2-Arylquinazolin-4(3H)-ones: A novel class of thymidine phosphorylase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2015.10.006

Thymidine phosphorylase (TP) over expression plays an important role in several pathological conditions, such as rheumatoid arthritis, chronic inflammatory diseases, psoriasis, and tumor angiogenesis. In this regard, a series of twenty-five 2-arylquinazolin-4(3H)-one derivatives 1-25 were evaluated for thymidine phosphorylase inhibitory activity. Six compounds 5, 6, 20, 2, 23, and 3 were found to be active against thymidine phosphorylase enzyme with IC₅₀ values in the range of 42.9-294.6 μM. 7-Deazaxanthine (IC₅₀ = 41.0 ± 1.63 μM) was used as a standard inhibitor. Compound 5 showed a significant activity (IC₅₀ = 42.9 ± 1.0 μM), comparable to the standard. The enzyme kinetic studies on the most active compounds 5, 6, and 20 were performed for the determination of their modes of inhibition, and dissociation constants K_i. All active compounds were found to be largely non-cytotoxic against the mouse fibroblast 3T3 cell line. This study identifies a novel class of thymidine phosphorylase inhibitors which may be further investigated as leads to develop therapeutic agents.

Full text of DOI:10.1016/j.bioorg.2015.10.006

Bioorganic Chemistry 63 (2015) 142–151
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
2-Arylquinazolin-4(3H)-ones: A novel class of thymidine phosphorylase
inhibitors
Sumaira Javaid a, Syed Muhammad Saad a, Shahnaz Perveen b, Khalid Mohammed Khan a,
,
⇑
M. Iqbal Choudhary a,c,
⇑
a H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
b PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280, Pakistan
c Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah 21412, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Thymidine phosphorylase (TP) over expression plays an important role in several pathological conditions,
such as rheumatoid arthritis, chronic inflammatory diseases, psoriasis, and tumor angiogenesis. In this
regard, a series of twenty-five 2-arylquinazolin-4(3H)-one derivatives 1–25 were evaluated for thymidine
phosphorylase inhibitory activity. Six compounds 5, 6, 20, 2, 23, and 3 were found to be active against
Received 10 September 2015
Revised 26 October 2015
Accepted 27 October 2015
Available online 29 October 2015
thymidine phosphorylase enzyme with IC50 values in the range of 42.9–294.6
(IC50 = 41.0 1.63 M) was used as a standard inhibitor. Compound 5 showed a significant activity
(IC50 = 42.9 1.0 M), comparable to the standard. The enzyme kinetic studies on the most active com-
lM. 7-Deazaxanthine
l
l
Keywords:
2-Arylquinazolin-4(3H)-ones
Thymidine phosphorylase inhibition
Tumor angiogenesis
pounds 5, 6, and 20 were performed for the determination of their modes of inhibition, and dissociation
constants Ki. All active compounds were found to be largely non-cytotoxic against the mouse fibroblast
3T3 cell line. This study identifies a novel class of thymidine phosphorylase inhibitors which may be fur-
ther investigated as leads to develop therapeutic agents.
Anticancer activity
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
bladder [13], and oesophageal cancers [14], showed increased level
of PD-ECGF/TP. Inhibition of TP enzyme is thus an important anti-
Thymidine phosphorylase (abbreviated as dThdPase or simply
TP) (EC 2.4.2.4) is a pyrimidine based nucleoside phosphorylase,
exists in both eukaryotic and prokaryotic domains [1,2]. This
enzyme catalyzes the initial step in the catabolism of thymidine
(dThd) nucleoside into thymine and 2-deoxy-D-ribose-1-
phosphate by reversible phosphorolysis, cleaving glycosidic bond
[3,4] (Scheme 1).
The dephosphorylated intermediate, 2-deoxy-D-ribose, plays a
key role in triggering the tumor angiogenesis and hence endorses
cancer metastasis [5–7]. TP is similar to platelet derived endothe-
lial cell growth factor (PD-ECGF) which is an angiogenic molecule
[1,8,9]. Primary sequence of TP is mostly conserved throughout
the evolution as mammalian TP which shares 39% sequence simi-
larity with TP of E. coli. The mammalian enzyme also shares 65%
similarity with the active site residues of E. coli enzyme [10].
