser59 is not one of the key residues of recognition
of the parent ligand LIH but it plays a crucial role in the recognition
of the tested compounds. Figure 2 showed the binding mode and
residues involved in the recognition of the most inactive com-
3.4. Molecular dynamic study
pound 31 (IC50 50.0
0.4 M) docked and minimized in the hDHFR binding pocket.
Since atomic-level detailedstructureof the relevantreceptors are
often not available, 3D flexible alignment (superposition) of putative
ligands can be used to deduce structural requirements for biological
activity.47–49 To probe similarity among 3D structures of the most
active hDHFR inhibitors 22, 34–36, 42 and 45 with minimal bias;
MOE/MMFF94 flexible alignment was employed.50,51 A common
feature of the MOE-generated alignments is the superposition of
lM) and the active compound 40 (IC50
l
The investigated compounds were subjected to molecular mod-
eling study to evaluate their recognition profile at hDHFR binding-
pocket, in comparison to the dihydrofolate inhibitor 6-(5-quin-
olylamino-methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine
(LIH). The tertiary complex of hDHFR, NADPH and LIH was used as
a reference for modeling and docking.42–45 At hDHFR pocket, LIH
formed bifurcated H-bonding with the key residue Glu30, besides
NH2 CH3
N
N
H
N
H2N
N
N
LIH
Figure 1. Binding mode for LIH docked and minimized in the hDHFR binding pocket, showing residues involved in its recognition.
Figure 2. The binding mode and residues involved in the recognition of the most inactive compound 31 (IC50 50.0 lM) and the most active compound 40 (IC50 0.4 lM)
docked and minimized in the hDHFR binding pocket.

2856
F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
the quinazolinone core of all compounds (Fig. 3a), in addition to six
points of similarities; namely, 4-carbonyl unit, N-1 of the quinazo-
line core, a hydrophilic area at position 6-, the p-system represented
on the carboxylic group. Similarly, hydrophobic mapping study of
the most active compounds showed a common features, which
are hydrophobic region (in green) distributed on both sides of
the aryl parts; and hydrophilic region (in red) located on 4-car-
bonyl, N-1, 6-NH of the quinazoline core (40, Fig. 4; left panel).
Those hydrophobic parts are responsible for the interaction with
the key amino acid residues inside the enzyme active pocket. On
the other hand, the hydrophobic distributions of the least active
compounds, (26, Fig. 4; left panel), lack such aromatic moieties
and hence the required lipophilicity, while the hydrophilic red re-
gion was located on the carboxylic acid fragment. The obtained
charge distribution, electrostatic, hydrophobic mapping and con-
formation of the active agents suggest a similar interaction of the
molecules with the potential protein-binding site.
Compound 36 served as the reference to which all conforma-
tions of each analogue were aligned For pharmacophore generation
and prediction.26,42,49 All structures were built de novo using 2D/
3D editor sketcher in MOE.50 The generated models for compound
36 and its analogues 22, 42 (Fig. 5, left panel) showed pharmaco-
phoric elements consists of aryl moieties at positions 2-, 3- and
6- as a hydrophobic triangle, in addition to a hydrophilic triangle;
in the middle of this hydrophobic area. The hydrophilic triangle
consists of two hydrogen bond acceptors, (4-carbonyl and N-1 of
the quinazoline core); and a hydrogen bond donor at the basic 6-
NH. On the contrary, models for compounds 26 and 27 (Fig. 5; right
panel) showed common elements only in the quinazoline core area
which attached to the non-favorable propionic acid fragment.
Since the two triangle elements of 36 plays an important role in
the binding mode, it was considered as the representative phar-
maophore map of DHFR inhibitors. Accordingly, the minimal struc-
ture requirements for DHFR inhibitors of this class of compounds
by aryl group and/or olefinic function located at positions 2- and 6-
and the 3-benzyl fragment. This alignment suggests the possible
existence of a region on the receptor suited for specific recognition
of such features. This hypothesis is supported by biological results
and docking studies. Adopting the same analogy, the most active
DHFR inhibitors 22, 34, 42 and the least active compounds 26 and
27 were subjected to flexible alignments (Fig. 3b). Analyses of the
least active molecule will help to understand the essential features
for a given activity. Compounds 26 and 27 were flexibly aligned in
a different fashion at position 2- of the quinazoline core, and the
hydrophilic propionic acid moiety; in addition to their lower lipo-
philicity as compared to the most active compoundsexplaining their
low biological activity.
In an attempt to figure out the reason behind the diminished
DHFR inhibition activity of compounds 26 and 27, electrostatic
and hydrophobic mapping was carried out for the lowest energy
conformers, to examine the similarity and dissimilarity in the elec-
tronic, electrostatic binding characteristics of the molecule surface
and the conformational properties (Fig. 4). Comparison of the elec-
trostatic mapping of the most active compounds, represented by
compound 40 ( Fig. 4; right panel), showed a common feature,
which are negative charged hydrogen bond acceptor–donor region
located on the 4-carbonyl, N-1, 6-NH (in red); and non-polar area
located on the aryl moiety attached to the quinazoline core (in
green). Such results indicated the structure and hence biological
similarity among those active analogues. On the contrary, the
low activity of the other compounds, represented by 26 (Fig. 4;
right panel), may be attributed to the difference in electrostatic
mapping in which the red hydrogen bond area was mainly located
consist of at least two aryl moieties or their equivalent p-systems
Figure 3. (a) Superposition of the most active compounds 36 (in brown), 34 (in blue), 35 (in green), 36 (in yellow), 42 (in cyan) and 45 (in red) using flexible alignments. (b)
Flexible alignment of the most active compounds 22 (red), 34 (cyan), 42 (yellow) and the least active compound 26 (blue) and 27 (green).
Figure 4. Electrostatic and hydrophobic map of the lowest energy conformers for the most active compound 40 and the least active compound 26, maps are color coded: red
for a hydrogen bond and a hydrophilic region, and green for a hydrophobic region.

F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
2857
Figure 5. The least active compounds 26 (yellow) and 27 (pink), mapped to the pharmacophore model for DHFRs (right panel). The left panel showed the most active
compounds 22 (cyan), 42 (green) and the least active 26 (yellow), 27 (pink) mapped to the pharmacophore features. Pharmacophore features are color coded: orange for
hydrophobics aromatic, blue for a hydrogen bonds donor, and red for a hydrogen bonds acceptor feature.
attached to the quinazoline ring which are essential for hydropho-
bic interaction with the enzyme’s hydrophobic pocket and at least
two H-bonding regions located in the quinazoline core which are
essential for hydrophilic interaction inside the receptor pocket.
For most of the active molecules, reasonable non-bonded spatial
distances among the hydrophilic triangle as well as the non-
bonded distances among the plane of the hydrophobic triangle is
needed. This pharmacophoric assumption was consistent with
the biological results and docking studies.
Research Laboratory, College of Pharmacy, King Saud University.
All of the new compounds were analyzed for C, H and N and agreed
with the proposed structures within 0.4% of the theoretical val-
ues. 1H, 13C NMR and other 2-D spectra were recorded on a Varian
XL 500 MHz FT spectrometer; 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 pre-
coated (0.25 mm) silica gel GF254 plates (E. Merck, Germany), com-
pounds were detected with 254 nm UV lamp. Silica gel (60–
230 mesh) was employed for routine column chromatography sep-
arations. DHFR inhibition activity experiments were performed at
Pharmacology Department, College of Pharmacy, King Saud Uni-
versity. Bovine liver DHFR enzyme, hDHFR enzyme, and metho-
trexate (MIX) were used in the assay (Sigma Chemical Co, USA).
