Synthesis of some novel 4-benzothiazol-2-yl-benzoyl-1H-pyrazoles, and evaluation as antiangiogenic agents

Article abstract of DOI:10.1007/s11164-015-2100-8

Some 4-(1,3-benzothiazol-2-yl)-benzoyl-1H-pyrazole derivatives have been synthesized by reaction of o-aminothiophenol (1) with different electrophilic and nucleophilic reagents. All of the newly synthesized compounds were evaluated for cytotoxicity against breast cancer cell line MCF-7. Two of the compounds were more potent than tamoxifen and three were as potent as tamoxifen. These results were consistent with percentage inhibition of human VEGF compared with control untreated cells.

Full text of DOI:10.1007/s11164-015-2100-8

Res Chem Intermed
DOI 10.1007/s11164-015-2100-8
Synthesis of some novel 4-benzothiazol-2-yl-benzoyl-
1H-pyrazoles, and evaluation as antiangiogenic agents
1
2
Eman Ali Abd El-Meguid
•
Mamdouh Moawad Ali
Received: 11 March 2015 / Accepted: 8 May 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract Some 4-(1,3-benzothiazol-2-yl)-benzoyl-1H-pyrazole derivatives have
been synthesized by reaction of o-aminothiophenol (1) with different electrophilic
and nucleophilic reagents. All of the newly synthesized compounds were evaluated
for cytotoxicity against breast cancer cell line MCF-7. Two of the compounds were
more potent than tamoxifen and three were as potent as tamoxifen. These results
were consistent with percentage inhibition of human VEGF compared with control
untreated cells.
Keywords 4-Benzothiazol-2-yl-benzoyl Á Pyrazole Á MCF-7 Á VEGFR
Introduction
Angiogenesis, formation of new blood vessels, is crucially important to cancer cell
survival and tumor growth, and is regarded as necessary for metastasis of all solid
tumors [1–3]. This process consists of formation of new capillaries by splitting of
pre-existing blood vessels. Among the other growth factors that are vital to
stimulation of angiogenesis, vascular endothelial growth factor (VEGF) is the most
important signaling protein [4]. The VEGF protein family consists of six members
(
VEGF-A, B, C, D, E and placenta growth factor). These proteins can bind to their
VEGF receptors, VEGFR1, VEGFR2, and VEGFR3, which are receptor tyrosine
kinases (RTK). VEGFR2 is regarded as an important receptor mediating all cellular
&
Eman Ali Abd El-Meguid
emannrc@yahoo.com
1
2
Division of Pharmaceutical Industries Research, Department of Chemistry of Natural and
Microbial Products, National Research Center, Cairo, Egypt
Division of Genetic Engineering and Biotechnology, Department of Biochemistry, National
Research Center, Cairo, Egypt
123

E. A. A. El-Meguid, M. M. Ali
responses to VEGF [5]. Because binding of VEGF to its family of receptors
(
VEGFR) is a crucial step that promotes the proliferation and survival of endothelial
cells and, consequently, cancer progression [6], the search for an effective anti-
VEGF/VEGFR drug has become a major interest of many research groups seeking
to new cancer therapy via inhibition of angiogenesis.
The benzothiazole ring system is a core structure of a variety of natural and
synthetic compounds with a broad range of biological activity [7–13] including
anticancer activity [14–19]. During the past few years, several attempts have been
made to modify their benzothiazole nucleus to improve their antitumor activity.
Such modification has resulted in identification of a variety of promising
benzothiazole derivatives with remarkable anticancer activity against malignant
cell lines. Pyrazole derivatives have also attracted the attention of organic chemists
because of their biological and chemotherapeutic importance. They are known to
have such biological activity as antitumor [20], antileukemic [21], anti-inflamma-
tory [22], analgesic [23], anticoagulant [24], and antimicrobial [25] activity.
Urea derivative A (Fig. 1) has good inhibitory activity against liver, breast, and
gastric cell lines and acts as an inhibitor of Raf-1 kinase [26]. Thiourea derivative
B (Fig. 1) has antiproliferative activity against two human monocytic cell lines (U
9
37 and THP-1) [27].
Semi(thiosemi)carbazide groups are present in many anti-tumor agents [28–30]
and there have been several reports [31–35] suggesting use of the 1,2,4-triazole
nucleus as a potential source of new anticancer agents. For example, the
CN
H C
3
N
S
NH
O
NH
CH3
A
F
F
N
S
S
N
H
N
H
B
Fig. 1 The structures of compounds a and b
1
23

Synthesis of some novel…
benzothiazole-1,2,4-triazole conjugate R115866 [35] has been reported to be a
potent anticancer agent against the MCF-7 cell line.
Stimulated by these observations, we report here the synthesis and anti-breast
cancer activity of some substituted 4-benzothiazol-2-yl-benzoyl-1H-pyrazoles
prepared from o-aminothiophenol.
Results and discussion
Chemistry
4
-(1,3-Benzothiazol-2-yl)benzonitrile (2) was prepared by reaction of o-aminoth-
iophenol (1) with 4-cyanobenzaldehyde in absolute ethanol, as reported elsewhere
36]. Acid hydrolysis of the carbonitrile group by stirring with 70 % sulfuric acid
[
gave 4-(1,3-benzothiazol-2-yl)benzoic acid (3). This was followed by esterification
and reaction with hydrazine hydrate to form the corresponding 4-(1,3-benzothiazol-
2
-yl)benzohydrazide (5). Heating of compound 5 with ethoxymethylenemalononi-
trile or ethyl (ethoxymethylene)cyanoacetate, under reflux in ethanol, yielded the
corresponding 5-amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole-4-car-
bonitrile (6) or ethyl 5-amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole-4-
carboxylate (7), respectively. Heating of 7 under reflux with hydrazine hydrate
furnished the corresponding carbohydrazide 8, as illustrated in Scheme 1.
Compound 9 was prepared by reaction of 6 with 5-nitroisatin in absolute ethanol
containing a few drops of glacial acetic acid. Compounds 10a and 10b were
0
synthesized by heating carbonitrile 6 with 4 -chlorophenyl isocyanate or phenyl
isothiocyanate under reflux in dry benzene. Stirring with 70 % sulfuric acid gave the
corresponding ureido/thioureido derivatives 11a and 11b, as illustrated in Scheme 2.
Indole derivative 12 was prepared by reaction of carbohydrazide 8 with
5
-nitroisatin in absolute ethanol containing a few drops of glacial acetic acid.
0
Compounds 13a and 13b were synthesized by heating 8 with 4 -chlorophenyl
isocyanate or phenyl isothiocyanate under reflux in dry benzene. Heating of 13a or
13b with sodium hydroxide under reflux for 4 h afforded the triazoles 14a or 14b,
respectively. Condensation of triazole 14a with the appropriate organohalogen
compound (methyl iodide, ethyl chloroformate, methyl 2-bromoacetate, ethyl
2
-bromoacetate or ethyl 2-bromopropanoate) in the presence of anhydrous sodium
carbonate, to abstract the halogen atom, and acetone as solvent afforded compounds
5a–e, usually in the pure state (Scheme 3).
1
Biological evaluation
Cytotoxicity against human breast cancer cell line MCF-7
The cytotoxicity of compounds 6–15e against the human breast cancer cell line
MCF-7 was tested by use of the method of Skehan et al. [37]. The cytotoxicity
results were compared with that of the standard drug tamoxifen. The activity of the
ureido and thioureido compounds 11a and 11b was better than or equal to that of
123