Various human tumors including colorectal [11], breast [12],
angiogenic/cancer approach. For the suppression of the angiogenic
and chemotactic activities of thymidine phosphorylase (dThdPase)
[15–19], different inhibitors of TP have been designed as potential
anticancer agents [20–24]. Currently only one TP inhibitor (tipira-
cil), in combination of trifluridine (a cytotoxin), is recently
approved by U.S. FDA for clinical use for the treatment of
metastatic colorectal cancer. This combination drug (trifluridine/
tipiracil), marketed as Lonsurf, is also associated with severe side
effects, such as myelosuppression, anemia, and neutropenia.
Therefore there is need to develop new TP inhibitory agents [25].
7-Deazaxanthine (7-DX) and 6-amino-5-bromouracil (6A5BU) are
the two classical inhibitors of TP, which are widely used as refer-
ence compounds for in vitro studies against this enzyme [26,27].
Our research group has been working for the discovery of
thymidine phosphorylase inhibitors. In this regard, we have
already reported 3-formylchromones [28] and 1,3,4-oxadiazoles
[29] as thymidine phosphorylase inhibitors. Recently triazines
and their fused pyrazolo and triazolo analogs have been reported
for their thymidine phosphorylase inhibitory activities [30–32].
Based on broad medicinal importance of 2-arylquinazolin-4(3H)-
ones, and their structural resemblance with 7-deazaxanthine
⇑
Corresponding authors at: H.E.J. Research Institute of Chemistry, International
Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270,
Pakistan (M.I. Choudhary).
E-mail addresses: hassaan2@super.net.pk, khalid.khan@iccs.edu (K.M. Khan),
hej@cyber.net.pk (M.I. Choudhary).
http://dx.doi.org/10.1016/j.bioorg.2015.10.006
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

S. Javaid et al. / Bioorganic Chemistry 63 (2015) 142–151
143
Scheme 1. Mechanism of thymidine phosphorylase catalyzed reaction.
Apparently substitution of hydroxyl groups to phenyl ring made
compounds active against TP. Compounds 2 and 3 substituted with
single hydroxyl group at para and meta position on phenyl ring
showed
234.0 1.5 and 294.6 2.9
a
weak TP inhibitory activity with IC50 values of
M, respectively. However, compound
l
4 with hydroxyl substitution at ortho position was found to be
inactive. Among the di-hydroxyl substituted derivatives, com-
pound 5 with hydroxyl groups at para and meta positions of phenyl
Fig. 1. Structures of 7-deazaxanthine and 2-arylquinazolin-4(3H)-ones.
ring showed an IC50 = 42.9 1.0
hydroxyls at ortho and meta position of phenyl ring gave an
IC50 = 59.5 1.9 M. TP inhibitory activity of compounds 5 and 6
were found comparable to the standard compound i.e.,
7-deazaxanthine (IC50 = 41.0 1.63 M).
SAR proposed that hydroxyl substitution on phenyl ring at
different positions, plays an important role in inducing TP
inhibition. The ability of inhibiting TP was found to be more in
di-hydroxylated analogs than mono-hydroxylated analogs. Better
activity of di-hydroxylated analogs than the mono-hydroxylated
analogs has been linked with the possible increase in polar interac-
tions with enzyme. Hydroxyl group, being strong electron
lM, and compound 6 with two
(Fig. 1), a library of 2-arylquinazolin-4(3H)-ones was synthesized
[33], and subjected to thymdine phosphorylase inhibition studies.
The results are presented in Table 1.
l
In addition to this, all active compounds were also screened
through MTT 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazo
lium bromide) spectrophotometric assay for the evaluation of their
cytoxicity (Table 2). In these studies, reduction of MTT dye to for-
mazan by mitochondrial enzyme was measured. The reduction of
MTT can only occur in metabolically active cell, so the activity level
is actually a measure of cell viability. These studies gave an idea
about the effect of test compounds on the cell viability [34].
l
donating, enhances the ability of phenyl rings to undergo
p–p
interactions with the aromatic amino acid residues, present in
active site or hydrophobic pocket of the TP enzyme. In addition
to this, hydroxyl groups could also form hydrogen bond with the
amino acid residues of the active site or hydrophobic pocket of
enzyme.
