Design, synthesis and biological evaluation of some novel substituted quinazolines as antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2014.04.029

A novel series of 6-chloro-2-p-tolylquinazolinone and substituted-(4- methylbenzamido)benzamide (1-20) were designed, synthesized and evaluated for their in-vitro antitumor activity. Compounds 3, 14 and 16 possessed remarkable broad-spectrum antitumor activity. Compound 16 was found to be a particularly active growth inhibitor of the renal cancer (GI₅₀ = 4.07 μM), CNS cancer (GI₅₀ = 7.41 μM), ovarian cancer (GI₅₀ = 7.41 μM) and non-small cell lung cancer (GI₅₀ = 7.94 μM). Compound 16 ranks as nearly 1.5-fold more potent (mean GI₅₀ = 15.8 μM) compared to 5-FU (mean GI₅₀ = 22.6 μM).

Full text of DOI:10.1016/j.ejmech.2014.04.029

European Journal of Medicinal Chemistry 79 (2014) 446e454
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of some novel substituted
quinazolines as antitumor agents
,
Amer M. Alanazi a, Alaa A.-M. Abdel-Aziz a b, Ibrahim A. Al-Suwaidan a,
,e,
Sami G. Abdel-Hamide c, Taghreed Z. Shawer d, Adel S. El-Azab a
*
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
b Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
c Department of Pharmaceutical Chemistry, College of Pharmacy, Salman Bin Abdulaziz University, Alkharj, Saudi Arabia
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
e Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 6-chloro-2-p-tolylquinazolinone and substituted-(4-methylbenzamido)benzamide
(1e20) were designed, synthesized and evaluated for their in-vitro antitumor activity. Compounds 3,
14 and 16 possessed remarkable broad-spectrum antitumor activity. Compound 16 was found to be a
Received 3 February 2014
Received in revised form
6 April 2014
Accepted 8 April 2014
Available online 12 April 2014
particularly active growth inhibitor of the renal cancer (GI50 ¼ 4.07
ovarian cancer (GI50 ¼ 7.41 M) and non-small cell lung cancer (GI50 ¼ 7.94
as nearly 1.5-fold more potent (mean GI50 ¼ 15.8 M) compared to 5-FU (mean GI50 ¼ 22.6
m
M), CNS cancer (GI50 ¼ 7.41
m
M),
m
mM). Compound 16 ranks
m
m
M).
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Synthesis
Quinazoline
In-vitro antitumor evaluation
Selective activity
NCI
1. Introduction
Many of chemotherapeutics currently used in cancer therapy are
agents which inhibit tumor growth by inhibiting the replication
and transcription of DNA. The practice of chemotherapy of cancer
suffers from various drawbacks viz. the participation of a number of
enzymes like ribonucleotides reductase (RNR), topoisomerase I
(Topo I) and topoisomerase II (Topo II) at different stages of
development of cancer [7], survival of cancer cells even under
anaerobic conditions [8], and ultimately the problem of multidrug
resistance [9e11] developed in the cancerous cells towards
chemotherapeutic agents.
Cancer is a disease characterized by a shift in the controlled
mechanisms that govern cell proliferation and differentiation [1].
Malignancy is caused by abnormalities in cells, which might be due
to inherited genes or caused by outside exposure of the body to
chemicals, radiation, or even infectious agents [2,3]. The develop-
ment of new anti-cancer therapeutic tools has advanced greatly in
the past decade; thus approaches for the treatment of cancer have
moved towards targeting the specific molecular alterations that
occur in tumor cells. This approach has been concentrated on the
development of ideal anticancer drugs that eradicate cancer cells
without harming normal tissues [4,5]. Unfortunately, no currently
available agents meet this criterion and clinical use of drugs in-
volves a weighing of benefits against toxicity in a search of favor-
able therapeutic index [6].
As an important pharmacophore, quinazoline has a variety of
biological activities [12e29].
FDA has approved several quinazoline derivatives as anticancer
drugs, such as Gefitinib, Erlotinib, Lapatinib and Vandetanib. Based
on the good performances of quinazoline derivatives in anticancer
application, development of novel quinazoline derivatives as anti-
cancer drugs is a promising field.
In the present study, we have designed a number of new qui-
nazoline and diamide derivatives (1e20) containing various sub-
stituent with different electronic environment which would affect
the lipophilicity, and hence the activity of the target molecules and
biologically evaluated there in-vitro antitumor activities. The
* Corresponding author. (Adel S. El-Azab), Department of Pharmaceutical
Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia.
E-mail addresses: adelazaba@yahoo.com, adelazaba@hotmail.com (A.S. El-
Azab).
http://dx.doi.org/10.1016/j.ejmech.2014.04.029
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

A.M. Alanazi et al. / European Journal of Medicinal Chemistry 79 (2014) 446e454
447
objective of forming these hybrids is an attempt to attain an active
antitumor agent with potentiated activity and selectivity toward
cancerous cells.
Furthermore, when compound 1 was reacted with hydrazine
hydrate in ethanol at room temperature and/or boiling ethanol
gave
N-(4-chloro-2-(hydrazinecarbonyl)phenyl)-4-
methylbenzamide (10) and/or 3-amino-6-chloro-2-p-tolylquina-
zolin-4(3H)-one (11) in 90% and 88% yield, respectively.
2. Results and discussion
As well, the reaction of compound 11 with chloroacetylchloride
and/or benzaldehyde provided 2-chloro-N-(6-chloro-4-oxo-2-p-
tolylquinazolin-3(4H)-yl)acetamide (12) and 3-(benzylidenea-
mino)-6-chloro-2-p-tolylquinazolin-4(3H)-one (16) in 89% and 92%
yield, respectively. 2-Amino-N-(6-chloro-4-oxo-2-p-tolylquinazo-
lin-3(4H)-yl)acetamide (13) and N-(6-chloro-4-oxo-2-p-tolylqui-
nazolin-3(4H)-yl)-2-hydrazinylacetamide (14) were obtained by
stirring of compound 12 with concentrated ammonia solution and/
or hydrazine hydrate at room temperature in relatively good yield.
In addition, 11-chloro-7-p-tolyl-2,3-dihydro-[1,2,4,5]tetraze-
pino[2,3-c]quinazolin-4(5H)-one (15) was achieved via boiling of
compound 14 with acetic acid in the presence of fused sodium
acetate in 46% yield (Scheme 2).
