Hybrid pharmacophore design and synthesis of isatin-benzothiazole analogs for their anti-breast cancer activity

Article abstract of DOI:10.1016/j.bmc.2009.08.068

A hybrid pharmacophore approach was used to design and synthesize isatin-benzothiazole analogs to examine their anti-breast cancer activity. The cytotoxicity of these compounds were determined using three different human breast tumor cell lines, MDA-MB231, MDA-MB468, MCF7, and two non-cancer breast epithelial cell lines, 184B5 and MCF10A. Although all compounds examined were quite effective on all the cancer cell lines examined, the compounds 4-bromo-1-diethylaminomethyl-1H-indole-2,3-dione (21) and 4-chloro-1- dimethylaminomethyl-3-(6-methyl-benzothiazol-2-ylimino)-1,3-dihydroindol-2-one (5e) emerged as the most active compounds of this series. Importantly, the cytotoxic effect of 21 was 10-15-fold higher on cancer than non-cancer cells, suggesting that this compound can be very effective for the control of breast cancer with low side effects. Since 21 showed effective cytotoxicity on MCF7 cells and arrested the cells at G2/M at a similar concentration, these two phenomena may be closely correlated. We conclude that the isatin-linked benzothiazole analog can serve as a prototype molecule for further development of a new class of anti-breast cancer agents.

Full text of DOI:10.1016/j.bmc.2009.08.068

Bioorganic & Medicinal Chemistry 17 (2009) 7585–7592
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Hybrid pharmacophore design and synthesis of isatin–benzothiazole analogs
for their anti-breast cancer activity
V. Raja Solomon a,b, Changkun Hu a, Hoyun Lee a,b,
*
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, 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:
Received 20 July 2009
Revised 27 August 2009
Accepted 30 August 2009
Available online 15 September 2009
A hybrid pharmacophore approach was used to design and synthesize isatin–benzothiazole analogs to
examine their anti-breast cancer activity. The cytotoxicity of these compounds were determined using
three different human breast tumor cell lines, MDA-MB231, MDA-MB468, MCF7, and two non-cancer
breast epithelial cell lines, 184B5 and MCF10A. Although all compounds examined were quite effective
on all the cancer cell lines examined, the compounds 4-bromo-1-diethylaminomethyl-1H-indole-2,3-
dione (2l) and 4-chloro-1-dimethylaminomethyl-3-(6-methyl-benzothiazol-2-ylimino)-1,3-dihydro-
indol-2-one (5e) emerged as the most active compounds of this series. Importantly, the cytotoxic effect
of 2l was 10–15-fold higher on cancer than non-cancer cells, suggesting that this compound can be very
effective for the control of breast cancer with low side effects. Since 2l showed effective cytotoxicity on
MCF7 cells and arrested the cells at G2/M at a similar concentration, these two phenomena may be clo-
sely correlated. We conclude that the isatin-linked benzothiazole analog can serve as a prototype mole-
cule for further development of a new class of anti-breast cancer agents.
Keywords:
Benzothiazole
Isatin
Schiff base
Breast cancer cells
Anticancer activity
Hybrid pharmacophore
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
derivatives of SU-5416 (semaxanib, Fig. 1) and SU-11248 (Suniti-
nib, Fig. 1) were reported having tyrosine kinase inhibitory and
The relative mortality rate caused by cancer is still very high in
the developed countries, as it accounts for more than 20% of all
deaths. Breast cancer is one of the most commonly diagnosed can-
cers and causes the second leading deaths in women.1 Although
chemotherapy is the mainstay of cancer therapy, the use of avail-
able chemotherapeutics is often limited mainly due to undesirable
side effects and a limited choice of available anticancer drugs. This
clearly underscores the need of developing novel chemotherapeu-
tic agents for more effective cancer treatments.2 Recently, we have
antiangiogenic properties.8 Besides, the structurally relevant
SU9516 (Fig. 1) was reported a potential inhibitor of cyclin-depen-
dent kinases (CDKs) that can induce apoptosis in colon carcinoma
cells.9 In addition, CDK inhibitory properties in isatin derived phen-
ylhydrazones have also been reported (Fig. 1, I).11 Based on this
information, Abadi et al. synthesized several 2-indolone imino
derivatives (Fig. 1, II) to examine their antitumor and antiangio-
genic properties.10 Vine et al. also synthesized several substituted
isatin analogs (Fig. 1, III), and screened against a panel of five hu-
man cancer cell lines.7 These authors concluded that substituted
isatins could be effective anticancer drugs.
Numerous papers have shown that the benzothiazole nucleus
possesses a potent anticancer activity against human cancer
(C.H., unpublished data).11–17 Importantly, we found that certain
compounds containing the benzothiazole nucleus killed cells in a
tumor-specific manner (C.H., unpublished data). Bradshaw et al.
also showed that the Phortress benzothiazole prodrug (Fig. 2)
had effective anticancer activity against xenografts in two different
rodent models.18
demonstrated that 10 lM chloroquine (CQ) significantly increases
cancer cell-killing effects when used in combination with radiation
or Akt inhibitors.3,4 Importantly, the CQ-mediated enhancement of
cell-killing by Akt inhibitors is cancer-specific.4 We also synthe-
sized several CQ-analogs and examined their cytotoxic effects on
MDA-MB 468 and MCF-7 breast cancer cell lines, and found that
some of these compounds are potentially more effective than
CQ.5 The present work is an extension of our ongoing efforts to-
ward developing new, effective anticancer agents by a hybrid phar-
macophore approach.
Isatin (1H-Indole-2,3-dione) is one of the most promising new
class of heterocyclic molecules having many interesting activity
profiles and well-tolerated in human subjects.6,7 The 2-oxoindoles
Based on these prior observations, we postulated that a com-
pound containing both isatin and benzothiazole pharmacophores
could be very effective for anticancer activity. The synthesis of isat-
in–benzothiazole conjugates could be possible by a pharmacophore
hybrid approach. Hybridization of two different bioactive molecules
with complementary pharmacophoric functions or with different
* Corresponding author. Tel.: +1 705 522 6237x2703; fax: +1 705 523 7326.
E-mail address: hlee@hrsrh.on.ca (H. Lee).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.068

7586
V. R. Solomon et al. / Bioorg. Med. Chem. 17 (2009) 7585–7592
O
H3C
O
H3C
N
H
N
N
CH3
H3CO
N
H
CH3
N
H
N
H
N
H
N
H
O
N
H
O
SU-9516
SU-5416, Semaxanib
SU-11248, Sunitinib
Br
COOH
R
N
O
O
X
NH
N
R
R'
N
H
N
H
N
H
O
N
H
O
X = N, CH
II
III
I
Figure 1. Isatin analogs with anticancer activity.
