Design, synthesis and in Vitro antiproliferative activity of novel isatin-quinazoline hybrids

Article abstract of DOI:10.1002/ardp.201400337

Using a molecular hybridization approach, a new series of isatin-quinazoline hybrids 15a-o was designed and synthesized via two different synthetic routes. The target compounds 15a-o were prepared by the reaction of quinazoline hydrazines 12a-e with indoline-2,3-diones 13a-c or by treating 4-chloroquinazoline derivatives 11a-e with isatin hydrazones 14a-c. The in vitro anticancer activity of the newly synthesized hybrids was evaluated against the liver HepG2, breast MCF-7 and colon HT-29 cancer cell lines. A distinctive selective growth inhibitory effect was observed towards the HepG2 cancer cell line. Compounds 15b, 15g and 15l displayed the highest potency, with IC50 values ranging from 1.0 ± 0.2 to 2.4 ± 0.4 mM, and they were able to induce apoptosis in HepG2 cells, as evidenced by enhanced expression of the pro-apoptotic protein Bax and reduced expression of the anti-apoptotic protein Bcl-2, in addition to increased caspase-3 levels.

Full text of DOI:10.1002/ardp.201400337

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Full Paper
Design, Synthesis and In Vitro Antiproliferative Activity of Novel
Isatin-Quinazoline Hybrids
Mohamed Fares1, Wagdy M. Eldehna1, and Sahar M. Abou-Seri2*, Hatem A. Abdel-Aziz3,4
Mohamed H. Aly5, and Mai F. Tolba6
,
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City,
Cairo, Egypt
2
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh,
Saudi Arabia
Department of Applied Organic Chemistry, National Research Center, Dokki, Giza, Egypt
Department of Pharmacology and Toxicology, Faculty of Pharmacy, British University in Egypt,
Cairo, Egypt
4
5
6
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University,
Cairo, Egypt
Using a molecular hybridization approach, a new series of isatin-quinazoline hybrids 15a–o was designed
and synthesized via two different synthetic routes. The target compounds 15a–o were prepared by the
reaction of quinazoline hydrazines 12a–e with indoline-2,3-diones 13a–c or by treating 4-chloroquin-
azoline derivatives 11a–e with isatin hydrazones 14a–c. The in vitro anticancer activity of the newly
synthesized hybrids was evaluated against the liver HepG2, breast MCF-7 and colon HT-29 cancer cell lines.
A distinctive selective growth inhibitory effect was observed towards the HepG2 cancer cell line.
Compounds 15b, 15g and 15l displayed the highest potency, with IC50 values ranging from 1.0 ꢀ 0.2 to
2.4 ꢀ 0.4 mM, and they were able to induce apoptosis in HepG2 cells, as evidenced by enhanced expression
of the pro-apoptotic protein Bax and reduced expression of the anti-apoptotic protein Bcl-2, in addition
to increased caspase-3 levels.
Keywords: Antiproliferative activity / Isatins / Molecular hybridization / Quinazolines
Received: August 31, 2014; Revised: November 30, 2014; Accepted: December 1, 2014
DOI 10.1002/ardp.201400337
to treat cancer [3–6]. The pharmacophore hybrid approach is
Introduction
an effective and commonly used strategy for the exploration
of novel active agents. Hybridization of two or more bioactive
fragments, each with complementary pharmacophoric func-
tion or with different modes of action, often showed a
synergistic effect and could be beneficial for the treatment of
cancer [7–9]. This “merged” pharmacophore may address
the active site of various targets and offers the possibility of
overcoming drug resistance.
Regardless of the notable improvement in survival among
cancer patients over the past three decades [1], the growing
incidence of drug resistance and unwanted side effects of
cancer chemotherapeutics underlie the need to develop new
classes of chemotherapeutic agents for cancer treatment [2].
A
combinational chemotherapeutic drug with different
mechanisms of action is one method that is being adopted
Isatin (1H-indole-2,3-dione) and its derivatives demonstrate
a broad spectrum of biological activities [10, 11]. Cytotoxic
and antineoplastic properties of isatins are widely reported
[12]. These reports were substantiated by FDA approval of
the tyrosine kinase inhibitor sunitinib 1 for the treatment
of gastrointestinal stromal cancers and metastatic renal cell
carcinoma [13, 14]. Additionally, the structurally relevant
SU9516 2 was reported to be a potential inhibitor of cyclin-
Correspondence: Dr. Hatem A. Abdel-Aziz, Department of
Pharmaceutical Chemistry, College of Pharmacy, King Saud
University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
E-mail: hatem_741@yahoo.com
Fax: þ966-146-76220
Additional correspondence: Dr. Sahar M. Abou-Seri,
E-mail: saharshaarawy_69@yahoo.com
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Figure 1. Structure of isatins 1–4 and quinazoline derivatives 5–9.
dependent kinases (CDKs) that can induce apoptosis in colon
carcinoma cells [15]. Moreover, isatin-3-arylhydrazones are
a newly emerging class of pharmacologically important
antineoplastic agents that are described as inhibitors of
CDK2 [16], protein tyrosine phosphatase Shp2 [17] and c-Met
kinase [18]. Other members of this class were reported to be
effective against colorectal and hepatocellular carcinoma by
virtue of their ability to inhibit tubulin polymerization,
activate caspase-3 and induce apoptosis [19]. Furthermore,
we have reported the synthesis of N,N0-hydrazino-bis-isatin
derivative 3, which has selective activity against multidrug-
resistant cancer cells [20], and 2H-chromene-3-carbohydra-
zide-isatins 4 with in vitro antiproliferative activity and a
good growth inhibitory profile against colon HT-29 cells [21]
(Fig. 1).
8b exhibited broad-spectrum antiproliferative activity toward
HepG2, MCF-7 and HeLa human cancer cells [30] (Fig. 1).
Encouraged by the previous reports and in continuation
to our efforts to develop effective anticancer agents based
on the isatin scaffold [20, 21, 31], a new series of isatin-
quinazoline conjugates 15a–o was designed via a hybrid
pharmacophore approach (Fig. 2).
Thus, the present study reports the synthesis and growth
inhibition evaluation of the new hybrids 15a–o against liver
HepG2, breast MCF-7, and colon HT-29 cancer cell lines, in
addition to providing some mechanistic insights for the
anticancer activity of the promising derivatives.
On the other hand, quinazoline derivatives have attracted
immense interest as novel class of cancer chemotherapeutic
agents with significant therapeutic efficacy against solid
tumors [22–24]. Quinazolines serve as the backbone for a
range of tyrosine kinase inhibitors including the reversible
EGFR inhibitor gefitinib 5 [23] and the dual EGFR-ErbB2
inhibitor afatinib 6 [24].
Quinazoline also forms the basis for compounds that inhibit
serine/threonine kinase CDKs [25] and thymidylate syn-
thase [26] or modulate aurora kinase activity [27]. In addition,
2-aryl quinazolines were recently identified as potential
antitumor agents. For example, quinazoline derivative 7
enhanced the inhibition of NF-kB function and initiated
apoptosis in the HepG2 liver cancer cell line [28]. Another
analog, 2-phenyl-4-anilinoquinazoline 8a proved to be a
potent and selective breast cancer resistance protein (BCRP)
inhibitor [29]. Meanwhile, the 2-p-tolylquinazoline derivative
Figure 2. Design of the targeted isatin-quinazoline conjugates
15a–o.
