Synthesis, biological evaluation and molecular modeling study of 2-(1,3,4-thiadiazolyl-thio and 4-methyl-thiazolyl-thio)-quinazolin-4-ones as a new class of DHFR inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.07.070

A new series of 2-(1,3,4-thiadiazolyl- or 4-methyl-thiazolyl)thio-6-substituted-quinazolin-4-one analogs was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 29, 34, and 39 proved to be the most active DHFR inhibitors with IC₅₀values range of 0.1-0.6 μM. Compounds 28, 31 and 33 showed remarkable broad-spectrum antimicrobial activity comparable to the known antibiotic Gentamicin. Compounds 26, 33, 39, 43, 44, 50, 55 and 63 showed broad spectrum antitumor activity with GI values range of 10.1-100%. Molecular modeling study concluded that recognition with key amino acid Glu30, Phe31 and Phe34 is essential for binding. ADMET properties prediction of the active compounds suggested that compounds 29 and 34 could be orally absorbed with diminished toxicity.

Full text of DOI:10.1016/j.bmcl.2014.07.070

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation and molecular modeling study
of 2-(1,3,4-thiadiazolyl-thio and 4-methyl-thiazolyl-thio)-
quinazolin-4-ones as a new class of DHFR inhibitors
c
d
Sarah T. Al-Rashood a, Ghada S. Hassan b, , Shahenda M. El-Messery , Mahmoud N. Nagi ,
⇑
El-Sayed E. Habib e, Fatmah A. M. Al-Omary a, Hussein I. El-Subbagh b,
⇑
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia
b Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, PO Box 35516, Mansoura, Egypt
c Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, PO Box 35516, Mansoura, Egypt
d Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
e Department of Pharmaceutics and Pharmaceutical Technology (Microbiology), College of Pharmacy, Taibah University, Almadinah Almunawwarah 344, Saudi Arabia
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-(1,3,4-thiadiazolyl- or 4-methyl-thiazolyl)thio-6-substituted-quinazolin-4-one analogs
was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, and antitumor
activities. Compounds 29, 34, and 39 proved to be the most active DHFR inhibitors with IC50 values range
Received 2 May 2014
Accepted 28 July 2014
Available online xxxx
of 0.1–0.6 lM. Compounds 28, 31 and 33 showed remarkable broad-spectrum antimicrobial activity
comparable to the known antibiotic Gentamicin. Compounds 26, 33, 39, 43, 44, 50, 55 and 63 showed
broad spectrum antitumor activity with GI values range of 10.1–100%. Molecular modeling study con-
cluded that recognition with key amino acid Glu30, Phe31 and Phe34 is essential for binding. ADMET
properties prediction of the active compounds suggested that compounds 29 and 34 could be orally
absorbed with diminished toxicity.
Keywords:
Synthesis
Quinazolin-4-ones
DHFR inhibition
Antimicrobial testing
Antitumor screening
Molecular modeling study
Ó 2014 Elsevier Ltd. All rights reserved.
Dihydrofolate reductase (DHFR) catalyzes the reduction of
dihydro folate to tetrahydrofolate which couples with thymidylate
synthase in the reductive methylation of deoxyuridine to deoxythy-
midine. The inhibition of DHFR activity leads to cellular deficiency
of tetrahydrofolate cofactors that result in cell death.1,2 DHFR
inhibition has long been identified as an important target for the
development of chemotherapeutic agents against bacterial and
parasitic infections as well as cancer.3 DHFR inhibitors are broadly
classified as either classical or non-classical antifolates. Literature
citations revealed numerous compounds which categorized under
the non-classical DHFR inhibitors such as: Trimethoprim (TMP, A),
trimetrexate (TMQ, B) and piritrexim (PTX, C), beside others belong
to the quinazoline heterocycle,4–12 (Chart 1).
allocation of compounds D–I as active DHFR inhibitors with IC50
values around 0.4 M. (Chart 1). Compounds D and F characterized
by bearing 2-thioallylic hydrophobic
-system,14 while com-
pounds G–I characterized by bearing 2-thiazolyl-thio
-system.15
Molecular modeling studies of this class of compounds revealed
the importance of the main pharmacophoric groups (the
4-carbonyl fragment, the basic nitrogen atom at N-1, and the
l
p
p
hydrophobic
p-system regions) as well as of their relative spatial
distances. The substitution pattern and spatial considerations of
the
p-systems in regard to the quinazoline nucleus proved to be
critical for DHFR inhibition.13–17
In continuation to our previous efforts,13–30 a new series of qui-
nazolin-4-one analogs was designed bearing 2-(1,3,4-thiadiazolyl-
Recently, a new series of 2,3,6-substituted-quinazolin-4-ones
was designed, synthesized, and evaluated for their in vitro DHFR
inhibition in our laboratories.13–17 The type of 2-, 3- or 6-substitu-
ent on the studied quinazolines manipulated and affected the mag-
nitude of the DHFR inhibition activity. This study allowed the
or 4-methyl-thiazolyl-)thio-functions as hydrophobic p-system
regions replacing the 2-thioalkyl or 2-thioallyl function of the lead
compounds D–F; and as isosters of the prototypes G–I to explore
the scope and limitations of this new class of DHFR inhibitors. In
addition, 6-chloro, 6-methyl, or 6,7-dimethoxy functions, repre-
senting electron donating and electron withdrawing substituents;
a phenyl or benzyl group at position 3- were introduced to the
quinazolin-4-one nucleus in resemblance to the leads D–I. Most
of the functions designed to be accommodated on the quinazoline
⇑
Corresponding authors. Tel.: +20 50 2247800; fax: +20 50 2247900.