Compounds 9–12 and 15–17 were previously reported.24–26 The
in vitro antimicrobial 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, P. aeuroginosa IFO 3448, and Candida albicans IFO 0583)
were employed. In vitro antitumor activity was conducted at the
NCI, Bethesda, MD. All modeling experiments were conducted with
Hyperchem 6.03 package from HYPERCUBE and POWERFIT1.0 program
from ‘Micro Simulations’ running on a PC computer. The docking
of the candidates into hDHFR pocket was performed with PowerFit
software. Starting coordinate of hDHFR enzyme in tertiary complex
with reduced-nicotinamide adenine dinucleotide phosphate
(NADPH) and LIH, code ID 1DLS, was obtained from the Protein
Data Bank of Brookhaven National Laboratory (Fig. 1).44–46
4. Conclusion
Compounds 22, 33–37, 39–43, and 45 proved to be the most ac-
tive inhibitors of DHFR with IC50 range of 0.4–1.0 lM. Structure–
activity relationship studies revealed that, the type of substituent
at positions 2- and 6- of the studied quinazolin-4-ones manipulate
the DHFR inhibition activity. Compound 18 showed broad-spec-
trum antimicrobial potency comparable to the known antibiotic
gentamicin. Compounds 23, 35, and 43–45 exert their antibacterial
activity through DHFR inhibition. Compounds 34 and 36 showed
antitumor activity at GI50 (MG-MID) concentration of 11.2, and
24.2
lM, respectively, and TGI (MG-MID) concentration of 86.2
and 81.9
l
M, respectively. Compound 34 is almost twofold more
active than the used positive control 5-FU. Molecular modeling
study was performed for the investigated compounds to evaluate
their recognition profiles at hDHFR binding-pocket. It is concluded
that recognition with key amino acid Glu30 and Ser59 are essential
for binding and biological activities. Flexible alignment; electro-
static, hydrophobic mappings; and pharmacophore prediction
study were performed. Two main triangles featured a pharmaco-
phore model. This model justifies the importance of the main phar-
macophoric groups (the 4-carbonyl fragment, the basic nitrogen
5.1. General procedure for preparation of 3-benzyl-2-ethylthio-
atom at N-1, and the hydrophobic
of their relative spatial distances. The substitution pattern and spa-
tial considerations of the -systems in regard to the quinazoline
nucleus proved to be critical for biological activity. Therefore, the
obtained pharmacophoric model could be useful for the develop-
ment of new DHFR inhibitors.
p-system regions) as well as
6-methyl or nitro-quinazolin-4(3H)-one (13 and 14)
p
A mixture of the 2-thioxo-quinazoline analogues 9 or 10
(0.01 mol), ethyl iodide (5 ml) and anhydrous potassium carbonate
(2 g) in acetone (50 ml) was heated under reflux for 8 h. The sepa-
rated solid was filtered while hot, washed with acetone, the filtrate
was then evaporated and the obtained residue was dried and
recrystallized from the suitable solvents.
5. Experimental
5.1.1. 3-Benzyl-2-ethylthio-6-methyl-quinazolin-4(3H)-one (13)
The crude product was recrystallized from EtOH (65%): mp
134–135 °C; 1H NMR (CDCl3) d 1.42 (t, 3H, J = 7.5 Hz, CH2CH3),
Melting points (°C) were determined on Mettler FP80 melting
point apparatus and are uncorrected. Microanalyses were per-
formed on a Perkin–Elmer 240 elemental analyzer at the Central

2858
F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
2.48 (s, 3H, CH3Ph), 3.26–3.30 (q, 2H, J = 7.5 Hz, –CH2CH3), 5.41 (s,
2H, CH2Ph), 7.28–7.39 (m, 5 h, ArH), 7.48–7.54 (m, 2H, ArH), 8.07
(s, 1H, ArH). 13C NMR d 13.9, 21.2, 26.8, 47.2, 119.1, 126, 126.5,
127.6, 127.7, 128.5, 135.7, 135.8, 135.9, 145.7, 155.8, 162.1; MS
m/e (310, 12%). Anal. (C18H18N2OS) C, H, N.
chromatographed on silica gel using 20% EtOAc/hexane as eluant
to afford 20 and the disulfide 21. An analytical pure sample of 20
was obtained and recrystallized from EtOAc (82%): mp 128–
130 °C; 1H NMR (CDCl3) d 3.99 (d, 2H, J = 12 Hz, CH2–CH@CH2),
5.23–5.41 (dd, 2H, J = 9.5 Hz, –CH2–CH@CH2), 5.42 (s, 2H, –CH2Ph),
5.95–6.03 (m, 1H, –CH2–CH@CH2), 7.28–7.40 (m, 5H, ArH), 7.66 (d,
1H, J = 9 Hz, ArH), 8.48–8.51 (dd, 1H, J = 2.5, 9 Hz, ArH), 9.14 (d, 1H,
J = 2.5 Hz, ArH). 13C NMR d 35.6, 47.8, 119.2, 119.6, 124.2, 127.5,
127.8, 128.1, 128.6, 128.7, 131.8, 134.7, 144.8, 151.0, 160.8,
161.2; MS m/e (353, 10%). Anal. (C18H15N3O3S) C, H, N.
5.1.2. 3-Benzyl-2-ethylthio-6-nitro-quinazolin-4(3H)-one (14)
The crude product was recrystallized from EtOH (72%): mp
149–150 °C; 1H NMR (CDCl3) d 1.45 (t, 3H, J = 7.5 Hz, two eclipsed
triplets for the two isomers S–CH2CH3 and N–CH2CH3), 3.30–3.34
(q, 2H, J = 7.5 Hz, for the S–CH2CH3 isomer), 3.47–3.51(q, 2H,
J = 7.5 Hz, for the N–CH2CH3 isomer), 5.41 (s, 2H, –CH2Ph), 7.28–
7.39 (m, 5H, ArH), 7.65 (d, 1H, J = 9 Hz, ArH), 8.47–8.50 (dd, 1H,
J = 2.5, 9 Hz, ArH), 9.13 (d, 1H, J = 2.5 Hz, 1H). 13C NMR d 8.2 (N-iso-
mer), 13.8, 27.3, 47.7, 53.3 (N-isomer), 119.2, 124.2, 127.5, 127.7,
128.0, 128.6, 128.7, 134.8, 144.6, 151.2, 160.8, 161.9; MS m/e
(341, 18%). Anal. (C17H15N3O3S) C, H, N.
5.6. Bis(1-allyl-6-nitro-quinazolin-4(1H)-one-2-yl)-disulfide
hydrochloride (21)
A light yellow oil, which was converted to its salt by the use of
ethereal HCl, identified to be the disulfide compound 21 (10%): 1H
NMR (CDCl3) d 4.86 (d, 4H, J = 12 Hz, –CH2–CH@CH2), 5.31–5.47
(ddd, 4H, J = 18.5, 1 Hz, –CH2–CH@CH2), 6.03–6.11 (m, 2H, –CH2–
CH@CH2), 6.45 (s, 1H, exchangeable), 6.69 (d, 2H, J = 9 Hz, ArH),
8.15–8.17 (dd, 2H, J = 2.5, 9 Hz, ArH), 8.89 (d, 2H, J = 2.5 Hz, ArH).