E. A. A. El-Meguid, M. M. Ali
NH
2
N
S
N
a
b
CN
COOH
S
SH
3
1
2
c
N
S
N
d
CONHNH
2
COOC
2
H
5
S
5
4
e
f
2 5
COOC H
N
N
S
O
N
S
N
N
NH
2
NH
2
O
N
6
CN
7
g
O
N
NH
2
N
S
N
H
NH
2
N
O
8
Scheme 1 Reagents: a 4-cyanobenzaldehyde, EtOH; b 70 % H
EtOH;
g NH
2
SO
4
; c EtOH, H
f ethyl (ethoxymethylene)cyanoacetate, EtOH;
2
SO
4
; d NH
2
NH
2
ÁH
2
O,
e
NH
ethoxymethylenemalononitrile, EtOH;
ÁH O, EtOH
2
2
2
tamoxifen (IC50 = 0.01–0.02 lmol/ml). The activity of carbohydrazide 8 was equal
to that of tamoxifen, and indole derivative 12 was more potent than the reference
drug. Cyclization of semi(thio)carbazide derivatives 13a and 13b to prepare the
triazole derivatives 14a and 14b, respectively led to a slight increase of anticancer
activity against the MCF-7 cell line. Condensation of compound 14a with different
alkyl halides to prepare compounds 15a–e led to increased activity for compound
1
5a only, which was equipotent with tamoxifen; the other derivatives had no
activity. The other compounds had moderate to weak activity against the MCF-7
cell line (Table 1).
The promising anticancer compounds were not toxic to a normal cell line (human
normal melanocyte, HFB4).
In-vitro VEGF inhibition in human breast cancer cell line MCF-7
Angiogenesis is essential for tumor progression because tumor masses cannot grow
3
larger than 2 mm without nourishment from blood vessels. Vascularization is,
moreover, required for the process of extravasation in metastasis [38]. Hence,
establishment of a chemotherapeutic strategy to block angiogenesis has attracted
1
23

Synthesis of some novel…
N
S
O
N
O
a
N
N
S
O
NH
NH
2
N
N
CN
N
9
CN
O
2
N
6
N
O
H
N
b
H
N
S
N
N
R
X
CN
1
0a,b
c
N
S
O
H
N
H
N
N
N
R
X
COOH
1
1a,b
a, R = Cl, X = O
b, R = H, X = S
0
Scheme 2 Reagents: a 5-nitroisatin, EtOH, glacial acetic acid; b 4 -chlorophenyl isocyanate or phenyl
isothiocyanate, benzene; c 70 % H SO
2
4
much attention in recent years. Alteration of cellular adaptation to hypoxia is also
fundamental to cancer treatment, because angiogenesis or other metabolic
modifications will be stimulated to maintain tumor cell survival [39]. Vascular
endothelial growth factor (VEGF) has been identified as the most important
angiogenic factor for tumor progression; it is released by a variety of tumor cells
and is overexpressed in a variety of human cancers. Drugs that inhibit production of
VEGF or block its receptor signaling significantly inhibit tumor growth [40, 41].
In this biological in-vitro study double-antibody sandwich enzyme-linked
immunosorbent assay (ELISA) was used to determine the level of human VEGF
in human breast cancer cell line MCF-7 treated with compounds 6–15e and in
untreated cells. The results (Table 1) showed that compound 12 was more potent
than tamoxifen (98 %) against human VEGF, with 99 % inhibition compared with
control untreated cells. The potency of compounds 8, 11a, 11b, and 15a was similar
to that of tamoxifen (91.67–97.32 %). These results were consistent with the
compounds’ cytotoxicity against MCF-7 cell line (the compounds had excellent
activity with IC50 ranging from 0.01 to 0.02 lmol/ml).
Experimental
General
Melting points (°C) were determined in open capillary tubes, with use of silicone oil
and Gallenkamp equipment (Ultraporter, Walsall, UK). H NMR spectra were
1
123

E. A. A. El-Meguid, M. M. Ali
O
N
N
O
N
N
H
S
N
a
NH
2
O
NH
R
O
N
N
NH
2
N
S
N
H
12
O N
2
NH
2
O
O
N
N
H
N
8
N
H
b
S
N
NH
NH
2
X
O
13a,b
a, R = Cl, X = O
b, R = H, X = S
c
NH
N
X
N
N
S
N
N
NH
2
O
1
4a,b
a, R = Cl, X = O
b, R = H, X = S
R
d
R'
N
N
O
N
N
N
S
N
NH
2
O
1
5a-e
5a, R' = CH
5b, R' = COOCH
5c, R' = CH
5d, R' = CH
5e, R' = CH(CH
Cl
1
1
1
1
1
3
2
CH
3
COOCH
3
COOCH
2
2
2 3
CH
3
)COOCH
2
CH
3
0
Scheme 3 Reagents: a 5-nitroisatin, EtOH, glacial acetic acid; b 4 -chlorophenyl isocyanate or phenyl
isothiocyanate, benzene; c NaOH; d methyl iodide, ethyl chloroformate, methyl 2-bromoacetate, ethyl
2 3
2-bromoacetate, or ethyl 2-bromopropanoate, Na CO , acetone
acquired with a Jeol (Canada) 270-MHz spectrometer, with DMSO-d as solvent
6
and tetramethylsilane as an internal standard. Mass Spectra were obtained with a
GCS-QP1000EX spectrometer (Schimadzu Scientific Instruments, Italy) at 70 eV.
IR spectra were recorded with a Philips model PU 9712 Infracord spectrophotome-
ter (PerkinElmer, Waltham, Masssachusetts 02451, USA) as KBr discs. Elemental
analysis was performed at the Microanalytical Laboratory of the National Research
Center. All the reactions were monitored by thin-layer chromatography (TLC) on
silica gel with chloroform as mobile phase.
4
-(1,3-Benzothiazol-2-yl)benzonitrile (2) 4-Cyanobenzaldehye (1.05 g, 0.21 mol)
and o-aminothiophenol 1 (1 ml, 0.21 mol) were dissolved in ethanol. This mixture
was heated under reflux for 5 h then cooled to room temperature. Water was then
added slowly to the mixture with stirring. The suspension was maintained at -5 °C
1
23

