Design and synthesis of anti-breast cancer agents from 4-piperazinylquinoline: A hybrid pharmacophore approach

Article abstract of DOI:10.1016/j.bmc.2010.01.001

A novel class of 4-piperazinylquinoline derivatives based on the isatin scaffold were designed by molecular hybridization approach and synthesized for biological evaluation. Subsequently, the compounds were examined for their cytotoxic effects on two human breast tumor cell lines, MDA-MB468 and MCF7, and two non-cancer breast epithelial cell lines, 184B5 and MCF10A. Although all compounds examined were quite effective on the breast cancer cell lines examined, the compound 4-bromo-1-[4-(7-chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-1H-indole-2,3-dione (5b) and N¹-[4-(7-trifluoromethyl-quinolin-4-yl)]-piperazin-1-ylmethyl-4-chloro-1H-indole-2,3-dione-3-thiosemicarbazone (8a) emerged as the most active among this series. It appeared that both 5b and 8a caused apoptosis to MCF7 cancer cells, but not MCF10A non-cancer cells. Thus, 4-piperazinylquinoline linked isatin analog can serve as the prototype molecule for further development of a new class of anti-breast cancer agents.

Full text of DOI:10.1016/j.bmc.2010.01.001

Bioorganic & Medicinal Chemistry 18 (2010) 1563–1572
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of anti-breast cancer agents from
4-piperazinylquinoline: A hybrid pharmacophore approach
a
a,b,c,
V. Raja Solomon a,b, , Changkun Hu , Hoyun Lee
*
*
a Tumour Biology Group, Northeastern Ontario Regional Cancer Program at the Sudbury Regional Hospital, 41 Ramsey Lake Road, Sudbury, Ontario, Canada P3E 5J1
b Department of Biology, Laurentian University, 935 Ramsey Lake Road, Sudbury, Ontario, Canada P3E 2C6
c Northern Ontario School of Medicine, Laurentian University Campus, 935 Ramsey Lake Road, Sudbury, Ontario, Canada P3E 2C6
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of 4-piperazinylquinoline derivatives based on the isatin scaffold were designed by molec-
ular hybridization approach and synthesized for biological evaluation. Subsequently, the compounds
were examined for their cytotoxic effects on two human breast tumor cell lines, MDA-MB468 and
MCF7, and two non-cancer breast epithelial cell lines, 184B5 and MCF10A. Although all compounds
examined were quite effective on the breast cancer cell lines examined, the compound 4-bromo-1-[4-
(7-chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-1H-indole-2,3-dione (5b) and N1-[4-(7-trifluoromethyl-
quinolin-4-yl)]-piperazin-1-ylmethyl-4-chloro-1H-indole-2,3-dione-3-thiosemicarbazone (8a) emerged
as the most active among this series. It appeared that both 5b and 8a caused apoptosis to MCF7 cancer
cells, but not MCF10A non-cancer cells. Thus, 4-piperazinylquinoline linked isatin analog can serve as the
prototype molecule for further development of a new class of anti-breast cancer agents.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 November 2009
Revised 28 December 2009
Accepted 3 January 2010
Available online 11 January 2010
Keywords:
4-Piperazinylquinoline
Isatin
Mannich base
Breast cancer cells
Anti-breast cancer activity
Hybrid pharmacophore approach
1. Introduction
the CQ-mediated enhancement of cell killing by Akt inhibitors is
cancer-specific.3,4 In continuation of our efforts to develop effective
The growing incidence of drug resistance to cancer chemother-
apeutic agent represents a serious medical problem.1 Therefore,
there is an urgent need to develop new classes of chemotherapeutic
agent to treat cancer.2 A combinational chemotherapeutic drug
with different mechanisms of action is one of the methods that
are being adopted to treat cancer.3–6 Besides the exploitation of
new targets, there is another approach combining two or more
pharmacophores into a single molecule. Therefore, a single mole-
cule containing more than one pharmacophore, each with different
mode of action, could be beneficial for the treatment of cancer.7–9
These ‘merged’ pharmacophore may be addressing the active site
of different targets and offer the possibility to overcome drug resis-
tance. In addition, this approach can also reduce unwanted side
effects.3,4,6,7 The success of this hybridization approach has already
been applied in developing novel antibacterial and antimalarial
agents to overcome drug resistance.9–13
anticancer drugs, we synthesized several CQ-analogs (Fig. 1, I–III),
and examined their cytotoxic effects on MDA-MB468 and MCF7
breast cancer cell lines. We found that some of these compounds
are very effective.15 The lysosomotropic CQ is accumulated in the
lysosomes, raises intra-lysosomal pH, and interferes with auto-
phagosome degradation in the lysosomes. This unique property
of CQ and its analogs may be important for the enhancement of cell
killing by cancer therapeutic agents in a variety of different
tumors.16 Consistent with data published in the literatures and
our results suggested that the 4-aminoquinoline ring system pos-
sesses potent anticancer activity.3,4,15,16
Published data showed that isatin (1H-Indole-2,3-dione) is one
of the most effective, newly emerging class of heterocyclic
molecules that possesses many interesting and beneficial proper-
ties. Importantly, isatin is well tolerated by humans.17 Several
laboratories reported that the isatin scaffold clearly shows antican-
cer activity against various human tumor cell lines (Fig. 2. I).8,17–19
We have also found that certain isatin-benzothiazole analogs
(Fig. 2. I, II) have anti-breast cancer activity.8
Recently, we have demonstrated that 10 lM chloroquine (Fig. 1,
CQ) significantly increases cancer cell killing effects when used in
combination with radiation or Akt inhibitors.3,4,14 Importantly,
A pharmacophore of thiosemicarbazones is known to have
anticancer and antiviral activity.20–22 For example, Methisazone
(N-methylisatin-b-thiosemicarbazone, Fig. 3) was effective as
prophylaxis against smallpox and vaccinia viruses, and 3-amino-
pyridine-2-carboxaldehyde thiosemicarbazone (Fig. 3, I) is
* Corresponding authors. Tel.: +1 705 522 6237x2703; fax: +1 705 523 7326
(V.R.S.).
E-mail addresses: rsolomon@hrsrh.on.ca (V.R. Solomon), hlee@hrsrh.on.ca (H.
Lee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.001

1564
V. R. Solomon et al. / Bioorg. Med. Chem. 18 (2010) 1563–1572
N
N
N
HN
HN
HN
N
HN
N
F
N
Cl
N
Cl
F
CQ
I
II
III
Figure 1. Chemical structures of 4-aminoquinolines reported for anticancer activity.
Schemes 1 and 2. The intermediate compounds 7-substituted-4-
piperazin-1-yl-quinoline (3–4) were prepared by aromatic nucleo-
philic substitution on 7-substituted-4-chloro-quinoline with
excess of piperazine and triethylamine in neat condition (without
solvent). Here, we performed aromatic nucleophilic substitution
reaction in excess of amine to avoid the use of phenol as a solvent,
which is toxic and prone to polymerization. Upon preparation of
the required precursor of secondary amine (3–4), we proceeded
with the Mannich reaction. The Mannich base analogs 5a–e,
6a–e, 7a–e and 8a–e were prepared by condensing the active
hydrogen atom of istain with formaldehyde and secondary amino
function of amino component. A Mannich reaction can be per-
formed via one pot multi-component protocol requiring isatin, an
aldehyde component and an amine, or via a preformed iminium
ion. Accordingly, molar equivalent amount of paraformaldehyde
and an amino component (compound 3 or 4) were dissolved in eth-
anol and iminium ion formed in situ was then reacted with isatin
and substituted isatin (Scheme 2, 9a–e) in ethanol reflux for 4 h
to furnish the desired N-Mannich isatin derivatives (Scheme 1,
5a–e, 6a–e; Scheme 2, 7a–e and 8a–e). In Scheme 2, the isatin-3-
thiosemicarbazones derivatives 9a–e were synthesized by conden-
sation of isatin with thiosemicarbazide in ethanol reflux for 2 h. In
Scheme 1, the final product 7a–e and 8a–e were obtained by con-
densation of N-Mannich isatin derivatives with thiosemicarbazide
in ethanol reflux for 3 h. The compounds reported in this manu-
script have been thoroughly characterized by elemental analysis
and spectral data.
N
R
O
R
S
CH3
N
O
N
N
O
R2
N
R1
R2
N
R1
I
II
Figure 2. Isatin analogs reported for anti-breast cancer activity from this
laboratory.
currently being evaluated by clinical trials against several
malignancies including leukemia.21,22 Hall et al.23 recently have
developed a set of isatin-b-thiosemicarbazones (Fig. 3, II) through
bioinformatics screening, and found selective activity on the mul-
tidrug resistant KB-3-1 and KB-VI cells.