2.1. Chemistry
6-Chloro-2-p-tolyl-4H-benzo[d][1,3]oxazin-4-one (1) was pre-
pared by the reaction of 5-chloroanthranilic acid with 4-
methylbenzoyl chloride in pyridine followed by boiling with ace-
tic anhydride [27]. Treatment of 1 with formamide furnished 6-
chloro-2-p-tolylquinazolin-4(3H)-one (2) in 73% yields. Boiling of
compound 1 with various amines in anhydrous pyridine afforded 5-
chloro-2-(4-methylbenzamido)-N-substituted benzamide (3e5) in
90e94% yields.
Additionally, reaction of 6-chloro-2-ptolyl-4H-benzo[d][1,3]
oxazin-4-one (1) with ethyl 2-aminoacetate hydrochloride in
boiling pyridine gave
a
mixture of ethyl 2-(5-chloro-2-(4-
Compound 10 showed a characteristic (CO) band of diamide
moiety at 1671, 1668 cmꢀ1 and (NH) absorption band at 3277, 3172,
3168 cmꢀ1 as well as 1H NMR showed a singlet signal at 11.20 and
doublet signal at 8.11 ppm corresponding to (CONH) group. Addi-
tionally compound 11 revealed (NH2) and (CO) groups at 3266, 3121
and 1680 cmꢀ1 respectively, in addition to singlet signal at
5.70 ppm due to the presence of (NH2) group in 1H NMR spectrum.
1H NMR spectra of compounds 12 and 16 were characterized by the
disappearance of (NH2) singlet signal at 5.70 ppm of compound 11
with appearance of new singlet signal at 9.01 ppm and 11.67 ppm
for (HNCO) and (HC]N) moieties respectively, as well as the
presence of additional aliphatic carbon at 40.4 ppm for compound
12 in 13C NMR spectrum.
methylbenzamido)benzamido)acetate (6) and ethyl 2-(6-chloro-
4-oxo-2-p-tolylquinazolin-3(4H)-yl)acetate (7) in 34% and 60%
yields, respectively.
Moreover, 2-(6-chloro-4-oxo-2-p-tolylquinazolin-3(4H)-yl)ace-
tohydrazide (8) and 5-chloro-N-(2-hydrazinyl-2-oxoethyl)-2-(4-
methylbenzamido)benzamide (9) were obtained in a good yield
(80% and 86%, respectively) via the reaction of compound 6 or 7
with hydrazine hydrate in boiling ethanol (Scheme 1).
IR spectra of compounds 3e5 showed absorption bands at
3173e3182 cmꢀ1 and 1668e1670 cmꢀ1 due to stretching vibration
of the (CONH) group, moreover, 1H NMR spectra showed the
appearance of two (CONH) as a singlet signal at d (12.46, 9.57 ppm),
(11.57, 10.64 ppm) and (11.39, 11.14 ppm) respectively. The presence
of a band at 1720e1716 cmꢀ1 due to the (CO) group of the ester
moiety as well as 1H NMR spectra showed the appearance of two
IR of compounds 13 showed absorption bands of (NH2) at 3269,
3175 cmꢀ1 and 1704, 1670 cmꢀ1 due to stretching vibration of (CO)
groups with the presence of (NH2) signal at 7.27 ppm in 1H NMR.
Compound 15 showed characteristic (CO) band at 1702 cmꢀ1 and
(NH) absorption band at 3167 cmꢀ1 due to the amide moiety as well
signals at
d 4.14e4.11 and 1.20e1.13 ppm of the ester function
confirmed compounds 6e7.
Scheme 1. Reactions of 6-chloro-2-p-tolyl-4H-benzo[d][1,3]oxazin-4-one.

448
A.M. Alanazi et al. / European Journal of Medicinal Chemistry 79 (2014) 446e454
Scheme 2. The synthesis and reactions of 3-amino-6-chloro-2-p-tolylquinazolin-4(3H)-one.
as 1H NMR showed a singlet signals at 11.23 and 3.24 ppm corre-
sponding to (CONH and COCH2) groups.
singlet signals at 9.69 and 4.92 ppm for (NH & NH2) respectively.
Compounds 19 and 20 were approved by the disappearance of
singlet signals at 9.69 and 4.92 ppm for (NH & NH2) groups with
appearance of new typical singlet proton of (SH) at 14.60 ppm for
compound 20.
On the other hand, 6-chloro-4-hydrazinyl-2-p-tolylquinazoline
(18) was achieved by reaction of compound 2 with Lawsson reagent
in boiling toluene followed by boiling with hydrazine hydrate in
62% overall yield. Correspondingly, reaction of compound 18 with
carbon disulfide in ethanol containing potassium hydroxide and/or
sodium nitrite in the presence of dilute hydrochloric acid produced
9-chloro-5-p-tolyltetrazolo[1,5-c]quinazoline (19) and 9-chloro-5-
p-tolyl-[1,2,4]triazolo[4,3-c]quinazoline-3-thiol (20) in 61% and
64% yield, respectively (Scheme 3).
2.2. Antitumor activity
The selected synthesized compounds were submitted and
evaluated in vitro at the single concentration of 10 mM toward a
panel of approximately 60 cancer cell lines at the National Cancer
Institute (NCI). The human tumor cell lines were derived from nine
different cancer types: leukemia, melanoma, lung, colon, CNS,
ovarian, renal, prostate, and breast cancers. Primary anticancer
assays were performed according to the US NCI protocol (http://
dtp.nci.nih.gov), as described elsewhere [30e33]. The compounds
were added at the single concentration, and the cell culture was
incubated for 48 h. The end point determinations were made with a
protein binding dye, sulforhodamine B (SRB). The data reported as
Compound 17 revealed a characteristic (CS) band at 1206 cmꢀ1
with disappearance of (CO) group at 1668 cmꢀ1of compound 2.
Likewise, 1H NMR of compound 17 showed fading singlet proton of
amide moiety (CONH) at 12.41 ppm through the presence of singlet
signal thioamide group (CSNH) at 13.98 ppm with existence of (CS)
peak at 187.2 ppm in 13C NMR. Compound 18 was confirmed by the
loss of singlet signal of thioamide group (CSNH) at 13.98 ppm and
(CS) peak at 187.2 ppm in NMR spectrum with the presence of new
Scheme 3. The synthesis and reactions of 6-chloro-4-hydrazinyl-2-p-tolylquinazoline.

A.M. Alanazi et al. / European Journal of Medicinal Chemistry 79 (2014) 446e454
449
mean-graph of the percent growth of the treated cells, and pre-
sented as percentage growth inhibition (GI %) caused by the test
compounds (Table 1).