(such as dimethyl amine, diethyl amine, diphenyl amine, morpho-
line, and piperdine) were dissolved in ethanol and iminium ion
formed in situ was then reacted with isatin and substituted isatin
(Scheme 1) in ethanol reflux for 4 h to furnish the desired N-Man-
nich isatin derivatives (2a–o). The intermediate compound 6-
methyl-benzothiazol-2-ylamine was synthesized from 4-methy-
laniline by a reported procedure, in which 4-methylaniline was
treated with potassium thiocynate and bromine in glacial acetic
acid.24 The compounds 5a–o were synthesized by a Schiff base
reaction, in which an aromatic amine such as 6-methyl-ben-
zothiazol-2-ylamine and a carbonyl compound (isatin Mannich
base analog) undergoes nucleophilic addition, forming a hemiami-
nal. This reaction was followed by a dehydration to generate a sta-
ble imine. The compounds 5a–o were synthesized by condensation
of the Mannich base analogs 2a–o with 6-methyl-benzothiazol-2-
ylamine in ethanol reflux in the presence of glacial acetic acid. The
final products reported in this study were thoroughly character-
ized by elemental analysis and spectral data.
N
N
CH3
R2
O
S
R1
S
NH2
N
H
NH2
IV
Phortress
Figure 2. Benzothiazole analogs with anticancer activity.
mechanisms of action often showed synergistic effects.19–23 There-
fore, we synthesized hybrid compounds by linking the main struc-
tural unit of the isatin ring system with the benzothiazole ring
system by a Schiff base reaction (Fig. 3, 5a–o), and then examined
their cytotoxic effects on three human breast tumor and two match-
ing non-cancer cell lines.
2. Result and discussion
2.1. Chemistry
2.2. In vitro cytotoxicity
The compounds 2a–o and 5a–o described in this study were
prepared as outlined in Scheme 1. The isatin Mannich base analogs
2a–o were prepared by condensing the active hydrogen atom of is-
tain with formaldehyde and secondary amino function of amino
component. A Mannich reaction can be performed via one pot mul-
ti-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
Isatin Mannich bases were reported for a wide variety of biolog-
ical activities such as antibacterial, antifungal, and anti-HIV activ-
ity. So far, there is no evidence in literature if isatin Mannich
base analogs (2a–o) have anti-breast cancer activity. Therefore,
we synthesized 15 isatin Mannich base analogs (2a–o) and 15
Schiff base compounds (5a–o), and then examined their potential
as anticancer drugs.
All the compounds synthesized were evaluated for their cyto-
toxic effects on three breast cancer cell lines, MDA-MB468, MDA-
MB231 and MCF7, and two non-cancer breast epithelial cell lines,
184B5 and MCF10A. Each compound stored at 20 mM was diluted
O
N
R1
N
O
R
H2N
S
CH3
from 200 lM to 0.0128 lM by fivefold serial dilutions. Cells were
N
R1
treated with each compound for 48 h, followed by measuring cell
growth rates by SRB-based spectrophotometry as described previ-
ously.4,25 The reading of SRB staining is known to accurately re-
flect the levels of total cellular macromolecules/cell growth/
proliferation.25 The GI50 concentration for each compound was
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 com-
pound, 50% growth inhibition (GI50) was calculated from Sigmoi-
dal dose–response curves and presented in Table 1. The data for
CQ and cisplatin were included as references. The resultant data
showed that isatin Mannich base (2a–o) and Schiff base com-
pounds (5a–o) had significant cytotoxic effects on the three
breast cancer cell lines examined.
Molecular
Hybridization
N
N
R1
S
CH3
O
R
N
5a-5o
N
R1
Figure 3. A design for synthesis of isatin–benzothiazole analogs (5a–o).

V. R. Solomon et al. / Bioorg. Med. Chem. 17 (2009) 7585–7592
7587
NH2
H3C
3
b
N
N
R
R
R
O
O
O
N
CH3
H3C
S
NH2
S
4
a
N
H
N
O
R1
c
N
O
R1
N
N
R2
R2
R = H, Cl, Br
R = H, Cl, Br
5a-o
1
2a-o
Scheme 1. Synthesis of isatin Mannich base and Schiff Base analogs (2a–o and 5a–o). Reagents and conditions: (a) Mannich reaction (HCHO and secondary amine, ethanol
reflux, 4–6 h (b) KSCN, Br2/AcOH; (c) 1% acetic acid in ethanol reflux, 6–8 h.
Table 1
Cytotoxicity of novel Mannich isatin and Schiff base analogs (2a–o and 5a–o) on human breast cancer and non-cancer cellsa
Log Pc
a,b
Compound code
R
R1R2
GI50
(lM)
MDA-MB231
MDA-MB468
MCF7
184B5
MCF10A
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
H
H
H
H
(CH3)2
(CH2CH3)2
(C6H5)2
Piperidinyl
Morpholinyl
(CH3)2
(CH2CH3)2
(C6H5)2
Piperidinyl
Morpholinyl
(CH3)2
(CH2CH3)2
(C6H5)2
Piperidinyl
Morpholinyl
(CH3)2
(CH2CH3)2
(C6H5)2
Piperidinyl
Morpholinyl
(CH3)2
(CH2CH3)2
(C6H5)2
Piperidinyl
Morpholinyl
(CH3)2
(CH2CH3)2
(C6H5)2
65.63 1.81
60.61 1.53
24.82 0.75
38.03 0.84
57.64 1.20
138.46 2.02
44.48 1.01
31.42 0.92
26.48 0.65
61.50 1.76
93.72 1.59
20.22 0.52
51.12 1.24
30.15 0.68
29.72 0.72
19.15 0.42
32.68 0.93
34.09 0.88
34.49 0.97
19.76 0.23
10.92 0.11
20.43 0.35
53.96 1.05
55.80 1.23
37.15 0.69
49.02 1.22
28.65 0.82
75.13 1.89
38.25 0.98
94.64 1.85
22.52 1.44
23.65 0.23
50.15 1.75
35.92 1.34
21.46 0.44
24.05 0.53
28.23 0.38
107.12 1.78
28.32 0.32
14.35 0.27
82.84 1.68
61.50 1.52
49.02 1.42
11.68 0.12
63.25 1.42
35.29 0.84
54.03 1.39
35.45 0.75
63.53 1.04
17.66 0.21
65.61 1.55
17.61 0.19
28.09 0.26
27.33 0.34
85.04 1.54
19.78 0.21
24.03 0.31
27.35 0.41
43.06 0.45
82.33 1.87
24.03 0.24
24.82 0.35
28.58 1.25
31.02 0.45
40.48 0.98
44.05 1.12
23.73 0.31
41.25 1.31
52.30 1.56
87.84 1.89
34.05 0.76
29.67 0.52
51.24 1.44
29.19 0.16
57.64 0.98
20.20 0.15
110.20 2.04
31.45 0.55
65.62 0.89
29.18 0.23
74.77 0.85
39.15 0.45
37.83 0.32
14.56 0.21
20.67 0.34
19.17 0.22
24.32 0.39
30.94 0.40
21.46 0.15
17.95 0.29
30.15 0.56