2. Results and discussion
2.1. Chemistry
All twenty-five derivatives of 2-arylquinazolin-4(3H)-ones (1–
25) were re-synthesized [33] for the evaluation thymidine phos-
phorylase inhibition (Scheme 2). Structures of the synthetic com-
pounds were deduced by 1H NMR and EI mass spectrometry. All
compounds furnished satisfactory HR-EIMS and elemental analy-
ses. Melting points and Rf values were also recorded.
Alkoxylated derivatives (compounds 8–12) did not show any
inhibitory activity but when integrated with hydroxyl group, as
in compounds 20 (IC50 = 168.8 1.8
lM), and 23 (IC50 = 256.0
2.0 M), were found to be active. SAR proposed that position of
l
hydroxyl and alkoxy groups on phenyl ring affects the ability of
compounds to inhibit enzyme, as revealed by the TP inhibitory
activity of compounds 20–23 (Table 1). Hydroxyl and alkoxy
groups being electron donating were proposed to be involved in
hydrogen bonding and/or hydrophobic interactions with amino
acid residues present in the active site or hydrophobic pocket of TP.
Compounds with electron withdrawing groups such as chloro
(compounds 13–16) or nitro (compounds 17–19) groups, did not
show any activity. Metyhylated, and di-aminomethylated deriva-
tives (compounds 7, and 25, respectively), were also found to be
inactive.
2.2. Enzyme inhibition studies
Differently substituted 2-arylquinazolin-4(3H)-ones (1–25)
were evaluated for thymidine phosphorylase inhibition versus the
standard, 7-deazxanthine (IC50 = 41.0 1.63 lM). Among twenty-
five compounds, six compounds (2, 3, 5, 6, 20, and 23) were found
to have TP inhibitory activity (Table 1). Compounds 5 and 6 with
IC50 values of 42.9 1.0 and 59.5 1.9
significant TP inhibitory activity in comparison to the standard.
Compounds 20 (IC50 = 168.8 1.8 M), 2 (IC50 = 234.0 1.5 M),
23 (IC50 = 256.0 2.0 M), and (IC50 = 294.6 2.9 M) were
found to have moderate levels of TP inhibitory activity.
lM, respectively showed
l
3
l
l
l
2.3. Cytotoxicity evaluation
Structure-activity relationship (SAR) studies suggested that TP
inhibitory activity depends mainly on the nature of substitution
on the phenyl moiety, attached to C-2 of the quinazolin-4(3H)-
one nucleus, as compound 1 (parent compound) without substitu-
tion on phenyl ring was found to be inactive.
All active compounds were found to be non-cytotoxic against
the mouse fibroblast 3T3 cell lines, except compound 6 which
showed a weak cytotoxic activity, in comparison to the standard
i.e. cycloheximide (Table 2).

144
S. Javaid et al. / Bioorganic Chemistry 63 (2015) 142–151
Table 1
Thymidine phosphorylase inhibitory activity of 2-arylquinazoline-4-(3H)-ones (1–25).
Compound
Structure
IC50 SEMa
N.A.b
(lM)
1
2
234.0 1.5
3
294.6 2.9
4
N.A.b
5
42.9 1.0
6
59.5 1.9
7
N.A.b
8
N.A.b

S. Javaid et al. / Bioorganic Chemistry 63 (2015) 142–151
145
Table 1 (continued)
Compound
Structure
IC50 SEMa
N.A.b
(lM)
9
10
N.A.b
11
N.A.b
12
N.A.b
13
14
15
N.A.b
N.A.b
N.A.b
16
N.A.b
(continued on next page)

146
S. Javaid et al. / Bioorganic Chemistry 63 (2015) 142–151
Table 1 (continued)
Compound
Structure
IC50 SEMa
N.A.b
(lM)
17
18
19
20
N.A.b
N.A.b
168.8 1.8
21
N.A.b
22
N.A.b
23
256.0 2.0
24
N.A.b

S. Javaid et al. / Bioorganic Chemistry 63 (2015) 142–151
147
Table 1 (continued)
Compound
Structure
IC50 SEMa
N.A.b
(lM)
25
7-Deazaxanthine (Standard)
41.0 1.63
a
SEM is the Standard Error of the Mean.
N.A. means Not Active.
b
plotted against the different concentrations of compounds. Kinetic
studies on compounds 5 and 6 revealed that they inhibit the TP in
non-competitive manner. These two compounds may interact
either with the hydrophobic pocket or at allosteric site of enzyme.