Synthesis of some novel…
Table 1 Effect of the
Compound
IC50
VEGF
synthesized compounds on the
human breast cancer cell line
MCF-7 IC50 (lmol/ml) and the
VEGF level (pg/ml) in breast
cancer cell line MCF-7
6
7
8
9
0.06
0.08
0.02
NA
4500.80 ± 460.60 (14.27 %)
5160.90 ± 550.00 (1.70 %)
322.98 ± 35.50 (93.85 %)
–
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
1a
1b
2
0.08
0.09
0.02
0.01
0.01
0.07
0.09
0.05
0.07
0.02
NA
5060.90 ± 550.00 (3.70 %)
5200.30 ± 535.90 (0.95 %)
224.98 ± 35.50 (95.98 %)
120.90 ± 38.00 (97.32 %)
106.75 ± 36.00 (99 %)
4914.83 ± 500.00 (6.38 %)
5220.70 ± 540.80 (0.60 %)
4170.82 ± 460.90 (19.13 %)
4938.83 ± 500.00 (6.88 %)
395.70 ± 210.80 (91.67 %)
–
3a
3b
4a
4b
5a
5b
5c
Data are expressed as means
from four independent
experiments
NA
–
15d
NA
–
Values in parentheses indicated
percentage changes compared
with control cancer cells
15e
NA
–
DMSO
Tamoxifen
–
5250.00
0.02
110.75 (98 %)
NA no activity
overnight. The product was washed repeatedly with ethanol–water (1:1) then
recrystallized from acetone. Yield = 1.2 g (64 %), mp = 150–2 °C. Analysis for
C H N S (236.2): Calcd.: C, 69.0; H, 4.5; N, 12.4; S, 14.2; Fd.: C, 69.2; H, 4.6; N,
1
4 8 2
1
2.5; S, 14.3.
4-(1,3-Benzothiazol-2-yl)benzoic acid (3) A mixture of 2 (2 g, 0.01 mol) and
30 ml 70 % sulfuric acid was stirred in a 100-ml three-necked flask at 140 °C for
5 h, then poured into 150 ml water. The resulting precipitate was isolated by
filtration. Recrystallization from dilute ethanol afforded white crystals.
Yield = 0.8 g (74 %), mp = 250–3 °C. Analysis for C H NO S (255.3): Calcd.:
1
4
9
2
C, 65.9; H, 3.6; N, 5.5; S, 12.6; Fd.: C, 66.0; H, 3.6; N, 5.6; S, 12.8. MS: m/z (%):
2
?
55 (M , 100).
Ethyl 4-(1,3-benzothiazol-2-yl)benzoate (4) A few drops of concentrated sulfuric
acid were added to a solution of compound 3 (2 g; 0.073 mol) in absolute ethanol
and the mixture was heated under reflux for 4 h. The crude product was filtered, air-
dried, and crystallized from ethanol. Yield = 2.1 g (94 %), mp = 102–6 °C.
Analysis for C H NO S (283.3): Calcd.: C, 67.8; H, 4.6; N, 4.9; S, 11.3; Fd.: C,
1
6
13
2
?
1
6
8.0; H, 4.6; N, 5.0; S, 11.5. MS: m/z (%): 283 (M , 15). H NMR: d, ppm (DMSO-
d ); 1.32 (t, 3H, CH ); 4.32 (q, 2H, CH CH ); 7.55 (mm, 2H, H ? H
6
6
3
2
3
5
123