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Scheme 1. Synthesis of compounds 12a–e. Reagents and conditions: (i) Ar-CHO/DMF/I2/K2CO3/70–90°C/4–5 h. (ii) POCl3/N,N-
dimethylaniline/reflux 6 h. (iii) NH2NH2.H2O/EtOH/reflux 4 h.
Results and discussion
NMR of compounds 12a–e revealed two D2O-exchangeable
singlet signals of NH and NH2 groups in the regions d 9.56–9.62
and 4.86–4.91 ppm, respectively.
Chemistry
The synthetic pathway employed to obtain the target 3-(2-(2-
phenylquinazolin-4-yl)hydrazono)indolin-2-one derivatives
15a–o started by the preparation of 4-quinazolinones 10a–e
from 2-amino-benzamide (9) using a modified Niementowski
reaction [29] (Scheme 1). The key intermediates 4-chloroqui-
nazolines 11a–e were obtained via chlorination of the
2-arylquinazolinone derivatives 10a–e with phosphorous
oxychloride in the presence of a catalytic amount of N,N-
dimethylaniline as an acid binder [32–34]. Subsequent
reaction of compounds 11a–e with refluxing hydrazine
hydrate led to the 4-hydrazinyl-2-substituted quinazoline
derivatives 12a–e with 70–79% yield (Scheme 1). IR spectra of
the latter products showed the absorption bands due to the
NH and NH2 groups in the region d 3273–3302 cmꢁ1. The 1H
Preparation of the targeted compounds 15a–o was
achieved via two synthetic routes. First, the appropriate
quinazoline hydrazines 12a–e reacted with indoline-2,3-
diones 13a–c, in refluxing ethanol in the presence of a
catalytic amount of glacial acetic acid to give 15a–o with 79–
91% yield (Scheme 2). Alternatively, indoline-2,3-diones 13a–c
were refluxed with hydrazine hydrate in absolute methanol
to afford the corresponding hydrazine derivatives 14a–c [31a],
which, upon reaction with the appropriate 4-chloroquinazo-
line derivatives 11a–e in refluxing isopropanol, furnished the
target compounds 15a–o with 60–82% yield (Scheme 2). The
IR spectra of 15a–o revealed the stretching band of NH around
3400 cmꢁ1. Furthermore, 1H NMR spectra of these compounds
showed a D2O-exchangeable signal of NH proton of the isatin
Scheme 2. Synthesis of compounds 15a–o. Reagents and conditions: (i) EtOH/AcOH (catalytic)/reflux 0.5 h. (ii) NH2NH2.H2O/CH3OH/
reflux 0.5 h; (iii) 11a–e/isopropanol/reflux 5 h.
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moiety in the region d 11.25–11.48 ppm whereas the proton of
the hydrazine function (¼N–NH–) appeared as two singlet
signals resonating in the regions d 10.61–10.79 and 13.75–
13.90 ppm due to the hydrogen bonding.
Growth inhibition evaluation in MCF-7 cell line revealed that
compounds 15b, 15c, 15g, 15i, and 15n were the most potent
ones, with IC50 of 7.4 ꢀ 0.5, 3.3 ꢀ 0.4, 9.8 ꢀ 0.8, 2.1 ꢀ 0.19, and
5.5 ꢀ 0.42 mM, respectively. IC50 values of the positive control
Dox was 0.24 ꢀ 0.03 mM. Investigations of the antiprolifer-
ative activity against HT-29 cells indicated that this cell line
was the least sensitive to the cytotoxicity of the new hybrids.
Only compounds 15b and 15g were moderately active, with
IC50 of 14.3 ꢀ 1.1 and 26.0 ꢀ 2.3 mM, respectively, while the
analog 15f exhibited weak potency (IC50 ¼ 79.0 ꢀ 6.1 mM)
relative to the reference drug Dox (IC50 ¼ 7.7 ꢀ 0.6 mM) in this
cell type.
From the aforementioned results, it was clear that the
evaluated hybrids displayed distinctive selectivity towards
HepG2 cancer cells, and only compounds 15b and 15g
displayed broad spectrum anticancer activity against the
three tested cell lines but with preferential potency in the
HepG2 cell line. Also, it can be observed that the new isatin-
quinazoline hybrids 15b, 15g, and 15l demonstrated en-
hanced cytotoxic activity against HepG2 cells compared to
sunitinib 1 and could be considered as good candidates for
future investigation.
Biological evaluations
In vitro antiproliferative activity
The newly synthesized isatin-quinazoline hybrids 15a–-o were
evaluated for their growth inhibition potential against three
aggressive cancer cell lines, namely HepG2 hepatocellular
carcinoma, MCF-7 breast cancer and HT-29 colon cancer cells
using the sulforhodamine B (SRB) colorimetric assay [35].
Doxorubicin (Dox) was used as positive control as it is one of
the most potent anticancer agents that possesses broad-
spectrum against wide range of solid tumors and is known to
exhibit potent pro-apoptic effects [36, 37]. Moreover, the
reported activity of sunitinib 1 was included for compari-
son [38, 39]. The antiproliferative activities are expressed
as the median growth inhibitory concentration (IC50) and
are provided in Table 1. From the results, it is evident
that most of the tested compounds displayed potent to fair
growth inhibitory activity against the HepG2 cancer cell
line. Compounds 15b, 15g, and 15l showed concentration-
dependent cytotoxicity with IC50 values of 1.0 ꢀ 0.2, 1.8 ꢀ 0.4,
and 2.4 ꢀ 0.4 mM, respectively. The tested compounds were
found to be more potent and efficacious than doxorubicin
(IC50 ¼ 2.9 ꢀ 0.5 mM). Moreover, compounds 15a, 15c, 15d,
15e, 15h, and 15i exhibited high potency at a low micromolar
level. These results are in line with a previous study indicating
that bis-Schiff base derivatives of isatin have antiproliferative
activity against HepG2 cells [40].
Effects on mitochondrial apoptosis pathway proteins Bax
and Bcl-2
Hepatocellular carcinoma (HCC) is the fifth most frequent
cancer worldwide and the third most common cause of
cancer-related death [41]. Screening of the synthesized
compounds in HepG2 cancer cells revealed that compounds
15b, 15g, and 15l showed the highest potency for
cytotoxicity compared to the other tested compounds.
Previous studies indicated that induction of apoptosis is
one of the main mechanisms behind the anticancer activities
of several isatin and quinazoline derivatives [42–46].
Therefore, we investigated the potential pro-apoptotic
effects of these promising isatin-quinazoline hybrids in an
attempt to define the underlying mechanism for their
antiproliferative activity.
Table 1. In vitro antiproliferative activities of the synthe-
sized compounds against hepatocellular carcinoma (HepG2),
colon cancer (HT-29), and breast cancer (MCF-7) cell lines.
IC50 (mM)a)
Compound 15
HepG2
HT-29
MCF-7
Exposure of HepG2 hepatocellular carcinoma cells to
compounds 15b, 15g, or 15l for 48 h resulted in a significant
increase in the expression of the pro-apoptotic protein Bax by
approximately 2.6-, 1.8-, and 2.2-fold, respectively, compared
to the control (Fig. 3A). However, the positive control Dox
significantly boosted Bax level by about 3.6 compared to
untreated cells. Bax is a member of the Bcl-2 family but has
pro-apoptotic effects. Reduced expression of Bax is known to
be associated with resistance to chemotherapy in many types
of cancers including liver cancer [47].