Compound 20 showed a competitive mode of inhibition, indicating
its interaction with the amino acid residues of the enzyme’s active
site (Table 3 and Figs. 2–4). In competitive-type of inhibition, the
Vmax of enzyme was not affected, while the Km increased, in case
of non-competitive type of inhibition, the Km of enzyme was not
affected, while the Vmax of enzyme decreased (Figs. 2–4).
Table 2
Cytotoxic data of active 2-arylquinazoline-4-(3H)-ones.
Compound
IC50 SEMa
(lM)
N.C.b
N.C.b
N.C.b
2
3
5
6
20
23
22.80 0.34
N.C.b
N.C.b
Cycloheximide
0.20 0.05
a
SEM: Standard Error Mean.
N.C.: Non-Cytotoxic.
b
3. Conclusion
In conclusion, 2-arylquinazolin-4(3H)-one derivatives (1–25)
were evaluated for thymidine phosphorylase inhibitory activity.
Six compounds 5, 6, 20, 2, 23, and 3 with IC50 values in the range
of 42.9–294.6
phorylase. 7-Deazxanthine (IC50 = 41.0 1.63
l
M, were found to be active against thymidine phos-
M) was used as a
l
standard in the study. Further studies, based on mode of action
of our designed inhibitors, may help in development of therapeutic
agents including anticancer drugs, based on quinazolinone
skeleton.
Scheme 2. Synthesis of 2-aryl-4(3H)-quinazolinones (1–25).
Table 3
Kinetic data of active 2-arylquinazoline-4-(3H)-ones (5, 6, and 20).
4. Experimental
Compound
Kia SEMb
(lM)
Type of inhibition
4.1. Method and materials
5
6
20
52.33 0.001
172.0 0.0006
140.0 0.001
49.1 0.002
Non-competitive
Non-competitive
Competitive
Thymidine phosphorylase enzyme (from E. coli, EC Number
2.4.2.4), thymidine, and potassium phosphate monobasic were
purchased from Sigma Aldrich, USA, 7-Deazaxanthine (2,4-
Dihydroxypyrrolo[2,3-d]pyrimidine,7H-pyrrolo[2,3-d]pyrimidine-
2,4-diol) was purchased from Santa Cruz Biotechnology Inc., USA,
dimethylsulfoxide (DMSO) was purchased from Fisher Scientific,
Germany.
7-Deazaxanthine
Non-competitive
a
Ki is Dissociation constant.
SEM is the Standard Error of the Mean.
b
2.4. Kinetic studies
Mechanism of inhibition of most active compounds was deter-
mined by kinetic studies, using thymidine as a substrate. Inhibition
type was deduced by Lineweaver–Burk plot, where the reciprocal
of the rate of the reaction were plotted against the reciprocal of
substrate concentration, to monitor the effect of inhibitor on both
Km and Vmax. The Ki values were determined by plotting the slope of
each line in the Lineweaver–Burk plots against the different
concentrations of compounds. The Ki values were re-confirmed
by Dixon plot in which the reciprocal of the rate of reaction were
4.2. Thymidine phosphorylase inhibition assay
Since human TP is not easily accessible, we used commercially
available recombinant E. coli TP (expressed in Escherichia coli)
enzyme. Primary sequence of TP is mostly conserved throughout
evolution as mammalian TP is reported to share 39% sequence
similarity with the TP of E. coli. The mammalian enzyme also
shared 65–70% similarity with the active site residues, and three
dimensional structure of E. coli TP enzyme [10].

148
S. Javaid et al. / Bioorganic Chemistry 63 (2015) 142–151
0.6
0.4
0.2
0
0.06
0.04
0.02
0
-0.02
-0.04
-0.2
-0.4
-100
-50
0
50
100
-6
-4
-2
0
2
4
6
1/Substrate (Thymidine)
Inhibitor (Compound 5)
0.4
0.2
0
-0.2
-0.4
-120 -80
-40
0
40
80
120
1/Inhibitor (Compound 5)
Fig. 2. The inhibition of thymidine phosphorylase by compound 5. (A) is the Lineweaver–Burk plot of reciprocal rate of reaction (velocities) versus reciprocal of substrate
(thymidine) in the absence (.), and in the presence of 31.25 M ( ), 62.5 M (j), and 125 M (h) of compound 5. (B) is secondary replot of Lineweaver–Burk plot between
l
D
l
l
the slopes of each line on Lineweaver–Burk plot versus different concentrations of compound 5. (C) is the Dixon plot of reciprocal of rate of reaction (velocities) versus different
concentrations of compound 5.