E. A. A. El-Meguid, M. M. Ali
benzothiazole); 7.59 (dd, 2H, H ? H benzene); 8.03 (dd, 2H, H ? H benzene);
2
6
3
5
8
.12–8.23 (dd, 2H, H ? H benzothiazole).
4 7
4
-(1,3-Benzothiazol-2-yl)benzohydrazide (5) Hydrazine hydrate (98 %; 2 ml) was
added to a solution of ester 4 (1 g; 0.033 mol) in ethanol and the mixture was heated
for 5 h on a water-bath then cooled. The crude product was isolated by filtration,
washed with water, dried, and crystallized from ethanol. Yield = 0.8 g (84 %),
mp = 234–8 °C. Analysis for C H N OS (269.3): Calcd.: C, 62.4; H, 4.1; N,
1
4 11 3
-
1
1
5.6; S, 11.9; Fd.: C, 62.5; H, 4.2; N, 15.7; S, 12.0. IR (cm ): 3453 (NH), 3314
?
1
(
NH ), 1684 (CO). MS: m/z (%): 269 (M , 16). H NMR: d, ppm (DMSO-d ); 7.55
2
6
(
H ? H benzene); 8.12–8.23 (dd, 2H, H ? H benzothiazole); 9.96 (s, 2H, NH ,
mm, 2H, H ? H benzothiazole); 7.66 (dd, 2H, H ? H benzene); 8.01 (dd, 2H,
5
6
2
6
3
5
4
7
2
exchangeable with D O); 10.45 (s, 1H, NH, exchangeable with D O).
2
2
General procedure for preparation of 5-amino-1-[4-(1,3-benzothiazol-2-yl)ben-
zoyl]-1H-pyrazole-4-substituted (6, 7) Ethoxymethylenemalononitrile or ethyl
ethoxymethylenecyanoacetate (0.01 mol) was added to a solution of hydrazide 5
(
1 g, 0.01 mol) in 30 ml ethanol and the mixture was heated under reflux for 6–8 h.
The reaction mixture was cooled to room temperature and the solvent was removed
under reduced pressure. The solid product was recrystallized from ethanol.
5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole-4-carbonitrile (6) Yield =
0
H, 3.2; N, 20.3; S, 9.3; Fd.: C, 62.5; H, 3.3; N, 20.2; S, 9.4. IR (cm ): 3424 (NH ),
.7 g (55 %); mp = 266–8 °C. Analysis for C H N OS (345.4): Calcd.: C, 62.6;
1
8 11 5
-
1
2
?
1
2
7
217 (CN), 1677 (CO). MS: m/z (%): 345 (M , 57). H NMR: d, ppm (DMSO-d );
6
.55 (mm, 2H, H ? H benzothiazole); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H,
5
6
H ? H benzene); 7.87 (dd, 2H, H ? H benzene); 8.12–8.23 (dd, 2H, H ? H
7
2
6
3
5
4
benzothiazole); 10.76 (s, 2H, NH2).
Ethyl-5-amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole-4-carboxylate (7)
Yield = 0.9 g (62 %); mp = [300 °C. Analysis for C H N O S (392.4): Calcd.:
C, 61.2; H, 4.1; N, 14.3; S, 8.2; Fd.: C, 61.3; H, 4.5; N, 14.6; S, 8.4. IR (cm ): 3224
2
0 16 4 3
-
1
?
1
(
NH ), 1677 (CO), 1662 (CO). MS: m/z (%): 392 (M , 57). H NMR: d, ppm
2
(
benzothiazole); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H, H ? H benzene); 7.87 (dd,
DMSO-d ); 1.19 (t, 3H, CH ); 3.30 (q, 2H, CH ); 7.55 (mm, 2H, H ? H
6
6
3
2
5
2
6
2H, H ? H benzene); 8.12–8.23 (dd, 2H, H ? H benzothiazole); 10.77 (s, 2H,
3 5 4 7
NH2).
5
-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole-4-carbohydrazide
Hydrazine hydrate (98 %; 2 ml) was added to a solution of ester compound 7
1 g; 0.033 mol) in ethanol, and the mixture was heated for 5 h on a water-bath. The
(8)
(
reaction mixture was cooled. The crude product was filtered, washed with water,
dried, then crystallized from ethanol. Yield = 0.8 g (83 %), mp = 182–5 °C.
Analysis for C H N O S (378.4): Calcd.: C, 57.1; H, 3.7; N, 22.2; S, 8.5; Fd.: C,
1
8 14 6 2
-
1
5
7.2; H, 3.9; N, 22.1; S, 8.6. IR (cm ): 3343 (NH), 3165 (NH ), 1677 (CO), 1662
2
1
23

Synthesis of some novel…
?
.
?
1
(
CO). MS: m/z (%): 379 (M , 16); 378 (M , 32). H NMR: d, ppm (DMSO-d );
6
3
D O); 7.55 (mm, 2H, H ? H benzothiazole); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H,
.96 (s, 2H, NH , exchangeable with D O); 4.94 (s, 2H, NH , exchangeable with
2
2
2
2
5
6
H ? H benzene); 7.87 (dd, 2H, H ? H benzene); 8.12–8.23 (dd, 2H, H ? H
7
2
6
3
5
4
benzothiazole); 10.73 (s, 1H, NH, exchangeable with D O).
2
1
-[4-(1,3-Benzothiazol-2-yl)benzoyl]-5-(5-nitro-2-oxo-1,2-dihydroindol-3-ylidenea-
mino)-1H-pyrazole-4-carbonitrile (9) 5-Nitroisatin (0.9 g, 0.018 mol) and few
drops of glacial acetic acid were added to a solution of compound 6 (1 g;
0
6
.018 mol) in absolute ethanol (30 ml) and the mixture was heated under reflux for
h. The solvent was removed by distillation and the solid product was crystallized
from ethyl acetate–petroleum ether (95:5). Yield = 1.4 g (93 %); mp = 272–5 °C.
Analysis for C H N O S (519.5): Calcd.: C, 60.1; H, 2.5; N, 18.9; S, 6.2; Fd.: C,
6
2
6 13 7 4
-
1
0.0; H, 2.5; N, 19.0; S, 6.2. IR (cm ): 3335 (NH), 2217 (CN), 1705 (CO), 1673
?
.
?
1
(
CO). MS: m/z (%): 520 (M , 62); 519 (M , 69). H NMR: d, ppm (DMSO-d );
6
7
H ? H benzene); 7.87 (dd, 2H, H ? H benzene); 7.91 (d, 1H, H indole);
.55 (mm, 2H, H ? H benzothiazole); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H,
5 6
2
6
3
5
7
8
indole); 10.79 (s, 1H, NH, exchangeable with D O).
.12–8.23 (dd, 2H, H ? H benzothiazole); 8.27 (d, 1H, H indole); 8.50 (s, 1H, H
4 7 6 4
2
General procedure for preparation of 1-{1-[4-(1,3-benzothiazol-2-yl)benzoyl]-4-
cyano-1H-pyrazol-5-yl}-3-(un)substituted-phenylurea/thiourea (10a, b) A mixture
0
of 6 (1 g, 0.01 mol) and 4 -chlorophenyl isocyanate or phenyl isothiocyanate
(0.01 mol) in dry benzene was heated under reflux for 6 h. The solid material
obtained on cooling was isolated by filtration and recrystallized from methanol.
1
-{1-[4-(1,3-Benzothiazol-2-yl)benzoyl]-4-cyano-1H-pyrazol-5-yl}-3-(4-chlorophe-
nyl)urea (10a) Yield = 1.2 g (83 %); mp = [300 °C. Analysis for C H ClN
2
5
15
6-
O S (498.9): Calcd.: C, 60.2; H, 3.0; N, 16.8; S, 6.4; Fd.: C, 60.1; H, 2.9; N, 16.9; S,
6
2
-
.4. IR (cm ): 3395 (NH), 3228 (NH), 2217 (CN), 1689 (CO), 1662 (CO). MS: m/
1
?
?
1
z (%): 500 (M , 3.3); 498 (M , 10). H NMR: d, ppm (DMSO-d ); 6.91 (s, 2H,
6
2
NH, exchangeable with D O); 7.25 (dd, 2H, H ? H chlorophenyl); 7.55 (mm,
2 3 5
2
pyrazole); 7.67 (dd, 2H, H ? H benzene); 7.87 (dd, 2H, H ? H benzene);
H, H ? H benzothiazole); 7.58 (dd, 2H, H ? H chlorophenyl); 7.61 (s, 1H,
5
6
2
6
2
6
3
5
8
.12–8.23 (dd, 2H, H ? H benzothiazole).
4 7
1
-{1-[4-(1,3-Benzothiazol-2-yl)benzoyl]-4-cyano-1H-pyrazol-5-yl}-3-phenylthiou-
rea (10b) Yield = 1.2 g (86 %); mp = [300 °C. Analysis for C H N OS
2
5
16
6
2
(
480.6): Calcd.: C, 62.5; H, 3.4; N, 17.5; S, 13.3; Fd.: C, 62.4; H, 3.3; N, 17.4; S,
-
3.4. IR (cm ): 3401 (NH), 3296 (NH), 2215 (CN), 1667 (CO). MS: m/z (%): 481
1
1
?
.
?
1
(M , 16); 480 (M , 30). H NMR: d, ppm (DMSO-d ); 5.02 (s, 2H, 2NH,
exchangeable with D O); 6.45 (dd, 2H, H ? H phenyl); 6.68 (m, 1H, H phenyl);
6
2
2
6
4
7
1
8
.08 (mm, 2H, H ? H phenyl); 7.55 (mm, 2H, H ? H benzothiazole); 7.61 (s,
3 5 5 6
H, pyrazole); 7.67 (dd, 2H, H ? H benzene); 7.87 (dd, 2H, H ? H benzene);
2
6
3
5
1
3
.12–8.23 (dd, 2H, H ? H benzothiazole). C-NMR d, ppm (DMSO-d ): showed
4
7
6
123