E-mail addresses: ghadak25@yahoo.com (G.S. Hassan), subbagh@yahoo.com
(H.I. El-Subbagh).
http://dx.doi.org/10.1016/j.bmcl.2014.07.070
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Al-Rashood, S. T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.070

2
S. T. Al-Rashood et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
OCH3
OCH3
NH2 CH3
OCH3
OCH3
NH2
N
NH2 CH3
N
OCH3
OCH3
N
N
N
N
H
OCH3
H2N
N
N
H2N
H2N
OCH3
A
B
. TMQ
, TMP
O
C
, PTX
O
Bn
N
Bn
O
N
H
N
S
N
Bn
S
N
N
N
N
N
CH3
S
E
F
D
O
O
N
O
CH3O
CH3O
CH3O
CH3O
H3C
N
N
S
N
S
N
S
N
S
COOEt
S
S
N
N
N
H2N
H2N
H2N
I
H
G
Chart 1. Structures of some literature antifolate lead compounds.
R1
R2
COOH
NH2
+
R3
NCS
1: R1= CH3, R2= H
4: R3 = H
5: R3 = CH3
2:
R1= Cl, R2= H
6:
R3 = OCH3
3: R1= R2= OCH3
7: R3 = Cl
8: R3 = OC6H5
EtOH, Et3N
R3
O
R1
R2
N
S
N
H
H3C
Br
9-23
N N
N
Br
NH2
NH2
S
S
24
40
DMF
DMF
K2CO3
K2CO3
R3
R3
O
O
N
R1
R2
R1
R2
N
N
S
N
S
S
CH3
S
N
N
N
H2N
H2N
25-39
41-55
Scheme 1. Synthesis of the target compounds 25–39 and 41–55.
ring such as thioether, heteroaryl groups are known to contribute
to DHFR inhibition activity.31,32 The aim of this study is to locate
novel synthetic lead compound(s), and their in vitro testing as
DHFR inhibitor(s). Compounds possessing DHFR inhibition activity
are candidates for treating cancer and bacterial infections. Accord-
ingly, the obtained derivatives were tested for their in vitro antimi-
crobial activity against a panel of 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.33–36
The synthetic strategy to obtain the target compounds is
depicted in Schemes 1 and 2. The starting materials 6-methyl-3-
substituted-2-thioxo-2,3-dihydro-quinazolin-4(1H)-ones (9–13),
6-chloro-3-sub-stituted-2-thioxo-2,3-dihydro-quinazolin-4(1H)-
ones (14–18), 6,7-dimethoxy-3-substituted-2-thioxo-2,3-dihydro-
quinazolin-4(1H)-ones (19–23) and 3-benzyl-6-substituted or
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S. T. Al-Rashood et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
6,7-disubstituted-2-thioxo-2,3-dihydro-quinazolin-4(1H)-ones
(57–59) were prepared adopting reported procedures.29,30 The
2-thioxo-function of the starting materials 9–23 was then
alkylated using either 2-amino-5-bromo-1,3,4-thiadiazole (24), or
2-amino-5-bromo-4-methylthiazole (40) in dimethylformamide
(DMF) in presence of potassium carbonate to afford 2-(5-amino-
1,3,4-thiadiazol-2-yl-thio)-3-(4-substituted-phenyl)-6-substituted
or 6,7-disub-stituted-quinazolin-4(3H)-ones (25–39) and 2-(2-
amino-4-methylthiazol-5-yl-thio)-3-(4-substituted-phenyl)-6-
substituted or 6,7-disubstituted-quinazolin-4(3H)-ones (41–55)
respectively, Scheme 1. Similarly, the 2-thioxo-function of the
starting materials 57–59 was reacted with 24 or 40 under the same
conditions to afford 2-(5-amino-1,3,4-thiadiazol-2-yl-thio)-3-
benzyl-6-substituted or 6,7-disubstituted-quinazolin-4(3H)-ones
(60–62) and 2-(2-amino-4-methylthiazol-5-ylthio)-3-benzyl-
6-substituted or 6,7-disubstituted-quinazolin-4(3H)-ones (63–65)
respectively, Scheme 2. Structure elucidation of the synthesized
intermediates and final products was attained by the aid of ele-
mentary analyses, 1H & 13C NMR spectroscopy, and mass spec-
trometry. The characteristic 2-amino function attached to the
thiadiazole nucleus in 25–39 and 60–62 appeared as exchangeable
singlet in their respective 1H NMR spectra, while the 3H singlet
peak at d 2.5 d ppm indicates the presence of the 4-methyl amino-
thiazole moiety in compounds 41–55 and 63–65. The 13C-NMR
spectra of the synthesized compounds were in agreement with
the proposed structures. For instance, compound 27 showed 18
absorption carbon peaks, the carbonyl and imine carbons of the
The synthesized compounds (25–39, 41–55, and 60–65) were
evaluated as inhibitors of bovine liver DHFR using reported proce-
dure.37 Results were reported as IC50 values (Tables 2). Compounds
28, 29, 32–34, 36, 39, 50 and 53 proved to be the most active DHFR
inhibitors with IC50 values range of 0.1–1.0
27, 41, 45–47, 54, 60, 63, 64 were considered of moderate activity
with IC50 range of 1.0–5.0 M, the rest of the tested compounds
were considered to be inactive with IC50 > 5 M. Methotrexate
(IC50 0.008 M) was used as a positive control.