30.15 0.59
39.07 .62
73.69 1.56
41.69 1.02
33.72 0.84
68.04 1.24
78.67 1.34
130.41 2.35
22.52 0.19
96.81 1.79
93.72 1.89
79.82 1.65
77.92 1.87
171.85 2.18
98.45 1.88
85.22 1.55
79.51 1.38
15.62 0.21
27.54 0.31
27.20 0.35
34.92 0.36
30.62 0.29
5.77 0.02
87.84 1.87
67.78 1.52
82.33 1.88
63.52 1.72
74.77 1.65
34.32 0.54
70.25 0.69
85.23 1.25
29.93 0.54
76.13 1.13
25.54 0.35
150.43 1.98
73.64 1.36
73.01 1.54
41.75 1.32
72.08 1.87
82.33 1.98
34.99 0.56
77.16 1.42
70.67 1.24
78.48 1.56
67.78 1.01
113.23 1.98
85.76 1.34
66.90 0.54
82.33 1.08
13.41 0.11
20.38 0.23
34.04 0.45
38.92 0.56
38.49 0.65
7.02 0.01
43.89 0.89
70.01 1.02
30.15 0.44
62.31 0.89
21.80 0.12
16.11 0.21
67.88 0.89
18.37 0.54
33.24 0.23
81.26 1.45
51.51 0.65
0.85
1.53
4.34
1.58
0.45
1.41
2.09
4.90
2.14
1.01
1.68
2.36
5.17
2.41
1.28
4.66
5.33
8.15
5.39
4.26
5.21
5.89
8.70
5.95
4.81
5.48
6.16
8.98
6.22
5.08
—
H
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Piperidinyl
Morpholinyl
12.01 0.11
38.44 1.20
25.77 0.38
Chloroquine
Cisplatin
—
GI50 was calculated from dose–response curves.
a
Sigmoidal dose–response curves (variable slope) were generated using GraphPad Prism V. 4.02 (GraphPad Software Inc.).
Values are the mean of triplicates of at least two independent experiments.
Log P were calculated using ChemDraw Ultra V.8.0 (CambridgeSoft Corporation).
b
c
Among the thirty isatin–benzothiazole compounds examined,
fourteen compounds showed GI50 in the range of 11.68–
28.32 M, nine compounds 35.29–61.50 M, and remaining seven
compounds showed above 63.25 M on the MDA-MB468 breast
cancer cells (Table 1). The GI50 of these 30 compounds on MDA-
MB231 cells was as follows: 10 compounds showed between
above 61.50
GI50 in the range of 12.01–30.94
57.64 M, the remaining three above 65.62 l
in the GI50 values may be attributable to such factors as the nature
of the N-substitution at the isatin ring system and the halogen sub-
stitution on the 4th position of isatin ring system, and the genetic
and biochemical background of the cell lines.
l
M. As for MCF-7 cells, fifteen compounds showed
M, 12 compounds at 31.45–
M. The differences
l
l
l
l
l
10.92 and 30.15 lM, 15 compounds at 31.42–60.61 lM, the rest

7588
V. R. Solomon et al. / Bioorg. Med. Chem. 17 (2009) 7585–7592
The structure–activity relationship studies appeared to suggest
We did not find any significant correlation between GI50 values
and log P values of the 30 compounds 2a–o and 5a–o (Table 1).
Therefore, the difference in liphophilicity may not be a significant
factor for the difference in cytotoxicity of the hybrid compounds
examined in our work.
We carried out cell cycle analysis by flow cytometry of the two
most promising compounds 2l and 5e to gain insight into their
mechanism of action. MCF7 and MCF10A cells were treated with
either the compound 2l or 5e at two different concentrations (20
that the introduction of a hydrophobic substituent of chloro (2g)
and bromo (2l) group in the 4th position of 1-diethylamino-
methyl-1H-indole-2,3-dione (2b) leads to an increase in cytotoxic
activity on MDA-MB231, MDA-MB468, and MCF7 cells, in compar-
ison to unsubstituted compound (2b). The most active compound
among the isatin Mannich base series was 2l, which exhibited
GI50 values of 20.22, 11.68 and 20.2 lM on MDA-MB231, MDA-
MB468, and MCF7 cells, respectively. This compound showed
in vitro cytotoxicity similar to that of cisplatin. However, 2l can
be significantly more advantageous over cisplatin, since 2l prefer-
entially inhibited cancer cell growth. For example, GI50 values of 2l
for cancer cells were 20.22 (MDA-MB231), 11.68 (MDA-MB468,
or 40 lM) for 24 h. MCF7 cells treated with 20 lM of compound
2l showed 38% and 8% in G2/M and S phase, respectively, while a
non-treat control showed 12% and 17% in G2/M and S phase,
respectively (Fig. 4, panels a and b). In contrast, the treatment of
and 20.20
were 171.85 (185B5) and 113.23
inhibition value of 2l on cancer cells was 5.6-fold (i.e.,
113.23 M/20.22 M) to 14.7-fold (i.e., 171.85 M/20.20 M)
higher than non-cancer cells (Table 1). In contrast, the cytotoxic ef-
fects of cisplatin on cancer cells and non-cancer cells were similar
(Table 1). This data certainly reveals the great potential of the com-
pound 2l as an effective anticancer agent, since it may have a sim-
ilar antitumor activity with cisplatin but with much lower side
effects. Considering the fact that undesirable side effect is often
the rate limiting factor for chemotherapy, the compound 2l shows
great promise.
l
M (MCF7), while its GI50 values for non-cancer cells
the non-cancer MCF10A with 20 lM of 2l did not alter cell cycle
l
M (MCF10A). Thus, the growth
distribution, compared to the non-treated control (f vs g in
Fig. 4). Since 2l could effectively arrest cell cycle and inhibit cancer
cell growth at a similar concentration, our data suggests that the
2l-mediated growth inhibition is directly relevant to its ability to
arrest cell cycle progression. This data is also consistent with pre-
vious reports that isatin and its analogs induced G2/M arrest.26–28
l
l
l
l
Unlike 2l, 5e did not induce cell cycle arrest at 20
MCF7 or MCF10A, although it did result in accumulation of small
number of MCF7 cells in G2/M at 40 M (Fig. 4, panel e). Since
GI50 values of the compound 5e for cancer cells were observed at
below 20 M (Table 1), the cytotoxic effects by 5e may not be
lM on either
l
l
Comparing with the Mannich base compounds 2a–o, the Schiff
base compounds 5a–o were generally more active. For example,
the GI50 values of 2a (Mannich base) and 5a (Schiff base) were
caused by cell cycle perturbation.