E. A. A. El-Meguid, M. M. Ali
the presence of 25 signals correspond to the 25 different carbon groups, signals
appeared at d 117.2 (CN), 122.4–130.7 (Ar-13CH), 133.2 (C=N), 134.7 (C=C),
1
37.0 (CS), 152.1 (C-NH ), 153.9 (C=N), 154.3 (C-N), 165.7 (C=C), 165.7 (C=N),
2
1
66.2 (C=O) and 179.9 (C=S).
General procedure for preparation of 1-[4-(1,3-benzothiazol-2-yl)benzoyl]-5-[3-
un)substituted-phenylureido/thioureido]-1H-pyrazole-4-carboxylic acid (11a,
(
b) A mixture of 10a or 10b (1 g, 0.01 mol) and 30 ml 70 % sulfuric acid was
stirred in a 100-ml three-necked flask at 140 °C for 5 h then poured into 150 ml
water. The resulting precipitate was isolated by filtration then recrystallized from
diluted ethanol.
1
-[4-(1,3-Benzothiazol-2-yl)benzoyl]-5-[3-(4-chlorophenyl)ureido)]-1H-pyrazole-4-
carboxylic acid (11a) Yield = 0.8 g (77 %); mp = [300 °C. Analysis for
C H ClN O S (517.9): Calcd.: C, 58.0; H, 3.1; N, 13.5; S, 6.2; Fd.: C, 58.1; H,
3
2
5
16
5 4
-
.1; N, 13.4; S, 6.2. IR (cm ): 3441 (NH), 3270 (NH), 3188 (OH), 1702 (CO),
1
?
?
1
1692 (CO), 1665 (CO). MS: m/z (%): 519 (M , 3.3); 517 (M , 10). H NMR: d,
ppm (DMSO-d ); 6.91 (s, 2H, 2NH, exchangeable with D O); 7.25 (dd, 2H,
6
2
H ? H chlorophenyl); 7.55 (mm, 2H, H ? H benzothiazole); 7.58 (dd, 2H,
3
5
5
6
H ? H chlorophenyl); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H, H ? H benzene);
2
2
6
6
7
.87 (dd, 2H, H ? H benzene); 8.12–8.23 (dd, 2H, H ? H benzothiazole); 11.01
3 5 4 7
(
s, H, OH, exchangeable with D O).
2
1
-[4-(1,3-Benzothiazol-2-yl)benzoyl]-5-(3-phenylthioureido)-1H-pyrazole-4-carbo-
xylic acid (11b) Yield = 0.9 g (87 %); mp = [300 °C. Analysis for C H N
2
5 17 5-
O S (499.6): Calcd.: C, 60.1; H, 3.4; N, 14.0; S, 12.8; Fd.: C, 60.1; H, 3.2; N, 13.8;
3
2
-
1
S, 12.9. IR (cm ): 3441 (NH), 3270 (NH), 3188 (OH), 2215 (CN), 1667 (CO). MS:
?
Á
?
1
m/z (%): 500 (M , 16); 499 (M , 30). H NMR: d, ppm (DMSO-d ); 4.13 (s, 2H,
6
2
phenyl); 7.08 (mm, 2H, H ? H phenyl); 7.55 (mm, 2H, H ? H benzothiazole);
NH, exchangeable with D O); 6.45 (dd, 2H, H ? H phenyl); 6.68 (m, 1H, H
2 2 6 4
3
5
5
6
7
benzene); 8.12–8.23 (dd, 2H, H ? H7 benzothiazole); 10.73 (s, 1H, OH,
.61 (s, 1H, pyrazole); 7.67 (dd, 2H, H ? H benzene); 7.87 (dd, 2H, H ? H
2 6 3 5
4
exchangeable with D O).
2
5
-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole-(5-nitro-2-oxo-1,2-dihy-
droindol-3-ylidene)-4-carbohydrazide (12) The method is the same as described
for preparation of indole 9 but with use of compound 8 instead of 6. Yield = 1.2 g
(
82 %); mp = 220–5 °C. Analysis for C H N O S (552.5): Calcd.: C, 56.5; H,
2
6 16 8 5
-1
.9; N, 20.3; S, 5.8; Fd.: C, 56.4; H, 2.8; N, 20.4; S, 5.8. IR (cm ): 3335 (NH),
2
3
206 (NH), 3195 (NH ), 1702 (CO), 1692 (CO), 1665 (CO). MS: m/z (%): 553
2
?
Á
?
1
(M , 12); 552 (M , 39). H NMR: d, ppm (DMSO-d ); 2.94 (s, 2H, NH ,
exchangeable with D O); 3.27 (s, 1H, NH, exchangeable with D O); 7.55 (mm, 2H,
6
2
2
2
H ? H benzothiazole); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H, H ? H benzene);
5
6
2
6
7
.87 (dd, 2H, H ? H benzene); 7.91 (d, 1H, H indole); 8.12–8.23 (dd, 2H,
3 5 7
1
23