Table 1
Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10
concentration.
m
M
With regard to broad spectrum antitumor activity; close ex-
amination of the data presented in Table 1, revealed that com-
pounds 3, 14 and 16 are the most active members of this study,
showing effectiveness toward numerous cell lines belong to
different tumor subpanels. Consequently, compound 16 was
selected in advanced assay against a panel of approximately 60
tumor cell lines at 10-fold dilutions of five concentrations (100, 10,
Subpanel tumor
cell lines
% Growth Inhibition (GI %)a
3
4
5
13
14
15
16
19
20
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
PRMI-8226
SR
Non-small cell lung cancer
A549/ATCC
EKVX
27
49
70
49
23
63
nt
nt
nt
nt
nt
nt
29
16
35
14
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
11
e
e
e
12
e
12
11
e
26
e
11
13
e
18
20
20
1, 0.1, and 0.01 mM) [26]. Dose response curves obtained from the
15
e
13
13
NCI’s in vitro disease-oriented human tumor cells line of compound
16 on nine cancer disease at five concentrations were shown in
Fig. 1. Based on the cytotoxicity assays, three antitumor activity
doseeresponse parameters were calculated for experimental
agents against each cell line: GI50, molar concentration of the
compound that inhibits 50% net cell growth; TGI, molar concen-
tration of the compound leading to total inhibition; LC50, molar
concentration of the compound leading to 50% net cell death.
Furthermore a mean graph midpoints (MG-MID) were calculated
for each of the parameters, giving an average activity parameter
overall cell lines for the tested compounds (Table 2). The NCI
screening data analysis indicated that compound 16 possessed
potent in vitro antiproliferative activity, with mean GI50 values
e
15
20
12
e
e
13
12
e
e
23
34
83
35
nt
11
12
e
87
40
98
14
L
11
24
23
52
47
11
51
e
11
11
e
e
22
15
11
e
HOP-62
NCI-H226
HOP-92
29
e
e
e
27
e
e
e
e
e
e
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
e
e
e
e
13
29
49
61
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
47
21
22
e
e
21
e
e
e
e
e
11
e
e
e
e
e
28
e
e
e
e
e
e
e
e
34
e
e
e
e
e
e
e
17
e
e
e
e
e
11
e
14
11
43
77
40
42
e
e
e
11
46
16
33
21
28
47
14
17
18
23
14
14
11
e
across the 60 cell lines 15.8
compound 16 was found to be a particularly active growth inhibitor
of the renal cancer (GI50 ¼ 4.07 M), CNS cancer (GI50 ¼ 7.41 M),
ovarian cancer (GI50 ¼ 7.41 M) and non-small cell lung cancer
(GI50 ¼ 7.94 M). Compound 16 ranks as nearly 1.5-fold more
potent (mean GI50 ¼ 15.8 M) compared with 5-FU (mean
GI50 ¼ 22.6 M).
mM. At the second assay (Table 2), the
12
e
m
m
e
m
e
e
13
22
e
e
28
54
22
25
58
e
e
e
e
e
40
22
25
74
88
e
e
e
e
e
e
m
SF-539
SNB-19
SNB-75
U251
16
e
29
13
28
14
15
e
18
11
e
m
m
33
19
24
e
15
e
On the other hand, Compound 4 yielded selective activities to-
ward CNS cancer cell lines, compound 5 showed selective activities
toward leukemia cancer cell lines, whereas compound 19 revealed
selective activities toward Non-small cell lung cancer. Compounds
13, 15, and 20 possess weak antitumor activity.
Regarding the activity toward individual cell lines; compound 3
and 5 showed selective activity against leukemia cell lines CCRF-
CEM and K-562 with GI values of 27, 29% and 70, 35% respec-
tively, whilst, HL-60(TB), MOLT-4, PRMI-8226 and SR cell lines were
sensitive to compounds 3 and 16 with GI values of 49, 26, 49, 18, 23,
20 and 63, 20% respectively.
Non-small cell lung; A549/ATCC and HOP-62 cell lines proved to
be selectively sensitive to 14, 16 and 19 with GI values of 23, 87, 47
and 83, 98, 51% respectively, compounds 3, 14 and 16 have fanatical
activity against EKVX and NCI-H522 cell lines with GI values of 20,
34, 40 and 47, 61 and 52% respectively. In addition, compound 14
and 20 proved to susceptible to NCI-H226 cell line with GI values of
35 and 27%, compounds 4, 5 and 19 have dedicated activity against
NCI-H522 cell lines with GI values of 21, 22 and 21%. Meanwhile,
NCI-H322M and NCI-H460 cell lines were susceptible to compound
14 and 16 with GI values of 29, 24 and 49 and 23% respectively,
while HOP-62 cell lines having a tendency to compound 4 with GI
values of 29% and compound 16 lethal to HOP-92 cell lines.
Respecting colon cancer; compounds 14 and 16 showed GI
values of 28, 34, 46, 47, 28 and 23% with colon COLO 205, HCT-116
and SW-620 cell line respectively, while 3 and 14 demonstrated
moderate activities against HT29 and KM12 cancer cell line with GI
value 77, 33, 40 and 21% respectively. On the other hand, compound
3 verified sensitivity in 43 and 42% to colon HCT-15 and SW-620
cancer cells.
e
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
21
12
26
98
49
17
22
15
30
e
e
e
e
11
e
e
e
e
e
e
27
14
17
e
e
e
e
e
e
e
e
e
e
11
35
29
34
e
e
e
12
e
e
e
e
e
e
e
11
e
19
e
13
e
e
e
e
e
e
23
11
23
18
e
e
e
13
e
39
e
e
e
12
33
e
e
e
16
23
18
16
e
e
14
12
22
e
e
e
e
11
e
e
e
41
45
80
18
23
24
84
e
e
e
e
e
e
e
43
42
86
18
73
30
64
e
e
e
e
e
e
e
e
e
11
e
21
e
11
14
e
e
13
28
e
e
e
e
e
e
12
e
12
e
e
e
e
22
46
42
35
11
33
61
25
e
30
16
74
40
e
e
e
21
14
23
13
e
e
39
e
34
e
11
e
19
e
17
18
e
14
14
e
20
e
14
e
25
e
15
e
e
e
e
e
32
26
33
16
e
e
e
e
e
e
e
19
e
24
e
13
11
18
24
24
e
15
e
23
11
e
e
33
28
e
e
30
27
18
e
e
e
DU-145
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
47
e
11
16
e
19
e
12
e
22
62
40
e
e
20
41
44
e
e
e
16
e
e
e
e
11
e
27
e
21
e
e
e
e
e
e
e
e
T-47D
MDA-MB-468
e
20
11
20
11
52
e
25
23
e
Concerning CNS cancer; compounds 14 and 16 showed selective
activity through CNS cancer SF-268, SNB-19 and U251 cell line with
GI values of 28, 40, 22, 25, 58 and 88%, respectively. Compounds 4,
5, 14 and 16 showed GI values of 29, 22, 54, 22% and 28, 24, 25 and
17
e
11
a
nt ¼ not tested; e, GI <10%; L, compound proved lethal to the cancer cell line.

