Synthesis and in vitro antitumor activity of new series of benzothiazole and pyrimido[2,1-b]benzothiazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.07.097

New series of benzothiazole and pyrimido[2,1-b]benzothiazole derivatives were synthesized and characterized by analytical and spectrometrical methods (IR, HRMS, ¹H and ¹³C NMR). Nineteen of the synthesized compounds were selected by

Full text of DOI:10.1016/j.ejmech.2014.07.097

European Journal of Medicinal Chemistry 85 (2014) 576e592
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro antitumor activity of new series of
benzothiazole and pyrimido[2,1-b]benzothiazole derivatives
Moustafa T. Gabr, Nadia S. El-Gohary*, Eman R. El-Bendary, Mohamed M. El-Kerdawy
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
New series of benzothiazole and pyrimido[2,1-b]benzothiazole derivatives were synthesized and char-
acterized by analytical and spectrometrical methods (IR, HRMS, 1H and 13C NMR). Nineteen of the
synthesized compounds were selected by the National Cancer Institute (NCI), USA, to be screened for
Received 10 June 2014
Received in revised form
24 July 2014
Accepted 25 July 2014
Available online 29 July 2014
their antitumor activity at a single dose (10
compounds, 4, 6, 10, 14, 17 and 20 were selected for further evaluation at five dose level screening.
Compounds 17 (GI50 ¼ 0.44 M, TGI ¼ 1.2 M and LC50 MG-MID ¼ 6.6 M) and 4 (GI50 ¼ 0.77 M,
M) were proved to be the most active members in this study.
mM) against a panel of 60 cancer cell lines. The most active
m
m
m
m
TGI ¼ 2.08
m
M and LC50 MG-MID ¼ 11.74
m
Keywords:
Benzothiazoles
Pyrimidobenzothiazoles
Synthesis
3D and 2D pharmacophoric maps for the structural features of both compounds were studied.
© 2014 Elsevier Masson SAS. All rights reserved.
Antitumor activity
Pharmacophore study
1. Introduction
region of the kinase enzyme. Benzothiazole derivatives constitute
an important class of therapeutic agents in medicinal chemistry.
Cancer is the second most common cause of death after heart
disease [1]. During the past decades, cancer continued to be a
worldwide killer [2]. Despite the extensive research and rapid
progress in cancer treatment, there is a growing demand for new
therapies. It is important to identify new agents and new targets for
the treatment of cancer. Receptor protein tyrosine kinases play an
important role in signal transduction pathways that regulate cell
division and differentiation. Epidermal growth factor receptor
tyrosine kinase (EGFR-TK) (also known as ErbB-1 or HER-1) and the
related human epidermal growth factor receptor (also known as
ErbB-2 or HER-2) are among the growth factor receptor kinases that
have been identified as being important in cancer [3]. EGFR
dependent aberrant signaling is associated with cancer cell prolif-
eration, apoptosis, angiogenesis and metastasis [4]. A number of
small molecule EGFR kinase inhibitors have been evaluated in
cancer clinical trials. Anilinoquinazoline-containing compounds,
erlotinib (Tarceva®) [4,5] and gefitinib (Iressa®) [6] have been
approved for the chemotherapeutic treatment of patients with
advanced non-small cell lung cancer. They contain 4-(substituted
amino)pyrimidine pharmacophoric core that binds to the hinge
Literature survey revealed that this nucleus is associated with
diverse pharmacological effects, including antitumor [7e18] and
antioxidant [19,20] activities. Moreover, pyrimido[2,1-b]benzo-
thiazole derivatives have been extensively investigated for their
pharmacological uses [7,21e26], some of these compounds showed
antitumor activity [7,24e26]. In addition, pyrazole-containing
compounds have received considerable attention owing to their
diverse chemotherapeutic potentials, including antitumor activity
[27e35]. These compounds are able to inhibit different kinds of
kinases with powerful antitumor activity [36e38]. On the same
line, [1,3,4] oxadiazole-containing compounds exhibited antitumor
activity [39e42]. Taking all the above findings into consideration
and in searching for new compounds endowed with potent anti-
tumor activity, we report herein the synthesis of new series of
benzothiazole and pyrimido[2,1-b]benzothiazole derivatives and
evaluation of their antitumor activity.
2. Rational and design
Quinazolines have emerged as a versatile template for inhibition
of a diverse range of receptor tyrosine kinases. The most widely
studied of these receptors is the epidermal growth factor receptor
(EGFR); with the small-molecule inhibitor, gefitinib being the first
agent from this class to be approved for the treatment of non-small
cell lung cancer as EGFR-TK inhibitor [43,44]. Subsequent research
* Corresponding author.
E-mail addresses: dr.nadiaelgohary@yahoo.com, nadiaelgohary2005@gmail.com
(N.S. El-Gohary).
http://dx.doi.org/10.1016/j.ejmech.2014.07.097
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
577
aimed at further exploration of the SAR of this novel template led to
the discovery of highly selective compounds that target EGFR. Later
on, a variety of compounds of structurally diverse chemical classes
was proved to be highly potent and selective ATP-competitive in-
hibitors. Benzothiazoles act via competing with ATP for binding at
the catalytic domain of EGFR-TK [45]. The ATP-binding site (Fig. 1)
has the following features; Adenine region which contains two key
hydrogen bonds formed by the interaction of N1 and N6 of the
adenine ring. Many potent inhibitors use one of these hydrogen
bonds. Sugar pocket which is a hydrophilic region. Hydrophobic
regions and channels, though not used by ATP but plays an
important role in inhibitor selectivity and binding affinity. Phos-
phate binding region which is largely solvent exposed and can be
used for improving inhibitor selectivity [46].
Our strategy is directed toward designing a variety of ligands
which are structurally similar to the basic skeleton, 4-
anilinoquinazoline of tinibs (erlotinib and gefitinib) with diverse
chemical properties (Fig. 2). Accordingly, we replaced quinazoline
ring with benzothiazole since both rings are isosteric with the
adenine portion of ATP and can mimic the ATP competitive binding
regions of EGFR-TK. The manipulation strategy embraces the incor-
poration of pyrazole and other azole moieties into the benzothiazole
and pyrimido[2,1-b]benzothiazole ring systems in order to verify the
importance of these moieties for the antitumor activity (Fig. 3).
In our previous work [47,48], compounds 4, 6, 10, 14, 17 and 20
(with considerable activity against individual cell lines that over-
express EGFR) were further evaluated for their EGFR-TK inhibitory
activity at Reaction Biology Corporation, USA. Compound 4 dis-
played the highest EGFR-TK inhibitory activity at the single dose
screening; subsequently, it was tested using ten dose IC50 mode and
amino-6-chlorobenzothiazole (1) was allowed to react with chlor-
oacetyl chloride in carbon tetrachloride in the presence of trie-
thylamine to produce the chloroacetamide derivative
2 [7].
Reaction of 2 with hydrazine hydrate in ethanol gave the hydrazi-
nylacetamide analog 3 [7].
Concerning Scheme 2, condensation of 3 with diethyl ethox-
ymethylenemalonate in acetonitrile in the presence of anhydrous
potassium carbonate gave the targeted ethyl pyrazole-4-
carboxylate derivative 4. 1H NMR spectrum of this compound
showed the triplet-quartet pattern characteristic for the ethyl
protons at
NMR spectrum displayed two signals at
responding to ethyl carbons. Reacting
d
1.30 and 4.05e4.35 ppm, respectively. In addition, 13
14.6 and 61.0 ppm cor-
with ethyl ethox-
C
d
3
ymethylenecyanoacetate, ethoxymethylenemalononitrile or ethyl
acetoacetate in ethanol in the presence of anhydrous potassium
carbonate gave the ethyl pyrazole-4-carboxylate 5, pyrazole-4-
carbonitrile 6 or 4,5-dihydropyrazol-5-one 7, respectively. The
structure of compound 5 was confirmed by the presence of the
triplet-quartet pattern characteristic for the ethyl protons at
d 1.30
and 4.28e4.35 ppm, respectively. The structural assignments of
compound 6 were based on analytical and spectral data. For
example, IR spectrum showed a characteristic absorption band at
2215 cmꢀ1 corresponding to (C^N) group. The presence of the
(C^N) was further confirmed by the presence of a signal at
d
117.1 ppm in the 13C NMR spectrum.
Analogously, compound 8 was synthesized by direct fusion of 3
with diethyl malonate. The IR spectrum of this compound showed
three significant absorption bands at 1720, 1686 and 1654 cmꢀ1
corresponding to three (C]O) groups.
When compound 3 was allowed to react with acetylacetone or
benzoylacetone in glacial acetic acid, 3,5-dimethyl-1H-pyrazole 9
or 3-methyl-5-phenyl-1H-pyrazole 10 was obtained, respectively.
it exhibited IC50 value of 0.239
mM [48]. Compound 4 was also
examined over other 10 different human kinases to study its
selectivity against EGFR-TK [48].
1H NMR spectrum of 9 showed two singlets at
d
2.05 and 2.25 ppm
corresponding to two (CH3) groups. Moreover, 13C NMR spectrum of
the same compound revealed two signals at 20.7 and 23.2 ppm
corresponding to two (CH3) carbons. 1H NMR spectrum of 10
showed a characteristic singlet at 1.90 ppm corresponding to
d
3. Results and discussion
d
3.1. Chemistry
(CH3) group. In addition, HRMS of the same compound displayed a
significant peak at m/z [MꢀH]ꢀ 381.0661 which is in agreement
with its molecular formula C19H14ClN4OSꢀ.
A general approach to synthesize the designed compounds is
outlined in Schemes 1e5. In Scheme 1, the starting compound, 2-
Fig. 1. The molecular surface representation of the ATP-binding site which consists of adenine region, sugar pocket, hydrophobic regions I and II and phosphate binding region [46].