13C NMR d 65.8, 109.3, 116.21, 116.23, 119.0, 128.9, 129.3, 131.7,
137.5, 154.8, 166.5; MS m/e (524, 10%). Anal. (C22H17ClN6O6S2) C,
H, N.
5.2. 6-Amino-3-benzyl-2-methylthio-quinazolin-4(3H)-one (17)
A mixture of the 3-benzyl-2-methylthio-6-nitro-quinazolin-
4(3H)-one (12; 3.3 g, 0.01 mol), Fe powder pre-washed with dilute
HCl and water (0.5 g), concentrated HCl (10 ml), in ethanol (50 ml),
was heated under reflux for 0.5 h. The reaction mixture was cooled
and treated with concentrated ammonia (5 ml) to precipitate Fe
salts. The resulting mixture was filtered through Celite. Filtrate
was concentrated to give the crude products which were chro-
matographed on silica gel using 0.5% MeOH/CH2Cl2 as eluant. Pure
sample of the reported compound 1726 was obtained, along with a
material identified to be compound 18.
5.7. General procedure for preparation of 3-benzyl-2-
cinnamylthio-6-(methyl or nitro)-quinazolin-4(3H)-one (22
and 23)
A mixture of 2-thioxo-quinazoline analogues 9 or 10 (0.01 mol),
cinnamyl bromide (3.0 g, 0.015 mol) and anhydrous potassium car-
bonate (2 g) in DMF (50 ml) was heated under reflux for 8 h. Sol-
vent was then removed under reduced pressure. The obtained
residue was dissolved in CH2Cl2 and washed with 10% NaOH solu-
tion, then water. The organic layer was separated, dried and evap-
orated. The obtained residue was chromatographed on silica gel
using 25% EtOAc/hexane as eluant.
5.3. 6-Amino-3-benzyl-quinazolin-4(3H)-one (18)
The product was recrystallized from EtOH to yield brownish
crystals (15%): mp 168–169 °C; 1H NMR (CDCl3) d 3.95 (br s, 2H,
NH2), 5.10 (s, 2H, –CH2Ph), 7.00–7.02 (m, 1H, ArH), 7.19–7.26 (m,
5H, ArH), 7.42–7.45 (m, 2H, ArH), 7.84 (s, 1H, ArH). 13C NMR d
49.5, 109.2, 122.8, 123.3, 127.9, 128.1, 128.8, 128.9, 136.1, 140.8,
142.9, 146.1, 160.9; MS m/e (251, 11%). Anal. (C15H13N3O) C, H, N.
5.7.1. 3-Benzyl-2-cinnamylthio-6-methyl-quinazolin-4(3H)-one
(22)
Analytical pure gummy material of 22 was obtained (87%): 1H
NMR (CDCl3) d 2.50 (s, 3H, CH3Ph), 4.13 (d, 2H, J = 7.5 Hz, CH2–
CH@CHPh), 5.43 (s, 2H, –CH2Ph), 6.33–6.39 (m, 1H, –CH2–
CH@CH–Ph), 6.72 (d, 1H, J = 15.5 Hz, –CH2–CH@CH–Ph), 7.25–
7.56 (m, 12H, ArH), 8.10 (s, 1H, ArH). 13C NMR d 21.3, 35.1, 47.3,
113.9, 119.2, 123.8, 126.0, 126.4, 126.6, 127.6, 127.62, 128.4,
134.1, 134.7, 135.8, 135.8, 135.9, 136.6, 145.6, 155.2, 162.1; MS
m/e (398, 3%). Anal. (C25H22N2OS) C, H, N.
5.4. 2-Allylthio-3-benzyl-6-methyl-quinazolin-4(3H)-one (19)
A mixture of 3-benzyl-2-mercapto-6-methyl-quinazolin-4(3H)-
one (9; 2.8 g, 0.01 mol), allyl bromide (1.8 g, 1.3 ml, 0.015 mol) and
anhydrous potassium carbonate (2 g) in DMF (50 ml) was heated
under reflux for 7 h. Solvent was then removed under reduced
pressure, the obtained residue was dissolved in CH2Cl2 and washed
with 10% NaOH solution, then water. The organic layer was sepa-
rated, dried and evaporated. The obtained residue was chromato-
graphed on silica gel using 25% EtOAc/hexane as eluant. An
analytical pure sample of 19 was obtained and recrystallized from
EtOAc/hexane (89%): mp 109–10 °C; 1H NMR (CDCl3) d 2.45 (s, 3H,
CH3Ph), 3.94 (d, 2H, J = 6.5 Hz, –CH2–CH@CH2), 5.18–5.38 (dd, 2H,
J = 10 Hz, –CH2–CH@CH2), 5.39 (s, 2H, –CH2Ph), 5.96–6.03 (m, 1H,
–CH2–CH@CH2), 7.27–7.48 (m, 7H, ArH), 8.08 (m, 1H, ArH); MS
m/e (322, 8%). Anal. (C19H18N2OS) C, H, N.
5.7.2. 3-Benzyl-2-cinnamylthio-6-nitro-quinazolin-4(3H)-one (23)
The product was recrystallized from EtOAc/hexane to yield 23
(79%): mp 125–126 °C; 1H NMR (DMSO-d6)
d 4.18 (d, 2H,
J = 10 Hz, –CH2–CH@CH–Ph), 5.37 (s, 2H, –CH2Ph), 6.33–6.45 (m,
1H, CH2–CH@CHPh), 6.78 (d,1H, J = 15 Hz, –CH2–CH@CH–Ph),
7.21–7.50 (m, 10H, ArH), 7.87 (d,1H, J = 10 Hz, ArH), 8.57–8.59
(dd, 1H, J = 2.5, 10 Hz, ArH), 8.83 (d, 1H, J = 2.5 Hz, ArH). 13C NMR
d 34.6, 47.3, 118.8, 123.0, 123.5, 126.3, 126.8, 127.5, 127.8, 128.6,
128.9, 134.0, 135.0, 136.1, 144.2, 150.6, 160.2, 161.4; MS m/e
(M+2; 431, 10%). Anal. (C24H19N3O3S) C, H, N.
5.5. 2-Allylthio-3-benzyl-6-nitro-quinazolin-4(3H)-one (20)
A mixture of 3-benzyl-2-mercapto-6-nitro-quinazolin-4(3H)-
one (10; 3.1 g, 0.01 mol), allyl bromide (1.8 g, 1.3 ml, 0.015 mol)
and anhydrous potassium carbonate (2 g) in DMF (50 ml) was
heated under reflux for 12 h. Solvent was then removed under
reduced pressure, the obtained residue was dissolved in CH2Cl2
and washed with 10% NaOH solution, then water. The organic layer
was separated, dried and evaporated. The obtained residue was
5.8. General procedure for preparation of 2-(allylthio or
cinnamylthio)-6-amino-3-benzyl-quinazolin-4(3H)-one (24 and
25)
A mixture of 0.01 mol of 2-Allylthio-3-benzyl-6-nitro-quinazo-
lin-4(3H)-one (20), or 3-benzyl-2-cinnamylthio-6-nitro-quinazo-
lin-4(3H)-one (23), Fe powder pre-washed with dilute HCl and

F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
2859
water (0.5 g), concentrated HCl (10 ml), in ethanol (50 ml), was
heated under reflux for 0.5 h. The reaction mixture was cooled
and treated with concentrated ammonia (5 ml) to precipitate Fe
salts. The resulting mixture was filtered through Celite. Filtrate
was concentrated to give the crude products, which were chro-
matographed on silica gel using 0.5% MeOH/CH2Cl2 as eluant.