lM, while compounds
l
l
l
The synthesized compounds (25–39, 41–55, and 60–65) were
tested for their in vitro antimicrobial activity against a panel of stan-
dard 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 fun-
gus Candida albicans. The primary screen was carried out using the
agar disc-diffusion method using Müller-Hinton agar medium.38,39
The broad spectrum antibiotic Gentamicin (100
lg/disc), and the
DHFR inhibitor Sulphacetamide (100 g/disc) were used as positive
l
controls. The obtained results revealed that the tested compounds
expressed varying degrees of activity against the tested microorgan-
isms (Table 2). Strong activity (>15 mm inhibition zone) against the
Gram-positive bacteria was observed for compounds 28, 31, 33, 45,
47 and 63–65 against S. aureus and B. subtilis. Compounds 28, 45 and
65 showed activity comparable to the used positive controls. In case
of Gram-negative bacteria, strong activity was observed for com-
pounds 28, 31 and 33 against E. coli. Compounds 28, 31 and 33
showed
a remarkable broad-spectrum activity against both
quinazolinone ring resonated at
respectively, while different aromatic carbons resonated at d
125.8–159.2 ppm (Table 1).
d
175.9 and 160.2 ppm,
Gram-positive and Gram-negative bacteria. Compounds 45, 47
and 63–65 appeared to be selective and active against gram positive
bacteria. The minimal inhibitory concentrations (MICs) for the most
NCS
R1
R2
COOH
NH2
+
1: R1= CH3, R2= H
2: R1= Cl, R2= H
56
3
: R1= R2= OCH3
EtOH, Et3N
O
R1
R2
N
N
H
S
H3C
N
57-59
N N
Br
NH2
Br
NH2
S
24
S
40
DMF
DMF
K2CO3
K2CO3
O
N
O
N
R1
R2
R1
R2
N
S
N
S
S
CH3
S
N
N
N
H2N
H2N
6
63-65
Scheme 2. Synthesis of the target compounds 60–62 and 63–65.
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4
S. T. Al-Rashood et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1
Physicochemical properties of the synthesized compounds 25–39, 41–55, and 60–65
R3
R3
O
N
O
N
O
N
O
N
R1
R2
R1
R2
R1
R2
R1
R2
N
S
N
S
N
S
N
S
S
S
S
S
CH3
CH3
N
N
N
N
N
N
H2N
H2N
63-65
H2N
H2N
25-39
60-62
41-55
Compd
R1
R2
R3
Yield %
Mp °C
Molecular formulaea
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
60
61
62
63
64
65
CH3
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
CH3
CH3
CH3
CH3
Cl
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
54
66
52
60
74
44
59
49
75
79
45
55
68
51
70
62
54
59
50
55
64
43
59
58
69
58
55
49
62
68
41
53
39
68
72
56
153–5
173–6
151–3
133–5
192–4
174–6
126–8
162–4
135–7
182–4
166–8
156–8
110–3
121–4
133–5
167–9
143–5
182–4
166–8
132–4
160–2
154–6
133–5
188–90
196–8
154–7
122–4
135–7
158–60
194–6
160–2
144–9
141–3
175–7
122–4
162–4
C17H13N5OS2
C18H15N5OS2
CH3
OCH3
Cl
OC6H5
H
CH3
OCH3
Cl
OC6H5
H
CH3
OCH3
Cl
OC6H5
H
CH3
OCH3
Cl
OC6H5
H
CH3
OCH3
Cl
OC6H5
H
CH3
OCH3
Cl
OC6H5
—
C18H15N5O2S2
C17H12ClN5OS2
C23H17N5O2S2
C16H10ClN5OS2
C17H12ClN5OS2
C17H12ClN5O2S2
C16H9Cl2N5OS2
C22H14ClN5O2S2
C18H15N5O3S2
C19H17N5O3S2
C19H17N5O4S2
C18H14ClN5O3S2
C24H19N5O4S2
C19H16N4OS2
C20H18N4OS2
C20H18N4O2S2
C19H15ClN4OS2
C25H20N4O2S2
C18H13ClN4OS2
C19H15ClN4OS2
C19H15ClN4O2S2
C18H12Cl2N4OS2
C24H17ClN4O2S2
C20H18N4O3S2
C21H20N4O3S2
C21H20N4O4S2
C20H17ClN4O3S2
C26H22N4O4S2
C18H15N5OS2
Cl
Cl
Cl
Cl
H
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
Cl
OCH3
CH3
Cl
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
OCH3
H
—
—
—
—
C17H12ClN5OS2
C19H17N5O3S2
C20H18N4OS2
C19H15ClN4OS2
C21H20N4O3S2
H
OCH3
OCH3
—
a
Compounds analyzed for C,H,N,; results were within 0.4 % of the theoretical values for the given formulae.
active compounds were carried out using the micro-dilution
susceptibility method. Comparing the potency of the active
antibacterial compounds and their DHFR inhibition revealed that
compounds 28 and 33 might exert their activity through DHFR
inhibition.