578
M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
Fig. 2. Reported antitumor quinazolines and proposed analogues incorporating different aryl and heterocyclic moieties; (A) benzothiazole derivatives, (B) pyrimido[2,1-b]ben-
zothiazole derivatives.
Fig. 3. Incorporation of pyrazole and other azole moieties into the benzothiazole and pyrimido[2,1-b]benzothiazole ring systems.
Scheme 1. Synthesis of compound 3.

M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
579
Scheme 2. Synthesis of compounds 4e10.
Concerning
Scheme
3,
reaction
of
2-amino-6-
with ethyl ethoxymethylenecyanoacetate or ethyl acetoacetate in
ethanol in the presence of anhydrous potassium carbonate resulted
in the formation of ethyl pyrazole-4-carboxylate 15 or 4,5-
dihydropyrazol-5-one 16, respectively. 1H NMR spectrum of com-
pound 15 showed the triplet-quartet pattern characteristic for the
chlorobenzothiazole (1) with dimethyl acetylenedicarboxylate in
refluxing methanol gave the targeted methyl pyrimido[2,1-b]ben-
zothiazole-4-carboxylate derivative 11 [7]. Reaction of 11 with hy-
drazine hydrate in ethanol produced the corresponding hydrazide
12 [7].
Referring to Scheme 4, condensing 12 with acetylacetone in
glacial acetic acid afforded 3,5-dimethyl-1H-pyrazole derivative 13.
1H NMR spectrum of this compound showed two characteristic
ethyl protons at
d
1.23 and 4.08e4.25 ppm, respectively. 1H NMR
2.15 and
spectrum of 16 showed two characteristic singlets at
d
3.73 ppm corresponding to (CH3) group and (pyrazoline-H),
respectively. Moreover, 13C NMR spectrum of the same compound
singlets at
d
2.42 and 2.95 ppm corresponding to two (CH3) groups.
19.4 and
revealed two signals at d 20.2 and 60.3 ppm corresponding to (CH3)
carbon and (pyrazoline C4), respectively.
In addition, 13C NMR spectrum showed two signals at
d
20.2 ppm corresponding to the two (CH3) carbons. Heating 12 with
diethyl malonate at 200 ꢁC yielded the pyrazolidine-3,5-dione de-
rivative 14. 1H NMR spectrum of this compound showed a char-
Reacting 12 with ethyl cyanoacetate in ethanol produced 2,3-
dihydropyrazol-3-one derivative 17. Reaction of hydrazide 12
with diethyl ethoxymethylenemalonate in acetonitrile in the
presence of anhydrous potassium carbonate produced ethyl 5-
hydroxy-1H-pyrazole-4-carboxylate analog 18. IR spectrum of this
compound showed two characteristic absorption bands at 3377
acteristic singlet at
Furthermore, 13C NMR spectrum showed a signal at
corresponding to the (pyrazolidine C-4). Condensing compound 12
d
4.15 ppm corresponding to (pyrazolidine-H).
d
51.6 ppm

580
M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
Scheme 3. Synthesis of compound 12.
Scheme 4. Synthesis of compounds 13e18.
and 1715 cmꢀ1 for (OH) and (COOC2H5) groups, respectively.
Moreover, 1H NMR spectrum showed the triplet-quartet pattern
derivative 19. 13C NMR spectrum of this compound showed three
characteristic signals at 161.2, 172.8 and 187.6 ppm corre-
d
characteristic for the ethyl protons at
respectively.
Regarding Scheme 5, reaction of 12 with carbon disulfide in
ethanol at room temperature in the presence of potassium hy-
droxide afforded the potassium hydrazine-1-carbodithioate
d
1.20 and 4.03e4.16 ppm,
sponding to two (C]O) and (C]S) carbons, respectively. On the
other hand, heating 12 with carbon disulfide in ethanol in the
presence of potassium hydroxide underwent cyclization to afford
[1,3,4]oxadiazole-5-thione analog 20. The spectroscopic data of
compound 20 were consistent with its structure. For example, 13
C

M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
581
Scheme 5. Synthesis of compounds 19e24.
NMR spectrum showed a characteristic signal at
d
183.4 ppm
melanoma, ovarian, renal, prostate and breast cancer cells. The data
reported as mean-graph of the percentage growth of the treated
cells, and presented as percentage growth inhibition (GI%). The
obtained results of the tested compounds (Tables 1 and 2) showed
distinctive potential pattern of selectivity as well as broad spectrum
antitumor activity. The mean inhibition percentages for each of the
tested compounds over the full panel of cell lines are illustrated in
Fig. 4.
corresponding to (C]S) carbon. In addition, HRMS exhibited a
significant peak at m/z [MꢀH]ꢀ 334.9551 which is in agreement
with its molecular formula C12H4ClN4O2Sꢀ2 . Heating 19 or 20 with
hydrazine hydrate in ethanol resulted in the formation of com-
pound 21. 1H NMR spectrum of this compound showed two sig-
nificant singlets at d 4.15 and 12.55 ppm indicating (NH2) and (NH)
groups, respectively. When compound 12 was allowed to react
with phenyl isothiocyanate in ethanol, 2-(8-chloro-2-oxo-2H-
pyrimido[2,1-b]benzothiazole-4-carbonyl)-N-(phenyl)hydrazine-
1-carbothioamide (22) was obtained. 1H NMR spectrum of 22
Regarding the activity toward individual cell lines, compounds
4, 10, 14 and 17 exhibited lethal effects (>100% inhibition) against
nearly all tested leukemia cell lines, whereas compounds 4 and 17
displayed promising activity against almost all colon, melanoma,
renal and breast cancer cell lines. In addition, compounds 4, 10 and
17 exhibited excellent activity against most of the tested ovarian
cancer cell lines. Close examination of the data presented in
Tables 1 and 2, revealed that compounds 4 and 17 are the most
active members in this study, showing lethal effects against
numerous cell lines belonging to different tumor subpanels. The
percentage inhibition of compounds 4 and 17 over each cell line of
the NCI-60 cell lines panel is illustrated in Fig. 5. The six most active
compounds, 4, 6, 10, 14, 17 and 20 passed this primary anticancer
assay and consequently they were carried over to the five dose
screen against a panel of about 60 cancer cell lines. Results indi-
cated that these compounds possess considerable activity against
non-small cell lung cancer (NCI-H522), colon cancer (HCT-116,
HCT-15 and HT29) and breast cancer (MDA-MB-468 and MDA-MB-
231/ATCC) cell lines in which EGFR is overexpressed in varying
levels. These results support the postulation that these compounds
may act via inhibition of EGFR-TK. Moreover, the broad spectrum
activity of compound 4 against various cell lines encouraged
further investigation of its inhibitory activity of additional targets.
Previously, we examined compound 4 over EGFR-TK as well as
other 10 different human kinases. Results indicated that compound
showed three singlets at
sponding to three (NH) groups. Furthermore, 13C NMR spectrum of
the same compound showed a significant signal at 187.7 ppm
d 10.20, 11.78 and 13.63 ppm corre-
d
corresponding to (C]S) carbon. Heating 22 in pyridine produced
5-phenylamino-[1,3,4]oxadiazole derivative 23. On the other hand,
cyclization of 22 into the corresponding 5-phenylamino-[1,3,4]
thiadiazole derivative 24 was accomplished via its stirring with
concentrated sulfuric acid at room temperature. HRMS of com-
pound 24 revealed a peak at m/z [MꢀH]ꢀ 410.0023 which is in
agreement with its molecular formula C18H9ClN5OS2- .
3.2. Antitumor screening
3.2.1. Preliminary in vitro antitumor screening
Out of the newly synthesized compounds, nineteen derivatives,
4e10 and 13e24 were selected by the National Cancer Institute
(NCI) in vitro disease-oriented human cells screening panel assay to
be evaluated for their antitumor activity in accordance with the
current protocol of the Drug Evaluation Branch, NCI, Bethesda
[49e51]. A single dose (10 mM) of the tested compounds was used
in the full NCI-60 cell lines panel assay which includes nine tumor
subpanels; namely, leukemia, non-small cell lung, colon, CNS,