450
A.M. Alanazi et al. / European Journal of Medicinal Chemistry 79 (2014) 446e454
Fig. 1. Dose response curves (% growth verses sample concentration at NCI fixed protocol,
compound (16) on nine cancer disease.
mM) obtained from the NCI’s in vitro disease-oriented human tumor cells line of
74% to CNS cancer SF-539, SNB-75 cell lines, respectively, while
compound 3 showed activities against SNB-75 cell lines with GI
values of 33%.
GI values of 23, 20 and 25%. Additionally, Renal UO-31 cancer cell
line sensitive to compounds 3 and 19 with GI values of 24 and 24%.
Prostate PC-3 cell lines proved to be selectively sensitive to
compounds 3, 5,14 and 16 with GI value of 24, 23, 33 and 30%, at the
same time as compounds 14 and 16 active against prostate DU-
145 cell line with GI value of 28 and 27%.
Pertaining to breast cancer; breast MDA-MB-231/ATCC cell line
convinced responsive to compounds 14, 16 and 19 with GI value of
62, 41and 27%, respectively. Compounds 4, 5, 14, 16 and 19 showed
GI effectiveness against breast T-47D cell line with values of 20, 20,
52, 25 and 23%, concomitantly, breast cancer MCF7 and HS 578Tcell
lines demonstrated sensitivity to compounds 3, 14 and 16 with GI
values of 47, 22, 20% and 21, 40 and 44%, respectively.
Regarding Melanoma; compounds 3 and 16 are active against
M14, MDA-MB-435 and UACC-62 cell lines with GI values of 26, 29,
98, 34, 30 and 23% respectively, whereas, LOX IMVI cell lines sen-
sitive to compounds 3 and 14 with GI values of 21% and 27%.
Compounds 3 and 5 are active against SK-MEL-5 cell lines with GI
values of 22 and 39%, while Compounds 14 and 20 showed mod-
erate activity towards UACC-257 cell lines with GI values of 23% and
33%. Melanoma SK-MEL-2, SK-MEL-28 and MALME-3M cell lines
showed selective activity to compounds 3, 14 and 16 with GI values
of 49%, 23 and 35%.
Respecting ovarian cancer; compounds 3, 5, 14 and 16 showed
activities against Ovarian OVCAR-4 cell line with GI values of 21, 22,
80 and 86%. Ovarian NCI/ADR-RES cell line responsive to com-
pounds 3, 14 and 16 with GI values of 28, 24 and 30%, while com-
pounds 14 and 16 showed remarkable potency against IGROV1,
OVCAR-3, OVCAR-8 and SK-OV-3 cell line with GI values of 41, 43,
45, 42, 23, 73, 84 and 64% respectively.
Relating to renal cancer; renal 786-0, ACHN, CAKI-1, SN12C, TK-
10 and UO-31 cell line sensitive to compounds 14 and 16 with GI
values of 22, 30, 42, 74, 35, 40, 33, 32, 61, 26, 25 and 33% respectively.
Compounds 3, 5, 14 and 15 showed certain activity against renal
A498 cell lines with GI values of 21, 39, 46 and 34%, whilst com-
pounds 3, 5 and 19 have certain sensitivity to CAKI-1 cell lines with
2.3. Structure activity correlations
Compounds of the present investigation belong to N-
substituted-5-chloro-2-(4-methylbenzamido)benzamide and 3-
substituted-6-chloro-2-p-tolylquinazolin-4(3H)-one. This study
revealed that compounds 13 and 15 devoid of any significant
antitumor potency.
Structureeactivitycorrelation revealed that reaction of 6-chloro-
2-p-tolyl-4H-benzo[d][1,3]oxazin-4-one (1) with various amines
gave N-substituted benzamide (3e5) analogs with variable potency,
therefore N-benzyl-5-chloro-2-(4-methylbenzamido)benzamide
(3) possessed broad spectrum antitumor activity compared to N-

A.M. Alanazi et al. / European Journal of Medicinal Chemistry 79 (2014) 446e454
451
Table 2
Compound 16 median growth inhibitory (GI50
,
m
M), total growth inhibitory (TGI,
m
M) and median lethal (LC50
,
mM) concentration of in-vitro subpanel tumor cell lines.
Compound
Activity
Subpanel tumor cell lines
MG-MIDa
Leukemia
NSC lung
cancer
Colon
cancer
CNS
cancer
Melanoma
Ovarian
cancer
Renal
cancer
Prostate
cancer
Breast
cancer
b
16
GI50
7.94
43.65
35.48
7.41
4.07
69.9
13.6
15.8
7.41
72.1
TGI
LC50
GI50
83.1
97.7
22.60
b
5-FU
15.1
8.4
70.6
61.4
45.6
22.7
76.4
b
b
TGI
LC50
a
Full panel mean-graph midpoint (
Compounds showed values >100
m
m
M).
M.
b
phenyl benzamide (4) or N-pyridyl benzamide (5), also N-pyridyl
benzamide 4 more active than N-phenyl benzamide 5.
Perkin-Elmer spectrometer. 1H NMR and 13C NMR was recorded in
DMSO-d6 on a Jeol 500 MHz instrument using TMS as internal
Replacement of 3-amino group of compound 11 with 2-
aminoacetamide produced compound 13 with insignificant anti-
tumor activity. Switch of 2-aminoacetamide group at position 3 of
compound 13 with 2-hydrazinylacetamide at position 3 afforded
compound 14 with dramatically advanced the antitumor activity;
on the other hand, cyclization of compound 14 gave compound 15
with severely failure of the antitumor activity.
Exchange of 3-amino group of compound 11 with benzylidine
moiety gave compound 16 enhanced the antitumor activity as
compared to compound 13-15.
Cyclization of compound 18 produced compounds 9-chloro-5-
p-tolyltetrazolo[1,5-c]quinazoline (19) and 9-chloro-5-p-tolyl-
[1,2,4]triazolo[4,3-c]quinazoline-3-thiol (20) with variable anti-
tumor activity, consequently substitution of triazole moiety of
compound 20 into tetrazole moiety gave compound 19 accompa-
nied with mild improved the antitumor activity.
standard (chemical shifts in d ppm). Microanalytical data (C, H, and
N) were performed on Perkin-Elmer 240 analyzer and they agreed
with proposed structures within ꢁ0.4% of the calculated values.