Synthesis of some novel…
H ? H benzothiazole); 8.27 (d, 1H, H indole); 8.50 (s, 1H, H indole); 10.00 (s,
4
7
6
4
1
H, NH, exchangeable with D O).
2
General procedure for preparation of {5-amino-1-[4-(1,3-benzothiazol-2-yl)ben-
zoyl]-1H-pyrazole}-4-(un)substituted phenylsemicarbazide/thiosemicarbazide (13a,
b) The method is the same as described for preparation of urea and thiourea 10a
and 10b but with use of compound 8 instead of 6.
{
5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole}-4-(4-chlorophenyl)
semicarbazide (13a) Yield = 1.3 g (93 %); mp = 245–8 °C. Analysis for C25-
H ClN O S (531.9): Calcd.: C, 56.4; H, 3.4; N, 18.4; S, 6.0; Fd.: C, 56.4; H, 3.6; N,
-
1
8
7 3
1
1
8.4; S, 6.0. IR (cm ): 3395 (NH), 3228 (NH ), 1720 (CO), 1692 (CO), 1674 (CO).
2
?
?
1
MS: m/z (%): 533 (M , 3.3); 531 (M , 10). H NMR: d, ppm (DMSO-d ); 6.53 (s,
6
2
H, NH , exchangeable with D O); 7.25 (dd, 2H, H ? H chlorophenyl); 7.55
2 2 3 5
(
mm, 2H, H ? H benzothiazole); 7.58 (dd, 2H, H ? H chlorophenyl); 7.61 (s,
5 6 2 6
1
8
H, pyrazole); 7.67 (dd, 2H, H ? H benzene); 7.87 (dd, 2H, H ? H benzene);
2 6 3 5
.12–8.23 (dd, 2H, H ? H benzothiazole); 8.87 (s, 1H, NH, exchangeable with
4
7
D O); 9.02 (s, 1H, NH, exchangeable with D O); 10.46 (s, 1H, NH, exchangeable
2
2
with D O).
2
{5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole}-4-phenyl thiosemicar-
bazide (13b) Yield = 1.2 g (88 %); mp = 270–4 °C. Analysis for C H N O S
2
5 19 7 2 2
(
513.6): Calcd.: C, 58.5; H, 3.7; N, 19.1; S, 12.5; Fd.: C, 58.4; H, 3.7; N, 19.0; S,
-
1
1
2.6. IR (cm ): 3395 (NH), 3228 (NH ), 1683 (CO), 1667 (CO). MS: m/z (%): 514
2
?
Á
?
1
(M , 22); 513 (M , 35). H NMR: d, ppm (DMSO-d ); 6.30 (s, 2H, NH ,
exchangeable with D O); 6.45 (dd, 2H, H ? H phenyl); 6.68 (m, 1H, H phenyl);
6
2
2
2
6
4
7
1
8
.08 (mm, 2H, H ? H phenyl); 7.55 (mm, 2H, H ? H benzothiazole); 7.61 (s,
3 5 5 6
H, pyrazole); 7.67 (dd, 2H, H ? H benzene); 7.87 (dd, 2H, H ? H benzene);
2
6
3
5
.12–8.23 (dd, 2H, H ? H benzothiazole); 8.57 (s, 1H, NH, exchangeable with
4
7
D O); 8.92 (s, 1H, NH, exchangeable with D O); 10.00 (s, 1H, NH, exchangeable
2
2
with D O).
2
General procedure for preparation of 5-{5-amino-1-[4-(1,3-benzothiazol-2-yl)ben-
zoyl]-1H-pyrazole}-4-[4-(un)substituted phenyl]-[1,2,4]-triazol-3-one/thione
14a,b) A mixture of compound 13a or 13b (0.01 mol) and sodium hydroxide
40 mg, 2 M solution) was heated under reflux for 4 h. After cooling, the solution
(
(
was acidified with hydrochloric acid and the precipitate was isolated by filtration.
The product was then crystallized from methanol.
5
2
-{5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole}-4-(4-chlorophenyl)-
,4-dihydro-[1,2,4]-triazol-3-one (14a) Yield = 0.8 g (82 %), mp = 220–2 °C.
Analysis for C H ClN O S (513.9): Calcd.: C, 58.4; H, 3.1; N, 19.1; S, 6.2; Fd.:
2
-
C, 58.5; H, 3.3; N, 19.0; S, 6.2. IR (cm ): 3395 (NH), 3228 (NH ), 1685 (CO),
5
16
7 2
1
2
?
?
1
1
d ); 7.25 (dd, 2H, H ? H chlorophenyl); 7.55 (mm, 2H, H ? H benzothiazole);
669 (CO). MS: m/z (%): 515 (M , 3.3); 513 (M , 10). H NMR: d, ppm (DMSO-
6
3
5
5
6
123