582
M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
4 has moderate selectivity against EGFR-TK [48]. It is worth
mentioning that the ongoing biological screening of compound 4
will shed the light on its broad spectrum antitumor activity.
Table 1
Percentage growth inhibition (GI %) of compounds 4e10 at concentration (10
over in vitro subpanel tumor cell lines.
mM)
3.2.1.1. Structureeactivity relationship (SAR) studies. Structure ac-
tivity correlation based on the number of cell lines that were
proved to be sensitive toward each of the tested compounds is
discussed:
Subpanel tumor cell lines
% Growth inhibition (GI %)a
4
5
6
7
8
9
10
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
Non-small cell lung cancer
A549/ATCC
HOP-62
L
L
L
L
L
L
5.9
e
L
L
35.1
55.6
51.0
76.8
19.7
7.5
9.7
9.7
13.5
3.6
22.4
3.0
6.3
38.3
13.4
44.0
65.1
38.0
51.1
L
L
96.5
L
L
L
Concerning compounds 4e10, combination of the pyrazole
moiety with the benzothiazole nucleus increased the antitumor
activity (compounds 4, 6, 9 and 10). The presence of 2-hydroxy
ester substituents on the pyrazole ring (compound 4) greatly
enhanced the antitumor activity against all tested cancer cell lines
compared to the 2-amino ester analog (compound 5). In addition,
replacement of the ester group at 4-position of the pyrazole moiety
with the cyano group increased the antitumor activity against most
of the tested cancer cell lines (compare 6 versus 5). Compound 8
containing 3,5-dioxopyrazole moiety exhibited higher activity
against most of the tested cancer cell lines compared to compound
8.5
8.0
7.1
10.6
e
24.4
0.91
L
L
nt
L
L
3.5
8.6
nt
18.6
nt
22.0
13.4
nt
5.2
nt
9.5
e
10.2
7.0
73.9
21.0
6.6
6.4
0.69
4.9
16.0
12.0
nt
15.7
nt
23.2
8.7
42.8
39.8
55.4
nt
64.2
nt
HOP-92
49.0
14.2
9.0
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM 12
SW-620
CNS cancer
SF-268
92.3
L
L
2.2
4.4
7.3
e
7.6
10.2
12.9
e
0.8
L
48.7
L
7
containing 3-methyl-5-oxopyrazole moiety, this might be
attributed to the additional hydrogen bonding site in compound 8.
Furthermore, replacement of 5-methyl substituent on the pyrazole
moiety with 5-phenyl substituent in attempt to fit into the addi-
tional hydrophobic pocket of EGFR-TK active site resulted in higher
antitumor activity (compound 10 versus 9).
L
L
L
L
L
L
L
e
e
e
e
e
79.1
65.6
84.0
84.7
95.8
33.8
79.3
e
e
56.3
e
e
10.0
33.2
27.0
36.3
21.3
0.36
7.0
7.5
e
11.5
18.0
94.7
20.6
9.1
1.3
3.7
e
6.5
e
Taking into account the structure of pyrimido[2,1-b]benzothia-
zoles 13e24, it is assumed that combination of 3-oxopyrazole
moiety with the pyrimido[2,1-b]benzothiazole nucleus enhanced
the antitumor activity over the other substituted pyrazole moieties
(compounds 14 and 17 versus 13, 15, 16 and 18). Furthermore,
compound 16 containing 3-methyl-5-oxopyrazole moiety exhibi-
ted higher activity compared to compound 13 containing 3,5-
dimethylpyrazole moiety, this might be attributed to the addi-
tional hydrogen bonding site in compound 16. In addition, com-
pound 14 containing 3,5-dioxopyrazole moiety exhibited higher
activity against most of the tested cancer cell lines compared to
compound 16 containing 3-methyl-5-oxopyrazole moiety, this
might be attributed to the additional hydrogen bonding site in the
3,5-dioxopyrazole derivative 14. Furthermore, combination of
[1,3,4]oxadiazole moiety with the pyrimido[2,1-b]benzothiazole
nucleus increased the activity over the analogs with [1,3,4]thia-
diazole moiety (compare 20 and 23 versus 24). Trials to improve the
binding affinity toward the target receptor by promoting hydrogen
bonding interaction of compound 20 via introducing an additional
amino group to the bioisosteric [1,2,4]triazole derivative (com-
pound 21) or by favoring hydrophobic interaction via adding an
extra phenyl moiety (compound 23) resulted in decreased activity.
Comparing the antitumor activity of benzothiazole derivatives
4, 5, 7 and 9 with that of the corresponding pyrimido[2,1-b]ben-
zothiazole derivatives 18, 15, 16 and 13, respectively, we can
conclude that the benzothiazole derivatives have higher activity
against most of the tested cancer cell lines than the corresponding
pyrimido[2,1-b]benzothiazoles.
4.5
2.3
17.0
e
e
e
L
L
L
65.3
L
L
e
70.5
2.3
10.0
4.0
30.6
0.8
e
e
25.4
28.2
14.6
12.0
17.8
16.8
L
SF-295
SF-539
SNB-19
SNB-75
7.2
2.1
e
21.3
9.8
3.1
22.3
8.9
24.1
66.0
31.3
L
10.0
12.2
26.8
7.3
3.2
18.4
10.1
U251
58.1
Melanoma
LOX IMVI
M14
L
L
L
4.8
1.7
e
L
4.5
e
e
e
28.2
14.2
9.0
12.8
e
nt
2.7
e
e
68.1
58.5
L
27.0
nt
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGORV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
12.5
0.4
0.67
20.0
11.8
32.5
L
L
e
62.3
e
e
e
e
L
L
nt
nt
1.6
6.4
nt
8.4
8.3
3.9
e
nt
4.8
35.5
55.8
74.0
16.5
L
L
L
L
L
L
L
e
L
24.5
e
16.7
e
11.4
e
33.0
18.4
23.7
20.9
21.5
16.5
20.8
L
L
L
38.7
73.2
63.8
L
2.25
16.7
13.2
3.4
0.87
e
55.0
19.2
6.5
e
8.5
e
3.5
37.5
66.9
e
9.9
e
e
1.5
L
91.3
L
L
nt
L
e
L
e
1.15
23.3
4.3
32.3
0.44
6.8
e
19.6
52.0
18.2
28.3
29.4
19.5
49.7
L
A498
ACHN
CAKI-1
RXF 393
SN12C
UO-31
11.3
12.4
19.6
9.2
4.2
31.5
20.7
6.8
30.6
3.2
21.3
40.2
10.6
83.3
92.0
31.3
53.1
L
52.2
65.6
7.6
3.7
L
L
36.1
Prostate cancer
PC-3
DU-145
Breast cancer
MCF-7
MDA-MB-231/ATCC
HS 578T
BT-549
MDA-MB-468
T-47D
81.7
L
20.4
e
15.6
2.8
18.6
e
4.9
e
46.5
9.0
43.9
22.5
3.2.2. Full in vitro five dose antitumor assay
About 60 cell lines of nine tumor subpanels; namely, leukemia,
non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate
and breast cancer cell lines were incubated with five concentra-
L
L
L
L
L
L
18.0
15.4
3.9
e
3.7
34.9
e
91.4
27.3
10.6
e
16.4
6.6
10.3
6.2
nt
15.4
33.6
42.7
36.3
29.7
23.6
nt
79.0
94.8
56.0
L
L
nt
tions (0.01e100 mM) for each compound and were used to create
e
log concentration e % growth inhibition curves. Three response
parameters (GI50, TGI, and LC50) were calculated for each cell line.
The GI50 value (growth inhibitory activity) corresponds to the
concentration of the compounds causing 50% decrease in cell
growth, the TGI value (cytostatic activity) is the concentration of
the compounds resulting in total growth inhibition and the LC50
8.3
nt
20.0
L
nt
63.4
Bold values used to point out the active compounds and lethal effect.
a
e, Percentage growth >100%; nt, not tested; L, lethal.