(t, 2H, J = 6.5 Hz, –S–CH2–CH2–CO–), 3.51 (t, 2H, J = 6.5 Hz, –S–
CH2–CH2–CO–), 5.39 (s, 2H, –CH2Ph), 7.28–7.38 (m, 5H, ArH),
7.49–7.55 (m, 2H, ArH), 8.08 (s, 1H, ArH), 11.10 (br s, 1H, exchange-
able OH). 13C NMR d 21.2, 26.3, 30.9, 47.3, 119.1, 126.0, 126.6,
127.6, 127.7, 128.4, 135.6, 136.0, 136.1, 145.5, 154.9, 162.1,
177.3. MS m/e (354, 1%). Anal. (C19H18N2O3S) C, H, N.
5.8.1. 2-Allylthio-6-amino-3-benzyl-quinazolin-4(3H)-one (24)
The product was recrystallized from MeOH to give 24 (45%): mp
136–137 °C; 1H NMR (CDCl3) d 3.91–3.93 (m, 4H, NH2 & CH2–
CH@CH2), 5.16–5.36 (dd, 2H, J = 10 Hz, –CH2–CH@CH2), 5.39 (s,
2H, –CH2Ph), 5.95–6.03 (m, 1H, –CH2–CH@CH2), 7.09–7.11 (m,
1H, ArH), 7.27–7.37 (m, 5H, ArH), 7.43–7.46 (m, 2H, ArH); MS m/
e (323, 52%). Anal. (C18H17N3OS) C, H, N.
5.10.2. 3-(3-Benzyl-6-nitro-4-oxo-3,4-dihydro-quinazolin-2-yl-
thio)-propanoic acid (28)
The product was recrystallized from EtOAc/hexane to afford 28
(80%): mp 198–199 °C; 1H NMR (DMSO-d6) d 2.76 (t, 2H, J = 6.5 Hz,
–S–CH2–CH2–CO–), 3.45 (t, 2H, J = 6.5 Hz, S–CH2–CH2–CO–), 5.32
(s, 2H, –CH2Ph), 7.28–7.33 (m, 5H, ArH), 7.72 (d, 1H, J = 9 Hz,
ArH), 8.08 (dd, 1H, J = 2.5, 9 Hz ArH), 8.78 (d, 1H, J = 2.5 Hz, ArH),
12.45 (br s, 1H, exchangeable OH). 13C NMR d 27.9, 33.6, 47.7,
119.2, 123.4, 127.3, 128.0, 128.2, 129.1, 129.3, 135.4, 144.6,
151.1, 160.6, 162.2, 173.1. MS m/e (M-1; 384, 1%). Anal.
(C18H15N3O5S) C, H, N.
5.8.2. 6-Amino-3-benzyl-2-cinnamylthio-quinazolin-4(3H)-one
(25)
The product was recrystallized from MeOH/H2O to afford 25
(52%): mp 157–158 °C; 1H NMR (CDCl3) 3.93 (br s, 2H, NH2), 4.10
(d, 2H, J = 7 Hz, –CH2–CH@CH–Ph), 5.41 (s, 2H, CH2Ph), 6.32–6.38
(m, 1H, –CH2–CH@CH–Ph), 6.69 (d, 1H, J = 15.5 Hz, –CH2–CH@CH–
Ph), 7.10–7.12 (m, 1H, ArH), 7.25–7.37 (m, 10H, ArH), 7.47–7.49
(m, 2H, ArH); MS m/e (399, 10%). Anal. (C24H21N3OS) C, H, N.
5.11. General procedure for preparation of 3-benzyl-1-cinnamoyl-
6-(methyl or nitro)-2-thioxo-2,3-dihydro-quinazolin-4(1H)-one
(29 and 30)
2-Thioxo-quinazoline analogues 9 or 10 (0.01 mol) was dis-
solved in toluene (30 ml), stirred in ice bath, and cinnamoyl chlo-
ride (6.7 g, 0.04 mol) was added dropwise over a period of
15 min. Stirring was continued in ice bath for 1 h then boiled under
reflux for 2 h. Solvent was then removed under reduced pressure;
the obtained residue was dissolved in CH2Cl2 and washed with 10%
NaOH solution, then water, separated and dried over MgSO4, then
evaporated in vacuo. The obtained residue was chromatographed
on silica gel using 0.5% MeOH/CH2Cl2 as eluant.
5.9. Ethyl 3-(3-benzyl-6-nitro-4-oxo-3,4-dihydro-quinazolin-2-
yl-thio)-propanoate (26)
To
a mixture of 3-benzyl-2-mercapto-6-nitro-quinazolin-
4(3H)-one (10, 3.1 g, 0.01 mol), and Et3N (2 ml), in CH2Cl2
(30 ml), stirred in ice bath, acryloyl chloride (3.2 ml, 0.04 mol)
was added dropwise over a period of 15 min. Stirring was contin-
ued in ice bath for 1 h then at room temperature overnight. Solvent
was then removed under reduced pressure; the obtained residue
was dissolved in CH2Cl2 and washed with 10% NaOH solution,
water, separated and dried over MgSO4 then evaporated in vacuo.
The obtained residue was chromatographed on silica gel using 10%
EtOAc/hexane as eluant. An analytical pure sample of 26 was ob-
tained and recrystallized from hexane/CH2Cl2 (94%): mp 100–
2 °C;. 1H NMR (CDCl3) d 1.19 (t, 3H, J = 7.5 Hz, –CH2CH3), 2.78 (t,
2H, J = 7.5 Hz, –S–CH2–CH2–CO–), 3.47 (t, 2H, J = 7.5 Hz, –S–CH2–
CH2–CO–), 4.08–4.12 (q, 2H, J = 7.5 Hz, CH2CH3), 5.30 (s, 2H,
–CH2Ph), 7.22–7.29 (m, 5H, ArH), 7.57 (d, 1H, J = 9 Hz, ArH),
8.40–8.42 (dd, 1H, J = 2.5, 9 Hz, ArH), 9.05 (d, 1H, J = 2.5 Hz, ArH).
13C NMR d 14.2, 27.7, 33.7, 47.7, 60.9, 119.3, 124.2, 127.5, 127.8,
128.1, 128.6, 128.7, 134.6, 144.8, 151.0, 160.7, 161.2, 171.4 (ester
C@O). MS m/e (M+2; 415, 3%). Anal. (C20H19N3O5S) C, H, N.