The synthesized compounds (25–39, 41–55, and 60–65) were
subjected to the National Cancer Institute (NCI) in vitro disease-
oriented human cells screening panel assay for in vitro antitumor
55 with GI values of 55.7% and 46.1%, respectively; RPMI-8226 cell
line toward 39 with GI value of 43.7%, also SR cell line toward com-
pound 39 and 44 with GI values of 57.3% and 49.2%, respectively;
while compounds 50 and 55 proved lethal to this particular cell
line. Non-small cell lung cancer HOP-92 proved sensitive toward
compounds 32, 33, 38, 43, 44 and 63 with GI values of 42.1%,
53.7%, 40.9%, 42.3%, 43.3% and 50.2%, respectively. Compound 50
showed GI values of 66.7%, 44.6% and 44.1% toward HCT-116,
HCT-15, and HT29, respectively; while compound 55 showed GI
value of 52.7% against HCT-116 Colon cancer. OVCAR-4 ovarian
cancer cell line proved sensitive toward compounds 50 and 55
with GI values of 59.1% and 49.3%, respectively. Regarding renal
cancer cell lines, compound 44 showed GI values of 53.0% and
46.0% against A498 and UO-31, respectively; while compound 50
showed GI value of 60.2% against A498. Finally, concerning pros-
tate cancer cell lines, compound 50 showed GI values of 51.1%
against PC-3 cell line (Tables 3 and 4). Comparing the potency of
the active antitumor compounds and their DHFR inhibition
revealed that compounds 33, 39 and 50 might exert their
antitumor activity through DHFR inhibition.
activity. A single dose (10 lM) of the test compounds were used
in the full NCI 60 cell lines panel assay which includes nine tumor
subpanels namely; Leukemia, Non-small cell lung, Colon, CNS, Mel-
anoma, Ovarian, Renal, Prostate, and Breast cancer cells.33–36 The
data reported as mean-graph of the percent growth of the treated
cells, and presented as percentage growth inhibition (GI %) caused
by the test compounds. Compounds 26, 33, 38, 39, 43, 44, 50, 55
and 63 showed broad spectrum potency toward several tumor cell
lines with GI values range of 10.1–100%. Concerning activity
toward individual cell lines, leukemia CCRF-CEM and HL-60(TB)
cell lines showed sensitivity toward compound 50 with GI values
of 58.5% and 40.6%, respectively; MOLT-4 cell line toward 50 and
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S. T. Al-Rashood et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
Table 2
DHFR inhibition (IC50, lM), and antimicrobial activity results of compounds 25–39, 41–55, and 60–65
Compd
DHFR inhibition
Inhibition Zone (mm)
S. aureus
B. subtilis
E. coli
P. aeuroginosa
C albicans
25
26
7
8
—
—
—
—
—
—
—
—
—
—
27
3
—
—
—
—
—
28
29
30
0.7
0.1
6
24 (2.0)
—
—
26 (2.0)
—
—
18 (2.0)
—
—
12
—
—
—
—
—
31
32
33
34
35
36
8
0.8
1
0.4
10
1
18 (8.0)
—
18 (8.0)
—
—
14
20 (4.0)
—
20 (4.0)
—
—
15
18 (4.0)
—
16 (8.0)
—
—
—
14
—
12
—
—
—
10
—
—
—
—
—
37
7
—
—
—
—
—
38
6
—
—
—
—
—
39
41
0.6
5
—
—
—
—
—
—
—
—
—
—
42
7
—
—
—
—
—
43
7
—
—
—
—
—
44
7
—
—
—
—
—
45
2
26 (2.0)
22 (2.0)
14
—
—
46
5
—
—
—
—
—
47
5
18 (8.0)
16 (8.0)
—
—
—
48
8
—
—
—
—
—
49
8
—
—
—
—
—
50
1
—
—
—
—
—
51
7
—
—
—
—
—
52
8
—
—
—
—
—
53
1
—
—
—
—
—
54
2
18
14
—
—
—
55
6
—
—
—
—
—
60
2
—
—
—
—
—
61
8
—
—
12
—
—
62
6
—
14
—
—
—
63
64
65
5
5
6
—
—
18 (4.0)
18 (4.0)
20 (4.0)
27 (2.0)
20 (2.0)
21 (2.0)
19 (4.0)
24 (2.0)
25 (2.0)
22 (2.0)
—
12
—
18 (2.0)
18 (2.0)
—
—
—
12
—
—
19 (1.0)
25 (2.0)
Gentamicin
Sulphacetamide
21 (0.5)
28 (1.0)
(—) 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.