Moreover, treatment of HepG2 cells with compounds 15b,
15g, or 15l significantly reduced the expression levels of the
anti-apoptotic protein Bcl-2 by approximately 57, 69, and
60%, respectively, compared to the control (Fig. 3B). The
effect of the tested agents on Bcl-2 was more potent
compared to Dox that elicited 60% reduction in Bcl-2 level
compared to control untreated cells. Bcl-2 family proteins
have been shown to be an important factor for apoptosis
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
Dox
18.5 ꢀ 1.7
1.0 ꢀ 0.2
15.3 ꢀ 1.1
13.5 ꢀ 1.3
13.0 ꢀ 2
28.2 ꢀ 1.4
1.8 ꢀ 0.4
6.4 ꢀ 0.8
17.3 ꢀ 1.5
25.0 ꢀ 2.1
59.5 ꢀ 4.9
2.4 ꢀ 0.4
44.3 ꢀ 3.7
>100
>100
>100
14.3 ꢀ 1.1
7.4 ꢀ 0.5
3.3 ꢀ 0.4
>100
>100
>100
>100
>100
>100
79 ꢀ 6.1
26 ꢀ 2.3
>100
>100
>100
>100
>100
9.8 ꢀ 0.8
>100
2.1 ꢀ 0.19
>100
>100
>100
>100
>100
>100
>100
5.5 ꢀ 0.42
23.6 ꢀ 1.9
2.9 ꢀ 0.5
12.0
>100
7.7 ꢀ 0.6
0.24 ꢀ 0.03
5
Sunitinib [38, 39]
1.6
a) IC50 valuesarethemean ꢀ S.D. ofthreeseparateexperiments.
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Figure 3. Effect of compounds 15b, 15g, and 15l on the the expression levels of (A) Bax, (B) Bcl-2, (C) Bax/Bcl-2 ratio and (D) active
caspase-3 in HepG2 cancer cells treated with the compounds at their IC50 concentrations. Data are the mean ꢀ S.D. of three separate
experiments. *Significantly different from control at P < 0.05.
resistance in HCC. Bcl-2 is known to inhibit apoptosis and to
promote the process of tumorigenesis and chemoresist-
ance [41]. The ability of the tested compounds to significantly
down-regulate Bcl-2 levels indicates their potential to
enhance apoptosis.
The ratio of Bax (apoptosis inducer) to Bcl-2 (apoptosis
suppressor) is considered a key indicator of therapeutic
response to chemotherapy [48]. The tested compounds 15b,
15g, or 15l showed significant elevation in Bax/Bcl-2 ratio
by about 7 folds compared to control (Fig. 3C) which
supports their ability to promote the therapeutic response
in HepG2 cells. The reference drug Dox triggered a significant
increase in Bax/Bcl-2 ratio by about 9 folds compared to
control.
by approximately 6.6-, 5.3-, and 6.4-fold, respectively,
compared to the control (Fig. 3D). It is noteworthy that
the tested compounds resulted in more induction in active
caspase-3 compared to the reference drug. Dox treatment
resulted in significant increase in caspase-3 levels by about 6
folds compared to control. Active caspase-3 indicates that cells
are undergoing apoptosis because this caspase is activated at
the end of the apoptosis cascade [50].
In conclusion, compounds 15b, 15g, and 15l have potent
apoptotic potential through, at least in part, inducing the
mitochondrial apoptotic pathway. They enhanced the expres-
sion of the pro-apoptotic protein Bax and reduced the
expression of the anti-apoptotic protein Bcl-2 in addition to
boosting caspase-3 levels.
Effects on the level of active caspase-3
(key executor of apoptosis)
Conclusion
Reduced expression of Bcl-2 leads to increased levels of free
Bax, which in turn leads to activation of the caspase cascade
leading to apoptosis [49]. In the current study, compounds
15b, 15g, and 15l elevated the Bax/Bcl-2 ratio. Therefore, the
subsequent step was to assess the levels of active caspase-3, a
cysteine protease, which is a key executor of apoptosis [50].
Treatment of HepG2 cells with compounds 15b, 15g, and 15l
produced a significant increase in the level of active caspase-3
We have presented two synthetic approaches for the synthesis
of the novel isatin-quinazoline hybrids 15a–o. The anticancer
activity of the newly synthesized compounds was evaluated
against liver HepG2, breast MCF-7, and colon HT-29 cancer cell
lines. Most of the compounds exhibited relatively potent and
selective growth inhibition against the HepG2 human HCC cell
line. Compounds 15b, 15g, and 15l (IC50 ¼ 1.0 ꢀ 0.2, 1.8 ꢀ 0.4,
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and 2.4 ꢀ 0.4 mM, respectively) were found to be more potent
than doxorubicin (IC50 ¼ 2.9 ꢀ 0.5 mM). Compounds 15b, 15g,
and 15l were studied further to gain mechanistic insights into
their antiproliferative activity by assessing their effect on
the intrinsic apoptotic pathway in HepG2 cells. The results
indicated that the tested compounds induced the activation
of caspase-3. Furthermore, the Bcl-2/Bax ratio, which is crucial
for the activation of the mitochondrial apoptotic pathway,
was significantly reduced in cells treated with compounds
15b, 15g, and 15l. These results indicate that compounds 15b,
15g and 15l exert an antiproliferative effect in HepG2 liver
cancer cells, which is, at least in part, via activation of the
mitochondrial apoptotic pathway.
chloroquinazolines 11a–e (5 mmol) in refluxing ethanol
(20 mL). The reaction mixture was refluxed for 4 h and left
to cool. The precipitated solid was filtered, dried and
recrystallized from acetonitrile to give 12a–e, respectively.
4-Hydrazinyl-2-phenylquinazoline (12a)
The physical properties and spectral data of this compound
are identical with those that have been reported [51].
4-Hydrazinyl-2-(4-methoxyphenyl)quinazoline (12b)
Pale yellow powder; yield (73%); m.p. 292ꢁ294°C; IR n 3300
(NH, NH2) cmꢁ1 1H NMR (DMSO-d6) d ppm: 3.84 (s, 3H,
;
-OCH3), 4.90 (s, 2H, NH2, D2O exchangeable), 7.00 (d,
J ¼ 8.7 Hz, 2H, Ar-H), 7.37–7.42 (m, 1H, Ar-H), 7.70 (m, 2H,
Ar-H), 8.14–8.21 (m, 1H, Ar-H), 8.5 (d, J ¼ 8.7 Hz, 2H, Ar-H), 9.57
(s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz) d
ppm: 55.7, 113.9, 122.7, 125.3, 126.3, 127.8, 129.9, 130.2,
131.4, 132.9, 135.0, 159.3, 161.5; Anal. calcd. for C15H14N4O
(266.30): C, 67.65; H, 5.30; N, 21.04. Found: C, 67.89; H, 5.37;
N, 21.18.
Experimental
Chemistry
Melting points were measured with a Gallenkamp apparatus
and were uncorrected. IR spectra were recorded on
Shimadzu FT-IR 8101 PC infrared spectrophotometer. The
NMR spectra were recorded on a 300 NMR spectrometer.