The present work is an extension of our ongoing efforts towards
developing effective anticancer agents by a hybrid pharmacophore
approach using the 4-piperazinylquinoline scaffold. There are
accumulating lines of evidence that hybridization of two or more
different bioactive molecules with complementary pharmaco-
phoric functions or with different mechanisms of action often
renders synergistic effects.8–13 Encouraged by these previous
reports, we designed three different set of compounds (Fig. 4,
Schemes 1 and 2) by hybridizing two or more different pharmaco-
phores in the aim of prospecting their anti-breast cancer potential-
ity. We then synthesized hybrid compounds by linking the main
structural unit of the 4-piperazinylquinoline ring system with the
isatin ring system by a Mannich base reaction, and examined their
cytotoxic effects on two human breast tumor and two matching
non-cancer cell lines.
2.2. In vitro cytotoxicity
All the compounds synthesized were evaluated for their cyto-
toxicity on two breast cancer cell lines, MDA-MB468 (a PTEN
defective, presumably p53 positive, EGFR positive breast adenocar-
cinoma cell line) and MCF7 (p53+/-, invasive ductal breast carci-
noma) and two non-cancer breast epithelial cell lines, 184B5 and
MCF10A. Each compound stored at 20 mM was diluted from
100 lM to 0.0064 lM by fivefold serial dilutions. Cells were trea-
2. Results and discussion
2.1. Chemistry
ted with each compound for 48 h, followed by measuring cell
growth rates by SRB-based spectrophotometry as described previ-
ously.3,9,24,25 The reading of SRB staining is known to accurately re-
flect the levels of total cellular macromolecules/cell growth/
proliferation.24 The GI50 concentration for each compound was
The compounds 7a–e and 8a–8e described in this study were
synthesized by two different synthetic methods as outlined in
S
S
N
NH2
N
H
R1
NH2
N
N
N
H
N
H
S
R
N
CH3
O
N
N
H
NH2
O
N
H
Methisazone
I
II
Figure 3. Chemical structures of biologically active thiosemicarbazones.

V. R. Solomon et al. / Bioorg. Med. Chem. 18 (2010) 1563–1572
1565
H
N
NH2
N
O
S
R
N
R
O
N
n
N
N
H
N
H
N
HN
N
Molecular
R
N
R1
N
O
R2
Hybridization
S
N
R1
X
X
N
7a-e X = Cl
Molecular
8a-e X = CF3
Molecular
Hybridization
Hybridization
S
O
O
N NH
NH2
R1
N
N
O
H
N
N
R
9a-e
X
N
5a-e X = Cl
6a-e X = CF3
Figure 4. The design of hybrid compounds.
H
N
NH2
O
O
N
O
S
N
N
H
N
N
N
R
c
N
N
R
N
Cl
N
a
b
X
N
X
X
N
X
N
1
2
X = Cl
X = CF3
5a-e X = Cl
6a-e X = CF3
7a-e X = Cl
8a-e X = CF3
3
4
X = Cl
X = CF3
Scheme 1. Synthesis of 4-piperazinylquinoline analogs by Mannich base reaction. Reagents and conditions: (a) piperazine, 80 °C for 1 h and then 130 °C for 7 h; (b) 37%
formaldehyde solution, isatin and substituted isatin, ethanol reflux, 4 h; (c) thiosemicarbazide, ethanol reflux, 3 h.
calculated with reference to a control sample, which represents the
concentration that results in a 50% decrease in cell growth after
48 h incubation in the presence of the drug. For each compound,
21.15–40.36
57.6 M on MDA-MB468 cell line. In the MCF7 cells, the GI50 values
of nine compounds were in the range of 11.44–17.61 M, eleven
compounds 21.56–39.10 M, the remaining five compounds above
46.63, but not more than 66.78 M. The difference in the GI50 values
may be attributable to factors such as the nature of substitutions at
4-piperazinylquinoline ring system and halogen substitution on the
4th and 6th positions of isatin ring system, as well as the genetic and
biochemical background of the cell lines.
lM, and two compounds above 52.2, but not more than
l
l
50% growth inhibition (GI50
)
was calculated from Sigmoidal
l
dose–response curves that were generated with data obtained
from two independent experiments carried out each in triplicate
and presented in Table 1. The data for CQ and cisplatin were
included as references. The resultant data showed that all the
compounds had significant cytotoxic effects on the two breast can-
cer cell lines examined.
l
Compounds derived from 7-chloro substitution on 4-piperazi-
nylquinoline ring system hybridized with 4-bromo (5b) or 6-bro-
mo (5d) substituted isatin ring system showed an increased
Among the twenty-five compounds examined, eight compounds
showed GI50 ranged 10.34–20.60 lM, fifteen compounds at

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V. R. Solomon et al. / Bioorg. Med. Chem. 18 (2010) 1563–1572
H
N
NH2
N
O
S
R
N
N
N
S
O
N NH
O
NH2
a
b
R1
R1
X
N
N
H
O
N
H
7a-e X = Cl
8a-e X = CF3
9a-e
Scheme 2. Synthesis of 4-piperazinylquinoline analogs by Mannich base reaction. Reagents and conditions: (a) thiosemicarbazide, ethanol reflux, 2 h; (b) 37% formaldehyde
solution, compound 3 or 4, ethanol reflux, 4 h.
Table 1
Cytotoxicity of 4-piperazinylquinoline derivatives on human breast cancer cells
Compd No.a
X
R
GI50
(lM)
b,c
MDA-MB 468
MCF7
184B5
MCF10A
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
Cl
Cl
Cl
Cl
4-Cl
4-Br
6-Cl
6-Br
H
4-Cl
4-Br
6-Cl
6-Br
H
4-Cl
4-Br
6-Cl
6-Br
H
4-Cl
4-Br
6-Cl
6-Br
H
37.79 1.81
15.88 0.15
36.62 1.51
35.75 1.41
37.93 1.71
28.41 1.21
52.20 1.98
26.06 1.02
24.82 1.21
15.27 0.56
57.61 1.91
40.36 1.71
26.48 1.02
11.22 0.23
20.60 0.65
23.04 0.86
30.42 0.98
11.03 0.65
39.59 1.23
10.34 0.23
24.04 0.75
21.15 1.23
17.66 0.89
18.63 0.95
35.45 1.56
28.58 1.25
31.02 0.45
39.11 1.21
15.12 0.34
26.09 0.89
17.61 0.95
48.29 1.68
16.93 0.68
66.78 1.92
15.77 0.45
28.06 0.78
33.99 0.96
57.53 1.29
46.63 2.01
22.52 0.36
11.44 0.25
23.03 0.85
21.56 0.69
50.63 1.24
24.57 0.54
35.45 0.98
13.91 0.87
30.15 1.02
14.78 0.83
17.23 0.97
17.88 0.85
36.93 1.34
38.44 1.20
25.77 0.38
77.71 2.23
49.02 1.68
47.46 1.75
33.22 1.47
71.68 1.97
47.43 1.23
49.67 1.54
25.25 0.68
21.82 0.58
43.91 1.32
20.70 0.68
46.36 1.34
41.33 1.65
13.14 0.45
28.82 0.89
87.84 1.85
26.41 0.75
20.13 0.35
21.36 0.52
20.17 0.42
25.93 0.86
22.56 0.67
40.82 1.58
34.85 1.23
60.42 1.85
76.13 1.13
25.54 0.35
25.64 0.35
65.62 1.68
67.78 1.79
17.95 0.21
45.94 2.01
24.03 1.02
37.91 1.32
32.77 1.41
24.45 0.56
70.02 1.94
19.78 0.31
29.38 0.49
64.61 1.97
22.09 0.32
27.72 0.34
51.73 1.02
30.55 0.89
38.80 0.77
26.95 0.63
22.57 0.79
28.69 0.89
21.36 0.56
48.54 1.68
31.99 1.25
88.78 1.95
81.26 1.45
51.51 0.65
Cl
CF3
CF3
CF3
CF3
CF3
Cl
Cl
Cl
Cl
Cl
CF3
CF3
CF3
CF3
CF3
—
—
—
—
—
4-Cl
4-Br
6-Cl
6-Br
H
9e
Chloroquine
Cisplatin
a
b
c
For chemical structures of these compounds, see Figure 4 and Schemes 1 and 2.
GI50 values were calculated from Sigmoidal dose–response curves (variable slope), which were generated with GraphPad Prism V. 4.02 (GraphPad Software Inc.).