TP inhibition assay was performed spectrophotometrically. The
method of Bera et al. was followed with slight modifications [35].
4.4. Cytotoxicity studies
Reaction mixture of 200
l
L, contained 20
l
L of enzyme (0.058
MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium
bromide) spectrophotometric assay in 96-well plate was used
[33], to evaluate the cytotoxic activity of active compounds.
unit/well), 150
lL
of potassium phosphate buffer (pH 7.0,
50 mM), and 10
l
L of test compound (0.5 mM). The reaction
mixture was then incubated for 10 min at 30 °C. After 10 min,
substrate (thymidine, kmax; 265 nm) (20 L, 1.5 mM) was added,
and change in absorbance was observed for 10 min at 290 nm in
96-well ELISA plate reader (Spectramax, Molecular Devices, CA,
USA). Every experiment was run in triplicate. 7-Deazaxanthine
was used as positive control.
l
4.5. Statistical analysis
Reactions for above mentioned biological activities were carried
out in triplicate. Results were then processed using SoftMax Pro 4.8
software (Molecular Devices, CA, USA) and then by Microsoft Excel.
Percent inhibition for above mentioned biological activities was
calculated by following formula:
4.3. Kinetic studies
Percent Inhibition ¼ 100 ꢀ ðODtest compound=ODcontrolÞ ꢁ 100
Kinetic studies were carried out to deduce the mechanism of
inhibition. Potential inhibitor could interact with enzyme in com-
petitive, non-competitive, mixed or uncompetitive manner. In
where OD is the optical density.
Results were presented as means standard error mean from
triplicate (n = 3) observation. IC50 values were determined by using
EZ-FIT, Enzyme kinetics software by Perrella Scientific, Inc., USA.
Grafit 7.0 version was used to determine the kinetics parameter.
The software was purchased from the Erithacus Software Ltd.,
Wilmington House, High Street, East Grinstead, West Sussex
RH19 3AU, UK.
kinetic assay, the enzyme (0.058U/200 lL) was incubated with dif-
ferent concentrations of inhibitor for 10 min at 30 °C. The reaction
was then initiated by adding different concentrations (0.1875–
1.5 mM) of substrate (thymidine). Degradation of thymidine was
measured continuously at 290 nm for 10 min on a ELISA plate
reader [35].

S. Javaid et al. / Bioorganic Chemistry 63 (2015) 142–151
149
0.04
0.02
0
0.4
0.2
0
-0.2
-0.4
-0.02
-0.04
-10 -8 -6 -4 -2
0
2
4
6
8
10
-240 -180 -120 -60
0
60 120 180 240
1/Substrate (Compound 6)
1/Inhibitor (Compound 6)
0.3
0.15
0
-0.15
-0.3
-200
-100
0
100
200
1/Inhibitor (Compound 6)
Fig. 3. The inhibition of thymidine phosphorylase by compound 6. (A) is the Lineweaver–Burk plot of reciprocal rate of reaction (velocities) versus reciprocal of substrate
(thymidine) in the absence (.), and in the presence of 31.25 M ( ), 62.5 M (j), 125 M (h), 250 M (d) of compound 6. (B) is secondary replot of Lineweaver–Burk plot
l
D
l
l
l
between the slopes of each line on Lineweaver–Burk plot versus different concentrations of compound 6. (C) is Dixon plot of reciprocal rate of reaction (velocities) verses
different concentrations of compound 6.
4.6. General procedure for the synthesis of compounds 1-25
C
C
14H10N2O2 [M]+ 238.0742; found 238.0746; Anal. Calcd for
14H10N2O2: C, 70.58; H, 4.23; N, 11.76; O, 13.43; found: C,
2-Aminobenzamide (1 eq.), substituted benzaldehydes (1.1 eq)
and CuCl2ꢂ2H2O (4 eq.) were mixed in ethanol (15 mL) and refluxed
for 16 h. The reaction was periodically monitored by thin layer
chromatography. Water was added to the reaction mixtures until
the appearance of precipitates. These precipitates were filtered,
washed with water, and dried under vacuum. Good yields of title
compounds were obtained [33].