5.11.1. 3-Benzyl-1-cinnamoyl-6-methyl-2-thioxo-2,3-dihydro-
quinazolin-4(1H)-one (29)
The product was recrystallized from hexane/CH2Cl2 to afford 29
(79%): mp 185–186 °C; 1H NMR (CDCl3) d 2.44 (s, 3H, CH3PH), 5.82
(s, 2H, –CH2Ph), 6.89 (d, 1H, J = 15.5 Hz, –CO–CH@CH–Ph), 7.00 (d,
1H, J = 7.5 Hz, ArH), 7.29–7.34 (m, 3H, ArH), 7.42–7.46 (m, 4H,
ArH), 7.58–7.59 (m, 4H, ArH), 7.86 (d,1H, J = 15.5 Hz, –CO–
CH@CH–Ph), 8.07 (s, 1H, ArH). 13C NMR 20.8, 48.8, 114.6, 116.5,
121.8, 127.4, 127.5, 128.4, 128.7, 128.8, 129.1, 131.6, 133.8,
135.6, 135.7, 163.1, 136.6, 147.9, 159.2, 166.1, 173.5 (C@S). MS
m/e (M+1; 413, 1%). Anal. (C25H20N2O2S) C, H, N.
5.11.2. 3-Benzyl-1-cinnamoyl-6-nitro-2-thioxo-2,3-dihydro-
quinazolin-4(1H)-one (30)
5.10. Generalprocedureforpreparationof3-(3-benzyl-6-(methyl
or nitro)-4-oxo-3,4-dihydro-quinazolin-2-yl-thio)-propanoic
acid (27 and 28)
The product was recrystallized from hexane/CH2Cl2 to afford 30
(61%): mp 220–222 °C; 1H NMR (CDCl3) d 5.81 (s, 2H, CH2Ph), 6.86
(d, 1H, J = 15.5 Hz, –CO–CH@CH–Ph), 7.22 (d, 1H, J = 9 Hz, ArH),
7.33–7.36 (m, 3H, ArH), 7.44–7.50 (m, 3H, ArH), 7.56–7.61 (m,
4H, ArH), 7.93 (d,1H, J = 15.5 Hz, –CO–CH@CH–Ph), 8.45 (d, 1H,
J = 9 Hz, ArH), 9.13 (s, 1H, ArH). 13C NMR; 49.2, 115.8, 116.8,
120.8, 125.7, 128.1, 128.5, 128.9, 129.0, 129.2, 130.0, 132.1,
133.4, 135.2, 141.0, 144.3, 149.3, 157.7, 165.3, 174.5 (C@S). MS
m/e (443, 1%). Anal. (C24H17N3O4S) C, H, N.
2-Thioxo-quinazoline analogues 9 or 10 (0.01 mol) was dis-
solved in toluene (30 ml), stirred in ice bath, and acryloyl chloride
(3.2 ml, 0.04 mol) was added dropwise over a period of 15 min.
Stirring was continued in ice bath for 1 h then at room temperature
overnight. Solvent was then removed under reduced pressure; the
obtained residue was suspended in water and pH was adjusted to
neutrality using 10% ammonia solution, and then extracted with
CH2Cl2, washed with water, separated and dried over MgSO4, then
evaporated in vacuo. The obtained residue was chromatographed
on silica gel using 0.5% MeOH/CH2Cl2 as eluant.
5.12. General procedure for preparation of N-(3-benzyl-2-
substituted-thio-4-oxo-3,4-dihydro-quinazolin-6-yl)-
acrylamides (31–33)
6-Amino-quinazolin-4(3H)-one (17, 24 or 25, 0.01 mol), was
dissolved in CH2Cl2 (30 ml). Et3N (2 ml) was added and the mixture
was stirred in ice bath. Acryloyl chloride (3.2 ml, 0.04 mol) was
added dropwise over a period of 15 min. Stirring was continued
in ice bath for 4 h. The CH2Cl2 solution was then washed succes-
5.10.1. 3-(3-Benzyl-6-methyl-4-oxo-3,4-dihydro-quinazolin-2-
yl-thio)-propanoic acid (27)
The product was recrystallized from EtOAc/hexane to afford 27
(72%): mp 146–147 °C; 1H NMR (CDCl3) d 2.48 (s, 3H, CH3PH), 2.94

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F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
sively with water and 10% NaOH solution. The organic layer was
then separated, dried over MgSO4, and then evaporated in vacuo.
The obtained residue was flash chromatographed on silica gel
using 30% CH2Cl2/hexane as eluant.
5.13.2. N-(2-Allylthio-3-benzyl-4-oxo-3,4-dihydro-quinazolin-
6-yl)-cinnamamide (35)
The product was recrystallized from EtOH to afford 35 (59%):
mp 105–106 °C; 1H NMR (DMSO-d6) d 3.93 (d, 2H, J = 6 Hz, S–
CH2–CH@CH2), 5.15–5.39 (dd, 2H, J = 10 Hz, S–CH2–CH@CH2),
5.34 (s, 2H, CH2Ph), 5.89–6.05 (m, 1H, –S–CH2–CH@CH2), 6.86 (d,
1H, J = 15 Hz, Ph–CH@CH–CO), 7.22–7.74 (m, 12H, ArH), 8.04–
8.12 (m, 1H, ArH), 8.57 (s, 1H, ArH), 10.55 (br s, 1H, –CONH–).
MS m/e (453, 10%). Anal. (C27H23N3O2S) C, H, N.
5.12.1. N-(3-Benzyl-2-methylthio-4-oxo-3,4-dihydro-quinazolin-
6-yl)-acrylamide (31)
The product was recrystallized from CH2Cl2/hexane to afford 31
(42%): mp 236–237 °C; 1H NMR (CDCl3) d 2.64 (s, 3H, S–CH3), 5.43
(s, 2H, CH2Ph), 5.82 (d, 1H, J = 9.5 Hz, CH2@CH–CO), 6.27–6.32 (dd,
1H, J = 9.5 Hz, CH2@CH–CO), 6.44 (d, 1H, J = 16.5 Hz, CH2@CH–CO),
7.28–7.39 (m, 5H, ArH), 7.53 (br s, 1H, –CONH–), 7.62 (d, 1H,
J = 8.5 Hz, ArH), 8.10–8.11 (m, 1H, ArH), 8.32–8.46 (m, 1H, ArH).
MS m/e (351, 10%). Anal. (C19H17N3O2S) C, H, N.
5.13.3. N-(3-Benzyl-2-cinnamylthio-4-oxo-3,4-dihydro-
quinazolin-6-yl)-cinnamamide (36)
The product was recrystallized from EtOH to afford 36 (65%):
mp 180–182 °C; 1H NMR (DMSO-d6) d 4.11 (d, 2H, J = 7 Hz, S–
CH2–CH@CH–Ph), 5.34 (s, 2H, CH2Ph), 6.31–6.44 (m, 1H, cinnamyl
H), 6.68–6.90 (m, 3H, cinnamoyl and cinnamyl-H), 7.23–7.69 (m,
17H, ArH), 8.58 (s, 1H, ArH), 10.61 (br s, 1H, –CONH–). MS m/e
(M-1; 528, 1%). Anal. (C33H27N3O2S) C, H, N.