Values are the mean of triplicates of at least two independent experiments.
cytotoxic effects on MDA-MB468 and MCF7 cells in comparison to
isatin ring system having 4-chloro (5a) or 6-chloro (5c) substitu-
tion (Table 1). Furthermore, compounds derived from 7-chloro
substitution on 4-piperazinylquinoline ring system hybridized
with 4-chloro (5a), 4-bromo (5b), 6-chloro (5c) and 6-bromo (5d)
substituted isatin ring system showed an increased cytotoxic
activity on MDA-MB468 and MCF7 cells in comparison to unsubsti-
tuted isatin compound 5e. This result suggests that hydrophobic
substitution (chloro and bromo) on isatin ring system favorable
for cytotoxic activity. However, the compounds derived from
bioisoteric replacement of chloro group with 7-trifluoromethyl
substitution on 4-piperazinylquinoline ring system hybridized
with 4-bromo (6b) or 6-bromo (6d) substituted isatin ring system
showed less cytotoxicity on two breast cancer cell lines examined,
as compared to isatin ring system having 4-chloro (6a) or 6-chloro
(6c) substitution. This result clearly indicates that a large steric
bulk (CF3) substitution on the 7th position of 4-piperazinylquino-
line ring system is not favorable for the enhancement of cytotoxic-
ity on breast cancer cells.
Similarly, compounds derived from 7-chloro substitution on 4-
piperazinylquinoline ring system hybridized with 4-bromo (7b) or
6-bromo (7d) substituted isatin-3-thiosemicarbazone ring system
showed an increase in cytotoxic effects on MDA-MB468 and
MCF7 cells, compared to the isatin ring system having 4-chloro
(7a) or 6-chloro (7c) substitution. Moreover, the compounds
derived from 7-trifluoromethyl substitution on 4-piperazinylquin-
oline ring system hybridized with 4-bromo (8b) and 6-bromo (8d)
substituted isatin-3-thiosemicarbazone ring system showed lower
cytotoxic activity on MDA-MB468 and MCF7 cells than the isatin
ring system having 4-chloro (8a) or 6-chloro (8c) substitution.
Compounds derived from thiosemicarbazone pharmacophore
with the isatin ring system with chloro (9a, 9c) or bromo (9c, 9d)

V. R. Solomon et al. / Bioorg. Med. Chem. 18 (2010) 1563–1572
1567
substitution on the 4th or 6th position showed good cytotoxic
activity in comparison to unsubstituted compound (9e) on two
breast cancer cell lines examined. This result also suggests that
the essential role of hydrophobic substituent on the isatin ring
system for cytotoxicity.
advantageous over cisplatin, since 5b preferentially inhibited
cancer cell growth. For example, GI50 values for non-cancer cells
were 49.02 (185B5) and 65.62
inhibition value of 5b on cancer cells was 3.2-fold (i.e., 49.02
15.5 M) to 4.2-fold (i.e., 65.62 M/15.5 M) higher than non-can-
lM (MCF10A). Thus, the growth
lM/
l
l
l
In general, the pharmacophore molecules that were generated
by the hybridization of the 4-piperazinylquinoline ring system
with isatin-3-thiosemicarbazones were more active than those
molecule generated from combining the 4-piperazinylquinoline
ring system and an isatin compound. For example, compounds
7d, 7e, 8a, 8b and 8e were more active than compounds 5d, 5e,
6a, 6b and 6e on the breast cancer cells examined. The GI50 values
cer cells (Table 1). Similarly, compound 8a was emerged as the
most active analog from 4-piperazinylquinoline-isatin-thiosemi-
carbazone series. The compound 8a was the most effective as its
GI50 values were 23.04 and 21.56 lM on MDA-MB468, and MCF7
cells, respectively (Table 1). This data demonstrates that 8a is
slightly more potent than cisplatin. The growth inhibition by 8a
on non-cancer cells was also quite high (87.84 and 51.73 lM for
of compound 7c were 26.48 and 22.52
MCF7, respectively. These values were compared with those
obtained with 5c (i.e., a 4-piperazinylquinoline ring system hybrid-
ized with isatin compound), which were 36.62 and 26.09 lM on
MDA-MB468 and MCF7 cells, respectively. This data suggests that
hybridization of the 4-piperazinylquinoline system with isatin-3-
thiosemicarbazones leads to enhanced cytotoxicity on human
breast cancer cells, and further validates the concept that antican-
cer activity can be enhanced by hybridization of two or more of
different pharmacophore functional groups.
To gain further insights into the potential of this new series of
compounds as an anticancer therapeutic agent, we evaluated
4-piperazinylquinoline-isatin analogs (5a–e and 6a–e), 4-piperazi-
nylquinoline-isatin-thiosemicarbazone analogs (7a–e and 8a–e),
and isatin-thiosemicarbazone analogs (9a–e) for cell killing effects
on the non-cancer 184B5 and MCF10A breast epithelial cell lines
(Table 1). Most of these analogs killed cancer cells more effectively
than non-cancer cells. However some of these compound 6b–e, 7a,
7c, 7e, 8c–e, and 9a–b show undistinguished cytotoxicity on
immortalized non-cancer cells. The compound 5a–e, 6a, 6e, 7b,
7d, 8a, 8b, 9c and 9d showed lesser cytotoxicity on 184B5 and
MCF10A cells in comparison to cancer cells. Among these
compound 5b, 8a and 9c showed two to four fold lesser cytotoxic-
ity on immortalized non-cancer cells in comparison to breast
cancer cells.
l
M on MDA-MB468 and
184B5 and MCF10A cell lines, respectively). Nevertheless, the cyto-
toxic effect of 8b on cancer cells was approximately threefold
greater than non-cancer cell lines (Table 1). The cell killing effect
of these compounds (5b and 8a) on 184B5and MCF10A, an immor-
talized ‘normal’ breast cell line was particularly low. It clearly
suggests that these compounds have selective cytotoxicidal activ-
ity against human breast cancer cells examined.
To gain a better understanding about the mechanisms of action
of these compounds, the most cytotoxic compound 5b and 8a were
further assayed for their effects on cell cycle (by flow cytometry).
MCF7 and MCF10A cells were treated with compound 5b and 8a
at two different concentrations (20 and 40 lM) for 24 h. In MCF7
cells, DMSO treated control group 19% and 13% cells were observed
in S and G2/M phase, respectively (Fig. 5a). In MCF7 cells treated
with, 20 and 40 lM compound 5b, 63 and 64% cells were in G1/
G0, 18 and 14% cells were in S phase and 14 and 12% cells were
in G2/M phase, respectively (Fig. 5b and c). At these concentrations
(20 and 40 lM), compound 5b induced dose-dependent apoptotic
cell death in MCF7 cells, and the percentage of apoptotic cells were
6 and 10% by 24 h, respectively. In the compound 8a treatment
group, 64 and 65% cells were in G1/G0, 17 and 16% cells were in
S phase, 12 and 11% cells were in G2/M phase, respectively
(Fig. 5d and e). The compound 8a also induced apoptosis in
MCF7 cells at these concentrations. The substantial apoptotic cell
deaths were observed at higher concentration of these compounds
(data not shown). Furthermore, MCF10A cells treated with
compounds 5b or 8a exhibited a decrease in cells in S phase
(Fig. 5g–j) and no significant changes was observed, compared to
the control (Fig. 5f). Together compounds 5b and 8a showed
effective cytotoxicidal activity on MCF7 breast cancer cells.
The most active compound among the 4-piperazinylquinoline-
isatin series was 5b, which exhibited GI50 values of 15.88 and
15.12 lM on MDA-MB468, and MCF7 cells, respectively. This
compound showed in vitro cytotoxicity approximately two fold
greater than cisplatin. However, 5b can be significantly more
Figure 5. The cytometric profile of the compounds 5b and 8a. MCF7 (panels a–e) and MCF10A (panels f–j) cells (2.0 ꢀ 106) collected at 24 h post-compound treatment, fixed
in 70% ethanol, stained with 100 lg/mL propidium iodide, and cell cycle progression was measured by cytometry (Beckmann Coulter Cytomics FC500). Panels a and f, are
negative controls (DMSO treated); samples were treated with 5b: b (20
lM), c (40 lM), g (20 lM), and h (40 lM). The following samples were treated with 8a: d (20 lM), e
(40 M), i (20 M), and j (40 M).
l
l
l

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V. R. Solomon et al. / Bioorg. Med. Chem. 18 (2010) 1563–1572
4.2.2. 4-Piperazin-1-yl-7-trifluoromethyl-quinoline (4)
3. Conclusion
Pale yellow white solid; yield 68%; 1H NMR (500 MHz, CDCl3): d
1.78 (br s, 1H, NH), 3.18 (s, 4H, N(CH2CH2)2NAr), 3.24 (s, 4H,
N(CH2CH2)2NAr), 7.07–7.08 (d, J = 5.0 Hz, 1H, Ar-H), 7.63–7.65 (d,
J = 10.0 Hz, 1H, Ar-H), 8.13–8.14 (d, J = 5.0 Hz, 1H, Ar-H), 8.34 (s,
1H, Ar-H), 8.81–8.82 (d, J = 5.0 Hz, 1H, Ar-H); 13C NMR (500 MHz,
CDCl3): d 52.15 (2C), 53.48 (2C), 110.64, 120.79, 125.14, 125.22,
127.76, 130.70, 130.96, 148.73, 152.20, 157.19; ES-MS m/z 282
[M+H]+. Anal. Calcd for C14H14F3N3: C, 59.78; H, 5.02; N, 14.94.