1H spectra were run at 300 MHz, and 13C spectra were run at
75 or 100 MHz in deuterated dimethylsulfoxide (DMSO-d6).
Chemical shifts (dH) are reported relative to TMS as the
internal standard. All coupling constant (J) values are given
in Hertz. Chemical shifts (dC) are reported relative to DMSO-
d6 as internal standards. The abbreviations used are as
follows: s, singlet; d, doublet; m, multiplet. Mass spectra
were measured on a GCMS-QP1000 EX spectrometer at
70 eV. Elemental analyses were carried out at the Regional
Center for Microbiology and Biotechnology, Al-Azhar
University, Cairo, Egypt. Analytical thin layer chromatogra-
phy (TLC) on silica gel plates containing a UV indicator was
routinely employed to follow the course of reactions and to
check the purity of the products. All reagents and solvents
were purified and dried by standard techniques.
2-(3,4-Dimethoxyphenyl)-4-hydrazinylquinazoline (12c)
Pale yellow powder; yield (77%); m.p. 180ꢁ182°C; IR n 3302
(NH, NH2) cmꢁ1 1H NMR (DMSO-d6) d ppm: 3.84 (s, 3H,
;
-OCH3), 3.89 (s, 3H, -OCH3), 4.88 (s, 2H, NH2, D2O exchange-
able), 7.04 (d, J ¼ 8.1 Hz, 1H, Ar-H), 7.39–7.42 (m, 1H, Ar-H),
7.72–7.73 (m, 2H, Ar-H), 8.15–8.19 (m, 3H, Ar-H), 9.56 (s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO-d6, 75 MHz) d ppm:
55.5 (2C), 111.1, 111.4, 112.6, 121.4, 122.2, 124.8, 127.4, 131.2,
132.5, 148.3, 149.5, 150.8, 159.0, 159.7; MS m/z (Rel. Int.) 297
[(M þ 1)þ, 17.6%], 296 [Mþ, 100.0%]; Anal. calcd. for
C
16H16N4O2 (296.33): C, 64.85; H, 5.44; N, 18.91. Found: C,
65.04; H, 5.49; N, 19.09.
2-(Benzo[d][1,3]dioxol-5-yl)-4-hydrazinylquinazoline
(12d)
Pale yellow fibers; yield (70%); m.p. 230ꢁ232°C; IR n 3302,
3277 (NH, NH2) cmꢁ1; 1H NMR (DMSO-d6) d ppm: 4.86 (s, 2H,
NH2, D2O exchangeable), 6.10 (s, 2H, -CH2), 7.00 (d, J ¼ 7.8 Hz,
1H, Ar-H), 7.39–7.42 (m, 1H, Ar-H), 7.71–7.72 (m, 2H, Ar-H),
8.07 (s, 1H, -Ar-H), 8.15–8.20 (m, 2H, Ar-H), 9.56 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz) d ppm: 101.8,
108.3, 108.5, 113.1, 122.7, 123.2, 125.4, 128.0, 133.0, 133.4,
147.9, 149.5, 149.9, 159.2, 160.3; Anal. calcd. for C15H12N4O2
(280.29): C, 64.28; H, 4.32; N, 19.99. Found: C, 64.53; H, 4.31;
N, 20.17.
General procedure for the preparation of
4-chloroquinazolines 11a–e
2-Substituted-quinazolin-4(3H)-ones 10a–e (0.01 mmol) were
added portion-wise to a cooled stirred mixture of phosphorus
oxychloride (15 mL) and N,N-dimethylaniline (0.5 mL). The
reaction mixture was refluxed for 6 h. Excess phosphorus
oxychloride was removed under vacuum, and the residue was
cooled, triturated with ice/water and alkalinized with 2 N
NaOH. The aqueous solution was extracted three times with
DCM. The combined DCM extracts were dried over Na2SO4
and filtered, and the solvent was evaporated under reduced
pressure. The obtained solid was crystallized from ethanol to
give 11a–e [32–34].
4-Hydrazinyl-2-(3,4,5-trimethoxyphenyl)quinazoline (12e)
Pale yellow powder; yield (79%); m.p. 235ꢁ237°C; IR n 3273–
3307 (NH, NH2) cmꢁ1; 1H NMR (DMSO-d6) d ppm: 3.74 (s, 3H,
-OCH3), 3.91 (s, 6H, -OCH3), 4.91 (s, 2H, NH2, D2O exchange-
able), 7.43–7.54 (m, 1H, Ar-H), 7.57 (s, 1H, Ar-H), 7.73–7.86
(m, 2H, Ar-H), 7.91 (s, 1H, Ar-H), 8.14–8.19 (m, 1H, Ar-H), 9.62
(s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz)
General procedure for the preparation of 4-hydrazinyl-2-
substituted quinazolines 12a–e
A solution of 99% hydrazine hydrate (1 mL, 20 mmol) in
d
ppm: 56.4, 60.6, 105.7, 113.2, 121.29, 125.6, 127.9, 128.1,
ethanol (5 mL) was added to
a stirred solution of 4-
133.0, 140.0, 149.9, 152.2, 159.3, 160.3; Anal. calcd. for
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Archiv der Pharmazie
C17H18N4O3 (326.36): C, 62.57; H, 5.56; N, 17.17. Found C,
62.82; H, 5.63; N, 17.45.
131.5, 134.3, 134.8, 138.4, 138.9, 154.9, 156.3, 156.8, 158.3,
159.9, 163.2, 165.4; Anal. calcd. for C22H14FN5O (383.39): C,
68.92; H, 3.68; N, 18.27. Found: C, 69.04; H, 3.65; N, 18.48.
General procedure for the preparation of
3-hydrazonoindolin-2-ones 14a–c
5-Chloro-3-(2-(2-phenylquinazolin-4-yl)hydrazono)-
indolin-2-one (15c)
To a stirred solution of 13a–c (10 mmol) in methanol (20 mL),
99% hydrazine hydrate (2.5 mL, 50 mmol) was added. Stirring
was continued at the refluxing temperature for 0.5 h. The
crystals were filtered, washed with methanol, dried and
recrystallized from the glacial acetic acid to afford 14a–c [52].
Orange powder; yield (88%); mp >300°C; IR n 3433 (NH), 1710
(C O) cmꢁ1; 1H NMR (DMSO-d6) d ppm: 6.90–7.03 (m, 1H, Ar-
–
–
H), 7.41–7.78 (m, 6H, Ar-H), 7.99–8.10 (m, 2H, Ar-H), 8.40–8.62
(m, 3H, Ar-H), 10.67 (s, 1H, NH, D2O exchangeable, E form),
11.48 (s, 1H, NH of isatin, D2O exchangeable), 13.87 (s, 1H, NH,
D2O exchangeable, Z form); 13C NMR (DMSO-d6, 75 MHz) d
ppm: 111.5, 112.7, 119.5, 120.9, 121.7, 123.6, 126.7, 127.3,
128.8, 130.3, 130.6, 131.7, 134.0, 137.3, 140.4, 151.8, 156.0,
158.9, 163.0, 165.3; MS m/z (Rel. Int.) 401 [(Mþ2)þ, 4.5], 399
[Mþ, 9.78], 294 [100]; Anal. calcd. for C22H14ClN5O (399.84): C,
66.09; H, 3.53; N, 17.52. Found: C, 66.25; H, 3.50; N, 17.61.