3. Conclusion
65.63 versus 19.15
lM (MDA-MB231), 50.15 versus 35.45
lM
(MDA-MB468), and 40.48 versus 29.18
l
M (MCF7), respectively
Here, we describe synthesis and examination of a new series of
isatin Mannich base and isatin-linked benzothiazole Schiff base ana-
logs. Some of these derivatives exhibited promising anti-breast
cancer activity. In particular, the compounds 2l (4-bromo-1-diethyl-
aminomethyl-1H-indole-2,3-dione) and 5e (4-Chloro-1-dimethyl-
aminomethyl-3-(6-methyl-benzothiazol-2-ylimino)-1,3-dihydro-
indol-2-one) emerged as promising compounds. The compound 2l is
particularly promising, since it could kill cancer cells 10–15 times
more effectively than non-cancer cells. This property of 2l may en-
able us to effectively control tumors with low side effects. The differ-
ential killing of cancer and non-cancer cells by 2l may be, at least in
part, due to its differential effects on cell cycle progression of cancer
and non-cancer cells. The compound 5e also showed a promise as it
requires lower concentration to achieve GI50 than 2l, although its
differential cytotoxic effects on cancer and non-cancer cells was
not as large as 2l. To further improve efficacy and specificity for can-
cer cell killing, additional structural modifications of isatin–benzo-
thiazole compounds are in progress.
(Table 1). Likewise, the Schiff base compounds 5a, 5e, 5f, 5g, 5j
and 5k were more active than isatin Mannich base compounds
2a, 2e, 2f, 2g, 2j and 2k on MDA-MB231, MDA-MB468, and MCF7
cells. This result suggests that hybridization of the isatin ring sys-
tem with the benzothiazole ring system can lead to enhanced cyto-
toxic effects on human breast cancer cells. Interestingly, Schiff base
compounds also showed stronger cytotoxic effects on non-cancer
cells than Mannich base compounds.
Among the Schiff base analogs, the compound 5e was the most
effective as its GI50 values were 19.76, 17.61 and 14.56 lM on
MDA-MB468, MDA-MB231 and MCF7 cells, respectively (Table 1).
This data demonstrates that 5e is more potent than cisplatin. Like
other isatin–benzothiazole hybrid compounds, the growth inhibi-
tion by 5e on non-cancer cells was also quite high (30.62 and
38.49 lM for 184B5 and MCF10A cell lines, respectively). Neverthe-
less, the cytotoxic effect of 5e on cancer cells was approximately
twofold greater than non-cancer cell lines (Table 1).
Figure 4. The compounds 2l and 5e arrest cell cycle progression at G2/M phase. Cells collected at 24 h post-compound treatment, fixed in 70% ethanol, stained with 100
mL propidium iodide, and cell cycle progression was measured by cytometry (Beckmann Coulter Cytomics FC500). Panels a–e and f–j show data generated from MCF7 and
MCF10A cells, respectively. Panels a and f are negative controls (DMSO treated). Samples were treated with 2l: b (20 M), c (40 M), g (20 M), and h (40 M). The following
samples were treated with 5e: d (20 M), 2 (40 M), i (20 M) and j (40 M).
lg/
l
l
l
l
l
l
l
l

V. R. Solomon et al. / Bioorg. Med. Chem. 17 (2009) 7585–7592
7589
4.1.5. 1-Morpholin-4-ylmethyl-1H-indole-2,3-dione (2e)
4. Experimental
This compound was obtained as a yellowish orange solid in 72%
yield. Mp 194–196 °C; IR (KBr, cmꢀ1): 1744, 1344, 1440, 1029, 997,
771, 731, 689; 1H NMR (200 MHz, CDCl3): d 2.55–2.62 (m, 4H, CH2
morpholinyl), 3.60–3.64 (m, 4H, CH2 morpholinyl), 4.43 (s, 2H,
CH2), 7.11–7.25 (m, 1H, Ar-H), 7.54–7.67 (m, 2H, Ar-H), 8.09 (s,
1H, Ar-H); 13C NMR (CDCl3): d 51.00 (2C), 62.58(2C), 66.65,
111.73, 117.57, 123.97, 125.30, 138.40, 151.43, 158.84, 183.08;
FAB-MS m/z 247 [M+H]+. Anal. Calcd for C13H14N2O3: C, 63.40; H,
5.73; N, 11.38. Found: C, 63.42; H, 5.75; N, 11.42.
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 limit of the calculated values. The 1H spectra
were recorded on a DPX-200 MHz Bruker FT-NMR spectrometer
using CDCl3 and DMSO-d6 as solvent. The chemical shifts were re-
ported 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 pro-
gress of the reaction was monitored on readymade silica-gel plates
(Merck) using chloroform/methanol (9:1) as solvent. 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 Al-
drich (USA) were used without further purification.
4.1.6. 4-Chloro-1-dimethylaminomethyl-1H-indole-2,3-dione
(2f)
This compound was obtained as gummy matter in 63% yield. 1H
NMR (200 MHz, CDCl3): d 2.36 (s, 6H, N(CH3)2), 4.40 (s, 2H, CH2),
7.02–7.22 (m, 2H, Ar-H), 7.54–7.58 (m, 1H, Ar-H); FAB-MS m/z
240 [M+H]+. Anal. Calcd for C11H11ClN2O2: C, 55.36; H, 4.65; N,
11.74. Found: C, 55.39; H, 4.68; N, 11.78.
4.1. General synthetic procedure for the preparation of
compounds 2a–o
4.1.7. 4-Chloro-1-diethylaminomethyl-1H-indole-2,3-dione
(2g)
This compound was obtained as a dark brownish red solid in
79% yield. Mp 98–100 °C; 1H NMR (200 MHz, CDCl3): d 1.04–1.11
(t, J = 7.0 Hz, 6H, N(CH2CH3)2), 2.61–2.72 (q, 4H, N(CH2CH3)2),
4.51 (s, 2H, CH2), 7.03–7.09 (m, 2H, Ar-H), 7.45–7.46 (m, 1H, Ar-
H); FAB-MS m/z 268 [M+H]+. Anal. Calcd for C13H15ClN2O2: C,
58.54; H, 5.67; N, 10.50. Found: C, 58.52; H, 5.65; N, 13.27.
To the solution of isatin or substituted isatin (0.9 g, 4.08 mmol)
in 5 mL of absolute ethanol was added to a mixture of secondary
amino compound (0.27 g, 1.35 mmol) and aqueous formaldehyde
37% (0.5 mL) also dissolved in 10 mL of absolute ethanol. The reac-
tion mixture was stirred for 3 h at room temperature, and then
refrigerated for 48 h to form crystals. The crystalline products were
separated by filtration, washed with hexane, and vacuum dried.