Mass spectra were recorded on a Shimadzu PQ-5000 GCeMS
apparatus. Solvent evaporation was performed under reduced
pressure using Buchan Rotatory Evaporator unless otherwise
stated. T.L.C. was performed on precoated silica gel plates (60- F254,
0.2 mm), manufactured by E.M. Sciences, Inc, and shortwave UV
(254) nm was used to detect the U.V. absorbing compounds
(CH2Cl2, EtOH 10:1 v/v). Compounds 1, 2, 11, 17 and 18 were pre-
pared according to reported procedure [27].
4.1.1. 6-Chloro-2-p-tolylquinazolin-4(3H)-one (2)
Benzoxazine (1) (10 mmol, 2.71 g) was heated under reflux in
formamide (50 ml) for 3 h. The solid obtained was filtered while hot
and dried, mp 300e302 ꢂC in 73% yield.
IR (KBr, cmꢀ1 : 3179 (NH), 1668 (CO); 1H NMR (DMSO-d6):
) n
3. Conclusion
d
12.41 (s, 1H, NHCO), 8.11 (s, 1H, Ar.), 8.04 (d, 1H, J ¼ 1.5 Hz), 7.82 (d,
2H, J ¼ 8.0 Hz), 7.68 (d, 1H, J ¼ 8.5 Hz), 7.37e7.22 (m, 2H), 2.33 (s,
3H, CH3). 13C NMR (DMSO-d6):
167.7, 160.2, 147.9, 146.3, 143.4,
A
novel series of 6-chloro-2-p-tolylquinazolinone and
substituted (4-methylbenzamido)benzamide (1e20) were
designed, synthesized and evaluated for their in-vitro antitumor
activity. A single dose (10 M) of the test compounds was used in
d
134.9, 129.9, 129.8, 128.2, 124.3, 125.3, 127.5, 21.6. MS: 252 ([Mꢀ28],
78% rel. abundance).
m
the National Cancer Institute (NCI) 60 cell lines panel assay. The
results of this study demonstrated that compound 4 yielded se-
lective activities toward CNS cancer cell lines, compound 5 showed
selective activities toward leukemia cancer cell lines, whereas
compound 19 revealed selective activities toward Non-small cell
lung cancer, in addition to, compounds 13, 15, and 20 possess weak
antitumor activity. On the other hand, 3, 14 and 16 possessed
remarkable broad-spectrum antitumor activity. Compound 16 was
carried over and tested against a panel of 60 different tumor cell
lines at a 5-log dose rang. Three response parameters, GI50, TGI and
LC50 were calculated for each cell line, using the known drug 5-
Fluorouracil (5-FU) as a positive control. Compound 16 was found
to be a particularly active growth inhibitor of the renal cancer
4.1.2. General procedure for the synthesis of compounds 3e5
A solution of benzoxazine (1) (2 mmol, 542 mg) and appropriate
amine (2.1 mmol) in pyridine (10 ml) was heated under reflux for
10e12 h. The reaction mixture was cooled and the solvent was
removed under reduced pressure; the residue was triturated with
water and filtered. The solid obtained was dried and recrystallized
from ethanol.
4.1.2.1. N-Benzyl-5-chloro-2-(ꢀ41-methylbenzamido)benzamide
(3).
Mp 270e272 ꢂC, IR (KBr, cm
) n
: 3182, 3173 (NH), 1670 (CO); 1H
NMR (DMSO-d6):
d
12.46 (s, 1H), 9.57 (t, 1H, J ¼ 5.5, 6.0 Hz), 8.68 (d,
1H, J ¼ 9.0 Hz), 7.99 (d, 1H, J ¼ 2.5 Hz), 7.64 (d, 2H, J ¼ 7.0 Hz), 7.62
(dd, 1H, J ¼ 2.0 Hz), 741e7.24 (m, 6H), 7.20 (d, 1H, J ¼ 6.0 Hz), 4.38
(GI50 ¼ 4.07
(GI50 ¼ 7.41
Compound 16 ranks as nearly 1.5-fold more potent (mean
GI50 ¼ 15.8 M) compared with 5-FU (mean GI50 ¼ 22.6 M).
m
M), CNS cancer (GI50 ¼ 7.41
m
M), ovarian cancer
(d, 2H, J ¼ 6.0 Hz), 2.33 (s, 3H). 13C NMR (DMSO-d6):
d 21.0, 42.7,
m
M) and non-small cell lung cancer (GI50 ¼ 7.94
mM).
121.4, 122.0, 126.5, 127.0, 127.3, 127.9, 128.3, 129.9, 131.4, 132.0,
138.5, 138.6, 142.3, 164.3, 167.3. MS: 378 ([M]þ, 31% rel. abundance).
m
m
4. Experimental
4.1.2.2. 5-Chloro-2-(4-methylbenzamido)-N-phenyl benzamide (4).
Mp 260e263 ꢂC, IR (KBr, cmꢀ1 : 3180, 3175 (NH), 1669 (CO); 1H
) n
4.1. Chemistry
NMR (DMSO-d6): d 11.57 (s, 1H), 10.64 (s, 1H), 8.47 (d, 1H,
J ¼ 9.0 Hz), 7.98 (d, 1H, J ¼ 2.0 Hz), 7.80 (d, 2H, J ¼ 8.0 Hz), 7.73e7.67
Melting points (corrected) were recorded on Barnstead 9100
Electrothermal melting apparatus. IR spectra were recorded on a
(m, 3H), 7.42e7.37 (m, 4H), 7.16 (t, 1H, J ¼ 7.0, 7.5 Hz), 2.41 (s, 3H).
13C NMR (DMSO-d6):
d 21.0, 121.1, 123.0, 124.4, 126.9, 127.1, 127.8,

452
A.M. Alanazi et al. / European Journal of Medicinal Chemistry 79 (2014) 446e454
128.6, 128.7, 129.4, 131.3, 131.8, 137.5, 138.2, 166.0. MS: 364 ([M]þ,
27% rel. abundance).
4.1.6. 2-Chloro-N-(6-chloro-4-oxo-2-p-tolylquinazolin-3(4H)-yl)
acetamide (12)
3-Amino-6-chloro-2-p-tolylquinazolin-4(3H)-one (11) (3 mmol,
855 mg) was stirred at room temperature with chloroacetyl-
chloride (3.1 mmol, 350 mg) in dichloromethane (10 ml) containing
triethylamine (5 mmol, 505 mg) for 12 h. The reaction mixture was
diluted with water and extract with chloroform, the extract was
washed successively with water and brine, dried over anhydrous
MgSO4, evaporated under reduced pressure and chromatographed
(SiO2, 20 g, elution with CHCl3eAcOEt, 10:1 v/v) afforded com-
pound 12, mp 217e218 ꢂC in 89% yield.