General procedure for the preparation of target
compounds 15a–o
Route A: Indoline-2,3-dione derivative 13a–c (1 mmol) was
added to a suspension of the appropriate 4-hydrazinyl-2-
substituted-quinazoline 12a–e (1 mmol) in absolute ethanol
(10 mL), and glacial acetic acid (0.5 mL) was added to the
mixture. The reaction mixture was refluxed for 0.5 h. The
precipitate formed was collected by filtration while hot,
washed with hot ethanol, dried and crystallized from EtOH/
DMF to afford compounds 15a–o with 79–91% yield.
Route B: The appropriate 3-hydrazonoindolin-2-one 14a–c
(1 mmol) was added to a stirred solution of 4-chloroquinazo-
line 11a–e (1 mmol) in refluxing 2-propanol (20 mL). The
reaction mixture was refluxed for 5 h. The obtained product
was filtered, washed with hot ethanol, dried and crystallized
from EtOH/DMF to afford compounds 15a–o with 60–82%
yield. The yields from route A (79–91%) are considered for
15a–o.
3-(2-(2-(4-Methoxyphenyl)quinazolin-4-yl)hydrazono)-
indolin-2-one (15d)
Orange powder; yield (88%); mp 277ꢁ278°C; IR n 3410 (NH),
ꢁ1
–
1716 (C O) cm
;
1H NMR (DMSO-d6) d ppm: 3.85 (s, 3H,
–
-OCH3, E form), 3.87 (s, 3H, –OCH3, Z form), 6.88 (d, J ¼ 8.4 Hz,
1H, Ar-H), 6.98 (d, J ¼ 7.8 Hz, 1H, Ar-H), 7.10–7.42 (m, 3H, Ar-
H), 7.67–7.82 (m, 3H, Ar-H), 7.93–8.44 (m, 2H, Ar-H), 8.51
(d, J ¼ 8.7 Hz, 2H, Ar-H)), 10.69 (s, 1H, NH, D2O exchangeable, E
form), 11.38 (s, 1H, NH of isatin, D2O exchangeable), 13.85
(s, 1H, NH, D2O exchangeable, Z form); 13C NMR (DMSO-d6,
75 MHz) d ppm: 55.2, 110.1, 111.1, 112.4, 113.8, 114.3, 120.4,
122.5, 126.6, 128.3, 129.9, 130.9, 133.8, 141.7, 151.8, 156.0,
158.9, 161.4, 162.2, 163.3, 165.2; MS m/z (Rel. Int.) 396
[(Mþ1)þ, 2.86], 395 [Mþ, 51.4], 205 [100]. Anal. calcd. for
3-(2-(2-Phenylquinazolin-4-yl)hydrazono)indolin-2-one
(15a)
Orange powder; yield (81%); mp 263ꢁ265°C; IR n 3421 (NH),
C23H17N5O2 (395.42): C, 69.86; H, 4.33; N, 17.71. Found: C,
69.97; H, 4.35; N, 17.93.
ꢁ1
–
1707 (C O) cm
;
1H NMR (DMSO-d6) d ppm: 6.89 (d,
–
J ¼ 7.2 Hz, 1H, Ar-H), 7.02–7.12 (m, 1H, Ar-H), 7.32–7.40 (m, 1H,
Ar-H), 7.56–8.08 (m, 8H, Ar-H), 8.45–8.57 (m, 2H, Ar-H), 10.67
(s, 1H, NH, D2O exchangeable, E form), 11.37 (s, 1H, NH of
isatin, D2O exchangeable), 13.9 (s, 1H, NH, D2O exchangeable,
Z form); 13C NMR (DMSO-d6, 100 MHz) d ppm: 110.7, 118.1,
122.5, 125.4, 127.9, 128.5 (2C), 129.4, 130.6, 132.2, 133.2,
134.5, 135.0, 144.1, 146.2, 152.5, 154.0, 155.5, 156.0, 166.1;
Anal. calcd. for C22H15N5O (365.40): C, 72.32; H, 4.14; N, 19.17.
Found: C, 72.49; H, 4.18; N, 19.32.
5-Fluoro-3-(2-(2-(4-methoxyphenyl)quinazolin-4-yl)-
hydrazono)indolin-2-one (15e)
Orange powder; yield (83%); mp 293ꢁ295°C; IR n 3412 (NH),
ꢁ1
–
1716 (C O) cm
;
1H NMR (DMSO-d6) d ppm: 3.86 (s, 3H,
–
-OCH3, E form), 3.87 (s, 3H, -OCH3, Z form), 6.85–7.24 (m, 4H,
Ar-H), 7.55–8.05 (m, 5H, Ar-H), 8.36–8.51 (m, 2H, Ar-H), 10.65
(s, 1H, NH, D2O exchangeable, E form), 11.33 (s, 1H, NH of
isatin, D2O exchangeable), 13.79 (s, 1H, NH, D2O exchange-
able, Z form); 13C NMR (DMSO-d6, 100 MHz) d ppm: 55.7, 107.8
(2JF-C ¼ 25.0 Hz), 111.2, 112.5 (3JF-C ¼ 8.0 Hz), 112.9, 114.2,
114.7, 117.4 (2JF-C ¼ 24.0 Hz), 121.8 (3JF-C ¼ 9.0 Hz), 124.0,
124.9 (1JF-C ¼ 170.0 Hz), 127.2, 130.2, 130.3, 138.4, 134.3,
152.3, 156.2, 159.2, 161.9, 163.8; Anal. calcd. for C23H16FN5O2
(413.41): C, 66.82; H, 3.90; N, 16.94. Found: C, 67.03; H, 33.87;
N, 17.07.
5-Fluoro-3-(2-(2-phenylquinazolin-4-yl)hydrazono)-
indolin-2-one (15b)
Red fibers; yield (79%); mp 286ꢁ288°C; IR n 3417 (NH), 1695
(C O) cmꢁ1; 1H NMR (DMSO-d6) d ppm: 6.85–6.92 (m, 1H, Ar-
–
–
H), 7.11–7.20 (dd, J ¼ 9.3 and 9.3, 1H, Ar-H), 7.43–7.57 (m, 3H,
Ar-H), 7.60 (d, J ¼ 7.5 Hz, 1H, Ar-H), 7.69 (d, J ¼ 7.5 Hz, 1H, Ar-
H), 7.77–7.96 (m, 1H, Ar-H), 8.04 (d, J ¼ 8.4 Hz, 1H, Ar-H), 8.37
(m, 2H, Ar-H), 8.50 (s, 1H, Ar-H), 10.67 (s, 1H, NH, E form), 11.38
(s, 1H, NH of isatin), 13.85 (s, 1H, NH, D2O exchangeable,
5-Chloro-3-(2-(2-(4-methoxyphenyl)quinazolin-4-yl)-
hydrazono)indolin-2-one (15f)
Z form); 13C NMR (DMSO-d6, 75 MHz) d ppm: 107.6 (2JF-C
¼
Yellow powder; yield (91%); mp 290ꢁ292°C; IR n 3460 (NH),
25.3 Hz), 112.2 (3JF-C ¼ 6.6 Hz), 117.9, 120.9 (3JF-C ¼ 9.15 Hz),
123.9 (1JF-C ¼ 163.7 Hz), 127.5 (2JF-C ¼ 23.1 Hz), 128.5, 128.8,
1693 (C O) cm
;
1H NMR (DMSO-d6) d ppm: 3.84 (s, 3H,
ꢁ1
–
–
-OCH3, E form), 3.85 (s, 3H, -OCH3, Z form), 6.84 (d, J ¼ 8.1 Hz,
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Archiv der Pharmazie
1H, Ar-H), 6.90 (d, J ¼ 8.1 Hz, 1H, Ar-H), 7.04 (d, J ¼ 7.8 Hz, 2H,
Ar-H), 7.11 (d, J ¼ 8.7 Hz, 1H, Ar-H), 7.31–7.37 (m, 1H, Ar-H),
7.53–8.43 (m, 5H, Ar-H), 10.75 (s, 1H, NH, E form), 11.44 (s, 1H,
NH of isatin), 13.75 (s, 1H, NH, Z form); 13C NMR (DMSO-d6,
75 MHz) d ppm: 55.3, 111.5, 112.0, 112.6, 113.9, 114.2, 120.1,
121.4, 125.4, 126.8, 127.2, 129.1, 130.6, 134.5, 140.6, 142.1,
155.9, 158.2, 162.0, 162.2, 165.2. Anal. calcd. for C23H16ClN5O2
(429.86): C, 64.27; H, 3.75; N, 16.29. Found: C, 64.35; H, 3.71; N,
16.44.