In the present investigation, the type of 2-, 3- or 6-substituent
on the studied quinazolines manipulated the DHFR inhibition
activity. Two different groups of quinazoline analogs were
synthesized differ in the type of the 2-thioether function attached
namely, 5-Amino-1,3,4-thiadiazole (25–39, 60–62) as isosters to
quinazoline nucleus in addition to the 4-position of the 3-phenyl
function proved to manipulate and contribute to the DHFR
inhibition activity in the following order: 2-(5-Amino-1,3,4-thia-
diazol-2-yl-thio)- > 2-(2-amino-4-methylthiazol-5-yl-thio)-; and
3-phenyl-> 3-benzyl-. The obtained antitumor results added
another piece of evidence which empathize the order of activity
what was concluded in the DHFR inhibition discussion that the
the lead compound G (IC50 0.8
(41–55, 63–65) as isosters to the lead compounds H and I (IC50 0.5
and 0.3 M, respectively). The 2-thioether function affects the
magnitude of DHFR inhibition. The order of activity proved to be
5-Amino-1,3,4-thiadiazole (IC50 0.4–1.0 M) > 2-amino-4-
methylthiazole (IC50 1.0–2.0 M). In the 6-methyl series, the
presence of 3-phenyl and 2-(5-Amino-1,3,4-thiadiazol-2-yl-thio)-
produced 25 with IC50 7.0 M with nine fold decrease in activity
compared to G. Replacing of the 3-phenyl function of 25 by
3-benzyl group produced 60 with IC50 2.0 M with almost four
lM); and 2-amino-4-methylthiazole
l
2-(5-Amino-1,3,4-thiadiazol-2-yl-thio)-series
represented
by
compounds 26, 33 and 39 is more active antitumors than the
l
2-(2-amino-4-methylthiazol-5-yl-thio)-series
represented
by
l
compounds 43, 44, 50, 55, and 63.
The DHFR inhibitory activity of the new synthesized com-
pounds 25–39, 41–55, and 60–65 was experimentally determined.
Compounds 29, 34 and 39 proved to be the most active members in
l
l
the present study (IC50 of 0.1, 0.4 and 0.6
lM, respectively) com-
folds increase in activity. The introduction of substituent at
position 4- of the 3-phenyl function of 25 increased the activity
producing the equipotent 3(4-chlorophenyl)-(28) with IC50
pared to the used positive control MTX (IC50, 0.008
l
M). Molecular
modeling study was essentially needed to understand and inter-
pret the unusual DHFR inhibitory pattern of this new class of com-
pounds. It was interesting to start a comparative modeling study of
the most active DHFR inhibitors 29, 34 and 39 and the least active
0.7
nyl)-29 with IC50 0.1
also observed in the other 6-chloro and 6,7-dimethoxy series
producing 2-(5-amino-1,3,4-thiadiazol-2-ylthio)-6-chloro-3-(4-
l
M and the eight fold more active analog 3(4-phenoxyphe-
lM compared to G. The same analogy was
compounds 35 and 52 (IC50 of 10 and 8 lM, respectively) against
MTX. 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 references for modeling
and docking.40–42 The binding of MTX to hDHFR is considered to be
phenoxyphenyl)-quinazolin-4(3H)-one (34), and 2-(5-amino-
1,3,4-thiadiazol-2-ylthio)-6,7-dimethoxy-3-(4-phenoxy-phenyl)-
quinazolin-4(3H)-one (39) with IC50 0.4, 0.6
lM, respectively. In
general, the type of substituent at positions 2-, 3-, and 6- of the
a
complex interaction where hDHFR undergo some kind of
Please cite this article in press as: Al-Rashood, S. T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.070

6
S. T. Al-Rashood et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 3
Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10
lM concentrations of compounds 26–39
Subpanel tumor cell lines
% Growth inhibitiona,b (GI %)
26
30
32
33
34
35
37
38
39
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
—
28.2
—
—
—
—
21.5
—
—
19.1
—
—
—
—
12.0
—
—
—
—
—
12.1
—
—
12.0
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
11.2
22.5
29.6
22.9
43.7
57.3
31.5
15.3
—
—
14.4
32.7
19.1
—
Non-small cell lung cancer
A549/ATCC
HOP-62
HOP-92
NCI-H226
—
—
21.1
—
—
—
—
28.6
—
—
—
—
42.1
13.4
—
11.1
16.7
53.7
—
—
—
—
—
—
—
15.4
—
—
—
—
33.7
—
—
—
—
40.9
—
—
15.2
—
—
13.4
23.6
NCI-H522
—
10.7
Colon cancer
HCT-116
HCT-15
HT29
11.6
—
—
—
—
—
—
—
—
—
—
—
12.7
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
11.5
21.4
17.6
20.7
KM12
—
—
CNS cancer
SF-295
SF-539
SNB-19
U251
—
15.6
—
—
—
—
—
11.0
—
—
20.6
—
—
—
—
—
—
10.6
—
—
—
—
—
—
12.1
—
11.9
11.5
—
—
—
—
—
—
11.0
—
Melanoma
MALME-3 M
MDA-MB-435
SK-MEL-28
SK-MEL-5
—
—
—
—
—
14.8
—
—
—
—
—
—
—
—
—
—
—
—
—
10.8
—
10.9
12.3
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
12.4
—
—
—
—
—
—
—
28.2
—
UACC-257
UACC-62
—
22.1
Ovarian cancer
OVCAR-4
—
—
—
—
—
—
—
—
34.6
Renal cancer
786-0
A498
CAKI-1
RXF 393
UO-31
—
—
—
—
—
—
—
36.4
—
—
21.1
15.4
30.6
—
—
24.6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
19.3
—
10.7
—
14.2
10.4
10.5
23.2
18.8
12.6
—
17.0
14.2
23.6
Prostate cancer
PC-3
11.8
—
—
14.1
—
10.9
—
11.2
23.2
Breast cancer
MCF7
MDA-MB-231/ATCC
BT-549
T-47D
MDA-MB-468
—
21.9
—
—
—
16.4
—
—
21.4
—
13.2
—
—
12.0
—
12.7
—
13.3
29.2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
10.9
13.7
—
14.8
—
—
38.4
22.6
10.4
a
GI < 10%; nt, not tested; L, compound proved lethal to the cancer cell line.