Found: C, 59.80; H, 4.99; N, 14.92.
We here describe hybrid pharmacophore design and synthesis
of a new series of 4-piperazinylquinoline derivatives. All the com-
pounds derived from 4-piperazinylquinoline exhibited promising
anticancer activity against human breast cancer cells in vitro. In
particular, the compound 4-bromo-1-[4-(7-chloro-quinolin-4-yl)-
piperazin-1-ylmethyl]-1H-indole-2,3-dione (5b) and N1-[4-(7-tri-
fluoromethyl-quinolin-4-yl)]-piperazin-1-ylmethyl-4-chloro-1H-
indole-2,3-dione-3-thiosemicarbazone (8a) emerged as the most
active compounds of the series. Data from flow cytometry studies
suggests that this class of compounds induce the cell death by
apoptosis. Furthermore, our data suggest that generating hybrid
compounds containing pharmacophore containing 4-piperazinyl-
quinoline-isatin derivatives are a promising new approach of
developing an effective anticancer agent.
4.3. General synthetic procedure for compounds 5a–e and 6a–e
To a solution of isatin or substituted isatin (3.35 mmol) in 5 mL
of 99.9% ethanol was added to a mixture of compound (3 or 4)
(3.35 mmol) and aqueous formaldehyde 37% (3.95 mmol) also dis-
solved in 10 mL of 99.9% ethanol. The reaction mixture was stirred
for 4 h at room temperature, refrigerated for 48 h to form crystals.
The crystalline products were separated by filtration, washed and
vacuum dried. Recrystallization from ethanol rendered desired
products in pure form.
4. Materials and methods
4.1. Experimental
4.3.1. 4-Chloro-1-[4-(7-chloro-quinolin-4-yl)-piperazin-1-
ylmethyl]-1H-indole-2,3-dione (5a)
Melting points (mp) were taken in open capillaries on the
Complab melting point apparatus. Elemental analysis was per-
formed on a Perkin–Elmer 2400 C, H, N analyzer and values were
within the acceptable limits of the calculated values. The 1H spec-
tra were recorded on a DPX-500 MHz Bruker FT-NMR spectrome-
ter using CDCl3 and DMSO-d6 as solvent. The chemical shifts were
reported as parts per million (d ppm) tetramethylsilane (TMS) as
an internal standard. Mass spectra were obtained on a JEOL-SX-
102 instrument using fast atom bombardment (FAB positive).
The progress of the reaction was monitored on readymade sil-
ica-gel plates (Merck) using chloroform/methanol (9:1) as sol-
vent. Iodine was used as a developing agent or by spraying
with the Dragendorff’s reagent. Chromatographic purification
was performed over a silica-gel (100–200 mesh). All chemicals
and reagents obtained from Aldrich (USA) were used without fur-
ther purification.
Red crystals, yield 68%; mp 118–120 °C; IR (KBr, cmꢁ1):
1725, 1625; 1H NMR (500 MHz, CDCl3): d 2.96 (s, 4H, N(CH2
CH2)2NAr), 3.25 (s, 4H, N(CH2CH2)2NAr), 4.64 (s, 2H, NCH2N),
6.84–6.85 (d, J = 5.0 Hz, 1H, Ar-H), 7.04–7.06 (d, J = 10.0 Hz, 1H,
Ar-H), 7.14–7.15 (d, J = 5.0 Hz, 1H, Ar-H), 7.44–7.45 (d,
J = 5.0 Hz, 1H, Ar-H), 7.54–7.57 (m, 1H, Ar-H), 7.92–7.94 (d,
J = 10.0 Hz, 1H, Ar-H), 8.06 (s, 1H, Ar-H), 8.73–8.74 (d,
J = 5.0 Hz, 1H, Ar-H); 13C NMR (500 MHz, CDCl3): d 50.23 (2C),
52.11 (2C), 62.09, 110.01, 111.14, 115.21, 121.87, 124.59,
126.24, 126.56, 128.49, 131.28, 134.03, 138.90, 150.07, 152.65,
153.34, 156.73, 158.94, 180.49; ES-MS m/z 441 [M+H]+. Anal.
Calcd for C22H18Cl2N4O2: C, 59.88; H, 4.11; N, 12.70. Found: C,
59.85; H, 4.09; N, 12.68.
4.3.2. 4-Bromo-1-[4-(7-chloro-quinolin-4-yl)-piperazin-1-
ylmethyl]-1H-indole-2,3-dione (5b)
4.2. Synthesis of 7-substituted-4-piperazin-1-yl-quinoline
Reddish orange crystals, yield 64%; mp 133–134 °C; IR (KBr,
cmꢁ1): 1722, 1621; 1H NMR (500 MHz, CDCl3): d 2.96 (s, 4H,
N(CH2CH2)2NAr), 3.25 (s, 4H, N(CH2CH2)2NAr), 4.64 (s, 2H, NCH2N),
6.83–6.84 (d, J = 5.0 Hz, 1H, Ar-H), 7.10–7.11 (d, J = 5.0 Hz, 1H, Ar-
H), 7.32–7.34 (d, J = 10.0 Hz, 1H, Ar-H), 7.43–7.47 (m, 2H, Ar-H),
7.91–7.93 (d, J = 10.0 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 8.73–8.74
(d, J = 5.0 Hz, 1H, Ar-H); 13C NMR (500 MHz, CDCl3): d 50.23 (2C),
52.11 (2C), 62.00, 109.99, 11.56, 116.80, 117.15, 119.64, 121.86,
126.23, 127.72, 128.48, 134.03, 138.84, 150.06, 152.63, 153.67,
156.72, 158.89, 181.01; ES-MS m/z 487 [M+H]+. Anal. Calcd for
C22H18BrClN4O2: C, 54.40; H, 3.73; N, 11.53. Found: C, 54.39; H,
3.75; N, 11.50.
A mixture of 7-substituted-4-chloro-quinoline (10.10 mmol),
piperazine (2.61 g, 30.30 mmol) and triethylamine (1.4 mL,
10.10 mmol) were heated slowly to 80 °C over 1 h while stirring.
The temperature was then increased to 130 °C for 7 h where it
was kept for while stirring continuously. The reaction mixture
was cooled to room temperature and taken up in dichloromethane.
The organic layer was washed with 5% aq NaHCO3, followed by
washing with water and then with brine. The organic layer was
dried over anhydrous Na2SO4 and solvent was removed under re-
duced pressure, and the residue was then precipitated by addition
of mixture of solvent hexane/chloroform (8:2).
4.3.3. 6-Chloro-1-[4-(7-chloro-quinolin-4-yl)-piperazin-1-
ylmethyl]-1H-indole-2,3-dione (5c)
4.2.1. 7-Chloro-4-piperazin-1-yl-quinoline (3)
Red crystals, yield 60%; mp 122–123 °C; IR (KBr, cmꢁ1): 1727,
1628; 1H NMR (500 MHz, CDCl3): d 2.90 (s, 4H, N(CH2CH2)2NAr),
3.27 (s, 4H, N(CH2CH2)2NAr), 4.62 (s, 2H, NCH2N), 6.85–6.86 (d,
J = 5.0 Hz, 1H, Ar-H), 7.15 (s, 1H, Ar-H), 7.17–7.19 (d, J = 10.0 Hz,
1H, Ar-H), 7.44–7.46 (d, J = 10.0 Hz, 1H, Ar-H), 7.59–7.60 (d,
J = 5.0 Hz, 1H, Ar-H), 7.92–7.94 (d, J = 10.0 Hz, 1H, Ar-H), 8.05 (s,
1H, Ar-H), 8.73–8.74 (d, J = 5.0 Hz, 1H, Ar-H); ES-MS m/z 441
[M+H]+. Anal. Calcd for C22H18Cl2N4O2: C, 59.88; H, 4.11; N,
12.70. Found: C, 59.90; H, 4.13; N, 12.68.
White solid; yield 69%; 1H NMR (500 MHz, CDCl3): d 2.31 (br s,
1H, NH), 3.15 (s, 4H, N(CH2CH2)2NAr), 3.18 (s, 4H, N(CH2CH2)2NAr),
6.80–6.81 (d, J = 5.0 Hz, 1H, Ar-H), 7.46–7.47 (d, J = 5.0 Hz, 1H, Ar-
H), 7.92–7.94 (d, J = 10.0 Hz, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 8.68–
8.69 (d, J = 5.0 Hz, 1H, Ar-H); 13C NMR (500 MHz, CDCl3): d 46.10
(2C), 53.58 (2C), 108.97, 121.97, 125.24, 126.09, 128.91, 134.84,
150.22, 151.99, 157.38; ES-MS m/z 248 [M+H]+. Anal. Calcd for
C13H14ClN3: C, 63.03; H, 5.70; N, 16.96. Found: C, 62.99; H, 5.65;
N, 16.97.