Recrystallization from ethanol rendered desired products in pure
form.
4.1.8. 4-Chloro-1-[(diphenylamino)-methyl]-1H-indole-2,3-
dione (2h)
This compound was obtained as a yellowish orange solid in 72%
yield. Mp 210–212 °C; 1H NMR (200 MHz, CDCl3): d 5.18 (s, 2H,
CH2), 6.83–6.87 (m, 2H, Ar-H), 6.97–7.01 (m, 2H, Ar-H), 7.08–7.12
(m, 2H, Ar-H), 7.17–7.22 (m, 2H, Ar-H), 7.44–7.48 (m, 2H, Ar-H),
7.52–7.63 (m, 2H, Ar-H), 7.90–7.92 (m, 1H, Ar-H); FAB-MS m/z
364 [M+H]+. Anal. Calcd for C21H15ClN2O2: C, 69.52; H, 4.17; N,
7.72. Found: C, 69.57; H, 4.15; N, 7.77.
4.1.1. 1-Dimethylaminomethyl-1H-indole-2,3-dione (2a)
This compound was obtained as gummy matter in 80% yield.
Mp 108–110 °C; 1H NMR (200 MHz, CDCl3):
d 2.31 (s, 6H,
N(CH3)2), 4.36 (s, 2H, CH2), 6.36–6.43 (m, 1H, Ar-H), 7.05–7.24
(m, 1H, Ar-H), 7.47–7.71 (m, 1H, Ar-H), 8.11 (s, 1H, Ar-H); FAB-
MS m/z 205 [M+H]+. Anal. Calcd for C11H12N2O2: C, 64.69; H,
5.92; N, 13.72. Found: C, 64.71; H, 5.95; N, 13.70.
4.1.9. 4-Chloro-1-piperidin-1-ylmethyl-1H-indole-2,3-dione
(2i)
4.1.2. 1-Diethylaminomethyl-1H-indole-2,3-dione (2b)
It was obtained as a reddish orange solid in 82% yield. Mp 176–
178 °C; 1H NMR (200 MHz, CDCl3): d 1.05–1.12 (t, J = 7.0 Hz, 6H,
N(CH2CH3)2), 2.62–2.73 (q, 4H, N(CH2CH3)2), 4.51 (s, 2H, CH2),
7.09–7.16 (m, 2H, Ar-H), 7.55–7.63 (m, 2H, Ar-H); FAB-MS m/z
233 [M+H]+. Anal. Calcd for C13H16N2O2: C, 67.22; H, 6.94; N,
12.06. Found: C, 67.22; H, 6.95; N, 12.07.
This compound was obtained as a dark brownish red solid in
75% yield. Mp 154–156 °C; 1H NMR (200 MHz, CDCl3): d 1.35–
1.39 (m, 2H, CH2 piperidinyl), 1.46–1.49 (m, 4H, CH2 piperidinyl),
2.43–2.54 (m, 4H, CH2 piperidinyl), 4.31 (s, 2H, CH2), 7.01–7.08
(m, 2H, Ar-H), 7.48–7.51 (m, 1H, Ar-H); FAB-MS m/z 280 [M+H]+.
Anal. Calcd for C14H15ClN2O2: C, 60.33; H, 5.42; N, 10.05. Found:
C, 60.29; H,12.70; N, 10.03.
4.1.3. 1-[(Diphenylamino)-methyl]-1H-indole-2,3-dione (2c)
It was was obtained as a pale orange solid in 72% yield. Mp 138–
140 °C; 1H NMR (200 MHz, CDCl3): d 5.16 (s, 2H, CH2), 6.39–6.93
(m, 2H, Ar-H), 7.05–7.24 (m, 5H, Ar-H), 7.48–7.69 (m, 6H, Ar-H),
8.05 (s, 1H, Ar-H); 13C NMR (CDCl3): d 63.40, 112.19, 112.67 (2C),
117.76 (2C), 118.27 (2C), 123.22 (2C), 123.99 (2C), 124.99 (2C),
125.15 (2C), 150.73, 151.18, 158.09, 183.99 (2C); FAB-MS m/z
329 [M+H]+. Anal. Calcd for C21H16N2O2: C, 76.81; H, 4.91; N,
8.53. Found: C, 76.80; H, 4.95; N, 8.55.
4.1.10. 4-Chloro-1-morpholin-4-ylmethyl-1H-indole-2,3-dione
(2j)
This compound was obtained as a yellowish orange solid in 69%
yield. Mp 184–186 °C; 1H NMR (200 MHz, CDCl3): d 2.51–2.55 (m,
4H, CH2 morpholinyl), 3.56–3.61 (m, 4H, CH2 morpholinyl), 4.34 (s,
2H, CH2), 6.99–7.12 (m, 2H, Ar-H), 7.48–7.51 (m, 1H, Ar-H); FAB-
MS m/z 282 [M+H]+. Anal. Calcd for C13H13ClN2O3: C, 55.62; H,
4.67; N, 9.98. Found: C, 55.65; H, 4.69; N, 10.00.
4.1.11. 4-Bromo-1-dimethylaminomethyl-1H-indole-2,3-dione
(2k)
4.1.4. 1-Piperidin-1-ylmethyl-1H-indole-2,3-dione (2d)
This compound was obtained as a orange red solid in 76% yield.
Mp 126–128 °C; IR (KBr, cmꢀ1): 1745, 1481, 1357, 970, 820, 767;
1H NMR (200 MHz, CDCl3): d 1.39–1.52 (m, 6H, CH2 piperidinyl),
2.52–2.57 (m, 4H, CH2 piperidinyl), 4.39 (s, 2H, CH2), 7.09–7.24
(m, 1H, Ar-H), 7.53–7.68 (m, 2H, Ar-H), 8.14 (s, 1H, Ar-H); FAB-
MS m/z 245 [M+H]+. Anal. Calcd for C14H16N2O2: C, 68.83; H,
6.60; N, 11.47. Found: C, 68.80; H, 6.58; N, 11.45.
This compound was obtained as a reddish orange solid in 76%
yield. Mp 152–154 °C; IR (KBr, cmꢀ1): 1735, 1445, 1331, 1015,
859, 782, 292; 1H NMR (200 MHz, CDCl3): d 2.35 (s, 6H, N(CH3)2),
5.19–5.23 (m, 2H, CH2), 7.21–7.30 (m, 2H, Ar-H), 7.31–7.49 (m,
1H, Ar-H); FAB-MS m/z 284 [M+H]+. Anal. Calcd for C11H11BrN2O2:
C, 46.66; H, 3.92; N, 9.89. Found: C, 46.68; H, 3.96; N, 9.91.