M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
583
Table 2
Percentage growth inhibition (GI %) of compounds 13e24 at concentration (10
m
M) over in vitro subpanel tumor cell lines.
Subpanel tumor cell lines
% Growth inhibition (GI %)a
13
14
15
16
17
18
19
20
21
22
23
24
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
9.7
e
L
L
75.7
L
L
L
5.2
e
11.0
3.5
11.3
17.6
3.4
L
L
L
L
L
L
14.8
10.9
32.6
11.8
12.8
11.0
13.3
e
87.5
89.1
86.2
99.6
85.7
86.2
10.4
0.7
2.0
0.5
6.4
e
8.5
e
38.4
19.0
50.4
43.5
49.3
71.2
40.6
9.2
18.1
15.0
4.4
1.9
0.4
9.2
e
13.2
6.7
8.2
8.3
5.0
11.2
12.0
e
3.7
9.1
e
22.6
14.5
10.8
Non-small cell lung cancer
A549/ATCC
HOP-62
0.07
7.4
34.6
18.5
3.8
e
29.3
23.4
nt
39.4
nt
5.2
21.2
L
e
8.1
8.1
nt
11.7
nt
6.5
0.5
26.5
68.0
27.3
nt
L
nt
27.9
76.6
L
12.6
e
6.0
e
90.0
60.8
nt
71.0
nt
63.8
93.7
L
e
e
23.8
10.8
91.4
36.2
38.6
15.7
36.1
41.2
e
e
e
e
e
HOP-92
nt
12.6
8.7
e
nt
14.7
nt
0.36
4.0
13.4
nt
16.5
nt
13.5
4.6
7.4
nt
11.6
nt
5.7
e
nt
2.0
nt
2.0
e
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
20.1
2.9
6.1
e
10.3
1.4
20.0
e
4.5
0.15
13.7
41.1
3.7
e
35.5
23.1
61.8
53.5
90.6
18.1
49.5
e
e
L
L
L
L
L
51.7
L
e
e
4.1
e
e
e
e
e
e
e
e
e
e
nt
6.8
5.0
e
e
e
e
59.7
90.4
80.5
28.5
85.8
70.5
e
18.7
58.0
38.0
50.4
31.9
31.0
e
1.2
e
12.3
5.6
5.2
3.5
e
e
2.0
12.0
6.1
0.65
e
1.5
5.3
e
7.1
12.0
7.0
e
18.2
e
HT29
KM 12
SW-620
e
e
2.6
e
15.4
e
2.7
e
e
2.6
CNS cancer
SF-268
SF-295
SF-539
SNB-19
e
98.2
21.1
22.3
14.5
L
e
e
90.0
1.8
L
49.2
58.7
62.1
e
3.8
nt
0.78
12.3
0.17
11.2
73.2
59.4
76.4
80.2
58.2
96.7
e
e
24.3
10.6
17.1
5.6
14.0
24.3
e
11.5
3.2
0.11
20.5
5.7
e
2.5
e
2.7
11.5
6.8
15.9
5.3
e
4.3
3.7
9.1
5.7
7.7
4.5
e
4.1
6.5
0.64
0.82
e
10.1
e
e
9.0
5.4
2.2
SNB-75
U251
17.1
e
31.5
13.5
Melanoma
LOX IMVI
M14
0.36
e
L
e
6.6
1.8
0.5
0.3
e
L
L
L
L
94.6
nt
L
e
4.5
0.95
5.4
0.34
e
nt
12.8
e
7.8
e
34.2
11.9
19.6
16.7
6.8
27.9
15.1
41.5
2.35
0.49
7.2
e
40.0
35.0
L
1.3
nt
1.62
e
e
74.7
73.5
81.6
67.8
nt
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGORV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
0.42
2.0
30.2
0.19
e
e
e
e
e
1.35
e
e
e
1.8
nt
e
nt
nt
9.8
5.4
0.75
1.0
3.9
9.9
e
nt
e
e
nt
e
nt
0.65
4.45
50.8
45.4
4.2
6.1
85.2
95.8
nt
e
L
6.85
e
e
L
2.1
e
9.2
0.8
5.3
e
90.4
L
L
1.8
e
e
76.6
83.7
77.8
52.6
77.3
84.0
42.0
9.8
e
e
66.4
38.4
42.4
15.8
14.9
22.8
12.2
e
e
84.8
30.2
11.0
44.4
41.3
71.3
1.4
7.1
8.0
3.1
e
e
e
5.1
1.7
3.9
6.1
3.0
e
e
e
7.4
14.1
4.8
e
9.6
6.2
0.5
e
L
e
e
e
99.3
89.2
51.7
4.7
e
e
e
e
e
e
0.8
e
e
0.49
e
3.0
e
e
e
69.1
0.94
35.5
71.4
27.1
30.8
L
3.7
20.7
3.9
6.8
nt
7.8
17.3
0.1
9.8
2.1
e
L
72.7
e
e
58.3
L
0.80
e
0.34
14.0
4.0
11.3
5.8
15.3
21.8
39.4
46.0
37.7
e
6.1
9.7
7.4
1.5
3.7
0.25
5.4
A498
ACHN
CAKI-1
RXF 393
SN12C
UO-31
e
7.1
12.0
8.5
nt
4.8
7.5
18.6
5.8
25.7
1.9
14.4
42.2
L
L
L
L
L
13.3
23.2
9.1
6.2
31.9
88.2
90.8
60.5
84.3
85.2
4.8
11.2
e
2.4
14.1
2.0
27.0
2.1
18.7
29.4
59.4
Prostate cancer
PC-3
DU-145
7.0
e
44.0
10.5
5.4
e
14.6
e
88.3
L
9.7
e
17.2
e
81.6
73.0
9.60
e
13.0
e
41.7
9.3
5.7
e
Breast cancer
MCF-7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
2.1
e
61.5
66.2
28.1
66.2
nt
7.6
e
11.5
e
L
L
85.2
L
nt
L
15.2
22.5
e
9.6
e
91.0
86.7
70.2
84.6
nt
8.3
18.3
4.2
e
14.2
8.7
e
55.6
67.8
24.5
15.0
60.8
nt
9.4
e
2.3
e
e
3.0
5.4
nt
e
e
3.4
5.2
19.7
e
1.06
26.3
nt
e
e
9.6
9.3
18.7
nt
8.2
nt
nt
7.5
nt
15.8
MDA-MB-468
L
17.8
L
Bold values used to point out the active compounds and lethal effect.
a
e, Percentage growth >100%; nt, not tested; L, lethal.

584
M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
Fig. 4. Mean inhibition percentages of the tested compounds at concentration (10 mM) over the NCI-60 cancer cell lines. The inhibition percentages were calculated by subtracting
the growth percentages from 100.
Fig. 5. % Growth inhibition expressed by compounds 4 and 17 at concentration (10 mM) over the NCI-60 cancer cell lines.
Table 3
Median growth inhibitory concentration (GI50
, mM) of the most active compounds over the most sensitive cell line of each tumor subpanel.
Comp. No.
Subpanel tumor cell lines
CCRF-CEMa
NCI-H522b
HT29c
SNB-75d
LOX IMVIe
IGROV1f
CAKI-1g
PC-3h
MDA-MB-468i
4
6
10
14
17
20
0.182
0.24
0.0575
0.0338
0.124
2.71
0.0573
0.191
0.154
0.0223
0.145
2.43
0.353
2.4
0.951
0.466
0.284
3.46
0.383
1.61
1.03
0.802
1.11
1.20
0.0319
0.199
0.0175
0.204
0.179
2.7
0.152
0.378
0.202
0.0428
1.47
0.548
2.62
0.0472
1.06
0.256
1.82
0.749
2.12
1.34
0.575
0.553
2.13
0.56
4.17
1.31
1.87
0.148
0.0691
2.29
Bold values represent GI50 values in the nanomolar range.
a
Leukemia cell line.
Non-small cell lung cancer cell line.
Colon cancer cell line.
CNS cancer cell line.
Melanoma cell line.
Ovarian cancer cell line.
Renal cancer cell line.
Prostate cancer cell line.
b
c
d
e
f
g
h
i
Breast cancer cell line.
value (cytotoxic activity) is the concentration of the compounds
causing 50% loss of initial cells at the end of the incubation period
(48 h). Full panel mean-graph midpoint values (MG-MID) for
certain agents are the average of individual GI50, TGI, or LC50 values
of all cell lines.
tested subpanel cancer cell lines (GI50
values < 100
of sensitivity against some individual cell lines (Table 3) as well as
broad spectrum antitumor activity (Tables 4 and 5).
,
TGI and LC50
m
M). These compounds showed a distinctive pattern
Concerning the sensitivity against some individual cell lines
In the present study, the six active compounds, 4, 6, 10, 14, 17
and 20 exhibited potential antitumor activity against most of the
(Table 3), compound 4 showed GI50 values of 0.0319 and 0.0573
against melanoma cancer (LOX IMVI) and non-small cell lung
mM