70.59; H, 4.25; N, 11.76 [33].
4.7.3. 2-(30,40-Dihydroxyphenyl)quinazolin-4(3H)-one (5)
Yield: 96%; m.p. 230 °C; Rf: 0.10 (ethyl acetate/hexane, 3:7); 1H
NMR: (400 MHz, DMSO-d6): dH 12.21 (s, 1H, NH), 9.64 (s, 1H,
40-OH), 9.29 (s, 1H, 30-OH), 8.10 (d, 1H, J5,6 = 8.0 Hz, H-5), 7.78 (d,
1H, J8,7 = 7.2 Hz, H-8), 7.66 (m, 2H, H-7, H-20), 7.54 (d, 1H,
J6 ,5 = 8.0 Hz, H-60), 7.46 (t, 1H, J6(5,7) = 7.6 Hz, H-6), 6.83 (d, 1H,
0
0
+
J5 ,6 = 8.0 Hz, H-50); EI-MS: m/z (rel. abund.%), 254 [M] (8), 146
0
0
4.7. Characterization data of the active compounds 2, 3, 5, 6, 20, and
23
(100), 119 (26); HREI-MS: m/z calcd for
C
14H10N2O3 [M]+
254.0691; found 254.0680; Anal. calcd for C14H10N2O3: C, 66.14;
H, 3.96; N, 11.02; O, 18.88; found: C, 66.12; H, 3.93; N, 11.05 [33].
4.7.1. 2-(40-Hydroxyphenyl)quinazolin-4(3H)-one (2)
Yield: 99%; m.p. >300 °C; Rf: 0.15 (ethyl acetate/hexane, 3:7); 1H
NMR: (400 MHz, DMSO-d6): dH 12.29 (s, 1H, NH), 10.14 (s, 1H,
4.7.4. 2-(20,50-Dihydroxyphenyl)quinazolin-4(3H)-one (6)
Yield: 95%; m.p. >300 °C; Rf: 0.18 (ethyl acetate/hexane, 3:7); 1H
NMR: (300 MHz, DMSO-d6): dH 12.60 (s, 1H, NH), 12.29 (s, 1H,
20-OH), 9.08 (s, 1H, 50-OH), 8.14 (d, 1H, J5,6 = 7.5 Hz, H-5), 7.86 (t,
1H, J7(6,8) = 7.5 Hz, H-7), 7.73 (d, 1H, J8,7 = 8.1 Hz, H-8), 7.60 (s,
1H, H-60), 7.54 (t, 1H, J6(5,7) = 7.2 Hz, H-6), 6.92 (m, 2H, H-30,
H-40); EI-MS: m/z (rel. abund.%), 254 [M]+ (100), 119 (36); HREI-
MS: m/z calcd for C14H10N2O3 [M]+ 254.0691; found 254.0692;
Anal. Calcd for C14H10N2O3: C, 66.14; H, 3.96; N, 11.02; O, 18.88;
found: C, 66.16; H, 3.95; N, 11.03 [33].
40-OH), 8.11 (m, 1H, H-5), 8.03 (d, 2H, J2 ,3 = J6 ,5 = 8.4 Hz, H-20,
H-60), 7.80 (t, 1H, J7(6,8) = 7.2 Hz, H-7), 7.67 (d, 1H, J8,7 = 8.0 Hz,
0
0
0
0
0
0
0
0
H-8), 7.47 (t, 1H, J6(5,7) = 7.2 Hz, H-6), 6.89 (d, 2H, J3 ,2 = J5 ,6 = 8.8
Hz, H-30, H-50); EI-MS: m/z (rel. abund.%), 238 [M]+ (100), 237
(5), 221 (5), 119 (83); HREI-MS: m/z calcd for C14H10N2O2 [M]+
238.0742; found 238.0738; Anal. Calcd for C14H10N2O2: C, 70.58;
H, 4.23; N, 11.76; O, 13.43; found: C, 70.57; H, 4.24; N, 11.77 [33].