5.12.2. N-(2-Allylthio-3-benzyl-4-oxo-3,4-dihydro-quinazolin-
6-yl)-acrylamide (32)
The product was recrystallized from CH2Cl2/hexane to afford 32
(56%): mp 186–7 °C; 1H NMR (CDCl3) d 3.94 (d, 2H, J = 6 Hz, –S–
CH2–CH@CH2), 5.19–5.38 (dd, 2H, J = 10 Hz, S–CH2–CH@CH2),
5.39 (s, 2H, –CH2Ph), 5.58 (d, 1H, J = 10 Hz, CH2@CH–CO), 5.96–
6.04 (m, 1H, –S–CH2–CH@CH2), 6.16–6.22 (m, 1H, –CH2@CH–CO),
6.40 (d, 1H, J = 17 Hz, –CH2@CH–CO), 7.29–7.30 (m, 5H, ArH),
7.56 (d, 1H, J = 9 Hz, ArH), 8.13 (s, 1H, ArH), 8.50 (br s, 1H, –CON
H–), 8.60 (s, 1H, ArH). 13C NMR d 35.3, 47.4, 116.5, 119.0, 119.4,
127.1, 127.2, 127.6, 127.7, 127.9, 128.6, 131.0, 132.5, 135.3,
136.4, 144.2, 155.1, 162.0, 163.9. MS m/e (M+1; 378, 1%). Anal.
(C21H19N3O2S) C, H, N.
5.14. General procedure for preparation of 6-(allylamino or
diallylamino)-3-benzyl-2-methylthio-quinazolin-4(3H)-one (37
and 38)
A
mixture of 6-amino-3-benzyl-2-methylthio-quinazolin-
4(3H)-one (17, 3 g, 0.01 mol), allyl bromide (1.8 g, 1.3 ml,
0.015 mol) and anhydrous potassium carbonate (2 g) in DMF
(50 ml) was heated under reflux for 10 h. Solvent was then re-
moved under reduced pressure, the obtained residue was chro-
matographed on silica gel using 25% EtOAc/hexane as eluant. An
analytical pure sample of 37 was obtained along with the 6-dially-
lamino- analogue 38.
5.12.3. (E)-N-(3-Benzyl-2-cinnamylthio-4-oxo-3,4-dihydro-
quinazolin-6-yl)-acryl-amide (33)
The product was recrystallized from CH2Cl2/hexane to afford 33
(38%): mp 221–222 °C; 1H NMR (DMSO-d6) d 4.10 (d, 2H, J = 7 Hz,
S–CH2–CH@CH–Ph), 5.33 (s, 2H, –CH2Ph), 5.80 (d, 1H, J = 6 Hz,
–CH2@CH–CO), 6.28–6.51 (m, 3H, acryloyl and cinnamyl-H), 6.75
(d, 1H, J = 9.5 Hz, –CH2@CH–CO), 7.23–7.40 (m, 10H, ArH), 7.67
(d, 1H, J = 9 Hz, ArH), 8.03–8.11 (s, 1H, J = 9 Hz, ArH), 8.53 (s, 1H,
ArH), 10.51 (br s, 1H, –CONH–). MS m/e (453, 1%). Anal.
(C27H23N3O2S) C, H, N.
5.14.1. 6-Allylamino-3-benzyl-2-methylthio-quinazolin-4(3H)-
one (37)
The product was recrystallized from CH2Cl2/hexane to afford
37(35%): mp 143–144 °C; 1H NMR (CDCl3) d 2.50 (s, 3H, S–CH3),
3.78 (d, 2H, J = 5.5 Hz, –CH2–CH@CH2), 4.18 (br s, 1H, NH–), 5.10–
5.24 (dd, 2H, J = 10.5 Hz, –CH2–CH@CH2), 5.31 (s, 2H, –CH2Ph),
5.85–5.91 (m, 1H, –CH2–CH@CH2), 6.94–6.96 (dd, 1H, J = 2.5 Hz,
ArH), 7.15–7.28 (m, 6H, ArH), 7.34 (d, 1H, J = 8.5 Hz, ArH). 13C
NMR d 15.1, 46.5, 47.3, 106.0, 116.6, 120.3, 122.3, 127.2, 127.5,
127.53, 128.5, 134.7, 136.0, 140.4, 146.1, 152.5, 162.1. MS m/e
(337, 22%). Anal. (C19H19N3OS) C, H, N.
5.13. General procedure for preparation of N-(3-benzyl-2-
substituted-thio-4-oxo-3,4-dihydro-quinazolin-6-yl)-
cinnamamides (34–36)
6-Amino-quinazolin-4(3H)-one (17, 24 or 25, 0.01 mol), was
dissolved in CH2Cl2 (30 ml). Potassium carbonate (1.0 g) was added
and the mixture was stirred in ice bath. Cinnamoyl chloride (6.7 g,
0.04 mol) was added dropwise over a period of 15 min. Stirring
was continued in ice bath for 8 h. The CH2Cl2 solution was the
washed successively with water, and 10% NaOH solution. The or-
ganic layer was then separated, dried over MgSO4, and then evap-
orated in vacuo. The obtained residue was flash chromatographed
on silica gel using 30% CH2Cl2/hexane as eluant.
5.14.2. 3-Benzyl-6-diallylamino-2-methylthio-quinazolin-
4(3H)-one (38)
A light brownish yellow gummy material of 38 was obtained
(52%): 1H NMR (CDCl3) d 2.40 (s, 3H, S–CH3), 3.83–3.84 (m, 4H,
–CH2–CH@CH2), 5.02–5.06 (m, 4H, –CH2–CH@CH2), 5.21 (s, 2H,
–CH2Ph), 5.68–5.75 (m, 2H, –CH2–CH@CH2), 6.95–6.97 (m, 1H,
ArH), 7.06–7.22 (m, 5H, ArH), 7.28–7.32 (m, 2H, ArH). 13C NMR d
15.1, 47.5, 53.0, 106.7, 116.5, 120.2, 120.7, 127.1, 127.5, 127.6,
128.5, 133.4, 136.2, 139.4, 146.8, 152.2, 162.2. MS m/e (377,
10%). Anal. (C22H23N3OS) C, H, N.
5.13.1. N-(3-Benzyl-2-methylthio-4-oxo-3,4-dihydro-
quinazolin-6-yl)-cinnamamide (34)
5.15. General procedure for preparation of 6-(allylamino or
diallylamino)-2-allylthio-3-benzyl-quinazolin-4(3H)-one (39
and 40)
The product was recrystallized from EtOH to afford 34 (68%):
mp 141–142 °C; 1H NMR (DMSO-d6) d 2.59 (s, 3H, S–CH3), 5.35
(s, 2H, –CH2Ph), 6.86 (d, 1H, J = 15.5 Hz, Ph–CH@CH–CO), 7.25–
7.34 (m, 5H, ArH), 7.43–7.47 (m, 3H, ArH), 7.60–7.72 (m, 4H,
ArH), 8.06–8.08 (m, 1H, ArH), 8.57 (s, 1H, ArH), 10.58 (br s, 1H,
–CONH–). 13C NMR d 14.6, 46.8, 115.2, 118.9, 121.9, 126.6, 126.7,
126.7, 127.3, 127.8, 128.5, 129.0, 129.8, 134.6, 135.7, 137.1,
140.6, 143.1, 155.9, 160.8, 163.7. MS m/e (427, 10%). Anal.
(C25H21N3O2S) C, H, N.