M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
585
cancer (NCI-H522) cell lines, respectively. In addition, compound 10
showed GI50 values of 0.0175, 0.0472 and 0.0575 M against mel-
anoma (LOX IMVI), renal cancer (CAKI-1) and leukemia (CCRF-CEM)
interaction through hydrogen bond donors which are represented
by red spheres, while compound 10 incorporates an extra hydro-
phobic feature represented by yellow (in web version) sphere
(Fig. 8).
m
cell lines, respectively. Furthermore, compound 14 showed GI50
values of 0.0223, 0.0338 and 0.0428
cancer (NCI-H522), leukemia (CCRF-CEM) and ovarian cancer
(IGROV1) cell lines, respectively.
With regard to broad spectrum antitumor activity, the results
revealed that the six active compounds, 4, 6, 10, 14, 17 and 20 have
effective growth inhibition GI50 (MG-MID) values of 0.77, 6.02, 1,
m
M against non-small cell lung
Similarly, the pharmacophoric map which was based on align-
ment of the structural features of pyrimido[2,1-b]benzothiazole
derivatives 14, 17 and 20 demonstrated the resemblance of the
geometric arrangement of their molecular features (Fig. 9).
4. Conclusion
1.04, 0.44 and 1.9
activity TGI (MG-MID) values of 2.08, 33.11, 2.63, 6.02, 1.2 and
8.3 M, respectively (Table 5). In addition, compounds 4, 10 and 17
exhibited moderate cytotoxic activity with LC50 (MG-MID) values of
11.74, 9.5 and 6.6 M, respectively (Table 5). Further interpretation
of the obtained data revealed that compounds 17 (GI50 ¼ 0.44
TGI ¼ 1.2 M and LC50 MG-MID ¼ 6.6 M) and 4 (GI50 ¼ 0.77
TGI ¼ 2.08 M and LC50 MG-MID ¼ 11.74
mM, respectively (Table 4), besides a cytostatic
In conclusion, new series of benzothiazole and pyrimido[2,1-b]
benzothiazole derivatives were synthesized. The most active
compounds, 4, 6, 10, 14, 17 and 20 displayed broad spectrum
antitumor activity against most of the tested subpanel cancer cell
lines at the GI50 and TGI levels, together with a mild to moderate
cytotoxic (LC50) activity. In addition, these compounds exhibited
promising antitumor activity against several cancer cell lines,
including colon cancer (HT29), non-small cell lung (NCI-H522),
and breast cancer (MDA-MB-468) cell lines that overexpress
epidermal growth factor receptor (EGFR); thus, these compounds
need further study to explore their mechanism of antitumor ac-
tivity. The benzothiazole derivative 4 and pyrimido[2,1-b]benzo-
thiazole derivative 17 are considered to be the most active
members in this study exhibiting remarkable growth inhibitory
activity, cytostatic and cytotoxic activities. These preliminary
encouraging results of biological screening of the newly synthe-
sized compounds could offer an excellent framework in this field
that may lead to the discovery of new potent antitumor agents.
m
m
m
m
M,
M,
m
m
m
mM) are the most active
members in this study with promising antitumor activity against all
the tested subpanel cancer cell lines at micromolar and sub-
micromolar concentration levels. Moreover, both compounds
revealed distinctive effectiveness against leukemia subpanel at
both the GI50 (0.27 and 0.24
mM, respectively) and TGI levels (1.4
and 0.87 M, respectively) (Tables 4 and 5).
m
3.3. 3D pharmacophore elucidation
3D pharmacophore is defined as
A
a critical geometric
arrangement of molecular features forming a necessary but not
sufficient condition for biological activity [52].
3D and 2D pharmacophoric maps for the structural features of
compounds 4 and 17 (the most active members in this study) were
created by LigandScout software [53] and presented in Figs. 6 and 7,
respectively. The investigated pharmacophoric features, included
hydrogen bond donors and acceptors as directed vectors, positive
and negative ionizable regions as well as lipophilic areas that are
represented by spheres. 3D and 2D pharmacophoric maps for
structural features of compounds 4 and 17 which provide the
arrangement of the pharmacophore contours are presented in
Figs. 6 and 7.
5. Experimental
5.1. Chemistry
All melting points (ꢁC) were recorded on Fisher-Johns melting
point apparatus and are uncorrected. The infrared spectra were
recorded using Nicolet Magna-IR Fourier-Transform 560 Spec-
trometer (v in cmꢀ1) at the Department of Chemistry, Georgia
State University, USA. Nuclear magnetic resonance (1H and 13C
NMR) spectra were obtained on Bruker Avance 400 MHz spec-
trometer using CDCl3 and DMSO-d6 as solvents at the Department
of Chemistry, Georgia State University, USA. The chemical shifts
Moreover, alignment of the structural features of benzothiazole
derivatives 4, 6 and 10 revealed that these compounds possess
similar arrangements of pharmacophoric characters. Furthermore,
compound 6 displayed additional site for hydrogen bonding
are expressed in
d ppm using tetramethylsilane (TMS) as internal
reference. Mass spectra were recorded on nano LC-Q-TOF micro
(Waters Micromass) spectrometer in positive or negative ion mode
as necessary at the Department of Chemistry, Georgia State Uni-
versity, USA. Microanalyses (C, H, N) were performed at the
Department of Chemistry, Georgia State University, and were in
agreement with the proposed structures. Reaction times were
monitored using TLC plates, Silica gel 60 F254 precoated (E. Merck)
and the spots were visualized by UV (366 nm). Chlor-
oform:methanol (9:1) was adopted as an elution solvent. Com-
pounds 2, 3, 11 and 12 were prepared adopting the reported
procedure [7].
Table 4
Mean GI50 values (
mM) of the most active compounds over in vitro subpanel tumor
cell lines.a
Comp. Subpanel tumor cell linesb
No.
MG-MIDc
I
II
III
IV
V
VI
VII
VIII
IX
4
6
10
14
17
20
0.27 0.99 1.29 1.01 1.1
1.36 0.93 1.16 1.08 0.77
2.95 17.5 13.4 4.4 23.4 11.4 9.07 8.11 11.58 6.02
1.02 1.55 1.52 1.8 1.5 1.19 0.97 1.93 1.34
1.02 1.88 1.86 2.13 1.9 1.74 1.71 1.77 1.62 1.04
0.24 1.01 0.51 1.11 0.61 0.81 0.37 0.54 0.55 0.44
3.01 2.21 2.38 2.5 2.61 2.26 2.24 2.88 0.89 1.9
1
5.1.1. Ethyl 1-[2-((6-chlorobenzothiazol-2-yl)amino)-2-oxoethyl]-
5-hydroxy-1H-pyrazole-4-carboxylate (4)
Bold values represent the best GI50 values against subpanel I cancer cell lines and
GI50 M) full panel mean-graph mid point.
Mean GI50 values were calculated by dividing the summation of GI50 values of
(m
a
A mixture of compound 3 (0.257 g, 0.001 mol), diethyl ethox-
ymethylenemalonate (0.216 g, 0.001 mol) and anhydrous potas-
sium carbonate (0.207 g, 0.0015 mol) in acetonitrile (15 mL) was
heated under reflux for 12 h. After cooling, the solution was acid-
ified with dilute hydrochloric acid and the precipitated dark yellow
solid was collected by filtration, dried and crystallized from
ethanol.
the compound over cell lines of the same cancer type by the number of cell lines in
the subpanel.
b
I: Leukemia; II: Non-Small Cell Lung Cancer; III: Colon Cancer; IV: CNS Cancer; V:
Melanoma; VI: Ovarian Cancer; VII: Renal Cancer; VIII: Prostate Cancer; IX: Breast
Cancer.
c
GI50
(
m
M) full panel mean-graph mid point (MG-MID) ¼ the average sensitivity
of all cell lines toward the test compounds.

586
M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
Table 5
Mean total growth inhibitory concentration (TGI,
m
M) and median lethal concentration (LC50
,
m
M) of the most active compounds over in vitro subpanel tumor cell lines.a
Comp. No.
Subpanel tumor cell linesb
MG-MIDe (MG-MID)f
I
II
III
IV
V
VI
VII
VIII
IX
4
6
10
14
17
20
1.4c (ꢀ)d
51.5 (ꢀ)
2.6 (ꢀ)
1.9 (37.5)
51.4 (ꢀ)
3.4 (5.8)
59.4 (ꢀ)
3.34 (7.2)
23.2 (ꢀ)
1.1 (3.1)
9.1 (35.1)
2.5 (52.1)
40.9 (ꢀ)
2.97 (5.5)
86.1 (ꢀ)
2.6 (52.7)
67.9 (ꢀ)
2.06 (4.4)
40.7 (ꢀ)
2.4 (6.6)
9.1 (51.2)
1.1 (2.9)
7.7 (34.2)
2.9 (5.5)
46.1 (85.3)
5.2 (16.9)
9.2 (67.4)
1.8 (4.7)
2.5 (52.8)
26.9 (ꢀ)
2.08 (11.74)
33.11 (93.3)
2.63 (9.5)
6.02 (56.2)
1.2 (6.6)
4.8 (18.7)
28.5 (79.1)
2.3 (4.8)
5.14 (5.8)
20.2 (68.4)
2.5 (24.1)
20.9 (45.9)
3.2 (18.8)
22.3 (93.8)
1.4 (3.9)
3.2 (16.1)
24.3 (72.1)
2.1 (4.5)
4.6 (36.1)
20.8 (ꢀ)
25.8 (ꢀ)
0.87 (ꢀ)
13.4 (92.9)
2.1 (76.6)
4.8 (81.7)
8.5 (47.2)
9.5 (42.9)
10.7 (66.5)
11.3 (81.4)
8.3 (44.6)
a
Mean TGI and LC50 values were calculated by dividing the summation of TGI and LC50 values of the compound over cell lines of the same cancer type by the number of cell
lines in the subpanel.
b
For subpanel tumor cell lines, see footnote (b) of Table 4.
Bold values represent the best mean TGI and LC50 values against subpanel tumor cell lines: I; III; V and VII.
c
d
Mean LC50 values are shown in parentheses, (ꢀ) for values > 100
m
M.
M) full panel mean-graph mid point (MG-MID) ¼ the average sensitivity of all cell lines toward the test compounds.
M) full panel mean-graph mid point (MG-MID) ¼ the average sensitivity of all cell lines toward the test compounds.
e
f
TGI (
LC50
m
(
m
Fig. 6. The 3D and 2D pharmacophoric maps of compound 4; (A) The 3D pharmacophoric map of compound 4. The pharmacophore color coding is red for hydrogen acceptor,
yellow for hydrophobic regions and green for hydrogen donors, (B) The 2D pharmacophoric map of compound 4. HBA, hydrogen bond acceptor; H, hydrophobic center; HBD,
hydrogen bond donor; AR, aryl. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Yield 45%, m.p. 195e196 ꢁC. IR (KBr, v, cmꢀ1): 3260 (OH), 3150
(NH),1710 (COOC2H5),1685 (C]O). 1H NMR: (CDCl3,
ppm): 1.30 (t,
Anal. calcd. for C15H13ClN4O4S (380.81): C, 47.31; H, 3.44; N, 14.71%.
Found: C, 47.56; H, 3.25; N, 14.47%.
d
3H, CH2CH3), 3.55 (s, 2H, COCH2NH), 4.05e4.35 (q, 2H, CH2CH3),
7.20e7.80 (m, 4H, AreH, pyrazole-H), 10.40 (s, 1H, OH), 11.50 (s, 1H,
5.1.2. Ethyl 5-amino-1-[2-((6-chlorobenzothiazol-2-yl)amino)-2-
oxoethyl]-1H-pyrazole-4-carboxylate (5)
NH). 13C NMR: (CDCl3,
d ppm): 14.6 (CH2CH3), 56.9 (COCH2NH), 61.0
(CH2CH3), 95.8 (pyrazole C-4),121.0,125.8,128.0,132.0,135.5,145.8,
157.2, 169.7 (AreC), 161.4 (C]O), 167.2 (C]O). HRMS: m/z (ESI)
calcd. for C15H12ClN4O4Sꢀ, [MꢀH]-: 379.0346; found: 379.0352.
A mixture of compound 3 (0.257 g, 0.001 mol), ethyl ethox-
ymethylenecyanoacetate (0.169 g, 0.001 mol) and anhydrous po-
tassium carbonate (0.207 g, 0.0015 mol) in ethanol (15 mL) was