V. R. Solomon et al. / Bioorg. Med. Chem. 18 (2010) 1563–1572
1569
4.3.4. 6-Bromo-1-[4-(7-chloro-quinolin-4-yl)-piperazin-1-
4.3.9. 6-Bromo-1-[4-(7-trifluoromethyl-quinolin-4-yl)-
ylmethyl]-1H-indole-2,3-dione (5d)
piperazin-1-ylmethyl]-1H-indole-2,3-dione (6d)
Orange crystals, yield 60%; mp 142–143 °C; IR (KBr, cmꢁ1):
Orange crystals, yield 72%; mp 168–170 °C; IR (KBr, cmꢁ1):
1732, 1632; 1H NMR (500 MHz, CDCl3):
d
2.96 (s, 4H,
1729, 1628; 1H NMR (500 MHz, CDCl3):
d 2.97 (s, 4H,
N(CH2CH2)2NAr), 3.27 (s, 4H, N(CH2CH2)2NAr), 4.60 (s, 2H, NCH2N),
6.85–6.86 (d, J = 5.0 Hz, 1H, Ar-H), 7.37 (s, 1H, Ar-H), 7.43–7.45 (d,
J = 10.0 Hz, 1H, Ar-H), 7.47–7.48 (d, J = 5.0 Hz, 1H, Ar-H), 7.53–7.54
(d, J = 5.0 Hz, 1H, Ar-H), 7.92–7.94 (d, J = 10.0 Hz, 1H, Ar-H), 8.10 (s,
1H, Ar-H), 8.67–8.68 (d, J = 5.0 Hz, 1H, Ar-H); ES-MS m/z 487
[M+H]+. Anal. Calcd for C22H18BrClN4O2: C, 54.40; H, 3.73; N,
11.53. Found: C, 54.38; H, 3.75; N, 11.55.
N(CH2CH2)2NAr), 3.32 (s, 4H, N(CH2CH2)2NAr), 4.62 (s, 2H, NCH2N),
6.95–6.96 (d, J = 5.0 Hz, 1H, Ar-H), 7.36 (s, 1H, Ar-H), 7.38–7.40 (d,
J = 10.0 Hz, 1H, Ar-H), 7.54–7.55 (d, J = 5.0 Hz, 1H, Ar-H), 7.67–7.68
(d, J = 5.0 Hz, 1H, Ar-H), 8.11–8.13 (d, J = 10.0 Hz, 1H, Ar-H), 8.37 (s,
1H, Ar-H), 8.83–8.84 (d, J = 5.0 Hz, 1H, Ar-H); ES-MS m/z 521
[M+H]+. Anal. Calcd for C23H18BrF3N4O2: C, 53.19; H, 3.49; N,
10.79. Found: C, 53.15; H, 3.45; N, 10.74.
4.3.5. 1-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-1H-
indole-2,3-dione (5e)
4.3.10. 1-[4-(7-Trifluoromethyl-quinolin-4-yl)-piperazin-1-
ylmethyl]-1H-indole-2,3-dione (6e)
Reddish orange crystals, yield 60%; mp 202–204 °C; IR (KBr,
cmꢁ1): 1738, 1619; 1H NMR (500 MHz, CDCl3): d 2.92 (s, 4H,
N(CH2CH2)2NAr), 3.21 (s, 4H, N(CH2CH2)2NAr), 4.60 (s, 2H, NCH2N),
6.82–6.83 (d, J = 5.0 Hz, 1H, Ar-H), 7.14–7.18 (m, 2H, Ar-H), 7.41–
7.43 (d, J = 10.0 Hz, 1H, Ar-H), 7.63–7.66 (m, 2H, Ar-H), 7.90–7.92
(d, J = 10.0 Hz, 1H, Ar-H), 8.03 (s, 1H, Ar-H), 8.70–8.71 (d,
J = 5.0 Hz, 1H, Ar-H); ES-MS m/z 407 [M+H]+. Anal. Calcd for
C22H19ClN4O2: C, 64.94; H, 4.71; N, 13.77. Found: C, 64.96; H,
4.70; N, 13.74.
Reddish orange crystals, yield 65%; mp 166–168 °C; IR (KBr,
cmꢁ1): 1727, 1630; 1H NMR (500 MHz, CDCl3): d 2.98 (s, 4H,
N(CH2CH2)2NAr), 3.28 (s, 4H, N(CH2CH2)2NAr), 4.65 (s, 2H, NCH2N),
6.93–6.94 (d, J = 5.0 Hz, 1H, Ar-H), 7.12–7.20 (m, 2H, Ar-H), 7.64–
7.67 (m, 3H, Ar-H), 8.10–812 (d, J = 10.0 Hz, 1H, Ar-H), 8.35 (s,
1H, Ar-H), 8.82–8.83 (d, J = 5.0 Hz, 1H, Ar-H); ES-MS m/z 441
[M+H]+. Anal. Calcd for C23H19F3N4O2: C, 62.72; H, 4.35; N, 12.72.
Found: C, 62.76; H, 4.34; N, 12.74.
4.4. General synthetic procedure for compounds 7a–e and 8a–e
(Scheme 1, Method A)
4.3.6. 4-Chloro-1-[4-(7-trifluoromethyl-quinolin-4-yl)-
piperazin-1-ylmethyl]-1H-indole-2,3-dione (6a)
Equimolar quantities of appropriate compound 5a–e and 6a–e
(0.95 mmol) and thiosemicarbazide (0.95 mmol) were dissolved
in warm 99.9% ethanol. The mixture was then continuously stirred
at 45 °C for 3 h. After standing for overnight at room temperature, a
crystalline product was formed. The product was then separated
by filtration, vacuum dried and recrystallized from mixture of sol-
vent hexane/dichloromethane (8:2).
Red crystals, yield 58%; mp 144–146 °C; IR (KBr, cmꢁ1): 1725,
1625; 1H NMR (500 MHz, CDCl3): d 2.97 (s, 4H, N(CH2CH2)2NAr),
3.28 (s, 4H, N(CH2CH2)2NAr), 4.66 (s, 2H, NCH2N), 6.87–6.88 (d,
J = 5.0 Hz, 1H, Ar-H), 7.05–7.07 (d, J = 10.0 Hz, 1H, Ar-H), 7.13–
7.15 (d, J = 10.0 Hz, 1H, Ar-H), 7.54–7.55 (d, J = 5.0 Hz, 1H, Ar-H),
7.65–7.67 (d, J = 10.0 Hz, 1H, Ar-H), 8.10–8.12 (d, J = 10.0 Hz, 1H,
Ar-H), 8.36 (s, 1H, Ar-H), 8.84–8.85 (d, J = 5.0 Hz, 1H, Ar-H); ES-
MS m/z 475 [M+H]+. Anal. Calcd for C23H18ClF3N4O2: C, 58.17;
H, 3.82; N, 11.80. Found: C, 58.14; H, 3.80; N, 11.76.
4.5. General synthetic procedure for compounds 7a–e and 8a–e
(Scheme 2, Method B)
4.3.7. 4-Bromo-1-[4-(7-trifluoromethyl-quinolin-4-yl)-
piperazin-1-ylmethyl]-1H-indole-2,3-dione (6b)
To a solution of substituted isatin-3-thiosemicarbazone deriva-
tive (2.36 mmol) in 5 mL of 99.9% ethanol was added a mixture of
compound 3 or 4 (2.36 mmol) and aqueous formaldehyde 37% W/V
(2.95 mmol) also dissolved in 10 mL ethanol (99.9%). The reaction
mixture was stirred at 45 °C for 4 h. The crystalline product was
separated by filtration, vacuum dried and recrystallized from mix-
ture of solvent hexane/chloroform (8:2).