E. A. A. El-Meguid, M. M. Ali
7
.58 (dd, 2H, H ? H chlorophenyl); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H,
2
6
H ? H benzene); 7.87 (dd, 2H, H ? H benzene); 8.12–8.23 (dd, 2H, H ? H
7
2
6
3
5
4
benzothiazole); 8.84 (s, 2H, NH ); 10.46 (s, 1H, NH).
2
5
-{5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole}-4-phenyl-2,4-dihy-
dro-[1,2,4]-triazol-3-thione (14b) Yield = 0.7 g (72 %), mp = 257–9 °C. Ana-
lysis for C H N OS (495.6): Calcd.: C, 60.6; H, 3.5; N, 19.8; S, 12.9; Fd.: C,
-
2
5
17
7
2
1
6
0.6; H, 3.4; N, 19.6; S, 13.0. IR (cm ): 3395 (NH), 3228 (NH ), 1667 (CO). MS:
2
?
Á
?
1
m/z (%): 496 (M , 9.8); 495 (M , 22). H NMR: d, ppm (DMSO-d ); 4.30 (s, 2H,
6
NH ); 6.45 (dd, 2H, H ? H phenyl); 6.68 (m, 1H, H phenyl); 7.08 (mm, 2H,
H ? H phenyl); 7.55 (mm, 2H, H ? H benzothiazole); 7.61 (s, 1H, pyrazole);
2
2
6
4
3
5
5
6
7
.67 (dd, 2H, H ? H benzene); 7.87 (dd, 2H, H ? H benzene); 8.12–8.23 (dd,
2 6 3 5
2
H, H ? H benzothiazole); 8.57 (s, 1H, NH).
4
7
General procedure for preparation of 5-{5-amino-1-[4-(1,3-benzothiazol-2-yl)ben-
zoyl]-1H-pyrazole}-4-(4-chlorophenyl)-2-substituted-2,4-dihydro-[1,2,4]-triazol-3-
one (15a–e) A mixture of compound 14a (2 g; 0.067 mol), the appropriate
organohalogen compound (methyl iodide, ethyl chloroformate, methyl 2-bromoac-
etate, ethyl 2-bromoacetate, or ethyl 2-bromopropanoate) (0.067 mol), anhydrous
sodium carbonate (4 g), and acetone (30 ml) was heated under reflux for 8 h. Most
of the solvent was removed by distillation, the residue was diluted with water, and
the product obtained was collected.
5
2
1
-{5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole}-4-(4-chlorophenyl)-
-methyl-2,4-dihydro-[1,2,4]-triazol-3-one (15a) Yield = 0.8 g (78 %), mp =
42–5 °C. Analysis for C H ClN O S (527.9): Calcd.: C, 59.2; H, 3.4; N, 18.6;
2
6
18
7 2
-
1
S, 6.1; Fd.: C, 59.3; H, 3.6; N, 18.5; S, 6.1. IR (cm ): 3290 (NH ), 1685 (CO),
2
?
?
1
1
d ); 2.71 (s, 3H, CH ); 7.25 (dd, 2H, H ? H chlorophenyl); 7.55 (mm, 2H,
666 (CO). MS: m/z (%): 529 (M , 33); 527 (M , 100). H NMR: d, ppm (DMSO-
6
3
3
5
H ? H benzothiazole); 7.58 (dd, 2H, H ? H chlorophenyl); 7.61 (s, 1H,
2
5
6
6
pyrazole); 7.67 (dd, 2H, H ? H benzene); 7.87 (dd, 2H, H ? H benzene);
2
6
3
5
8
.12–8.23 (dd, 2H, H ? H benzothiazole); 9.72 (s, 2H, NH ).
4 7 2
3
5
0
5
-{5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole}-4-(4-chlorophenyl)-
-oxo-4,5-dihydro-[1,2,4]-triazole-1-carboxylic acid ethyl ester (15b) Yield =
.9 g (79 %), mp = 92–5 °C. Analysis for C H ClN O S (585.5): Calcd.: C,
2
8
20
7 4
-
1
7.4; H, 3.4; N, 16.7; S, 5.5; Fd.: C, 57.5; H, 3.6; N, 16.6; S, 5.5. IR (cm ): 3220
?
?
(
NH ), 1710 (CO), 1685 (CO), 1662 (CO). MS: m/z (%): 587 (M , 3.3); 585 (M ,
2
1
1
0). H NMR: d, ppm (DMSO-d ); 1.31 (t, 3H, CH ); 4.11 (q, 2H, CH ); 7.25 (dd,
6
3
2
2
H ? H chlorophenyl); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H, H ? H benzene);
H, H ? H chlorophenyl); 7.55 (mm, 2H, H ? H benzothiazole); 7.58 (dd, 2H,
3
5
5
6
2
6
2
6
7
.87 (dd, 2H, H ? H benzene); 8.12–8.23 (dd, 2H, H ? H benzothiazole); 9.72
3 5 4 7
(
s, 2H, NH2).
1
23

Synthesis of some novel…
3
5
0
5
-{5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole}-4-(4-chlorophenyl)-
-oxo-4,5-dihydro-[1,2,4]-triazol-1-yl-acetic acid methyl ester (15c) Yield =
.9 g (79 %), mp = 102–5 °C. Analysis for C H ClN O S (586.0): Calcd.: C,
2
8
20
7 4
-
1
7.4; H, 3.4; N, 16.7; S, 5.5; Fd.: C, 57.5; H, 3.6; N, 16.6; S, 5.5. IR (cm ): 3246
?
?
(
NH ), 1708 (CO), 1685 (CO), 1662 (CO). MS: m/z (%): 588 (M , 3.3); 586 (M ,
2
1
1
0). H NMR: d, ppm (DMSO-d ); 3.71 (s, 3H, CH ); 4.11 (s, 2H, CH ); 7.25 (dd,
6
3
2
2
H ? H chlorophenyl); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H, H ? H benzene);
H, H ? H chlorophenyl); 7.55 (mm, 2H, H ? H benzothiazole); 7.58 (dd, 2H,
3
5
5
6
2
6
2
6
7
.87 (dd, 2H, H ? H benzene); 8.12–8.23 (dd, 2H, H ? H benzothiazole); 9.72
3 5 4 7
(
s, 2H, NH2).
3
5
-{5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole}-4-(4-chlorophenyl)-
-oxo-4,5-dihydro-[1,2,4]-triazol-1-yl-acetic acid ethyl ester (15d) Yield = 0.8 g
(
69 %), mp = 83–5 °C. Analysis for C H ClN O S (600.1): Calcd.: C, 58.1; H,
2
9
22
7 4
-
1
3
.7; N, 16.3; S, 5.3; Fd.: C, 58.0; H, 3.6; N, 16.4; S, 5.3. IR (cm ): 3246 (NH ),
2
?
710 (CO), 1685 (CO), 1662 (CO). MS: m/z (%): 602 (M , 3.3); 600 (M , 10).
?
1
1
H NMR: d, ppm (DMSO-d ); 1.31 (t, 3H, CH ); 4.11 (s, 2H, CH ); 4.15 (q, 2H,
6
3
2
CH ); 7.25 (dd, 2H, H ? H chlorophenyl); 7.55 (mm, 2H, H ? H benzothia-
zole); 7.58 (dd, 2H, H ? H chlorophenyl); 7.61 (s, 1H, pyrazole); 7.67 (dd, 2H,
2
3
5
5
6
2
6
H ? H benzene); 7.87 (dd, 2H, H ? H benzene); 8.12–8.23 (dd, 2H, H ? H
7
2
6
3
5
4
benzothiazole); 9.72 (s, 2H, NH2).
2
-{3-{5-Amino-1-[4-(1,3-benzothiazol-2-yl)benzoyl]-1H-pyrazole}-4-(4-chlorophe-
nyl)-5-oxo-4,5-dihydro-[1,2,4]-triazol-1-yl}-propionic acid ethyl ester (15e)
Yield = 0.85 g (71 %), mp = 95–9 °C. Analysis for C H ClN O S (614.1):
Calcd.: C, 58.7; H, 3.9; N, 16.0; S, 5.2; Fd.: C, 58.7; H, 3.9; N, 16.0; S, 5.2. IR
3
0
24
7 4
-
1
?
(
cm ): 3446 (NH ), 1710 (CO), 1685 (CO), 1662 (CO). MS: m/z (%): 616 (M ,
2
?
1
3
CH ); 4.15 (q, 2H, CH ); 4.61 (q, 1H, CH); 7.25 (dd, 2H, H ? H chlorophenyl);
.3); 614 (M , 10). H NMR: d, ppm (DMSO-d ); 1.31 (t, 3H, CH ); 1.73 (d, 3H,
6
3
3
2
3
5
7
.55 (mm, 2H, H ? H benzothiazole); 7.58 (dd, 2H, H ? H chlorophenyl); 7.61
5 6 2 6
(
benzene); 8.12–8.23 (dd, 2H, H ? H benzothiazole); 9.72 (s, 2H, NH ).
s, 1H, pyrazole); 7.67 (dd, 2H, H ? H6 benzene); 7.87 (dd, 2H, H ? H
2 3 5
4
7
2
Bioactivity materials and methods
Cytotoxicity against human breast cancer cell line MCF-7
Antitumor activity against MCF-7 was determined in the National Research Center,
Division of Genetic Engineering and Biotechnology, Department of Biochemistry,
Cairo, Egypt. Fetal bovine serum (FBS) and L-glutamine, were obtained from Gibco
Invitrogen (Scotland, UK). Dulbecco’s modified Eagle’s (DMEM) medium was
from Cambrex (New Jersey, USA). Dimethyl sulfoxide (DMSO), tamoxifen,
penicillin, streptomycin, and sulfo-rhodamine-B stain (SRB) were obtained from
123