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V. R. Solomon et al. / Bioorg. Med. Chem. 17 (2009) 7585–7592
4.1.12. 4-Bromo-1-diethylaminomethyl-1H-indole-2,3-dione (2l)
This compound was obtained as a yellowish orange solid in 77%
yield. Mp 128–130 °C; IR (KBr, cmꢀ1): 1741, 1446, 1326, 1160,
1442, 906, 870, 770, 693; 1H NMR (200 MHz, CDCl3): d 1.04–1.11
(t, J = 7.0 Hz, 6H, N(CH2CH3)2), 2.61–2.71 (q, 4H, N(CH2CH3)2),
4.51 (s, 2H, CH2), 7.19–7.31 (m, 2H, Ar-H), 7.36–7.40 (m, 1H, Ar-
H); FAB-MS m/z 312 [M+H]+. Anal. Calcd for C13H15BrN2O2: C,
50.18; H, 4.86; N, 9.00. Found: C, 50.16; H, 4.88; N, 8.98.
4.2.2. 1-Diethylaminomethyl-3-(6-methyl-benzothiazol-2-
ylimino)-1,3-dihydro-indol-2-one (5b)
This compound was obtained as a dark yellowish orange solid
in 60% yield. Mp 202–204 °C; 1H NMR (200 MHz, CDCl3): d 1.19–
1.25 (t, J = 7.0 Hz, 6H, N(CH2CH3)2), 2.40 (s, 3H, CH3), 2.49–2.53
(q, 4H, N(CH2CH3)2), 5.30–5.32 (m, 2H, CH2), 6.91–6.94 (m, 2H,
Ar-H), 6.96–7.40 (m, 2H, Ar-H), 7.51–7.62 (m, 2H, Ar-H), 8.65
(s, 1H, Ar-H); FAB-MS m/z 379 [M+H]+. Anal. Calcd for
C21H22N4OS: C, 66.64; H, 5.86; N, 14.80. Found: C, 66.62; H,
5.88; N, 14.81.
4.1.13. 4-Bromo-1-[(diphenylamino)-methyl]-1H-indole-2,3-
dione (2m)
This compound was obtained as a yellowish orange solid in 71%
yield. Mp 230–232 °C; IR (KBr, cmꢀ1): 1721, 1439, 1388, 970, 793,
676; 1H NMR (200 MHz, CDCl3): d 5.20–5.24 (m, 2H, CH2), 6.89–
6.91 (m, 2H, Ar-H), 7.20–7.31 (m, 2H, Ar-H), 7.33–7.37 (m, 2H,
Ar-H), 7.41–7.48 (m, 2H, Ar-H), 7.44–7.48 (m, 2H, Ar-H), 7.52–
7.63 (m, 2H, Ar-H), 7.64–7.66 (m, 1H, Ar-H); FAB-MS m/z 408
[M+H]+. Anal. Calcd for C21H15BrN2O2: C, 61.93; H, 3.71; N, 6.88.
Found: C, 61.91; H, 3.69; N, 6.86.
4.2.3. 1-[(Diphenylamino)-methyl]-3-(6-methyl-benzothiazol-
2-ylimino)-1,3-dihydro-indol-2-one (5c)
This compound was obtained as a reddish orange solid in 58%
yield. Mp 158–160 °C; IR (KBr, cmꢀ1): 1731, 1573, 1460, 1344,
920,910, 790, 760; 1H NMR (200 MHz, CDCl3): d 2.36 (s, 3H,
CH3), 5.39–5.43 (s, 2H, CH2), 6.88–6.93 (m, 2H, Ar-H), 7.04–7.15
(m, 6H, Ar-H), 7.37–7.63 (m, 6H, Ar-H), 7.93–7.97 (m, 2H, Ar-H),
8.65 (s, 1H, Ar-H); FAB-MS m/z 475 [M+H]+. Anal. Calcd for
C29H22N4OS: C, 73.39; H, 4.67; N, 11.81. Found: C, 73.41; H,
4.71; N, 11.80.
4.1.14. 4-Bromo-1-piperidin-1-ylmethyl-1H-indole-2,3-dione
(2n)
This compound was obtained as a reddish orange solid in 68%
yield. Mp 136–138 °C; 1H NMR (200 MHz, CDCl3): d 1.42–1.45
(m, 2H, CH2 piperidinyl), 1.49–1.57 (m, 4H, CH2 piperidinyl),
2.43–2.54 (m, 4H, CH2 piperidinyl), 5.19–5.22 (s, 2H, CH2), 7.13–
7.31 (m, 2H, Ar-H), 7.44–7.51 (m, 1H, Ar-H); FAB-MS m/z 324
[M+H]+. Anal. Calcd for C14H15BrN2O2: C, 52.03; H, 4.68;; N, 8.67.
Found: C, 52.05; H,4.64; N, 8.70.
4.2.4. 3-(6-Methyl-benzothiazol-2-ylimino)-1-piperidin-1-
ylmethyl-1,3-dihydro-indol-2-one (5d)
This compound was obtained as viscous gummy matter in 59%
yield; 1H NMR (200 MHz, CDCl3): d 1.52–1.71 (m, 6H, CH2 piperid-
inyl), 2.40 (s, 3H, CH3), 2.52–2.57 (m, 4H, CH2 piperidinyl), 5.53–
5.55 (m, 2H, CH2), 6.61–6.95 (m, 2H, Ar-H), 7.10–7.17 (m, 2H, Ar-
H), 7.30–7.76 (m, 2H, Ar-H), 8.29 (s, 1H, Ar-H); FAB-MS m/z 391
[M+H]+. Anal. Calcd for C22H22N4OS: C, 67.67; H, 5.68; N, 14.35.
Found: C, 67.65; H, 5.65; N, 14.34.
4.1.15. 4-Bromo-1-morpholin-4-ylmethyl-1H-indole-2,3-dione
(2o)
This compound was obtained as a orange red solid in 65% yield.
Mp 184–186 °C; 1H NMR (200 MHz, CDCl3): d 2.59–2.64 (m, 4H,
CH2 morpholinyl), 3.62–3.67 (m, 4H, CH2 morpholinyl), 4.35 (s,
2H, CH2), 7.15–7.31 (m, 2H, Ar-H), 7.45–7.53 (m, 1H, Ar-H); FAB-
MS m/z 326 [M+H]+. Anal. Calcd for C13H13BrN2O3: C, 48.02; H,
4.03; N, 8.62. Found: C, 48.07; H, 4.01; N, 8.59.
4.2.5. 3-(6-Methyl-benzothiazol-2-ylimino)-1-morpholin-4-
ylmethyl-1,3-dihydro-indol-2-one (5e)
This compound was obtained as gummy matter in 45% yield.