Reddish orange crystals, yield 62%; mp 152–154 °C; IR (KBr,
cmꢁ1): 1737, 1615; 1H NMR (500 MHz, CDCl3): d 2.92 (s, 4H,
N(CH2CH2)2NAr), 3.28 (s, 4H, N(CH2CH2)2NAr), 4.64 (s, 2H, NCH2N),
6.94–6.95 (d, J = 5.0 Hz, 1H, Ar-H), 7.10–7.11 (d, J = 5.0 Hz, 1H, Ar-
H), 7.33–7.35 (d, J = 10.0 Hz, 1H, Ar-H), 7.45–7.49 (m, 1H, Ar-H),
7.66–7.67 (d, J = 5.0 Hz, 1H, Ar-H), 8.10–8.12 (d, J = 10.0 Hz, 1H,
Ar-H), 8.37 (s, 1H, Ar-H), 8.82–8.83 (d, J = 5.0 Hz, 1H, Ar-H); 13C
NMR (500 MHz, CDCl3): d 49.61 (2C), 51.41 (2C), 61.43, 110.37,
110.73, 116.02, 119.27, 120.08, 122.60, 125.49, 126.75, 127.19,
129.12, 129.38, 138.07, 147.90, 152.04, 152.95, 155.86, 158.02,
180.26; ES-MS m/z 519 [M+H]+. Anal. Calcd for C23H18BrF3N4O2:
C, 53.19; H, 3.49; N, 10.79. Found: C, 53.23; H, 3.52; N, 10.85.
4.5.1. N1-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-yl]methyl-4-
chloro-1H-indole-2,3-dione-3-thiosemicarbazone (7a)
Yellowish orange crystals, yield 72%; mp 210–212 °C; IR (KBr,
cmꢁ1): 3687, 1620; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.88
(s, 4H, N(CH2CH2)2NAr), 3.42 (s, 4H, N(CH2CH2)2NAr), 4.70 (s, 2H,
NCH2N), 6.86 (s, 1H, Ar-H), 7.05–7.07 (d, J = 10.0 Hz, 1H, Ar-H),
7.12–7.14 (d, J = 10.0 Hz, 1H, Ar-H), 7.19 (s, 1H, Ar-H), 7.37–7.38
(d, J = 5.0 Hz, 1H, Ar-H), 7.44 (s, 1H, Ar-H), 7.54 (s, 1H, Ar-H), 8.43
(s, 1H, Ar-H), 8.74 (br s, 2H, NH2), 12.84 (br s, 1H, NH); ES-MS m/
z 514 [M+H]+; C23H21Cl2N7OS: C, 53.70; H, 4.11; N, 19.06; found:
C, 53.72; H, 4.14; N, 19.09.
4.3.8. 6-Chloro-1-[4-(7-trifluoromethyl-quinolin-4-yl)-
piperazin-1-ylmethyl]-1H-indole-2,3-dione (6c)
Red crystals, yield 58%; mp 162–164 °C; IR (KBr, cmꢁ1): 1735,
1633; 1H NMR (500 MHz, CDCl3): d 2.97 (s, 4H, N(CH2CH2)2NAr),
3.31 (s, 4H, N(CH2CH2)2NAr), 4.64 (s, 2H, NCH2N), 6.95–6.96 (d,
J = 5.0 Hz, 1H, Ar-H), 7.15 (s, 1H, Ar-H), 7.18–7.19 (d, J = 5.0 Hz,
1H, Ar-H), 7.62–7.63 (d, J = 5.0 Hz, 1H, Ar-H), 7.66–7.68 (d,
J = 10.0 Hz, 1H, Ar-H), 8.11–8.13 (d, J = 10.0 Hz, 1H, Ar-H), 8.37 (s,
1H, Ar-H), 8.83–8.84 (d, J = 5.0 Hz, 1H, Ar-H); 13C NMR (500 MHz,
CDCl3): d 49.63 (2C), 51.55 (2C), 61.50, 110.83, 112.15, 116.56,
120.39, 122.88, 123.08, 124.71, 125.05, 126.01, 126.83, 128.98,
129.83, 142.13, 147.94, 152.49, 152.57, 156.03, 181.74; ES-MS m/
z 475 [M+H]+. Anal. Calcd for C23H18ClF3N4O2: C, 58.17; H, 3.82;
N, 11.80. Found: C, 58.21; H, 3.79; N, 11.86.
4.5.2. N1-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-yl]methyl-4-
bromo-1H-indole-2,3-dione-3-thiosemicarbazone (7b)
Orange crystals, yield 70%; mp 215–217 °C; IR (KBr, cmꢁ1):
13690, 1618; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.78 (s,
4H, N(CH2CH2)2NAr), 2.98 (s, 4H, N(CH2CH2)2NAr), 4.68 (s, 2H,
NCH2N), 6.87 (s, 1H, Ar-H), 7.11–7.13 (d, J = 10.0 Hz, 1H, Ar-H),
7.54–7.58 (m, 2H, Ar-H), 7.88–7.90 (d, J = 10.0 Hz, 1H, Ar-H),
7.91–7.93 (d, J = 10.0 Hz, 1H, Ar-H), 8.00–8.02 (d, J = 10.0 Hz, 1H,
Ar-H), 8.63 (s, 1H, Ar-H), 8.88 (br s, 2H, NH2), 12.75 (br s, 1H,

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V. R. Solomon et al. / Bioorg. Med. Chem. 18 (2010) 1563–1572
NH); ES-MS m/z 590 [M+H]+; C23H21BrClN7OS: C, 49.43; H, 3.79; N,
17.54; found: C, 49.46; H, 3.82; N, 17.56.
4.5.7. N1-[4-(7-Trifluoromethyl-quinolin-4-yl)]-piperazin-1-
ylmethyl-4-bromo-1H-indole-2,3-dione-3-thiosemicarbazone
(8b)
4.5.3. N1-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-yl]methyl-6-
chloro-1H-indole-2,3-dione-3-thiosemicarbazone (7c)
Red crystals, yield 68%; mp 230–232 °C; IR (KBr, cmꢁ1):
3688, 1618; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.95 (s,
4H, N(CH2CH2)2NAr), 3.30 (s, 4H, N(CH2CH2)2NAr), 4.65 (s, 2H,
NCH2N), 6.96–6.97 (d, J = 5.0 Hz, 1H, Ar-H), 7.10–7.12 (d,
J = 10.0 Hz, 1H, Ar-H), 7.20 (s, 1H, Ar-H), 7.52–7.54 (d,
J = 10.0 Hz, 1H, Ar-H), 7.60–7.62 (d, J = 10.0 Hz, 1H, Ar-H),
7.65–7.67 (d, J = 10.0 Hz, 1H, Ar-H), 8.11–8.12 (d, J = 5.0 Hz,
1H, Ar-H), 8.70–8.71 (d, J = 5.0 Hz, 1H, Ar-H), 9.20 (br s, 2H,
NH2), 12.30 (br s, 1H, NH); ES-MS m/z 515 [M+H]+;
C23H21Cl2N7OS: C, 53.70; H, 4.11; N, 19.06; found: C, 53.74;
H, 4.14; N, 19.09.
Orange crystals, yield 64%; mp 216–218 °C; IR (KBr, cmꢁ1):
3690, 1645; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.94 (s,
4H, N(CH2CH2)2NAr), 3.20 (s, 4H, N(CH2CH2)2NAr), 4.72 (s, 2H,
NCH2N), 6.95 (s, 1H, Ar-H), 7.12–7.14 (d, J = 10.0 Hz, 1H, Ar-H),
7.27 (s, 1H, Ar-H), 7.37–7.42 (m, 2H, Ar-H), 7.73–7.77 (m, 1H,
Ar-H), 8.20–8.21 (d, J = 5.0 Hz, 1H, Ar-H), 8.80 (s, 1H, Ar-H), 9.38
(br s, 2H, NH2), 12.78 (br s, 1H, NH); ES-MS m/z 594 [M+H]+;
C24H21BrF3N7OS: C, 48.66; H, 3.57; N, 16.55; found: C, 48.68; H,
3.60; N, 16.59.
4.5.8. N1-[4-(7-Trifluoromethyl-quinolin-4-yl)]-piperazin-1-
ylmethyl-6-chloro-1H-indole-2,3-dione-3-thiosemicarbazone
(8c)
Orange red crystals, yield 70%; mp 208–210 °C; IR (KBr, cmꢁ1):
3685, 1639; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.91 (s, 4H,
N(CH2CH2)2NAr), 3.19 (s, 4H, N(CH2CH2)2NAr), 4.60 (s, 2H, NCH2N),
6.96–6.97 (d, J = 5.0 Hz, 1H, Ar-H), 7.08–7.10 (d, J = 10.0 Hz, 1H, Ar-
H), 7.25 (s, 1H, Ar-H), 7.54–7.56 (d, J = 10.0 Hz, 1H, Ar-H), 7.60–7.62
(d, J = 10.0 Hz, 1H, Ar-H), 7.65–7.67 (d, J = 10.0 Hz, 1H, Ar-H), 8.11–
8.12 (d, J = 5.0 Hz, 1H, Ar-H), 8.72–8.73 (d, J = 5.0 Hz, 1H, Ar-H),
8.96 (br s, 2H, NH2), 12.39 (br s, 1H, NH); ES-MS m/z 548
[M+H]+; C24H21ClF3N7OS: C, 52.60; H, 3.86; N, 17.89; found: C,
52.55; H, 3.83; N, 17.84.