M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
587
Fig. 7. The 3D and 2D pharmacophoric maps of compound 17; (A) The 3D pharmacophoric map of compound 17. The pharmacophore color coding is red for hydrogen acceptor,
yellow for hydrophobic regions and green for hydrogen donors, (B) The 2D pharmacophoric map of compound 17. HBA, hydrogen bond acceptor; H, hydrophobic center; HBD,
hydrogen bond donor; AR, aryl. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
heated under reflux for 10 h. The solution was acidified with dilute
hydrochloric acid and the precipitated yellow solid was collected by
filtration, dried and crystallized from ethanol.
heated under reflux for 8 h. The solvent was evaporated under
reduced pressure and the remaining solid was crystallized from
ethanol.
Yield 59%, m.p. 203e205 ꢁC. IR (KBr, v, cmꢀ1): 3315, 3210 (NH2,
Yield 49%, m.p. 187e189 ꢁC. IR (KBr, v, cmꢀ1): 3260, 3160 (NH2,
NH), 1715 (COOC2H5), 1675 (C]O). 1H NMR: (DMSO-d6,
d
ppm):
NH), 2215 (C^N), 1674 (C]O). 1H NMR: (DMSO-d6,
d
ppm): 3.95 (s,
2H, CH2), 5.25 (s, 2H, NH2), 7.35e8.15 (m, 4H, AreH, pyrazole-H),
11.35 (s, 1H, NH). 13C NMR: (DMSO-d6,
ppm): 63.0 (CH2), 80.8
1.30 (t, 3H, CH2CH3), 3.85 (s, 2H, COCH2NH), 4.28e4.35 (q, 2H,
CH2CH3), 6.40 (s, 1H, pyrazole-H), 7.25e8.25 (m, 5H, AreH, NH2),
d
11.40 (s, 1H, NH). 13C NMR: (DMSO-d6,
d
ppm): 15.0 (CH2CH3), 59.1
(pyrazole C-4), 117.1 (C^N), 119.0, 122.5, 126.0, 131.9, 135.6, 144.7,
161.2, 166.3, 181.8 (AreC), 168.6 (C]O). HRMS: m/z (ESI) calcd. for
(COCH2NH), 59.6 (CH2CH3), 118.5, 119.9, 120.1, 121.2, 126.1, 134.2,
150.6, 152.0, 163.7, 176.0 (AreC), 167.1 (C]O), 167.7 (C]O). HRMS:
m/z (ESI) calcd. for C15H13ClN5O3Sꢀ, [MꢀH]ꢀ: 378.0506; found:
378.0512. Anal. calcd. for C15H14ClN5O3S (379.82): C, 47.43; H, 3.72;
N, 18.44%. Found: C, 47.16; H, 3.62; N, 18.67%.
C
C
13H8ClN6OSꢀ, [MꢀH]-: 331.0247; found: 331.0254. Anal. calcd. for
13H9ClN6OS (332.77): C, 46.92; H, 2.73; N, 25.25%. Found: C, 47.18;
H, 2.89; N, 24.97%.
5.1.4. N-(6-Chlorobenzothiazol-2-yl)-2-(3-methyl-5-oxo-4,5-
dihydro-1H-pyrazol-1-yl)acetamide (7)
5.1.3. 2-(5-Amino-4-cyano-1H-pyrazol-1-yl)-N-(6-
chlorobenzothiazol-2-yl)acetamide (6)
A mixture of compound 3 (0.257 g, 0.001 mol), ethyl acetoace-
tate (0.13 g, 0.001 mol) and anhydrous potassium carbonate
(0.207 g, 0.0015 mol) in ethanol (15 mL) was heated under reflux
for 10 h. The reaction mixture was cooled and the precipitated
A
mixture of compound 3 (0.257 g, 0.001 mol), ethox-
ymethylenemalononitrile (0.122 g, 0.001 mol) and anhydrous po-
tassium carbonate (0.207 g, 0.0015 mol) in ethanol (15 mL) was

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M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
Fig. 8. The 3D alignment of the pharmacophoric features of benzothiazole derivatives 4, 6 and 10.
Fig. 9. The 3D alignment of the pharmacophoric features of pyrimido[2,1-b]benzothiazole derivatives 14, 17 and 20.
green solid was collected by filtration, washed with water, dried
and crystallized from ethanol.
5.1.5. N-(6-Chlorobenzothiazol-2-yl)-2-(3,5-dioxopyrazolidin-1-yl)
acetamide (8)
Yield 55%, m.p. 195e197 ꢁC. IR (KBr, v, cmꢀ1): 3277 (NH), 1710,
A mixture of compound 3 (0.257 g, 0.001 mol) and diethyl
malonate (0.641 g, 0.004 mol) was heated at 200 ꢁC in an oil bath
for 2 h then cooled. The obtained residue was triturated with
diethyl ether and filtered. The filtrate was evaporated and the ob-
tained solid was dried and crystallized from ethanol.
Yield 41%, m.p. 165e167 ꢁC. IR (KBr, v, cmꢀ1): 3150 (2NH), 1720,
1674 (2C]O). 1H NMR: (DMSO-d6,
d
ppm): 1.80 (s, 3H, CH3), 4.20 (s,
2H, pyrazoline-H), 4.90 (s, 2H, CH2), 7.20e7.70 (m, 3H, AreH), 11.50
(s, 1H, NH). 13C NMR: (DMSO-d6,
ppm): 22.5 (CH3), 43.4 (CH2),
d
56.0 (CH2), 119.0, 121.0, 125.0, 126.2, 133.0, 152.1, 180.0 (AreC), 167.6
(C]O), 176.5 (C]O). HRMS: m/z (ESI) calcd. for C13H10ClN4O2Sꢀ,
[MꢀH]-: 321.0291; found: 321.0298. Anal. calcd. for C13H11ClN4O2S
(322.77): C, 48.37; H, 3.44; N, 17.36%. Found: C, 48.69; H, 3.62; N,
17.15%.
1686, 1654 (3C]O). 1H NMR: (DMSO-d6,
pyrazolidine-H), 5.00 (s, 2H, CH2), 7.30e8.20 (m, 3H, AreH), 11.60
(s, 1H, NH), 12.10 (s, 1H, NH). 13C NMR: (DMSO-d6,
ppm): 53.7
d ppm): 2.90 (s, 2H,
d