1H, NH of isatin, D2O exchangeable), 13.79 (s, 1H, NH, D2O
exchangeable, Z form); Anal. calcd. for C24H18ClN5O3 (459.89):
C, 62.68; H, 3.95; N, 15.23. Found: C, 62.79; H, 3.91; N, 15.47.
3-(2-(2-(Benzo[d][1,3]dioxol-5-yl)quinazolin-4-yl)-
hydrazono)indolin-2-one (15j)
Orange powder; yield (80%); mp 269ꢁ271°C; IR n 3396 (NH),
1697 (C O) cmꢁ1; 1H NMR (DMSO-d6) d ppm: 6.13 (s, 2H, -CH2-,
–
–
E form), 6.17 (s, 2H, -CH2-, Z form), 6.88 (d, J ¼ 7.8 Hz, 1H, Ar-H),
6.97 (d, J ¼ 7.5 Hz, 1H, Ar-H), 7.07–7.13 (m, 1H, Ar-H), 7.15 (d,
J ¼ 7.8 Hz, 1H, Ar-H), 7.31–7.42 (m, 1H, Ar-H), 7.55–7.81 (m, 2H,
Ar-H), 7.92 (d, J ¼ 3.9 Hz, 1H, Ar-H), 7.99 (s, 1H, Ar-H), 8.15 (d,
J ¼ 9.9 Hz, 1H, Ar-H), 8.40 (d, J ¼ 7.8 Hz, 1H, Ar-H), 10.69 (s, 1H,
NH, D2O exchangeable, E form), 11.38 (s, 1H, NH of isatin, D2O
exchangeable), 13.85 (s, 1H, NH, D2O exchangeable, Z form);
13C NMR (DMSO-d6, 100 MHz) d ppm: 102.6, 108.0, 108.9,
109.0, 110.6, 118.1, 122.4, 125.0, 126.6, 127.0, 129.3, 130.4,
132.0, 134.5, 137.4, 138.6, 145.5, 146.1, 148.3, 150.9, 158.6,
163.0, 166.1; MS m/z (Rel. Int.) 410 [(Mþ1)þ, 6.12], 409 [Mþ,
12.33], 260 [100]; Anal. calcd. for C23H15N5O3 (409.41): C,
67.48; H, 3.69; N, 17.11. Found: C, 67.69; H, 3.72; N, 17.30.
3-(2-(2-(3,4-Dimethoxyphenyl)quinazolin-4-yl)hydrazono)-
indolin-2-one (15g)
Orange powder; yield (81%); mp 233ꢁ234°C; IR n 3327 (NH),
ꢁ1
–
1699 (C O) cm
;
1H NMR (DMSO-d6) d ppm: 3.86 (s, 3H,
–
-OCH3, E form), 3.88 (s, 3H, -OCH3, Z form), 3.90 (s, 3H, -OCH3,
E form), 3.92 (s, 3H, -OCH3, Z form), 6.89 (d, J ¼ 7.8 Hz, 1H, Ar-
H), 6.99 (d, J ¼ 7.8 Hz, 1H, Ar-H), 7.1–7.16 (m, 1H, Ar-H), 7.22 (d,
J ¼ 8.4 Hz, 2H, Ar-H), 7.35 (m, 1H, Ar-H), 7.73 (s, 1H, Ar-H),
7.84–7.97 (m, 2H, Ar-H), 8.14–8.47 (m, 2H, Ar-H), 10.69 (s, 1H,
NH, D2O exchangeable, E form), 11.39 (s, 1H, NH of isatin, D2O
exchangeable), 13.88 (s, 1H, NH, D2O exchangeable, Z form);
13C NMR (DMSO-d6, 75 MHz) d ppm: 55.4, 55.7, 110.1, 111.3,
117.6, 120.1, 121.6, 122.0, 122.5, 124.8, 126.7, 128.0, 128.3,
130.0, 131.0, 131.9, 133.9, 141.8, 145.5, 148.8, 152.0, 158.9,
162.7, 163.3; Anal. calcd. for C24H19N5O3 (425.45): C, 67.76; H,
4.50; N, 16.46. Found: C, 67.93; H, 4.58; N, 16.62.
3-(2-(2-(Benzo[d][1,3]dioxol-5-yl)quinazolin-4-yl)-
hydrazono)-5-fluoroindolin-2-one (15k)
Red powder; yield (86%); mp 272–274°C; IR n 3460 (NH), 1722
ꢁ1
–
(C O) cm
;
1H NMR (DMSO-d6) d ppm: 6.10 (s, 2H, -CH2-, E
–
form), 6.15 (s, 2H, -CH2-, Z form), 6.86–6.93 (m, 1H, Ar-H), 7.00
(d, J ¼ 8.4 Hz, 1H, Ar-H), 7.10–7.17 (m, 1H, Ar-H), 7.45 (d,
J ¼ 7.8 Hz, 1H, Ar-H), 7.53–7.78 (m, 2H, Ar-H), 7.86 (s, 1H, Ar-H),
7.92 (s, 1H, Ar-H), 8.10–8.45 (m, 2H, Ar-H), 10.60 (s, 1H, NH,
D2O exchangeable, E form), 11.25 (s, 1H, NH of isatin,
D2O exchangeable), 13.70 (s, 1H, NH, D2O exchangeable,
Z form); 13C NMR (DMSO-d6, 75 MHz) d ppm: 101.5, 107.2
(2JF-C ¼ 28.2 Hz), 108.0, 111.9 (3JF-C ¼ 7.7 Hz), 112.0, 112.4,
116.9 (2JF-C ¼ 24.2 Hz), 121.2 (2JF-C ¼ 9.1 Hz), 122.9, 126.7,
127.0, 128.1 (1JF-C ¼ 260 Hz), 131.6, 133.7, 137.8, 147.5,
151.7, 155.62, 156.8, 158.3, 163.3, 165.5, 170.9; Anal. calcd.
for C23H14FN5O3 (427.40): C, 64.64; H, 3.30; N, 16.39. Found: C,
64.78; H, 3.28; N, 16.52.