4.1.2.3. 5-Chloro-2-(4-methylbenzamiꢀd1o)-N-(pyridin-2-yl)benza-
) n: 3179, 3176 (NH), 1668
mide (5). Mp 257e259 ꢂC, IR (KBr, cm
(CO); 1H NMR (DMSO-d6):
d 11.39 (s, 1H), 11.14 (s, 1H), 8.40e8.36
(m, 2H), 8.08 (d, 1H, J ¼ 8.5 Hz), 7.98 (d, 1H, J ¼ 7.5 Hz), 7.86e7.83
(m, 1H), 7.80 (d, 2H, J ¼ 7.5 Hz), 7.64 (dd, 1H, J ¼ 2.5 Hz), 7.34 (d, 2H,
J ¼ 8.0 Hz), 7.20e7.18 (m, 1H), 2.48 (s, 3H). 13C NMR (DMSO-d6):
d
21.0,115.4,120.3,123.2,124.5,127.0,127.1,129.2,129.3,131.3,131.9,
137.4, 138.1, 142.2, 148.0, 151.6, 164.6, 166.6. MS: 365 ([M]þ, 24% rel.
abundance).
IR (KBr, cmꢀ1
d6):
) n
: 3183 (NH), 1708, 1668 (CO); 1H NMR (DMSO-
d
11.67 (s, 1H), 8.13 (d, 1H, J ¼ 2.5 Hz), 7.88 (dd, 1H, J ¼ 2.5 Hz),
7.76 (d, 1H, J ¼ 8.7 Hz), 7.58 (d, 2H, J ¼ 8.0 Hz), 7.29 (d, 2H,
4.1.3. General procedure for the synthesis of compounds 6e7
A solution of benzoxazine (1) (2 mmol, 542 mg) and ethyl 2-
aminoacetate hydrochloride (2.1 mmol, 292 mg) in pyridine
(10 ml) was heated under reflux for 10 h. The reaction mixture was
cooled and the solvent was removed under reduced pressure; the
residue was triturated with water and filtered. The solid obtained
was dried and chromatographed (SiO2, 20 g, elution with CHCl3)
afforded compounds 6 and 7.
J ¼ 8.0 Hz), 4.21 (dd, 2H, J ¼ 8.5 Hz), 2.49 (s, 3H). 13C NMR (DMSO-
d6):
d 21.0, 40.4, 121.8, 125.4, 128.5, 128.6, 129.9, 131.7, 135.3, 140.3,
145.3, 156.2, 158.3, 165.5. MS: 361 ([M]þ, 32%, 363 [Mþ2], 11% rel.
abundance).
4.1.7. 2-Amino-N-(6-chloro-4-oxo-2-p-tolylquinazolin-3(4H)-yl)
acetamide (13)
2-Chloro-N-(6-chloro-4-oxo-2-p-tolylquinazolin-3(4H)-yl)acet-
amide (12) (3 mmol, 1086 mg) was stirred at room temperature
with ammonia solution (5 ml) for 12 h. The reaction mixture was
filtered, washed with water, dried and recrystallized from ethanol,
mp 222e223 ꢂC in 74% yield.
4.1.3.1. Ethyl 2-(5-chloro-2-(4-methylbenzamido)benzamido)acetate
(6). Mp 233e235 ꢂC, IR (KBr, cmꢀ1
) n: 3179, 3178 (NH), 1716, 1661
(CO); 1H NMR (DMSO-d6):
d
12.20 (s, 1H), 9.50 (t, 1H, J ¼ 5.5,
IR (KBr, cmꢀ1
(DMSO-d6):
) n
: 3269, 3175 (NH), 1704, 1670 (CO); 1H NMR
12.95 (s, 1H), 8.85 (d, 2H, J ¼ 5.5 Hz), 8.68 (t, 1H,
5.75 Hz), 8.68 (d, 1H, J ¼ 9.0 Hz), 7.94 (d, 1H, J ¼ 2.5 Hz), 7.79 (d, 2H,
J ¼ 8.0 Hz), 7.68 (dd, 1H, J ¼ 2.5 Hz), 7.38 (d, 2H, J ¼ 8.0 Hz), 4.14 (dd,
2H, J ¼ 7.0 Hz), 4.06 (d, 2H, J ¼ 5.75 Hz), 2.38 (s, 3H), 1.20 (t, 3H,
d
J ¼ 2.5, 3.0 Hz), 8.18 (t, 1H, J ¼ 7.0, 7.5 Hz), 8.09 (d, 1H, J ¼ 2.5 Hz),
8.01 (dd, 1H, J ¼ 2.5 Hz), 7.77 (d, 1H, J ¼ 9.0 Hz), 7.58 (d, 1H,
J ¼ 8.0 Hz), 7.27 (d, 2H, J ¼ 8.0 Hz), 5.80 (d, 1H, J ¼ 15.5 Hz), 5.68 (d,
J ¼ 7.0 Hz). 13C NMR (DMSO-d6):
d 14.0, 21.0, 41.4, 60.7, 121.0, 122.0,
126.6, 127.0, 127.9, 129.5, 131.3, 132.3, 138.4, 142.5, 164.4, 168.0,
1H, J ¼ 15.5 Hz), 2.38 (s, 3H). 13C NMR (DMSO-d6):
d 21.0, 59.9, 121.7,
169.3. MS: 374 ([M]þ, 12% rel. abundance).
125.4, 127.9, 128.5, 128.6, 129.7, 130.0, 131.0, 140.4, 145.2, 145.7,
146.6, 155.8, 158.0, 164.2. MS: 342 ([M]þ, 18% rel. abundance).
4.1.3.2. Ethyl 2-(6-chloro-4-oxo-2-ꢀp1-tolylquinazolin-3(4H)-yl)ace-
tate (7). Mp 247e248 ꢂC, IR (KBr, cm
)
n
: 1720, 1668 (CO); 1H NMR
4.1.8. N-(6-Chloro-4-oxo-2-p-tolylquinazolin-3(4H)-yl)-2-
hydrazinylacetamide (14)
(DMSO-d6):
d 8.12e8.09 (m, 1H), 7.98e7.93 (m, 1H), 7.77e7.72 (m,
1H), 7.44 (d, 2H, J ¼ 7.5 Hz), 7.34 (d, 2H, J ¼ 7.0 Hz), 4.63 (s, 2H), 4.11
(q, 2H, J ¼ 6.5 Hz), 2.39 (s, 3H),1.13 (t, 3H, J ¼ 6.5 Hz). MS: 356 ([M]þ,
36% rel. abundance).