M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
589
(CH2), 60.8 (CH2),121.8,122.1,126.8,127.9,133.6,147.9,174.3 (AreC),
5.1.10. Ethyl 5-amino-1-(8-chloro-2-oxo-2H-pyrimido[2,1-b]
159.3 (C]O), 170.1 (C]O), 171.9 (C]O). HRMS: m/z (ESI) calcd. for
C
benzothiazole-4-carbonyl)-1H-pyrazole-4-carboxylate (15)
12H8ClN4O3Sꢀ, [MꢀH]ꢀ: 323.0084; found: 323.0089.
A mixture of compound 12 (0.295 g, 0.001 mol), ethyl ethox-
ymethylenecyanoacetate (0.169 g, 0.001 mol) and anhydrous po-
tassium carbonate (0.207 g, 0.0015 mol) in ethanol (15 mL) was
heated under reflux for 10 h. The solution was acidified with dilute
hydrochloric acid and the precipitated yellow solid was collected by
filtration, dried and crystallized from ethanol.
5.1.6. N-(6-Chlorobenzothiazol-2-yl)-2-(3,5-dimethyl-1H-pyrazol-
1-yl)acetamide (9)
A mixture of compound 3 (0.257 g, 0.001 mol) and acetylacetone
(0.1 g, 0.001 mol) in glacial acetic acid (15 mL) was heated under
reflux for 10 h. The reaction mixture was cooled and the precipi-
tated green solid was collected by filtration, washed with water,
dried and crystallized from ethanol.
Yield 48%, m.p. 195e197 ꢁC. IR (KBr, v, cmꢀ1): 3215, 3100 (NH2),
1710 (COOC2H5), 1670, 1655 (2C]O). 1H NMR: (DMSO-d6,
d ppm):
1.23 (t, 3H, CH2CH3), 4.08e4.25 (q, 2H, CH2CH3), 6.42e7.78 (m, 4H,
AreH, pyrimidine-H), 8.90 (s, 1H, pyrazole-H), 12.63 (s, 2H, NH2).
HRMS: m/z (ESI) calcd. for C17H11ClN5O4Sꢀ, [MꢀH]-: 416.0297;
found: 416.0299.
Yield 41%, m.p. 153e154 ꢁC. IR (KBr, v, cmꢀ1): 3215 (NH), 1670
(C]O). 1H NMR: (DMSO-d6,
d
ppm): 2.05 (s, 3H, CH3), 2.25 (s, 3H,
CH3), 5.15 (s, 2H, CH2), 7.35e8.15 (m, 4H, AreH, pyrazole-H), 12.45
(s, 1H, NH). 13C NMR: (DMSO-d6,
ppm): 20.7 (CH3), 23.2 (CH3),
d
63.2 (CH2), 101.8 (pyrazole C-4), 121.9, 122.1, 126.9, 127.9, 133.6,
136.0, 147.9, 159.3, 174.5 (AreC), 167.9 (C]O). HRMS: m/z (ESI)
calcd. for C14H12ClN4OSꢀ, [MꢀH]-: 319.0497; found: 319.0499.
5.1.11. 8-Chloro-4-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazole-1-
carbonyl)-2H-pyrimido[2,1-b]benzothiazol-2-one (16)
A mixture of compound 12 (0.295 g, 0.001 mol), ethyl acetoa-
cetate (0.13 g, 0.001 mol) and anhydrous potassium carbonate
(0.207 g, 0.0015 mol) in ethanol (15 mL) was heated under reflux
for 10 h. The reaction mixture was cooled and the precipitated
green solid was collected by filtration, washed with water, dried
and crystallized from ethanol.
5.1.7. N-(6-Chlorobenzothiazol-2-yl)-2-(3-methyl-5-phenyl-1H-
pyrazol-1-yl)acetamide (10)
A mixture of compound 3 (0.257 g, 0.001 mol) and benzoyla-
cetone (0.162 g, 0.001 mol) in glacial acetic acid (15 mL) was heated
under reflux for 8 h. The solvent was evaporated under reduced
pressure and the remaining solid was crystallized from ethanol.
Yield 66%, m.p. 201e203 ꢁC. IR (KBr, v, cmꢀ1): 3297 (NH), 1658
Yield 63%, m.p. 213e215 ꢁC. IR (KBr, v, cmꢀ1): 1720,1675 (3C]O).
1H NMR: (DMSO-d6,
d
ppm): 2.15 (s, 3H, CH3), 3.73 (s, 2H, pyrazoline-
H), 6.45 (d,1H, AreH), 6.85 (s,1H, pyrimidine-H), 6.97 (d,1H, AreH),
8.05 (s, 1H, AreH). 13C NMR: (DMSO-d6,
ppm): 20.2 (CH3), 60.3
(C]O). 1H NMR: (DMSO-d6,
d
ppm): 1.90 (s, 3H, CH3), 5.20 (s, 2H,
CH2), 7.45e8.20 (m, 9H, AreH, pyrazole-H), 12.40 (s, 1H, NH). 13C
NMR: (DMSO-d6, ppm): 21.5 (CH3), 60.0 (CH2), 115.5 (pyrazole C-
d
(pyrazoline C-4), 116.3, 122.8, 125.5, 127.2, 127.9, 128.1, 138.8, 153.3,
d
160.2,163.4 (AreC, pyrazoline C-3),163.5 (C]O),168.7 (C]O),184.0
4), 118.4, 121.8, 122.1, 126.8, 126.9, 127.9, 129.9, 133.6, 134.9, 147.8,
(C]O). HRMS: m/z (ESI) calcd. for
359.0084; found: 359.0097.
C
15H8ClN4O3Sꢀ, [MꢀH]ꢀ:
159.2, 172.4 (AreC), 170.1 (C]O). HRMS: m/z (ESI) calcd. for
C
19H14ClN4OSꢀ, [MꢀH]ꢀ: 381.0655; found: 381.0661. Anal. calcd.
for C19H15ClN4OS (382.87): C, 59.60; H, 3.95; N, 14.63%. Found: C,
59.36; H, 3.84; N, 14.82%.
5.1.12. 4-(5-Amino-3-oxo-2,3-dihydro-1H-pyrazole-1-carbonyl)-8-
chloro-2H-pyrimido[2,1-b]benzothiazol-2-one (17)
A mixture of compound 12 (0.295 g, 0.001 mol) and ethyl cya-
noacetate (0.113 g, 0.001 mol) in ethanol (15 mL) was heated under
reflux for 10 h. The reaction mixture was cooled and the precipi-
tated solid was collected by filtration, washed with water, dried and
crystallized from ethanol.
5.1.8. 8-Chloro-4-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-2H-
pyrimido[2,1-b]benzothiazol-2-one (13)
A mixture of compound 12 (0.295 g, 0.001 mol) and acetylace-
tone (0.1 g, 0.001 mol) in glacial acetic acid (15 mL) was heated
under reflux for 10 h. The reaction mixture was cooled and the
precipitated green solid was collected by filtration, washed with
water, dried and crystallized from ethanol.
Yield 48%, m.p. 145e147 ꢁC. IR (KBr, v, cmꢀ1): 3300, 3210 (NH2,
NH), 1685, 1660 (3C]O). 1H NMR: (DMSO-d6,
d ppm): 5.75 (s, 1H,
pyrazoline-H), 6.23 (s, 1H, pyrimidine-H), 7.13e7.86 (m, 5H, AreH,
NH2), 11.97 (s, 1H, NH). HRMS: m/z (ESI) calcd. for C14H7ClN5O3Sꢀ,
[MꢀH]-: 360.0063; found: 360.0045.
Yield 49%, m.p.186e188 ꢁC. IR (KBr, v, cmꢀ1): 1675,1660 (2C]O).
1H NMR: (DMSO-d6,
d
ppm): 2.42 (s, 3H, CH3), 2.95 (s, 3H, CH3),
6.78e7.77 (m, 5H, AreH, pyrazole-H, pyrimidine-H). 13C NMR:
(DMSO-d6, ppm): 19.4 (CH3), 20.2 (CH3), 112.7, 117.6, 122.1, 122.3,
d
5.1.13. Ethyl 1-(8-chloro-2-oxo-2H-pyrimido[2,1-b]benzothiazole-
4-carbonyl)-5-hydroxy-1H-pyrazole-4-carboxylate (18)
123.5, 126.9, 128.9, 137.1, 137.3, 163.5, 168.7 (AreC), 177.1 (C]O),
185.7 (C]O). HRMS: m/z (ESI) calcd. for C16H10ClN4O2Sꢀ, [MꢀH]ꢀ:
357.0291; found: 357.0298. Anal. calcd. for C16H11ClN4O2S (358.80):
C, 53.56; H, 3.09; N, 15.61%. Found: C, 53.31; H, 3.26; N, 15.87%.
A mixture of compound 12 (0.295 g, 0.001 mol), diethyl ethox-
ymethylenemalonate (0.216 g, 0.001 mol) and anhydrous potas-
sium carbonate (0.207 g, 0.0015 mol) in acetonitrile (15 mL) was
heated under reflux for 12 h. The solution was acidified with dilute
hydrochloric acid and the precipitated dark yellow solid was
collected by filtration, dried and crystallized from ethanol.
Yield 41%, m.p. 225e227 ꢁC. IR (KBr, v, cmꢀ1): 3377 (OH), 1715
5.1.9. 1-(8-Chloro-2-oxo-2H-pyrimido[2,1-b]benzothiazole-4-
carbonyl)pyrazolidine-3,5-dione (14)
A mixture of compound 12 (0.295 g, 0.001 mol) and diethyl
malonate (0.641 g, 0.004 mol) was heated at 200 ꢁC in an oil bath
for 2 h then cooled. The obtained residue was triturated with
diethyl ether and filtered. The filtrate was evaporated and the ob-
tained solid was dried and crystallized from ethanol.
(COOC2H5), 1690, 1665 (2C]O). 1H NMR: (DMSO-d6,
d ppm): 1.20 (t,
3H, CH2CH3), 4.03e4.16 (q, 2H, CH2CH3), 7.15e8.20 (m, 6H, AreH,
pyrazole-H, pyrimidine-H, OH). HRMS: m/z (ESI) calcd. for
C
17H10ClN4O5Sꢀ, [MꢀH]ꢀ: 417.0139; found: 417.0143. Anal. calcd.
Yield 42%, m.p. 211e212 ꢁC. IR (KBr, v, cmꢀ1): 3200 (NH),
for C17H11ClN4O5S (418.81): C, 48.75; H, 2.65; N, 13.38%. Found: C,
48.93; H, 2.84; N, 13.49%.
1710, 1692, 1660, 1650 (4C]O). 1H NMR: (DMSO-d6,
d
ppm): 4.15 (s,
2H, pyrazolidine-H), 6.80e8.25(m, 4H, AreH, pyrimidine-H),12.60(s,
1H, NH). 13C NMR: (DMSO-d6,
ppm): 51.6 (pyrazolidine C-4), 119.0,
d
5.1.14. Potassium 2-(8-chloro-2-oxo-2H-pyrimido[2,1-b]
121.8,122.4,129.3,129.5,131.2,143.2,157.2,162.8(AreC),162.9(C]O),
170.9 (C]O), 171.1 (C]O), 176.0 (C]O). HRMS: m/z (ESI) calcd. for
C
benzothiazole-4-carbonyl)hydrazine-1-carbodithioate (19)
A solution of potassium hydroxide (0.84 g, 0.015 mol) in water
(10 mL) was added to a mixture of compound 12 (2.95 g, 0.01 mol)
14H6ClN4O4Sꢀ, [MꢀH]ꢀ: 360.9877; found: 360.9884.