4.5.4. N1-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-yl]methyl-6-
bromo-1H-indole-2,3-dione-3-thiosemicarbazone (7d)
Red crystals, yield 72%; mp 215–217 °C; IR (KBr, cmꢁ1): 3688,
1620; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.90 (s, 4H,
N(CH2CH2)2NAr), 3.27 (s, 4H, N(CH2CH2)2NAr), 4.66 (s, 2H,
NCH2N), 6.91–6.92 (d, J = 5.0 Hz, 1H, Ar-H), 7.27–7.29 (d,
J = 10.0 Hz, 1H, Ar-H), 7.42–7.44 (d, J = 10.0 Hz, 1H, Ar-H), 7.47
(s, 1H, Ar-H), 7.63–7.65 (d, J = 10.0 Hz, 1H, Ar-H), 7.95–7.97 (d,
J = 10.0 Hz, 1H, Ar-H), 8.52–8.54 (d, J = 10.0 Hz, 1H, Ar-H), 8.64
(s, 1H, Ar-H), 9.04 (br s, 2H, NH2), 12.36 (br s, 1H, NH); 13C
NMR (500 MHz, CDCl3): d 50.33 (2C), 52.10 (2C), 61.55, 109.85,
114.53, 119.25, 120.95, 122.57, 124.39, 125.63, 126.29, 126.30,
126.79, 128.08, 130.64, 145.34, 148.46, 153.02, 156.90, 162.28,
179.17; ES-MS m/z 590 [M+H]+; C23H21BrClN7OS: C, 49.43; H,
3.79; N, 17.54; found: C, 49.40; H, 3.75; N, 17.50.
4.5.9. N1-[4-(7-Trifluoromethyl-quinolin-4-yl)]-piperazin-1-
ylmethyl-6-bromo-1H-indole-2,3-dione-3-thiosemicarbazone
(8d)
Red crystals, yield 61%; mp 218–220 °C; IR (KBr, cmꢁ1): 2689,
1635; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.80 (s, 4H,
N(CH2CH2)2NAr), 3.30 (s, 4H, N(CH2CH2)2NAr), 4.35 (s, 2H, NCH2N),
7.08 (s, 1H, Ar-H), 7.12–7.13 (d, J = 5.0 Hz, 1H, Ar-H), 7.30–7.31 (d,
J = 5.0 Hz, 1H, Ar-H), 7.39–7.41 (d, J = 10.0 Hz, 1H, Ar-H), 7.57–7.59
(d, J = 10.0 Hz, 1H, Ar-H), 7.75–7.77 (d, J = 10.0 Hz, 1H, Ar-H), 8.20–
8.21 (d, J = 5.0 Hz, 1H, Ar-H), 8.20–8.21 (d, J = 5.0 Hz, 1H, Ar-H),
9.15 (br s, 2H, NH2), 12.38 (br s, 1H, NH); 13C NMR (500 MHz,
CDCl3): d 50.31 (2C), 52.08 (2C), 61.51,111.35, 114.28, 114.55,
119.25, 120.95, 122.93, 124.39, 125.22, 125.63, 126.30, 126.60,
127.33, 130.63, 145.33, 148.46, 153.02, 156.55, 162.28, 179.18;
ES-MS m/z 593 [M+H]+; C24H21BrF3N7OS: C, 48.66; H, 3.57; N,
16.55; found: C, 48.68; H, 3.59; N, 16.58.
4.5.5. N1-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-yl]methyl-
1H-indole-2,3-dione-3-thiosemicarbazone (7e)
Orange crystals, yield 78%; mp 215–216 °C; IR (KBr, cmꢁ1):
3686, 1625; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.99 (s,
4H, N(CH2CH2)2NAr), 3.19 (s, 4H, N(CH2CH2)2NAr), 4.60 (s, 2H,
NCH2N), 6.98–6.99 (d, J = 5.0 Hz, 1H, Ar-H), 7.13–7.14 (d,
J = 5.0 Hz, 1H, Ar-H), 7.18–7.20 (d, J = 10.0 Hz, 1H, Ar-H),
7.35–7.36 (d, J = 5.0 Hz, 1H, Ar-H), 7.43–7.44 (d, J = 5.0 Hz, 1H,
Ar-H), 7.53–7.54 (d, J = 5.0 Hz, 1H, Ar-H), 7.98–8.00 (d,
J = 10.0 Hz, 1H, Ar-H), 8.68–8.69 (d, J = 5.0 Hz, 1H, Ar-H), 8.76
(s, 1H, Ar-H), 9.11 (br s, 2H, NH2), 12.48 (br s, 1H, NH); 13C
4.5.10. N1-[4-(7-Trifluoromethyl-quinolin-4-yl)]-piperazin-1-
ylmethyl-1H-indole-2,3-dione-3-thiosemicarbazone (8e)
Orange crystals, yield 63%; mp 211–212 °C; IR (KBr, cmꢁ1):
3688, 1642; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.90 (s, 4H,
N(CH2CH2)2NAr), 3.20 (s, 4H, N(CH2CH2)2NAr), 4.62 (s, 2H, NCH2N),
6.97–6.98 (d, J = 5.0 Hz, 1H, Ar-H), 7.11–7.12 (d, J = 5.0 Hz, 1H, Ar-
H), 7.20–7.22 (d, J = 10.0 Hz, 1H, Ar-H), 7.33–7.34 (d, J = 5.0 Hz, 1H,
Ar-H), 7.40–7.41 (d, J = 5.0 Hz, 1H, Ar-H), 7.97–7.99 (d, J = 10.0 Hz,
1H, Ar-H), 8.67–8.68 (d, J = 5.0 Hz, 1H, Ar-H), 8.79 (s, 1H, Ar-H),
9.01 (br s, 2H, NH2), 12.35 (br s, 1H, NH); 13C NMR (500 MHz,
CDCl3): d 50.44 (2C), 52.10 (2C), 61.40, 109.91, 111.59, 118.55,
119.75, 121.00, 121.62, 123.35, 126.46, 126.63, 128.38, 131.51,
131.576, 134.05, 144.29, 149.96, 152.55, 156.74, 162.37, 179.17;
ES-MS m/z 515 [M+H]+; C24H22F3N7OS: C, 56.13; H, 4.32; N,
19.09; found: C, 56.11; H, 4.28; N, 19.13.
NMR (500 MHz, CDCl3):
d 50.48 (2C), 52.12 (2C), 61.47,
109.94, 111.57, 119.85, 121.10, 121.79, 123.46, 126.26,
126.63, 128.38, 131.51, 131.576, 134.05, 144.29, 149.96,
152.55, 156.74, 162.37, 179.17; ES-MS m/z 481 [M+H]+;
C23H22ClN7OS: C, 57.55; H, 4.62; N, 20.43; found: C, 57.53;
H, 4.60; N, 20.39.
4.5.6. N1-[4-(7-Trifluoromethyl-quinolin-4-yl)]-piperazin-1-
ylmethyl-4-chloro-1H-indole-2,3-dione-3-thiosemicarbazone
(8a)
Red crystals, yield 62%; mp 228–230 °C; IR (KBr, cmꢁ1): 3689,
1655; 1H NMR (500 MHz, DMSO-d6 + CDCl3): d 2.94 (s, 4H,
N(CH2CH2)2NAr), 3.24 (s, 4H, N(CH2CH2)2NAr), 4.72 (s, 2H,
NCH2N), 7.13 (s, 1H, Ar-H), 7.20–7.21 (d, J = 5.0 Hz, 1H, Ar-H),
7.39 (s, 1H, Ar-H), 7.45–7.46 (d, J = 5.0 Hz, 1H, Ar-H), 7.76–7.78
(d, J = 10.0 Hz, 1H, Ar-H), 7.93 (s, 1H, Ar-H), 8.20–8.21 (d,
J = 5.0 Hz, 1H, Ar-H), 8.26 (s, 1H, Ar-H), 8.81 (br s, 2H, NH2),
12.78 (br s, 1H, NH); ES-MS m/z 548 [M+H]+; C24H21ClF3N7OS:
C, 52.60; H, 3.86; N, 17.89; found: C, 52.65; H, 3.82; N, 17.93.
4.6. General synthetic procedure for compounds (9a–e)
Equimolar quantities of isatin or substituted isatin (0.95 mmol)
and thiosemicarbazide (0.95 mmol) were dissolved in warm 99.9%
ethanol. The mixture was then continuously stirred at 45 °C for 2 h.
After standing for overnight at room temperature, a crystalline

V. R. Solomon et al. / Bioorg. Med. Chem. 18 (2010) 1563–1572
1571
product was formed. The product was then separated by filtration,
vacuum dried and recrystallized from mixture of solvent hexane/
dichlormethane (8:2).