2-Chloro-N-(6-chloro-4-oxo-2-p-tolylquinazolin-3(4H)-yl)acet-
amide (12) (3 mmol, 1.086 g) was stirred at room temperature with
hydrazine hydrate (5 ml) for 12 h. The reaction mixture was filtered,
washed with water, dried and recrystallized with ethanol, mp 243e
244 ꢂC in 75% yield.
4.1.4. N-(4-Chloro-2-(hydrazinecarbonyl)phenyl)-4-
methylbenzamide (10)
A mixture of benzoxazine (1) (10 mmol, 2.71 g) and hydrazine
hydrate (750 mg, 15 mmol) in (50 ml) absolute ethanol was stirred
at room temperature for 12 h. The reaction mixture was filtered and
dried, mp 224e226 ꢂC in 90% yield.
4.1.9. 11-Chloro-7-p-tolyl-2,3-dihydro-[1,2,4,5]tetrazepino[2,3-c]
quinazolin-4(5H)-one (15)
N-(6-Chloro-4-oxo-2-p-tolylquinazolin-3(4H)-yl)-2-
hydrazinylacetamide (14) (3 mmol, 1071 mg) was heated under
reflux with acetic acid (10 ml) containing fused sodium acetate
(1.0 g) for 12 h. The reaction mixture was cooled and the solvent
was removed under reduced pressure; the residue was triturated
with water and filtered. The solid obtained was washed with water,
dried and recrystallized from ethanol, mp > 300 ꢂC in 46% yield.
IR (KBr, cmꢀ1
(DMSO-d6):
) n
: 3277, 3172, 3168 (NH), 1671, 1668 (CO); 1H NMR
11.20 (s, 1H), 8.11 (d, 1H, J ¼ 2.5 Hz), 7.93 (dd, 1H,
d
J ¼ 2.5 Hz), 7.77 (d, 1H, J ¼ 9.0 Hz), 7.54 (d, 2H, J ¼ 7.0 Hz), 7.30 (d,
2H, J ¼ 8.0 Hz), 2.38 (s, 3H), 1.38 (s, 2H). 13C NMR (DMSO-d6):
d 21.0,
122.0, 125.4, 128.4, 128.5, 129.9, 130.3, 131.5, 135.3, 140.2, 145.4,
IR (KBr, cmꢀ1
) n
: 3167 (NH), 1702 (CO); 1H NMR (DMSO-d6):
156.6, 158.6, 168.0. MS: 303 ([M]þ, 17% rel. abundance).
d
11.23 (s, 1H), 8.15 (d, 1H, J ¼ 23.0 Hz), 7.90 (dd, 1H, J ¼ 24.0 Hz),
7.76 (t, 1H, J ¼ 87.0 Hz), 7.56 (d, 2H, J ¼ 80.5 Hz), 7.29 (d, 2H,
4.1.5. 3-Amino-6-chloro-2-p-tolylquinazolin-4(3H)-one (11)
Benzoxazine (1) (2 mmol, 543 mg) was heated under reflux in
hydrazine hydrate (5 ml) for 3 h. The solid obtained was filtered
while hot and dried, mp 208e210 ꢂC in 88% yield. IR (KBr, cmꢀ1) n:
3266, 3121 (NH), 1680 (CO); 1H NMR (DMSO-d6): d 8.10 (d, 1H,
J ¼ 2.5 Hz), 7.84 (dd, 1H, J ¼ 2.5 Hz), 7.74e7.71 (m, 3H), 7.29 (d, 2H,
J ¼ 8.0 Hz), 5.70 (s, NH2), 2.39 (s, 3H, CH3). 13C NMR (DMSO-d6):
d 160.7, 156.6, 145.9, 139.9, 134.8, 132.1, 131.2, 130.2, 130.1, 128.4,
125.3, 121.6, 21.4. MS: 285 ([M]þ, 60% rel. abundance and 287
[Mþ2] 20% rel. abundance).
J ¼ 79.5 Hz), 3.24 (s, 2H), 2.49 (s, 3H). 13C NMR (DMSO-d6):
d 21,
56.1, 121.9, 125.4, 128.4, 128.5, 129.4, 130.3, 131.5, 135.2, 140.1, 145.3,
156.6, 158.6, 168.6. MS: 339 ([M]þ, 29% rel. abundance).
4.1.10. 3-(Benzylideneamino)-6-chloro-2-p-tolylquinazolin-4(3H)-
one (16)
3-Amino-6-chloro-2-p-tolylquinazolin-4(3H)-one (11) (3 mmol,
855 mg) was stirred at room temperature with benzaldehyde
(3.1 mmol, 320 mg) in methanol (10 ml) for 12 h. The reaction
mixture was filtered and dried, mp 288e290 ꢂC in 92% yield.

A.M. Alanazi et al. / European Journal of Medicinal Chemistry 79 (2014) 446e454
453
IR (KBr, cmꢀ1
)
n
: 1671(CO); 1H NMR (DMSO-d6):
d
9.01 (s, 1H),
diseases, in accordance with the protocol of the Drug Evaluation
Branch, National Cancer Institute, Bethesda, MD [30e33].
8.14 (s, 1H), 7.91 (d, 1H, J ¼ 7.0 Hz), 7.80 (d, 1H, J ¼ 7.0 Hz), 7.75 (d,
2H, J ¼ 6.0 Hz), 7.61e7.52 (m, 5H), 7.25 (d, 2H, J ¼ 6.0 Hz), 2.34 (s,
3H).