Mp 124–126 °C; 1H NMR (200 MHz, DMSOd6 + CDCl3): d 2.36 (s,
3H, CH3), 2.51–2.67 (m, 4H, CH2 morpholinyl), 3.67–3.78 (m, 4H,
CH2 morpholinyl), 5.30–5.33 (m, 2H, CH2), 6.63–6.77 (m, 2H, Ar-
H), 7.15–7.22 (m, 2H, Ar-H), 7.37–7.48 (m, 2H, Ar-H), 8.65 (s, 1H,
Ar-H); 13C NMR (DMSOd6 + CDCl3): d 21.62, 22.99, 23.76, 28.93,
30.38, 68.16, 112.58, 119.01, 121.98, 123.72, 125.78, 128.16,
130.88, 132.47, 135.05, 137.08, 149.31, 150.47, 167.76 (2C),
170.55; FAB-MS m/z 393 [M+H]+. Anal. Calcd for C21H20N4O2S: C,
64.27; H, 5.14; N, 14.28. Found: C, 64.25; H, 5.16; N, 14.32.
4.2. General synthetic procedure for the preparation of
compounds 5a–o
Equimolar quantities of isatin derivatives 2a–o (3.65 mmol)
and 6-methyl-benzothiazol-2-ylamine (3.65 mmol) were dissolved
in 50 mL of absolute ethanol containing 0.5 mL of glacial acetic
acid. The reaction mixture was refluxed for 12–14 h, and was
cooled to room temperature, and then concentrated to dryness un-
der reduced pressure. The residue was taken up in dichlorometh-
ane and washed with 5% aqueous sodium hydrogen carbonate
(2x) and then with brine solution. The organic phase was then
dried over sodium sulfate, the filtrate was concentrated to dryness
under reduced pressure and the crude product was purified by col-
umn chromatography on silica gel using dichloromethane–
methanol.
4.2.6. 4-Chloro-1-dimethylaminomethyl-3-(6-methyl-
benzothiazol-2-ylimino)-1,3-dihydro-indol-2-one (5f)
This compound was obtained as a reddish orange solid in 57%
yield. Mp 134–136 °C; 1H NMR (200 MHz, CDCl3): d 2.35 (s, 6H,
N(CH3)2, 2.44 (s, 3H, CH3), 5.48 (s, 2H, CH2), 6.97–7.13 (m, 2H,
Ar-H), 7.23–7.45 (m, 3H, Ar-H), 7.95 (s, 1H, Ar-H); FAB-MS m/z
386 [M+H]+. Anal. Calcd for C19H17ClN4OS: C, 59.29; H, 4.45; N,
14.56. Found: C, 59.27; H, 4.43; N, 14.54.
4.2.7. 4-Chloro-1-diethylaminomethyl-3-(6-methyl-
4.2.1. 1-Dimethylaminomethyl-3-(6-methyl-benzothiazol-2-
ylimino)-1,3-dihydro-indol-2-one (5a)
benzothiazol-2-ylimino)-1,3-dihydro-indol-2-one (5g)
This compound was obtained as a orange red solid in 53% yield.
Mp 200–202 °C; IR (KBr, cmꢀ1): 1740, 1586, 1439, 1390, 921, 910,
877, 662; 1H NMR (200 MHz, CDCl3): d 1.01–1.06 (t, J = 7.0 Hz, 6H,
N(CH2CH3)2), 2.35 (s, 3H, CH3), 2.47–2.51 (q, 4H, N(CH2CH3)2), 5.36
(s, 2H, CH2), 6.80–6.96 (m, 2H, Ar-H), 7.33–7.57 (m, 3H, Ar-H), 7.98
(s, 1H, Ar-H); 13C NMR (CDCl3): d 10.8 (2C), 21.25, 49.12 (2C), 66.51,
120.96, 123.77, 124.23, 124.76, 125.02, 131.05, 131.39, 132.42,
138.84, 139.03, 149.90, 151.56, 164.85 (2C), 181.30; FAB-MS m/z
414 [M+H]+. Anal. Calcd for C21H21ClN4OS: C, 61.08; H, 5.13; N,
13.57. Found: C, 61.06; H, 5.15; N, 13.55.
This compound was obtained as a orange solid in 65% yield. Mp
188–190 °C; 1H NMR (200 MHz, CDCl3): d 2.35 (s, 6H, N(CH3)2),
2.39 (s, 3H, CH3), 5.38–5.45 (m, 2H, CH2), 7.04–7.12 (m, 2H, Ar-
H), 7.36–7.40 (m, 2H, Ar-H), 7.52–7.62 (m, 2H, Ar-H), 8.65 (s, 1H,
Ar-H); 13C NMR (CDCl3): d 21.24, 48.93 (2C), 66.75, 112.67,
120.93, 123.09, 123.83, 124.80, 125.00, 126.92, 131.03, 131.36,
138.52, 138.55, 149.96, 150.43, 151.17, 164.89; FAB-MS m/z 351
[M+H]+. Anal. Calcd for C19H18N4OS: C, 65.12; H, 5.18; N, 15.99.
Found: C, 65.15; H, 5.16; N, 15.96.

V. R. Solomon et al. / Bioorg. Med. Chem. 17 (2009) 7585–7592
7591
4.2.8. 4-Chloro-1-[(diphenylamino)-methyl]-3-(6-methyl-
benzothiazol-2-ylimino)-1,3-dihydro-indol-2-one (5h)
This compound was obtained as a yellowish orange solid in 61%
yield. Mp 190–192 °C; 1H NMR (200 MHz, CDCl3): d 2.51 (s, 3H,
CH3), 5.38 (s, 2H, CH2), 6.77–6.93 (m, 5H, Ar-H), 7.01–7.04 (m,
5H, Ar-H), 7.30–7.35 (m, 5H, Ar-H), 7.98 (s, 1H, Ar-H); FAB-MS m/
z 510 [M+H]+. Anal. Calcd for C29H21ClN4OS: C, 68.43; H, 4.16; N,
11.01. Found: C, 68.45; H, 4.15; N, 11.04.
7.01–7.19 (m, 2H, Ar-H), 7.31–7.38 (m, 3H, Ar-H), 8.33 (s, 1H, Ar-
H); FAB-MS m/z 470 [M+H]+. Anal. Calcd for C22H21BrN4OS: C,
56.29; H, 4.51; N, 11.94. Found: C, 56.31; H, 4.49; N, 11.92.
4.2.15. 4-Bromo-3-(6-methyl-benzothiazol-2-ylimino)-1-
morpholin-4-ylmethyl-1,3-dihydro-indol-2-one (5o)
This compound was obtained as a yellowish orange solid in 44%
yield. Mp 118–120 °C; IR (KBr, cmꢀ1): 1748, 1607, 1570, 1442, 937,
814, 780, 660; 1H NMR (200 MHz, DMSOd6): d 2.29–2.39 (m, 4H,
CH2 morpholinyl), 2.40 (s, 3H, CH3), 3.35–3.53 (m, 4H, CH2 mor-
pholinyl), 5.34–5.35 (m, 2H, CH2), 7.14–7.28 (m, 2H, Ar-H), 7.34–
7.50 (m, 3H, Ar-H), 8.41 (s, 1H, Ar-H); FAB-MS m/z 472 [M+H]+.