The final concentration of DMSO in the SRB-based cytotoxicity
assays did not exceed 0.1%. To rule out that the DMSO concentra-
tion used may affect cell cytotoxicity, culture medium containing
equivalent concentration of DMSO was used as a negative control
in all experiments. In all studies, the concentration of DMSO used
did not notably show any cytotoxicity.
4.6.1. 4-Chloro-1H-indole-2,3-dione-3-thiosemicarbazone (9a)
Yellow crystals, yield 72%; mp 160–162 °C; IR (KBr, cmꢁ1):
3689, 1665; 1H NMR (500 MHz, DMSO-d6): d 6.79 (s, 1H, Ar-H),
7.01 (s, 1H, Ar-H), 7.21–7.23 (d, J = 10.0 Hz, 1H, Ar-H), 9.10 (br s,
2H, NH2), 11.28 (br s, 1H, NH), 12.76 (br s, 1H, NH); ES-MS m/z
255 [M+H]+; C9H7ClN4OS: C, 42.44; H, 2.77; N, 22.00; found: C,
42.42; H, 2.79; N, 22.07.
4.9. SRB assay
Cytotoxic effects were determined by a sulforhodamine B
(SRB)-based protocol.3,8,24,25 For a typical screening experiment,
5000–10,000 cells were inoculated into100 lL medium per well
4.6.2. 4-Bromo-1H-indole-2,3-dione-3-thiosemicarbazone (9b)
Yellow crystals, yield 74%; mp 170–172 °C; IR (KBr, cmꢁ1):
of a 96-well microtiter plate as described previously.24,25 Briefly,
after the inoculation, the microtiter plate was incubated at 37 °C,
5% CO2, 95% air and 100% relative humidity for 24 h, prior to addi-
tion of experimental drugs. Some of the sample wells were fixed
3680, 1641; 1H NMR (500 MHz, DMSO-d6):
d 6.86–6.88 (d,
J = 10.0 Hz, 1H, Ar-H), 7.13–7.15 (d, J = 10.0 Hz, 1H, Ar-H), 7.57 (s,
1H, Ar-H), 9.14 (br s, 2H, NH2), 11.26 (br s, 1H, NH), 12.75 (br s,
1H, NH); ES-MS m/z 300 [M+H]+; C9H7BrN4OS: C, 36.13; H, 2.36;
N, 18.73; found: C, 36.15; H, 2.32; N, 18.76.
with 25 lL of 50% trichloroacetic acid (TCA) as a control of the cell
population for each cell line at the time of drug addition (Tz). An
aliquot of the frozen stock was thawed and diluted to the desired
final maximum test-concentration with complete medium. Two
to ten fold serial dilutions were made to provide a total of seven
drug concentrations (and a control [C]). Following addition of
drugs, the culture plate was incubated for additional 48 h. Cells
4.6.3. 6-Chloro-1H-indole-2,3-dione-3-thiosemicarbazone (9c)
Yellow crystals, yield 75%; mp 156–158 °C; IR (KBr, cmꢁ1):
3689, 1638; 1H NMR (500 MHz, DMSO-d6):
d 6.96–6.98 (d,
J = 10.0 Hz, 1H, Ar-H), 7.56–7.58 (d, J = 10.0 Hz, 1H, Ar-H), 8.30 (s,
1H, Ar-H), 8.90 (br s, 2H, NH2), 11.13 (br s, 1H, NH), 12.45 (br s,
1H, NH); ES-MS m/z 255 [M+H]+; C9H7ClN4OS: C, 42.44; H, 2.77;
N, 22.00; found: C, 42.42; H, 2.73; N, 22.05.
were fixed in situ by slowly adding 25 lL of cold 50% (w/v) TCA
(final concentration, 10% TCA), and were then incubated for
60 min at 4 °C. The supernatant was discarded, and the plate was
washed five times with tap water, followed by air-dry. 50 lL of
SRB solution at 0.4% (w/v) in 1% acetic acid was added to each well,
and the plate was incubated for >30 min at room temperature.
Unbound SRB was removed by five washes with tap water,
followed by air-drying. The cells ‘stained’ with SRB were solubi-
lized with 10 mM trizma base, and the absorbance was read on
an automated plate reader at a wavelength of 515–564 nm. The
relative growth rate (%) was calculated for each of the compound
concentrations according to the following formula:
4.6.4. 6-Bromo-1H-indole-2,3-dione-3-thiosemicarbazone (9d)
Yellow crystals, yield 76%; mp 180–181 °C; IR (KBr, cmꢁ1):
3682, 1635; 1H NMR (500 MHz, DMSO-d6):
d 7.13–7.14 (d,
J = 5.0 Hz, 1H, Ar-H), 7.49–7.50 (d, J = 5.0 Hz, 1H, Ar-H), 8.26 (s,
1H, Ar-H), 8.88 (br s, 2H, NH2), 11.02 (br s, 1H, NH), 12.52 (br s,
1H, NH); 13C NMR (500 MHz, CDCl3): d 114.28, 119.85, 122.93,
124.21, 125.63, 131.51, 144.00, 162.94, 179.18; ES-MS m/z 301
[M+H]+; C9H7BrN4OS: C, 36.13; H, 2.36; N, 18.73; found: C, 36.10;
H, 2.32; N, 18.75.
ðTi ꢁ TzÞ=ðC ꢁ TzÞ ꢀ 100
In the formula, time zero (Tz), control growth (C), and OD for
different concentration of tested compounds (Ti). The GI50 for each
compound was obtained from a non-linear Sigmoidal dose–re-
sponse (variable slope) curve which is fitted by GraphPad Prism
v.4.03 software. Values were calculated for each of these
parameters if the level of activity was reached. However, if the
effect was not reached or was exceeded, the value for that param-
eter was expressed as greater or less than the maximum or
minimum concentration tested.24,25
4.6.5. 1H-Indole-2,3-dione-3-thiosemicarbazone (9e)
Red crystals, yield 78%; mp 151–153 °C; IR (KBr, cmꢁ1): 3679,
1655; 1H NMR (500 MHz, DMSO-d6): d 6.87 (s, 1H, Ar-H), 6.98–
7.00 (d, J = 10.0 Hz, 1H, Ar-H), 7.24–7.26 (d, J = 10.0 Hz, 1H, Ar-H),
7.67 (s, 1H, Ar-H), 8.87 (br s, 2H, NH2), 10.95 (br s, 1H, NH),
12.48 ((br s, 1H, NH); 13C NMR (500 MHz, CDCl3): d 108.81,
117.83, 118.77, 120.10, 128.86, 129.83, 140.23, 160.53, 176.85;
ES-MS m/z 221 [M+H]+; C9H8N4OS: C, 49.08; H, 3.66; N, 25.44;
found: C, 49.04; H, 3.64; N, 25.48.
4.10. Flow cytometry
4.7. Cell lines
Cells (2.0 ꢀ 106) were harvested by centrifugation at 1000 rpm
on a bench-top centrifuge for 5 min, followed by fixation with
ice-cold ethanol (70%) for 30 min to overnight at ꢁ20 °C.4,8 The
ethanol was then removed by centrifugation, and cells were resus-
pended in 1X PBS solution, followed by centrifuge. The cell pellet
The human MDA-MB468, MDA-MB231 and MCF-7 breast
cancer cell lines were maintained in RPMI 1640 medium supple-
mented with 10% fetal bovine serum (Hyclone, Logan UT) and
2 mM L-glutamine. 184B5 and MCF10A immortalized breast cells
were maintained in mammary epithelial basal medium
supplemented with an MEGM mammary epithelial singlequot kit
(Cambrex). Cells were grown at 37 °C with 5% CO2, 95% air under
the humidified conditions.
was than stained with PI master mix (100
lg/mL RNase A,
100 g/mL PI, 0.3% Nonidet P-40 and 0.1% sodium citrate in dis-
l
tilled water) for 30 min at 37 °C. DNA content was measured using
a Beckmann Coulter Cytomics FC500 (Beckman Coulter, Fullerton,
CA), and the proportion of cells in G0/G1, S, and G2/M phases of cell
cycle was calculated on the basis of DNA distribution histograms
using CXP software.
4.8. Reagents
Chloroquine diphosphate and cisplatin were purchased from
Sigma–Aldrich Canada Ltd (Oakaville, ON, Canada). All the
compounds were dissolved in 10–20 mM dimethyl sulfoxide
(DMSO) and stored at ꢁ20 °C until use. The stock solution was
Acknowledgments
The authors wish to thank G. Ulibarri (Laurentian University)
for letting us using his laboratory equipment in the process of
diluted in culture medium (0.1–100 lM) immediately before use.

1572
V. R. Solomon et al. / Bioorg. Med. Chem. 18 (2010) 1563–1572
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Research and Innovation.
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