3-(2-(2-(3,4-Dimethoxyphenyl)quinazolin-4-yl)-
hydrazono)-5-fluoroindolin-2-one (15h)
Orange powder; yield (84%); mp 280ꢁ282°C; IR n 3412 (NH),
ꢁ1
–
1716 (C O) cm
;
1H NMR (DMSO-d6) d ppm: 3.87 (s, 3H,
–
-OCH3, E form), 3.88 (s, 3H, -OCH3, Z form), 3.90 (s, 3H, OCH3, E
form), 3.92 (s, 3H, -OCH3, Z form), 6.90 (m, 1H, Ar-H), 7.22 (d,
J ¼ 8.7 Hz, 1H, Ar-H), 7.64–7.86 (m, 4H, Ar-H), 7.98 (s, 1H, Ar-H),
8.13 (s, 1H, Ar-H), 8.21–8.45 (m, 2H, Ar-H), 10.70 (s, 1H, NH,
D2O exchangeable, E form), 11.43 (s, 1H, NH of isatin, D2O
exchangeable), 13.88 (s, 1H, NH, D2O exchangeable, Z form).
13C NMR (DMSO-d6, 75 MHz) d ppm: 55.4, 55.7, 107.2
(2JF-C ¼ 25.4 Hz), 110.7 (3JF-C ¼ 17.3 Hz), 111.4, 112.4, 116.9
(2JF-C ¼ 36.5 Hz), 121.0 (3JF-C ¼ 15.4 Hz), 125.8, 126.0, 126.8
(1JF-C ¼ 238 Hz), 127.3, 128.2, 129.9, 133.8, 134.5, 148.5, 151.2,
151.8, 158.8, 159.9, 162.3, 163.3, 165.3; MS m/z (Rel. Int.) 445
[(Mþ2)þ, 46.02%], 444 [(Mþ1)þ, 60.18%], 443 [Mþ, 83.19%],
315 [100%]; Anal. calcd. for C24H18FN5O3 (443.44): C, 65.01; H,
4.09; N, 15.79. Found: C, 65.17; H, 4.13; N, 15.88.
3-(2-(2-(Benzo[d][1,3]dioxol-5-yl)quinazolin-4-yl)-
hydrazono)-5-chloroindolin-2-one (15l)
Orange powder; yield (89%); mp 291ꢁ293°C; IR n 3423 (NH),
1716 (C O) cmꢁ1; 1H NMR (DMSO-d6) d ppm: 6.13 (s, 2H, -CH2-,
–
–
E form), 6.17 (s, 2H, -CH2-, Z form), 6.87 (d, J ¼ 8.1 Hz, 1H, Ar-H),
6.95 (d, J ¼ 8.4 Hz, 1H, Ar-H), 7.05 (d, J ¼ 7.8 Hz, 1H, Ar-H),
7.14 (d, J ¼ 8.1 Hz, 1H, Ar-H), 7.35–7.41 (m, 1H, Ar-H), 7.56–7.90
(m, 1H, Ar-H), 7.92 (s, 1H, Ar-H), 7.95 (s, 1H, Ar-H), 8.12 (d,
J ¼ 8.4 Hz, 1H, Ar-H), 8.46 (d, J ¼ 9.3 Hz, 1H, Ar-H), 10.79 (s, 1H,
NH, D2O exchangeable, E form), 11.46 (s, 1H, NH of isatin, D2O
exchangeable), 13.76 (s, 1H, NH, D2O exchangeable, Z form);
13C NMR (DMSO-d6, 100 MHz) d ppm: 102.0, 108.1, 108.6,
112.0, 112.9, 113.0, 120.4, 122.2, 123.5, 125.9, 127.2, 127.4,
128.7, 130.7, 132.1, 134.4, 140.8, 148.1, 150.0, 152.2, 156.2,
158.9, 163.5; Anal. calcd. for C23H14ClN5O3 (443.85): C, 62.24;
H, 3.18; N, 15.78. Found C, 62.37; H, 3.12; N, 15.94.
5-Chloro-3-(2-(2-(3,4-dimethoxyphenyl)quinazolin-4-yl)-
hydrazono)indolin-2-one (15i)
Orange powder; yield (90%); mp >300°C; IR n 3435 (NH), 1716
1
(C O) cmꢁ1; H NMR (DMSO-d6) d ppm: 3.86 (s, 3H, -OCH3, E
–
–
form), 3.88 (s, 3H, -OCH3, Z form), 3.90 (s, 3H, -OCH3, E form),
3.92 (s, 3H, -OCH3, Z form), 6.90 (d, J ¼ 8.4 Hz, 1H, Ar-H), 7.00
(d, J ¼ 8.7 Hz, 1H, Ar-H), 7.14 (d, J ¼ 8.7 Hz, 1H, Ar-H), 7.21 (d,
J ¼ 8.4 Hz, 1H, Ar-H), 7.41–7.45 (m, 1H, Ar-H), 7.72–7.85 (m, 1H,
Ar-H), 7.95 (s, 1H, Ar-H), 8.12 (s, 1H, Ar-H), 8.17–8.57 (m, 2H,
Ar-H), 10.76 (s, 1H, NH, D2O exchangeable, E form), 11.45 (s,
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Archiv der Pharmazie
3-(2-(2-(3,4,5-Trimethoxyphenyl)quinazolin-4-yl)-
hydrazono)indolin-2-one (15m)
5% CO2. The cells were maintained as a “monolayer culture”
by serial subculturing. Growth inhibition was determined
using the SRB method as previously described by Skehan
et al. [35]. Exponentially growing cells were collected using
0.25% trypsin-EDTA and seeded in 96-well plates at 1000–
2000 cells/well in supplemented DMEM medium. After 24 h,
cells were incubated for 72 h with various concentrations of
the tested compounds as well as doxorubicin as the reference
compound. Following 72 h of treatment, the cells were fixed
with 10% trichloroacetic acid for 1 h at 4°C. Wells were
stained for 10 min at room temperature with 0.4% SRB
dissolved in 1% acetic acid. The plates were air dried for 24 h,
and the dye was solubilized with Tris-HCl for 5 min on a shaker
at 1600 rpm. The optical density (OD) of each well was
measured spectrophotometrically at 564 nm with an ELISA
microplate reader (ChroMate-4300, FL, USA). The IC50 values
were calculated according to the equation for Boltzmann
sigmoidal concentration–response curve using the nonlinear
regression models (GraphPad Prism, version 5). The results
reported are means of at least three separate experiments.
Significant differences were analyzed by one-way ANOVA
wherein the differences were considered to be significant at
P < 0.05.