A mixture of 2-allylthio-6-amino-3-benzyl-quinazolin-4(3H)-
one (24, 3.2 g, 0.01 mol), allyl bromide (1.8 g, 1.3 ml, 0.015 mol)
and anhydrous potassium carbonate (2 g) in DMF (50 ml) was
heated under reflux for 10 h. Solvent was then removed under re-
duced pressure, the obtained residue was chromatographed on sil-
ica gel using 25% EtOAc/hexane as eluant. An analytical pure

F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
2861
sample of 39 was obtained, along with the 6-diallylamino- ana-
logue 40.
Ph), 6.67 (d, 1H, J = 15.5 Hz, –CH2–CH@CH–Ph), 7.08–7.10 (m, 1H,
ArH), 7.26–7.48 (m, 12H, ArH). 13C NMR d 15.1, 46.2, 47.3, 106.0,
120.4, 122.4, 126.2, 126.4, 127.3, 127.6, 127.7, 128.5, 128.6,
128.8, 132.1, 136.0, 136.5, 140.5, 146.0, 152.6, 162.1. MS m/e
(413, 1%). Anal. (C25H23N3OS) C, H, N.
5.15.1. 6-Allylamino-2-allylthio-3-benzyl-quinazolin-4(3H)-one
(39)
A light brownish yellow gummy material of 39 was obtained
(58%): 1H NMR (CDCl3) d 3.85–4.29 (m, 5H, –S–CH2–CH@CH2, –
NH–CH2–CH@CH2), 5.17–5.42 (m, 6H, –CH2Ph, –S–CH2–CH@CH2,
–NH–CH2–CH@CH2), 5.84–6.04 (m, 2H, –S–CH2–CH@CH2, –NH–
CH2–CH@CH2), 7.04–7.06 (m, 1H, ArH), 7.27–7.35 m, 6H, ArH),
7.53–7.56 (m, 1H, ArH). 13C NMR d 35.3, 46.5, 47.4, 105.6, 116.5,
119.0, 120.4, 122.4, 127.3, 127.5, 127.6, 128.5 132.5, 134.7, 136.0,
140.2, 146.3, 151.3, 162.2. MS m/e (363, 15%). Anal. (C21H21N3OS)
C, H, N.
5.17.2. 3-Benzyl-6-dicinnamylamino-2-methylthio-quinazolin-
4(3H)-one (43)
A light brownish yellow gum of 43 was obtained (45%): 1H NMR
(CDCl3) d 2.64 (s, 3H, S–CH3), 4.20–4.29 (m, 4H, –CH2–CH@CH–Ph),
5.46 (s, 2H, –CH2Ph), 6.35–6.40 (m, 2H, –CH2–CH@CH–Ph), 6.66 (d,
2H, J = 15.5 Hz, CH2–CH@CH–Ph), 7.29–7.47 (m, 16H, ArH), 7.60–
7.62 (m, 1H, ArH), 7.71–7.72 (m, 1H, ArH). 13C NMR 14.4, 15.2,
21.1, 47.4, 52.4, 60.4, 106.9, 120.4, 121.1, 125.2, 126.6, 127.6,
128.6, 131.9, 136.2, 136.9, 139.7, 146.9, 152.5, 162.2, 171.1. MS
m/e (529, 12%). Anal. (C34H31N3OS) C, H, N.
5.15.2. 2-Allylthio-3-benzyl-6-diallylamino-quinazolin-4(3H)-
one (40)
A light brownish yellow gummy material of 40 was obtained
5.18. General procedure for preparation of 2-allylthio-3-benzyl-
6-(cinnamylamino or dicinnamylamino)-quinazolin-4(3H)-one
(44 and 45)
(29%): 1H NMR (CDCl3) d 3.93–4.04 (m, 6H, –S–CH2–CH@CH2
&
-N–CH2–CH@CH2), 5.13–5.44 (m, 8H, –CH2Ph, S–CH2–CH@CH2 &-
N–CH2–CH@CH2), 5.87–6.05 (m, 3H, S–CH2–CH@CH2 & –N–CH2–
CH@CH2), 7.15–7.19 (m, 1H, ArH), 7.62–7.52 (m, 7H, ArH). 13C
NMR d 35.2, 47.4, 53.0, 106.7, 116.7, 118.6, 120.3, 120.7, 127.1,
127.5, 127.6, 128.5, 133.0, 133.3, 136.1, 139.3, 146.9, 151.0,
162.3. MS m/e (403, 8%). Anal. (C24H25N3OS) C, H, N.
A mixture of 2-allylthio-6-amino-3-benzyl-quinazolin-4(3H)-
one (24, 3.2 g, 0.01 mol), cinnamyl bromide (3.0 g, 0.015 mol) and
anhydrous potassium carbonate (2 g) in DMF (50 ml) was heated
under reflux for 10 h. Solvent was then removed under reduced
pressure, the obtained residue was chromatographed on silica gel
using 10% EtOAc/hexane as eluant. An analytical pure sample of
44 was obtained, along with the 6-dicinnamylamino analogue 45.
5.16. 6-Allyl-(methyl)-amino-3-benzyl-2-methylthio-
quinazolin-4(3H)-one (41)
To a solution of 6-allylamino-3-benzyl-2-methylthio-quinazo-
lin-4(3H)-one (37, 1.7 g, 0.005 mol) in 25 ml of acetonitrile, formal-
dehyde (0.4 g, 0.011 mol) was added with constant stirring.
NaCNBH3 (0.5 g, 0.008 mol) was then added, and the pH of the
mixture was adjusted to 2–3 using concentrated HCl. A bright yel-
low precipitate was obtained. The acetonitrile was evaporated un-
der reduced pressure and the obtained residue was suspended in
10 ml of water, and neutralized using NH4OH to afford 41, which
was filtered, dried and recrystallized from CH2Cl2/hexane (81%):
mp 123–125 °C; 1H NMR (DMSO-d6) d 2.55 (s, 3H, S–CH3), 3.01
(s, 3H, N–CH3), 4.05 (s, 2H, –N–CH2–CH@CH2), 5.10–5.16 (m, 2H,
–N–CH2–CH@CH2), 5.32 (s, 2H, –CH2Ph), 5.81–5.90 (m, 1H, –N–
CH2–CH@CH2), 7.19 (d, 1H, J = 3 Hz, ArH), 7.19–7.36 (m, 6H, ArH),
7.46 (d, 1H, J = 9 Hz, ArH). 13C NMR d 15.0, 38.6, 47.1, 54.7, 105.8,
116.7, 119.9, 121.3, 127.2, 127.4, 127.7, 129.0, 133.9, 136.6,
138.9, 147.7, 152.4, 161.5. MS m/e (351, 30%). Anal. (C20H21N3OS)
C, H, N.
5.18.1. 2-Allylthio-3-benzyl-6-cinnamylamino-quinazolin-
4(3H)-one (44)
A gummy material of 44 was obtained (39%): 1H NMR (CDCl3) d
3.81–4.04 (m, 2H, NH–CH2–CH@CH–Ph), 4.26–4.28 (m, 2H, –S–
CH2–CH@CH2), 5.18–5.43 (m, 4H, CH2Ph, –S–CH2–CH@CH2),
5.98–6.06 (m, 1H, –S–CH2–CH@CH2), 6.31–6.35 (m, 1H, CH2–
CH@CH–Ph), 6.61 (d, 1H, J = 16 Hz, –CH2–CH@CH–Ph) 7.28–7.67
(m, 14H, ArH & NH). 13C NMR d 36.1, 47.3, 52.1, 106.9, 118.6,
120.4, 121.9, 125.1, 126.5, 127.4, 127.55, 127.6, 127.62, 128.55,
128.6, 131.9, 132.6, 136.1, 136.8, 139.5, 147.0, 151.3, 162.2. MS
m/e (439, 10%). Anal. (C27H25N3OS) C, H, N.