E. A. A. El-Meguid, M. M. Ali
Sigma (St Louis, MO, USA). All other chemicals and reagents used in this study
were of analytical grade and purchased from Sigma–Aldrich (St Louis, MO, USA).
Antitumor activity was measured in vitro by use of the sulfo-rhodamine-B stain
(
SRB) assay and the standard procedure reported elsewhere [37]. Ninety-six-well
4
microtiter plates were inoculated with cells (10 cells/well) 24 h before treatment
with the tested compounds, to enable attachment of cells to the walls of the plate.
Test compounds were dissolved in DMSO and diluted with saline to the appropriate
volume. Different concentrations of the compounds under test (0–100 lg/ml) were
added to the cells. Triplicate wells were prepared for each dose. Monolayers of cells
were incubated with the compounds for 48 h at 37 °C in an atmosphere of 5 % CO .
2
After 48 h the cells were fixed, washed, and stained for 30 min with 0.4 % (w/
v) SRB dissolved in 1 % acetic acid. Unbound dye was removed by four washes
with 1 % acetic acid, and attached stain was recovered with Tris–EDTA buffer.
Color intensity was measured in an ELISA reader and optical density was
determined at 492 nm. After the specified time the relationship between surviving
fraction and drug concentration was plotted to obtain the survival curve. The
concentration required for 50 % inhibition of cell viability (IC ) was calculated;
5
0
the results are given in Table 1.
In-vitro VEGF inhibition of human breast cancer cell line MCF-7
The effect of the compounds on the level of human vascular endothelial growth
factor (VEGF) was determined in human breast cancer cell line MCF-7 obtained
from the American Type Culture Collection (Rockville, MD, USA). The tumor cells
were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10 % heat-inactivated fetal calf serum (GIBCO), penicillin (100 U/ml), and
streptomycin (100 lg/ml), at 37 °C, in humidified atmosphere containing 5 % CO .
2
6
2
Cells at a concentration of 0.50 9 10 were grown in a 25 cm flask in 5 ml
complete culture medium.
The cells in the culture medium were treated with 20 ll of one-tenth of the IC50
values of the compounds and the standard reference drug, tamoxifen, dissolved in
DMSO, then incubated for 24 h at 37 °C, in a humidified 5 % CO atmosphere. The
2
cells were harvested and homogenates were prepared in saline by use of a tight
pestle homogenizer until complete cell disruption.
The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay
ELISA) for determination of the level of human VEGF in samples. The VEGF is
(
added to a monoclonal antibody enzyme well pre-coated with human VEGF
monoclonal antibody and incubated. VEGF antibodies labeled with biotin and
combined with Streptavidin-HRP to form an immune complex are then added and
the mixture is incubated then washed to remove uncombined enzyme. Chromogen
solutions A and B are then added and the color of the liquid changes to blue. Acid is
then added and the color finally becomes yellow. Optical density was determined at
4
50 nm. Color intensity and concentration of human VEGF in the sample are
positively correlated. Levels (pg/ml) of human VEGF in the samples were
calculated from the standard curve as means from triplicate determinations.
1
23

Synthesis of some novel…
Conclusion
Our main objective in this study was the synthesis of new urea, indole, and triazole
derivatives of 4-(1,3-benzothiazol-2-yl)-benzoyl-1H-pyrazole and testing of the
compounds as anticancer agents working by inhibiting VEGF–VEGFR2 complex
formation, thus suppressing proliferation and survival of endothelial cells and
consequently preventing cancer progression.
Five of the compounds (8, 11a, 11b, 12, and 15a) had promising cytotoxic
activity against breast cancer cell line MCF-7; inhibition of human VEGF in MCF-7
cancer cell line was comparable with that of tamoxifen.
Acknowledgments The authors gratefully acknowledge financial support from the Microanalytical
1
Center, Faculty of Science, Cairo University, Egypt, by performing elemental analysis, IR, H NMR, and
mass spectroscopy.
Conflict of interest The authors have declared no conflicts of interest.
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