Yellow powder; yield (81%); mp 278ꢁ280°C; IR n 3340 (NH),
ꢁ1
–
1726 (C O) cm
;
1H NMR (DMSO-d6) d ppm: 3.75 (s, 3H,
–
-OCH3, E form), 3.78 (s, 3H, -OCH3, Z form), 3.90 (s, 3H, -OCH3, Z
form), 3.92 (s, 6H, -OCH3, E form), 3.95 (s, 3H, -OCH3, Z form),
6.89 (d, J ¼ 7.5 Hz, 1H, Ar-H), 6.99 (d, J ¼ 7.5 Hz, 1H, Ar-H),
6.07–7.17 (m, 1H, Ar-H), 7.38 (s, 1H, Ar-H), 6.38–7.86 (m, 3H,
Ar-H), 7.91 (s, 1H, Ar-H), 7.96–7.99 (m, 1H, Ar-H), 8.46 (d,
J ¼ 6.3 Hz, 1H, Ar-H), 10.64 (s, 1H, NH, D2O exchangeable, E
form), 11.33 (s, 1H, NH of isatin, D2O exchangeable), 13.88 (s,
1H, NH, D2O exchangeable, Z form); 13C NMR (DMSO-d6,
75 MHz) d ppm: 55.8, 60.0, 105.2, 111.0, 112.5, 120.1, 122.4,
123.0, 126.8, 128.3, 130.8, 132.8, 135.0, 141.7, 148.6, 151.5,
152.9, 155.7, 158.5, 162.2, 163.2, 171.9; Anal. calcd. for
C
25H21N5O4 (455.47): C, 65.93; H, 4.65; N, 15.38. Found C,
66.17; H, 4.72; N, 15.47.
5-Fluoro-3-(2-(2-(3,4,5-trimethoxyphenyl)quinazolin-4-yl)-
hydrazono)indolin-2-one (15n)
Orange powder; yield (88%); mp 287ꢁ288°C; IR n 3396 (NH),
ꢁ1
–
1726 (C O) cm
;
1H NMR (DMSO-d6) d ppm: 3.78 (s, 3H,
–
-OCH3), 3.91 (s, 6H, -OCH3, E form), 3.93 (s, 6H, -OCH3, Z form),
6.90–6.99 (m, 1H, Ar-H), 7.18 (dd, J ¼ 5.4, 2.4 Hz, 1H, Ar-H), 7.24
(d, J ¼ 2.4 Hz, 1H, Ar-H), 7.36 (s, 1H, Ar-H), 7.47 (dd, J ¼ 5.4,
2.4 Hz, 1H, Ar-H), 7.68–7.73 (m, 1H, Ar-H), 7.87 (s, 1H, Ar-H),
7.97 (s, 1H, Ar-H), 8.52 (m, 1H, Ar-H), 10.61 (s, 1H, NH, D2O
exchangeable, E form), 11.32 (s, 1H, NH of isatin, D2O
exchangeable), 13.81 (s, 1H, NH, D2O exchangeable, Z form);
Anal. calcd. for C25H20FN5O4 (473.46): C, 63.42; H, 4.26; N,
14.79. Found C, 63.64; H, 4.23; N, 15.03.
Cell culture and lysate collection
Hepatocellular carcinoma human HepG2 cells were cultured
in RPMI þ 10% FBS þ 5% penicillin/streptomycin and were
incubated at humidity 5% CO2 and maintained at 37°C.
HepG2 cells were treated with the IC50 concentration of 15b,
15g, and 15l (IC50 ¼ 1.0, 1.8, and 2.4 mM, respectively). HepG2
cells were cultured as a monolayer in T-25 flasks and were
seeded to attain 30% confluency prior to treatment. The main
100 mM stock dissolved in DMSO was diluted with cell culture
medium in order to reach the previously determined IC50
concentration of each of the three preparations. After 72 h of
treatment with 15b, 15g, and 15l, HepG2 cells were collected
via trypsinization and centrifuged at 10,000 rpm. The pellet
was then rinsed with PBS and lysed in RIPA lysis buffer at
4°C for 45 min, then centrifuged at 14,000 rpm for 20 min to
remove the cellular debris. Lysates were then collected
and stored at ꢁ80°C for later protein determination and
immunoassays.
5-Chloro-3-(2-(2-(3,4,5-trimethoxyphenyl)quinazolin-4-yl)-
hydrazono)indolin-2-one (15o)
Yellow powder; yield (90%); mp >300°C; IR n 3385 (NH), 1722
1
(C O) cmꢁ1; H NMR (DMSO-d6) d ppm: 3.78 (s, 3H, -OCH3),
–
–
3.92 (s, 6H, -OCH3, E form), 3.94 (s, 6H, -OCH3, Z form), 6.90–
7.02 (m, 1H, Ar-H), 7.40 (dd, J ¼ 6.3, 2.4 Hz, 1H, Ar-H), 7.69 (s,
1H, Ar-H), 7.69–7.88 (m, 2H, Ar-H), 7.88 (s, 1H, Ar-H), 7.99 (s,
1H, Ar-H), 8.38–8.60 (m, 2H, Ar-H), 10.64 (s, 1H, NH, D2O
exchangeable, E form), 11.33 (s, 1H, NH of isatin, D2O
exchangeable), 13.88 (s, 1H, NH, D2O exchangeable, Z form);
MS m/z (Rel. Int.) 492 [(Mþ2)þ, 21.05], 491 [(Mþ1)þ, 58.25],
490 [(M)þ, 46.08], 90 [100]. Anal. calcd. for C25H20ClN5O4
(489.92): C, 61.29; H, 4.11; N, 14.30. Found C, 61.47; H, 4.15; N,
14.45.
ELISA immunoassay
The levels of the anti-apoptotic marker Bcl-2 as well as the
apoptotic markers Bax and caspase-3 were assessed using
ELISA colorimetric kits per the manufacturer’s instructions.
The main principle of sandwich ELISA is the quantification
of a specific protein through its containment in a sandwich
of specific antibodies conjugated to the colorimetric TMB
substrate, whose intensity is proportional to the protein
quantity and is measured spectrophotometrically.
The cell lysate was diluted 10 times, and 100 mL (50 mg
protein) was added to the wells of three separate microtiter
plates for the three ELISA kits that were precoated with
primary antibodies specific to caspase-3, Bax and Bcl-2,
Biological evaluation
In vitro antiproliferative activity
HepG2 liver cancer, MCF-7 breast cancer and HT-29 colon
cancer cell lines were obtained from the National Cancer
Institute (Cairo, Egypt). HepG2 cells were grown in DMEM
while MCF-7 and HT-29 were grown in RPMI-1640.
Media were supplemented with 10% heat-inactivated FBS,
50 units/mL of penicillin and 50 g/mL of streptomycin and
maintained at 37°C in a humidified atmosphere containing
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Archiv der Pharmazie
respectively. A secondary biotin-linked antibody specific
to the protein captured by the primary antibody was
added to bind the captured protein, forming a “sandwich”
of specific antibodies around the desired protein in the cell
lysate. The streptavidin-HRP complex was then used to
bind the biotin-linked secondary antibody through its
streptavidin portion. The HRP domain reacted with the
added TMB substrate, forming a colored product that was
measured at 450 nm by a plate reader (ChroMate-4300, FL,
USA) after the reaction was terminated by the addition of
stop solution.
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Statistical analysis
The data are presented as the mean ꢀ S.D. Comparisons were
carried out using one-way analysis of variance followed by
Tukey–Kramer’s test for post hoc analysis. Statistical signifi-
cance was considered to be P < 0.05. All statistical analyses
were performed using GraphPad InStat software, version 3.05
(GraphPad Software, La Jolla, CA).
The authors would like to extend their sincere appreciation
to the Deanship of Scientific Research at King Saud University
for its funding of this research through the Research Group
Project no. RGP-VPP-321. Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Cairo, Egypt, is highly appreciated for supporting this
research.
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