Compounds 25, 27–29, 31and 36 proved inactive.
b
isomerization or conformational change, resulting in tight binding,
in addition to ionic bonding of N1 and 2-NH2 to Glu30 (Fig. 1).43–47
The calculated binding free energies were used as the parameter of
selection for the cluster of docking posed to be evaluated in which
the binding mode of the lowest energy structure located in the top
docking cluster. The interaction of the most active derivative 29
with DHFR binding site shows key interaction with amino acid
Glu30 in resemblance to MTX which explain the high inhibitory
activity of this compound. Compound 34 binding mode revealed
a triad interaction with DHFR binding site via arene–arene interac-
tion with Phe31 and a side chain acceptor relationship with both
carbonyl group of the quinazoline core and one of the nitrogens
of the thiadiazole ring via ser59 and Try24, respectively. In case
of 39 a tight binding interactions played a role in its DHFR inhibi-
tory activity where a binary interaction of thiadiazole moiety
bounded to Phe34 and to Glu30 which is in resemblance to MTX.
The phenyl moiety of the quinazoline ring was embedded in a
hydrophobic pocket constructed by the side chains of Tyr22 resi-
due (Fig. 1). On the contrary, compound 35, the least active deriv-
ative compared to 29, lacks any interaction with the amino acids in
the binding site of DHFR enzyme. In case of compound 52, the only
interaction occurred was between one of the methoxy groups and
Arg77 residue which does not seem to have any impact on the
biological activity (Fig. 2). To probe similarity between the 3D
structures of the most active compound 29 and MTX, flexible align-
ment was employed. The initial approach was to employ MOE/
MMFF94 flexible alignment to automatically generate superposi-
tion of the compounds under investigation with minimal user
bias,48 200 conformers of each compound were generated and
minimized with a distance-dependant dielectric model. A low
energy set of 100 was selected for further analysis. The top scoring
alignment with the least strain energy is shown in Figure 3. There
Please cite this article in press as: Al-Rashood, S. T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.070

S. T. Al-Rashood et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
7
Table 4
Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10
lM concentrations of compounds 43–55, 60–64
Subpanel tumor cell lines
% Growth inhibitiona,b (GI %)
43
44
46
48
50
51
55
61
62
63
64
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
—
—
13.0
—
16.2
—
—
—
15.6
19.0
—
—
—
—
—
—
—
—
—
14.3
—
14.6
13.5
58.5
40.6
25.7
55.7
32.0
L
—
—
—
—
—
15.0
35.5
21.2
21.5
46.1
32.4
L
—
—
—
—
18.5
—
14.9
—
17.4
34.2
28.1
19.0
—
—
17.6
16.0
20.8
16.1
24.3
24.8
16.8
17.3
—
49.2
13.4
Non-small cell lung cancer
A549/ATCC
HOP-92
NCI-H226
NCI-H23
15.6
42.3
—
—
—
16.9
43.3
—
10.1
—
—
—
—
—
—
—
—
29.8
25.6
13.6
26.0
18.0
14.7
—
—
—
—
—
25.6
16.7
20.5
10.3
—
—
16.3
—
—
—
10.3
—
—
26.7
—
—
—
22.1
50.2
22.7
19.2
—
—
24.9
11.8
—
—
10.5
16.8
10.4
13.6
—
NCI-H460
NCI-H522
—
—
—
—
—
35.4
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
24.1
19.6
52.7
20.1
25.0
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
26.0
66.7
44.6
44.1
20.2
33.9
19.1
25.3
—
19.4
—
22.8
27.7
11.7
24.6
—
20.7
13.5
—
18.8
—
18.8
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
10.6
10.6
—
15.5
11.9
15.7
13.2
18.2
13.9
11.6
—
—
—
—
—
—
—
—
27.6
—
—
—
—
32.2
—
30.7
—
22.8
—
—
—
—
—
—
—
—
13.5
—
—
—
—
11.4
—
—
—
10.8
21.9
15.1
—
22.7
12.2
13.3
—
—
—
—
14.6
18.7
—
13.3
12.4
21.5
—
—
—
—
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-5
UACC-257
UACC-62
11.0
—
—
—
15.7
—
16.5
—
15.6
—
16.3
13.6
26.0
—
—
—
—
—
—
—
—
—
—
—
—
—
12.6
24.5
—
—
—
—
—
—
—
—
21.7
—
18.5
—
23.2
10.3
18.6
—
19.2
—
—
—
—
—
—
—
—
—
—
19.5
—
10.3
—
25.8
11.6
22.8
23.2
15.9
—
—
—
23.4
11.9
23.9
24.3
36.1
—
17.5
—
15.2
15.4
Ovarian cancer
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
—
—
—
12.1
10.8
17.5
—
—
—
—
—
—
13.8
—
—
—
—
—
—
—
—
—
—
49.3
—
15.0
24.8
—
—
—
—
—
—
—
—
—
—