Tested compounds were added to the culture at a single con-
centration (10ꢀ5 M), and the cultures were incubated for 48 h. End
point determinations were made with a protein binding dye, sul-
forhodamine B (SRB). The results for each tested compound were
reported as the growth percentage of the treated cells when
compared to that of the untreated control cells. The percentage
growth was evaluated spectrophotometrically versus controls not
treated with test agents. The cytotoxic and/or growth inhibitory
effects of the most active selected compounds were tested in vitro
against the full panel of about 60 human tumor cell lines at 10-fold
dilutions of five concentrations ranging from 10ꢀ4 to 10ꢀ8 M. A 48 h
continuous drug exposure protocol was followed, and an SRB
protein assay was used to estimate cell viability or growth. By use of
the seven absorbance measurements [time zero, (Tz), control
growth in the absence of drug (C), and test growth in the presence
of drug at the five concentration levels (Ti)], the percentage growth
was calculated at each of the drug concentrations levels. Percentage
growth inhibition was calculated as
4.1.11. 6-Chloro-2-p-tolylquinazolin-4(3H)-thione (17)
A
mixture of 6-chloro-2-p-tolylquinazolin-4(3H)-one (2)
(10 mmol, 2.70 g) and Lawsson reagent (5.0 g) was heated under
reflux in anhydrous toluene (70 ml) for 6 h. The reaction mixture
was filtered while hot, the solvent was evaporated and the residue
was chromatographed (SiO2, 30 g, elution with CHCl3) gave com-
pound 17. Mp 265e267 ꢂC in 80% yield. IR (KBr, cmꢀ1
)
n
: 3188 (NH),
1206 (CS); 1H NMR (DMSO-d6):
d 13.98 (s, 1H, Ar.), 8.49 (d, 1H,
J ¼ 1.5 Hz), 8.06 (d, 2H, J ¼ 7.5 Hz), 7.87e7.83 (m, 1H), 7.75e7.69 (m,
1H), 7.33 (d, 2H, J ¼ 7.5 Hz), 2.38 (s, 3H). 13C NMR (DMSO-d6):
d
187.2, 161.8, 152.3, 147.9, 143.6, 135.7, 132.6, 129.5, 128.9, 128.3,
126.4, 122.6, 21.5. MS: 286 ([M]þ, 39% rel. abundance).
4.1.12. 6-Chloro-4-hydrazino-2-p-tolylquinazoline (18)
6-Chloro-2-p-tolylquinazolin-4(3H)-thione (17) (2 mmol,
773 mg) was heated under reflux in (5 ml) hydrazine hydrate for 3 h
The solid obtained was filtered while hot, dried and recrystallized
from ethanol, mp 273e275 ꢂC in 62% yield.
½ðTi ꢀ TzÞ=ðC ꢀ TzÞ ꢃ 100ꢄfor concentration of which
Ti ꢅ Tz
1H NMR (DMSO-d6):
d
9.69 (s, 1H), 8.45 (d, 2H, J ¼ 8.0 Hz), 8.32
(s, 1H), 7.74 (d, 2H, J ¼ 11.0 Hz), 7.29 (d, 2H, J ¼ 8.0 Hz), 4.92 (s, 2H),
2.37 (s, 3H). 13C NMR (DMSO-d6):
160.2, 159.5, 148.8, 140.4, 136.0,
½ððTi ꢀ TzÞ=TzÞ ꢃ 100ꢄfor concentration of which
Ti ꢆ Tz
d
133.3, 130.1, 129.4, 129.2, 128.6, 122.2, 114.0, 21.5. MS: 285 ([Mþ1],
6% rel. abundance).
Three dose response parameters (GI50, TGI, LC50) were calcu-
lated for each compound. Growth inhibition of 50% (GI50) was
calculated from [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢃ 100 ¼ 50, which was the drug
concentration resulting in a 50% lower net protein increase in the
treated cells (measured by SRB staining) compared to the net
protein increase seen in the control cells. The drug concentration
resulting in total growth inhibition (TGI) was calculated from
Ti ¼ Tz. The LC50 (concentration of drug resulting in a 50% reduction
in the measured protein at the end of the drug treatment compared
to that at the beginning) indicating a net loss of cells following
treatment was calculated from [(Ti ꢀ Tz)/Tz] ꢃ 100 ¼ ꢀ50. Values
were calculated for each of these three parameters if the level of
activity was reached; however, if the effect was not reached or was
exceeded, the value for that parameter was expressed as more or
less for the maximum or minimum concentration tested. The
lowest values are obtained with the most sensitive cell lines. The
4.1.13. 9-Chloro-5-p-tolyltetrazolo[1,5-c]quinazoline (19)
A solution of 6-chloro-4-hydrazino-2-p-tolylquinazoline (18)
(568 mg, 2 mmol) in EtOHeAcOH (10:1) (11 ml) at 0 ꢂC, concen-
trated hydrochloric acid (1 ml) and NaNO2 solution (742 mg,
10 mmol) (1 ml) were added, the mixture was stirred for 2 h at the
same temperature and the stirring was continued for 10 h at room
temperature. The mixture was diluted with water, extracted with
EtOAc, the extract was washed successively with water and brine,
dried over anhydrous MgSO4, evaporated under reduced pressure
and chromatographed (SiO2, 50 g, elution with hexaneeAcOEt, 10:1
v/v) afforded compound 19, mp 277e278 ꢂC in 61% yield.
1H NMR (DMSO-d6):
J ¼ 5.5 Hz), 7.85 (dd, 1H, J ¼ 2.5 Hz), 7.74 (d, 1H, J ¼ 8.75 Hz), 7.53 (d,
1H, J ¼ 2.5 Hz), 7.38 (d, 2H, J ¼ 4.5 Hz), 2.39 (s, 3H). MS: 295 ([M]þ,
49% rel. abundance).
d
8.41 (d, 1H, J ¼ 8.0 Hz), 8.08 (d, 2H,
compounds having GI50 ꢆ 100
mM were declared to be active.
Acknowledgments
4.1.14. 9-Chloro-5-p-tolyl-[1,2,4]triazolo[4,3-c]quinazoline-3-thiol
(20)
The authors extend their appreciation to the Deanship of Sci-
entific Research at King Saud University for funding the work
through the research group project No. RGP-VPP-163. The authors
would like to express their gratitude and thanks to the National
Cancer Institute (NCI), Bethesda Maryland, USA, http://dtp.cancer.
gov/for doing the antitumor testing of the new compounds.
A solution of 6-chloro-4-hydrazino-2-p-tolylquinazoline (18)
(568 mg, 2 mmol), carbon disulphide (760 mg, 10 mmol) and po-
tassium hydroxide (170 mg, 3 mol) in ethanol (10 ml) was stirred at
room temperature for 4 h the reaction mixture was heated under
reflux for 6 h, cooled, the solvent was evaporated under reduced
pressure. The residual solid obtained was dissolved in water, acid-
ified with dilute hydrochloric acid and the precipitated solid was
filtered, washed with water, and crystallized from ethanol, mp
263e265 ꢂC in 64% yield.
Appendix A. Supplementary data
1H NMR (DMSO-d6):
d
14.60 (s, 1H), 8.47 (d, 1H, J ¼ 8.5 Hz),
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.029.
8.24e8.85 (m, 3H), 8.60e8.51 (m, 1H), 7.35 (dd, 2H, J ¼ 6.5 Hz), 2.40
(s, 3H). MS: 326 ([M]þ, 36% rel. abundance).
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