5.18.2. 2-Allylthio-3-benzyl-6-dicinnamylamino-quinazolin-
4(3H)-one (45)
A light brownish yellow gum of 45 was obtained (37%): 1H NMR
(CDCl3) d 3.95–4.28 (m, 6H,S–CH2–CH@CH2 & N–CH2–CH@CHPh),
5.19–5.44 (m, 4H, –CH2Ph, –S–CH2–CH@CH2), 5.98–6.05 (m, 1H, –
S–CH2–CH@CH2), 6.32–6.35 (m, 2H, –CH2–CH@CH–Ph), 6.61 (d,
2H, J = 16 Hz, –CH2–CH@CH–Ph), 7.15–7.61 (m, 18H, ArH), 13C
NMR 14.3, 29.8, 35.2, 47.4, 52.4, 106.9, 118.6, 121.0, 121.2, 125.1,
126.5, 127.4, 127.6, 128.56, 128.6,131.9, 132.9, 136.1, 136.8, 139.5,
147.0, 151.2,162.3. MS m/e (555, 15%). Anal. (C36H33N3OS) C, H, N.
5.17. General procedure for preparation of 3-benzyl-6-
(cinnamylamino or dicinnamylamino)-2-methylthio-
quinazolin-4(3H)-one (42 and 43)
A mixtureof 6-amino-3-benzyl-2-methylthio-quinazolin-4(3H)-
one (17, 3 g, 0.01 mol), cinnamyl bromide (3.0 g, 0.015 mol) and
anhydrous potassium carbonate (2 g) in DMF (50 ml) was heated
under reflux for 10 h. Solvent was then removed under reduced
pressure, the obtained residue was chromatographed on silica gel
using 10% EtOAc/hexane as eluant. An analytical pure sample of 42
was obtained, along with the 6-dicinnamylamino analogue 43.
5.19. Dihydrofolate reductase (DHFR) inhibition assay
The assay mixture contained 50
lM Tris–HCl buffer (pH 7.4),
50
l
M NADPH, 20
ll DMSO or the same volume of DMSO solution
containing the test compounds to a final concentration of 10ꢀ11 to
10ꢀ5 M, and 0.02 units of bovine liver DHFR or Human 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
5.17.1. 3-Benzyl-6-cinnamylamino-2-methylthio-quinazolin-
4(3H)-one (42)
was initiated by adding 25 lM FH2, the change in absorbance
A gummy material of 42 was obtained (34%): 1H NMR (CDCl3) d
2.61 (s, 3H, S–CH3), 4.03–4.16 (m, 2H, CH2–CH@CH–Ph), 4.2 (br s,
1H, NH), 5.42 (s, 2H, –CH2Ph), 6.34–6.38 (m, 1H, –CH2–CH@CH–
(DA/min) was measured at 340 nm. The activity under these con-
ditions was linear for 10 min.35 Results are reported as % inhibition
of enzymatic activity calculated using the following formula:

2862
F. A. M. Al-Omary et al. / Bioorg. Med. Chem. 18 (2010) 2849–2863
ꢀ
ꢁ
multiconformer method.53–55 Energy minima for investigated com-
D
A=mintest
% Inhibition ¼ 1 ꢀ
pounds were determined by a semi-empirical method AM156 (as
implemented in HyperChem 5.1). The conformations thus obtained
were confirmed as minima by vibrational analysis. Atom-centred
charges for each molecule were computed from the AM1 wave
functions (HyperChem 5.1) by the procedure of Orozco and Lu-
que,57 which provides derived charges that closely resemble those
D
A=minDMSO
The % inhibition values were plotted versus drug concentration (log
scale). The 50% inhibitory concentration (IC50) of each compound
was obtained using the GRAPH PAD PRISM program, version 3 (San Diego,
CA).
*
obtainable from ab initio 6-31G calculations.
5.20. Determination of in vitro antimicrobial activity
5.21.2. Flexible alignment and pharmacophore prediction
The investigated compounds were subjected to flexible align-
ment and pharmacophore prediction experiment using ‘Molecular
Operating Environment’ software (MOE of Chemical Computing
Group Inc., on a Core 2 duo 1.83 GHz workstation).50 The molecules
were built using the Builder module of MOE. Their geometry was
optimized by using the MMFF94 forcefield followed by a flexible
alignment using systematic conformational search. Lowest energy
aligned conformation(s) were identified through the analysis mod-
ule of DSV by Accelrys Inc.,58 and the distances among the pharma-
cophoric elements were measured.
The primary screen was carried out using the agar disc-diffu-
sion method36 using Müller–Hinton agar medium. Sterile filter pa-
per discs (8 mm diameter) were moistened with the compound
solution in dimethylsulfoxide of specific concentration 200
disc, the antibacterial antibiotic gentamicin, sulfacetamide
(100 g/disc) and the antifungal drug Clotrimazole (100 g/disc)
lg/
l
l
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 in case of bacteria and
at 25 °C for 48 h in case of C. albicans. The minimal inhibitory con-
centrations (MIC, lg/ml) for the test compounds against the same
Acknowledgments
microorganisms used in the primary screening were carried out
using the microdilution susceptibility method in Müller–Hinton
Broth.37 Test compounds, gentamicin, and Sulfacetamide were dis-
The financial support of College of Graduate Studies, King Saud
University, College of Pharmacy Research Center, and King Abdula-
ziz City for Science and Technology (Grant no. AT-14-19) are grate-
fully acknowledged. Thanks are due to the NCI, Bethesda, MD, for
performing the antitumor testing of the synthesized compounds.
solved in dimethylsulfoxide at concentration of 64
lg/ml. The two-
fold dilutions of the solution were prepared (64 till 0.5
lg/ml). 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 deter-
mined as the lowest concentration that completely inhibited visi-
ble growth of the microorganism as detected by unaided eye.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.03.019.
5.21. Docking and molecular dynamic simulations
References and notes
Lowest energy conformer of each new analogue ‘global-minima’
was docked into the hDHFR enzyme-binding domain. For each of the
quinazoline analogues, energy minimizations (EM) were performed
using 1000 steps of steepest descent, followed by conjugate gradient
minimization to a RMS energy gradient of 0.01 Kcal/mol Å. The ac-
tive site of the enzyme was defined using a radius of 8.0 Å around
LIH. MD simulations were carried out for each ligand, with the rest
of the enzyme kept fixed. The cofactor (NADPH) as a part of the en-
zyme structure was not fixed during the simulations. MD simulation
was performed using time steps of 0.001 pico-second (ps), a distance
dependant dielectric of 4.00, and a non-bonded cut-off distance of
8.0 Å, at 300 K. Complexes were first equilibrated for 10 ps and then
simulated for 40 ps. Trajectory frames were collected every 200
stepsfordetailedanalysisonthebasisofpotentialenergyandhydro-
gen bond interactions. These selected frames were minimized until
RMS deviation values of 0.01 kcal/mol Å were reached for the
active-site residues. Energy of binding was calculated as the differ-
ence between the energy of the complex and individual energies of
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