19.2
18.7
—
16.0
20.3
14.9
20.9
—
—
—
12.9
17.9
13.4
11.3
59.1
12.1
24.9
30.1
NCI/ADR-RES
Renal cancer
786-0
A498
12.0
24.6
—
11.5
28.0
—
29.3
53.0
—
11.2
28.5
12.8
—
—
—
—
—
—
—
—
32.6
—
24.3
—
13.1
13.4
—
31.5
60.2
21.5
26.5
13.4
26.1
11.1
38.3
—
—
—
—
—
—
—
32.3
20.0
18.0
18.5
—
14.1
20.3
—
—
25.9
—
—
—
—
—
27.2
—
18.7
—
—
—
—
—
21.5
—
—
—
—
—
—
—
—
36.4
21.9
19.8
13.7
17.8
—
ACHN
CAKI-1
RXF 393
SN12C
TK-10
—
26.2
—
35.5
—
35.5
UO-31
46.0
23.9
Prostate cancer
PC-3
15.9
28.6
—
14.7
51.1
—
34.9
12.8
—
34.5
15.8
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
—
25.0
—
20.6
—
—
—
18.0
—
—
—
14.3
—
11.5
—
—
—
—
12.6
10.7
18.1
36.8
24.5
23.4
21.7
—
—
—
—
25.7
—
11.6
28.7
22.7
19.9
—
—
—
—
—
—
—
—
—
—
—
—
10.7
14.2
19.4
—
23.9
30.1
—
—
—
—
14.7
—
13.0
12.6
33.1
12.4
—
MDA-MB-468
—
—
a
GI < 10%; nt, not tested; L, compound proved lethal to the cancer cell line.
Compounds 41, 42, 45, 47, 49, 52–54, 60 and 65 proved inactive.
b
is a good alignment between MTX and 29 explaining their resem-
blance in binding mode flipped profile into DHFR binding site.
Compound 29 (the most active compound) was chosen as the tem-
plate molecule, on which other molecules were aligned. Thirteen
low energy, maximally dissimilar structures were selected for
comparison to the other compounds. After assigning MMFF94
charges to all molecules, flexible alignment was ranked by overlays
of compounds 29, 34 and 39 based on electrostatic, steric field,
hydrophobic areas overlap, hydrogen bond acceptors and donors
overlap. From the highest scoring superposition, the limited set
Please cite this article in press as: Al-Rashood, S. T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.070

8
S. T. Al-Rashood et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Arg
Phe
Gln
35
70
34
Ile
60
Arg
28
Thr
56
Leu
67
Phe
34
O
Phe
31
Glu
30
N H2
Ser
59
-
O
Ser
59
Tyr
22
O
O
Phe
31
N
S
Thr
136
N
Glu
N
N
N
30
N
-
Pro
61
N
O
Ala
9
S
N
Lys
55
Asn
64
N
Ile
7
N
O
N
O
N
Ile
16
Ile
60
Val
8
Val
115
Thr
146
Gly
17
Gly
20
Asp
21
Tyr
121
MTX
29
Glu
30
Phe
Ala
34
9
Phe
31
H
S
Phe
31
Arg
28
Arg
28
N H
Phe
34
NH2
Ile
60
Glu
30
N
Trp
24
N
N
Asn
64
N
S
Ala
9
Tyr
22
Pro
61
N
S
N
O
O
O
Val
115
Gly
117
S
N
Gly
117
O
Ile
16
N
O
O
Tyr
22
Pro
61
Thr
146
Ser
59
Thr
56
Thr
56
Ile
16
Cl
Ile
60
Ser
59
Thr
146
Lys
55
34
39
Figure 1. The 2D binding mode and residues involved in the recognition for MTX, the most active compounds 29 (IC50 0.1 lM), 34 (IC50 0.4 lM) and 39 (IC50 0.6 lM) docked
and minimized in the DHFR binding pocket. The essential amino acid residues at the binding site are tagged in circles.
of conformers was used in the analysis of molecules with high flex-
ibility capable to achieving complete atom to atom superposition.
A common feature of the MOE-generated alignments was that the
three structures showed matched quinazoline and phenoxy rings,
and slight differences in the distance the between methyl and thia-
diazole moieties (0.16 Å, Fig. 4a). Adopting the same methodology,
the most active DHFR inhibitors 29, 34, 39 and the least active
compounds 35 and 52 were subjected to flexible alignment analy-
sis (Fig. 4b). It is an important experiment to gain a clear vision of
the essential features for a given activity. It is clear that compounds
35 and 52 were flexibly aligned in a different manner when
compared with the active compound 29. Common feature of the
MOE-generated alignments showed that the super position is
occurred among the compounds where a deviation distance of
about 2.23 Å compared with the least active compounds. These
features explain the difference in activity among the two groups
and show the importance of the phenoxy moiety attached to qui-
nazoline N atom. An attempt to investigate the reasons behind
the diminished DHFR inhibition activity of compound 35, electro-
static mapping was carried out for its lowest energy conformer
to study the similarity and dissimilarity of the electrostatic binding
characteristics of the molecule surface and its conformational
properties, in comparison to the most active compound 29.