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M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
and carbon disulfide (5 mL, excess) in ethanol (20 mL). The mixture
was stirred at room temperature for 8 h. The precipitated dark
yellow solid was collected by filtration, washed with water, dried
and crystallized from ethanol.
5.1.18. 8-Chloro-4-(5-(phenylamino)-[1,3,4]oxadiazol-2-yl)-2H-
pyrimido[2,1-b]benzothiazol-2-one (23)
A mixture of compound 22 (0.43 g, 0.001 mol) and pyridine
(5 mL) was heated under reflux for 6 h. The mixture was poured
onto ice-water and the precipitated dark yellow solid was collected
by filtration, washed with water, dried and crystallized from
ethanol.
Yield 65%, m.p. 167e169 ꢁC. IR (KBr, v, cmꢀ1): 3100 (2NH), 1690,
1655 (2C]O). 1H NMR: (DMSO-d6,
d
ppm): 7.05e7.65 (m, 3H,
AreH), 8.20 (s, 1H, pyrimidine-H), 11.15 (s, 1H, NH), 12.65 (s, 1H,
NH). 13C NMR: (DMSO-d6,
ppm): 108.3, 118.5, 121.2, 124.6, 126.5,
d
Yield 56%, m.p. 178e179 ꢁC. IR (KBr, v, cmꢀ1): 3110 (NH), 1690
129.5, 138.7, 158.4, 161.2 (AreC), 161.5 (C]O), 172.8 (C]O), 187.6
(C]O). 1H NMR: (DMSO-d6,
d
ppm): 6.50e7.45 (m, 8H, AreH), 8.75
(s, 1H, pyrimidine-H), 10.65 (s, 1H, NH). 13C NMR: (DMSO-d6,
ppm): 118.4, 118.6, 120.6, 121.2, 124.1, 128.9, 129.1, 129.5, 132.0,
(C]S). HRMS: m/z (ESI) calcd for
406.8978; found: 406.8984.
C
12H5ClKN4O2S-3, [MꢀH]ꢀ:
d
140.2, 140.8, 162.7, 167.5 (AreC), 181.5 (C]O). HRMS: m/z (ESI)
calcd. for C18H9ClN5O2Sꢀ, [MꢀH]ꢀ: 394.0244; found: 394.0251.
Anal. calcd. for C18H10ClN5O2S (395.82): C, 54.62; H, 2.55; N, 17.69%.
Found: C, 54.41; H, 2.72; N, 17.86%.
5.1.15. 8-Chloro-4-(5-thioxo-4,5-dihydro-[1,3,4]oxadiazol-2-yl)-
2H-pyrimido[2,1-b]benzothiazol-2-one (20)
A solution of potassium hydroxide (0.84 g, 0.015 mol) in water
(10 mL) was added to a mixture of compound 12 (2.95 g, 0.01 mol)
and carbon disulfide (5 mL, excess) in ethanol (20 mL). The mixture
was heated under reflux for 10 h. The precipitated dark orange solid
was collected by filtration, washed with water, dried and crystal-
lized from ethanol.
5.1.19. 8-Chloro-4-(5-(phenylamino)-[1,3,4]thiadiazol-2-yl)-2H-
pyrimido[2,1-b]benzothiazol-2-one (24)
A mixture of compound 22 (0.43 g, 0.001 mol) and concentrated
sulfuric acid (5 mL) was stirred at room temperature for 8 h. The
mixture was poured onto ice-water and the precipitated dark yel-
low solid was collected by filtration, washed with water, dried and
crystallized from ethanol.
Yield 51%, m.p. 190e192 ꢁC. IR (KBr, v, cmꢀ1): 3175 (NH), 1690
(C]O). 1H NMR: (DMSO-d6,
d
ppm): 6.95e7.35 (m, 3H, AreH), 8.25
(s, 1H, pyrimidine-H), 14.55 (s, 1H, NH). 13C NMR: (DMSO-d6,
ppm): 109.5, 114.8, 123.9, 127.9, 129.1, 132.1, 147.6, 157.0, 157.7,
d
Yield 54%, m.p. 193e195 ꢁC. IR (KBr, v, cmꢀ1): 3250 (NH), 1710
172.4 (AreC, oxadiazole C-2), 178.ꢀ6 (C]O), 183.4 (C]S). HRMS: m/z
(C]O). 1H NMR: (DMSO-d6,
d
ppm): 6.45e7.78 (m, 8H, AreH), 8.65
(s, 1H, pyrimidine-H), 11.05 (s, 1H, NH). 13C NMR: (DMSO-d6,
ppm): 108.7, 118.0, 121.9, 122.1, 123.5, 125.1, 128.8, 129.1, 129.4,
(ESI) calcd. for
334.9551.
C
12H4ClN4O2S2 , [MꢀH]ꢀ: 334.9542; found:
d
139.1,141.9, 143.2,151.5,161.8, 165.2 (AreC),174.5 (C]O). HRMS: m/
z (ESI) calcd for C18H9ClN5OS2ꢀ, [MꢀH]ꢀ: 410.0015; found: 410.0023.
5.1.16. 4-(4-Amino-5-thioxo-4,5-dihydro-1H-[1,2,4]triazol-3-yl)-8-
chloro-2H-pyrimido[2,1-b]benzothiazol-2-one (21)
5.1.16.1. Method A. A mixture of compound 19 (0.409 g, 0.001 mol)
and hydrazine hydrate 99% (0.5 g, 0.01 mol) in ethanol (30 mL) was
heated under reflux for 14 h. The reaction mixture was cooled and
the precipitated solid was collected by filtration, washed with
water, dried and crystallized from ethanol.
5.2. Biological screening
5.2.1. Preliminary in vitro antitumor screening
Nineteen derivatives, 4e10 and 13e24 were selected by the
National Cancer Institute (NCI) in vitro disease-oriented human
cells screening panel assay to be evaluated for their in vitro anti-
tumor activity. Preliminary in vitro single dose antitumor assay was
performed using the full NCI-60 cell lines in accordance with the
current protocol of the Drug Evaluation Branch, NCI, Bethesda
[49e51]. These cell lines were incubated with a single concentra-
5.1.16.2. Method B. A mixture of compound 20 (0.337 g, 0.001 mol)
and hydrazine hydrate 99% (0.5 g, 0.01 mol) in ethanol (30 mL) was
heated under reflux for 8 h. The reaction mixture was cooled and
the precipitated solid was collected by filtration, washed with
water, dried and crystallized from ethanol.
tion (10 mM) of each of the tested compounds. A 48 h continuous
Yield 38% (method A); 51% (method B), m.p. 202e204 ꢁC. IR
drug exposure protocol was used, and sulforhodamine B (SRB)
assay was employed to estimate cell viability or growth. The data
reported as mean-graph of the percentage growth of the treated
cells, and presented as percentage growth inhibition (GI%) (Tables 1
and 2).
(KBr, v, cmꢀ1): 3233, 3115 (NH2, NH), 1685 (C]O). 1H NMR: (DMSO-
d6,
d
ppm): 4.15 (s, 2H, NH2), 6.30e7.15 (m, 3H, AreH), 7.85 (s, 1H,
ppm):
pyrimidine-H), 12.55 (s, 1H, NH). 13C NMR: (DMSO-d6,
d
102.4, 114.0, 117.1, 120.4, 124.9, 131.5, 144.7, 161.2, 164.7, 168.7 (AreC,
triazole C-3), 176.3 (C]O), 185.9 (C]S). HRMS: m/z (ESI) calcd. for
C
12H6ClN6OS-2, [MꢀH]-: 348.9811; found: 348.9818.
5.2.2. NCI full in vitro five dose antitumor assay
The human tumor cell lines of the cancer screening panel were
grown in Roswell Park Memorial Institute (RPMI) 1640 medium
5.1.17. 2-(8-Chloro-2-oxo-2H-pyrimido[2,1-b]benzothiazole-4-
carbonyl)-N-(phenyl)hydrazine-1-carbothioamide (22)
A mixture of compound 12 (0.295 g, 0.001 mol) and phenyl
isothiocyanate (0.135 g, 0.001 mol) in ethanol (15 mL) was heated
under reflux for 10 h. The precipitated yellowish white solid was
collected by filtration, washed with water, dried and crystallized
from ethanol.
containing 5% fetal bovine serum and 2 mM L-glutamine. For a
typical screening experiment, cells were inoculated into multiwell
microtiter plates at plating densities ranging from 5000 to
40,000 cells/well depending on the doubling time of individual cell
lines. After cell inoculation, the microtiter plates were incubated at
37 ꢁC, 5% CO2, 95% air and 100% relative humidity for 24 h and then
two plates of each cell line were fixed in situ with trichloroacetic
acid (TCA) to represent a measurement of the cell population for
each cell line at the time of compound addition. The tested com-
pounds were solubilized in DMSO at 400-fold the desired final
maximum test concentration and stored frozen prior to use. At the
time of compound addition, an aliquot of frozen concentrate was
thawed and diluted to twice the desired final maximum test con-
centration with complete medium containing 50 mg/mL genta-
micin. Additional 4-, 10-fold or 1/2 log serial dilutions were made to
Yield 54%, m.p. 206e208 ꢁC. IR (KBr, v, cmꢀ1): 3330, 3210, 3115
(3NH), 1695, 1670 (2C]O). 1H NMR: (DMSO-d6,
d
ppm): 6.20e7.40
(m, 9H, AreH, pyrimidine-H),10.20 (s,1H, NH),11.78 (s,1H, NH),13.63
(s, 1H, NH). 13C NMR: (DMSO-d6,
ppm): 110.2, 118.4, 123.5, 124.1,
d
124.9, 125.0, 128.9, 129.5, 138.3, 139.9, 161.0 (AreC), 163.7 (C]O),
174.3 (C]O), 187.7 (C]S). HRMS: m/z (ESI) calcd. for C18H11ClN5O2S2- ,
[MꢀH]-: 428.0121; found: 428.0129. Anal. calcd. for C18H12ClN5O2S2
(429.90): C, 50.29; H, 2.81; N, 16.29%. Found: C, 50.52; H, 2.73; N,
16.12%.

M.T. Gabr et al. / European Journal of Medicinal Chemistry 85 (2014) 576e592
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The authors would like to express their sincere thanks to the
staff members of the Department of Health and Human Services,
National Cancer Institute (NCI), Bethesda, Maryland, USA, for per-
forming the antitumor screening of the newly synthesized com-
pounds. Thanks also to Georgia State University, USA, for carrying
out the spectral and elemental analyses.
Appendix A. Supplementary data
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