4.7.2. 2-(30-Hydroxyphenyl)quinazolin-4(3H)-one (3)
Yield: 88%; m.p. 260–262 °C; Rf: 0.21 (ethyl acetate/hexane,
3:7); 1H NMR: (400 MHz, DMSO-d6): dH 12.42 (s, 1H, NH), 9.74
(s, 1H, 30-OH), 8.14 (m, 1H, H-5), 7.84 (m, 1H, H-7), 7.72 (d, 1H,
J8,7 = 8.0 Hz, H-8), 7.59 (m, 2H, H-6, H-20), 7.52 (m, 1H, H-60), 7.34
(m, 1H, H-50), 6.97 (m, 1H, H-40); EI-MS: m/z (rel. abund.%), 238
[M]+ (82), 237 (9), 221 (7), 119 (100); HREI-MS: m/z calcd for
4.7.5. 2-(30-Hydroxy-40-methoxyphenyl)quinazolin-4(3H)-one (20)
Yield: 100%; m.p. 202–204 °C; Rf: 0.10 (ethyl acetate/hexane,
3:7); 1H NMR: (400 MHz, DMSO-d6): dH 12.30 (s, 1H, NH), 9.35
(s, 1H, 30-OH), 8.09 (s, 1H, H-5), 7.77 (m, 5H, H-7, H-8, H-6, H-60,
H-50), 7.04 (s, 1H, H-20), 3.85 (s, 3H, 40-OCH3); EI-MS: m/z

150
S. Javaid et al. / Bioorganic Chemistry 63 (2015) 142–151
0.04
0.2
0.1
0
0.02
0
-0.1
-0.2
-0.02
-0.04
-6
-4
-2
0
2
4
6
-300 -200 -100
0
100
200
300
1/Inhibitor (Compound 20)
1/Substrate(Thymidine)
0.4
0.2
0
-0.2
-0.4
-400
-200
0
200
400
1/Inhibitor (Compound 20)
Fig. 4. The inhibition of thymidine phosphorylase by compound 20. (A) is the Lineweaver–Burk plot of reciprocal rate of reaction (velocities) versus reciprocal of substrate
(thymidine) in the absence (.), and in the presence of 50 M ( ), 100 M (N), and 200 M ( ) of compound 20. (B) is secondary replot of Lineweaver–Burk plot between the
l
l
l
D
r
slopes of each line on Lineweaver–Burk plot versus different concentrations of compound 20. (C) is the Dixon plot of reciprocal of rate of reaction (velocities) versus different
concentrations of compound 20.
(rel. abund.%), 268 [M]+ (100), 253 (26), 119 (82); HREI-MS: m/z
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calcd for C15H12N2O3 [M]+ 268.0848; found 268.0845; Anal. Calcd
for C15H12N2O3: C, 67.16; H, 4.51; N, 10.44; O, 17.89; found: C,
67.15; H, 4.52; N, 10.44.
4.7.6. 2-(30-Ethoxy-40-hydroxyphenyl)quinazolin-4(3H)-one (23)
Yield: 98%; m.p. 229–230 °C; Rf: 0.21 (ethyl acetate/hexane,
3:7); 1H NMR: (400 MHz, DMSO-d6): dH 12.32 (s, 1H, NH), 9.67
(s, 1H, 40-OH), 8.11 (d, 1H, J5,6 = 7.6 Hz, H-5), 7.77 (m, 3H, H-7, H-
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198.
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1316.
8, H-20), 7.68 (d, 1H, J6 ,5 = 8.0 Hz, H-60), 7.47 (t, 1H, J6
0
0
(5,7) = 7.2 Hz, H-6), 6.91 (d, 1H, J5 ,6 = 8.4 Hz, H-50), 4.16 (q, 2H,
J(CH2,CH3) = 6.8 Hz, CH2), 1.39 (t, 3H, J(CH3,CH2) = 7.2 Hz, CH3); EI-
MS: m/z (rel. abund.%), 282 [M]+ (100), 267 (29), 254 (81), 238
0
0
(12), 119 (47); HREI-MS: m/z calcd for
C
16H14N2O3 [M]+
282.1004; found 282.1005; Anal. Calcd for C16H14N2O3: C, 68.07;
H, 5.00; N, 9.92; O, 17.00; found: C, 68.05; H, 5.04; N, 9.94 [33].
Acknowledgments
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(2009) 903–953.
The authors acknowledge the financial support of Higher Edu-
cation Commission (HEC) Pakistan, Project No. 20-2073 under
National Research Program for Universities.
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