Figure 5a showed some common features of 29, having more
Ile
60
Lys
54
N
H2
N
Met
52
S
N
Ser
NH2
59
S
N
O
Phe
31
O
N
O
S
Ser
76
N
O
N
N
O
Val
115
Gly
53
S
Ser
119
Arg
77
Asn
64
Leu
75
Glu
78
Thr
56
Lys
55
O
Tyr
22
Pro
61
Phe
34
Asp
21
Ile
Glu
123
16
35
52
Figure 2. The 2D binding mode and residues involved in the recognition for the inactive compounds 35 (IC50 10.0 lM) and 52 (IC50 8.0 lM) docked and minimized in the
DHFR binding pocket. The essential amino acid residues at the binding site are tagged in circles.
Please cite this article in press as: Al-Rashood, S. T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.070

S. T. Al-Rashood et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
9
Table 5
Pharmacokinetic parameters of compounds 29, 34 and 39
Compd
Mwt
TPSA
94.95
96.14
253.31
LogP
Lip. don
Lip. acc
Lip. V
b.rotN
29
34
39
459.55
479.94
505.57
5.06
4.35
5.46
2
2
2
7
7
9
5.66
6.00
5.37
5
5
7
TPSA: Polar surface area, LogP: Calculated lipophilicity, Lip.don: Number of
hydrogen bond donors, Lip.acc: Number of hydrogen bond acceptors, Lip.V: Number
of violations of Lipinski rule, b.rotN: No. of rotatable bonds.
performed for the determination of topological polar surface area
(TPSA), and the ‘rule of five’ formulated by Lipinski50 for the activ-
ity prediction of an orally administered drug, if it has no more than
one violation of the following rules: (i) ClogP (partition coefficient
between water and octanol) <5; (ii) number of hydrogen bond
donors sites 65; (iii) number of hydrogen bond acceptor sites
610; (iv), molecular weight <500; (v) No. of rotatable bonds <5.
In addition, the total polar surface area (TPSA) is another key
property linked to drug bioavailability; the passively absorbed
molecules with TPSA >140 have low oral bioavailability.51 All cal-
culated descriptors were obtained using the MOE package, and
the results are listed in Table 5. The obtained results revealed that
the CLogP are around 5.0 for compounds 29 and 39 and less than
5.0 for compound 34, the molecular weight was less than 500,
except for compound 39 (505.57), hydrogen bond acceptor <10
and hydrogen bond donors <5 which fulfill Lipinski’s rule. Also,
the percent absorption of compounds 39 total surface area with
253.31 shows the lowest bioavailability among all tested com-
pounds. From these data, it could be suggested that compounds
Figure 3. Flexible alignment of the most active compound 29 (pink) and MTX
(brown).
hydrophobic regions distributed all over the molecule, on the con-
trary the least active DHFR inhibitor which showed scattered mild
Polar Regions (Fig. 5b). The obtained hydrophobic mapping confor-
mations suggest a distinct varied interaction of the active and inac-
tive molecules with the potential protein binding site. Oral
bioavailability plays an important role in the development of
bioactive molecules into therapeutic agents. Many potential
therapeutic agents fail to reach the clinic because of their unfavor-
able absorption, distribution, metabolism, elimination and toxic
(ADMET) factors.49 Therefore, a computational study for the
prediction of ADMET properties of compounds 29, 34 and 39 was
Figure 4. (a) Flexible alignment of the most active compounds 29 (pink), 34 (cyan) and 39 (orange). (b) Flexible alignment of the most active compounds 29 (pink), 34 (cyan)
and 39 (orange) against inactive compounds 35 (yellow) and 52 (brown).
Figure 5. Surface map for (a) the most active compound 29; (b) the least active compound 35. Pink: hydrogen bond, blue: mild polar, green: hydrophobic region.
Please cite this article in press as: Al-Rashood, S. T.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.07.070

10
S. T. Al-Rashood et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
O
O
O
N
O
N
O
N
H3C
Cl
CH3O
CH3O
N
S
N
S
N
S
S
S
S
N
N
N
N
N
N
H2N
H2N
H2N
39, IC50 0.6 µM
34, IC50 0.4 µM
29, IC50 0.1 µM
Figure 6. Structures of the active DHFR inhibitors 29, 34 and 39.
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with diminished toxicity among the investigated compounds.
In conclusion, compounds 29, 34 and 39 (Fig. 6) proved to be
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in general, the type of substituent at positions 2-, 3-, and 6- of
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the DHFR inhibition activity in the following order: 2-(5-
Amino-1,3,4-thiadiazol-2-yl-thio)- > 2-(2-amino-4-methylthiazol-
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a remarkable broad-spectrum activity against both
Gram-positive and Gram-negative bacteria. Compounds 28 and
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