Anal. Calcd for C21H19BrN4O2S: C, 53.51; H, 4.06; N, 11.89. Found:
C,53.55; H, 4.10; N, 11.92.
4.2.9. 4-Chloro-3-(6-methyl-benzothiazol-2-ylimino)-1-
piperidin-1-ylmethyl-1,3-dihydro-indol-2-one (5i)
This compound was obtained as gummy matter in 50% yield. 1H
NMR (200 MHz, CDCl3): d 1.55–1.60 (m, 2H, CH2 piperidinyl), 1.66–
1.69 (m, 4H, CH2 piperidinyl), 2.35 (s, 3H, CH3), 2.38–2.42 (m, 4H,
CH2 piperidinyl), 5.36 (s, 2H, CH2), 7.15–7.46 (m, 2H, Ar-H), 7.62–
7.99 (m, 3H, Ar-H), 8.35 (s, 1H, Ar-H); FAB-MS m/z 426 [M+H]+.
Anal. Calcd for C22H21ClN4OS: C, 62.18; H, 4.98; N, 13.18. Found:
C, 62.20; H, 5.01; N, 13.20.
5. Materials and methods
5.1. Cell lines
4.2.10. 4-Chloro-3-(6-methyl-benzothiazol-2-ylimino)-1-
morpholin-4-ylmethyl-1,3-dihydro-indol-2-one (5j)
This compound was obtained as a yellowish orange solid in 49%
yield. Mp 218–220 °C; IR (KBr, cmꢀ1): 1741, 1590, 1439, 914, 793,
676; 1H NMR (200 MHz, DMSOd6): d 2.18–2.27 (m, 4H, CH2 mor-
pholinyl), 2.35 (s, 3H, CH3), 3.38–3.44 (m, 4H, CH2 morpholinyl),
4.02–4.06 (m, 2H, CH2), 7.15–7.34 (m, 2H, Ar-H), 7.47–7.51 (m,
3H, Ar-H), 8.15 (s, 1H, Ar-H); FAB-MS m/z 428 [M+H]+. Anal. Calcd
for C21H19ClN4O2S: C, 59.08; H, 4.49; N, 13.12. Found: C, 58.06; H,
4.51; N, 13.10.
The human MDA-MB468, MDA-MB231 and MCF-7 breast can-
cer 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 supple-
mented with an MEGM mammary epithelial singlequot kit (Camb-
rex). Cells were grown at 37 °C with 5% CO2, 95% air under the
humidified conditions.
5.2. Reagents
4.2.11. 4-Bromo-1-dimethylaminomethyl-3-(6-methyl-
benzothiazol-2-ylimino)-1,3-dihydro-indol-2-one (5k)
This compound was obtained as a yellowish orange solid in 56%
yield. Mp 202–204 °C; IR (KBr, cmꢀ1): 1741, 1580, 1450, 1325, 970,
920, 787; 1H NMR (200 MHz, CDCl3): d 2.27 (s, 6H, N(CH3)2, 2.51 (s,
3H, CH3), 5.36–5.38 (m, 2H, CH2), 7.00–7.20 (m, 2H, Ar-H), 7.32–
7.40 (m, 3H, Ar-H), 8.57 (s, 1H, Ar-H); FAB-MS m/z 430 [M+H]+.
Anal. Calcd for C19H17BrN4OS: C, 53.15; H, 3.99; N, 13.05. Found:
C, 53.11; H, 3.96; N, 13.01.
Chloroquine diphosphate and cisplatin were purchased from
Sigma–Aldrich Canada Ltd (Oakaville, ON, Canada). All the com-
pounds were dissolved in 10–20 mM dimethyl sulfoxide (DMSO)
and stored at ꢀ20 °C until use. The stock solution was diluted in
culture medium (0.1–100 lM) immediately before use. The final
concentration of DMSO in the SRB-based cytotoxicity assays did
not exceed 0.1%. To rule out that the DMSO concentration used
may affect cell cytotoxicity, culture medium containing equivalent
concentration of DMSO was used as a negative control in all exper-
iments. In all studies, the concentration of DMSO used did not
notably show any cytotoxicity.
4.2.12. 4-Bromo-1-diethylaminomethyl-3-(6-methyl-
benzothiazol-2-ylimino)-1,3-dihydro-indol-2-one (5l)
This compound was obtained as a yellowish orange solid in 54%
yield. Mp 235–237 °C; 1H NMR (200 MHz, CDCl3): d 1.23–1.26 (t,
J = 7.0 Hz, 6H, N(CH2CH3)2), 2.36 (s, 3H, CH3), 2.41–2.47 (q, 4H,
N(CH2CH3)2), 5.40–5.43 (m, 2H, CH2), 7.15–7.35 (m, 2H, Ar-H),
7.36–7.51 (m, 3H, Ar-H), 8.63 (s, 1H, Ar-H); FAB-MS m/z 458
[M+H]+. Anal. Calcd for C21H21BrN4OS: C, 55.14; H, 4.63; N, 12.25.
Found: C, 55.17; H, 4.68; N, 12.29.
5.3. SRB assay
Cytotoxic effects were determined by a Sulphorhodamine B
(SRB)-based protocol.4,25 For
5000–10,000 cells were inoculated into100
a
typical screening experiment,
L medium per well
l
of a 96-well microtiter plate as described previously.25,29 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
4.2.13. 4-Bromo-1-[(diphenylamino)-methyl]-3-(6-methyl-
benzothiazol-2-ylimino)-1,3-dihydro-indol-2-one (5m)
This compound was obtained as a yellowish orange solid in 50%
yield. Mp 228–230 °C; 1H NMR (200 MHz, CDCl3): d 2.38 (s, 3H,
CH3), 5.34–5.38 (m, 2H, CH2), 6.59–7.01 (m, 5H, Ar-H), 7.15–7.35
(m, 5H, Ar-H), 7.38–7.41 (m, 3H, Ar-H), 7.57–7.65 (m, 2H, Ar-H),
8.61 (s, 1H, Ar-H); FAB-MS m/z 554 [M+H]+. Anal. Calcd for
C29H21BrN4OS: C, 62.93; H, 3.82; N, 10.12. Found: C, 62.90; H,
3.85; N, 10.09.
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 10-fold serial dilutions were made to provide a total of seven
drug concentrations (and a control [C]). Following the addition of
drugs, the culture plate was incubated for additional 48 h. Cells
were fixed in situ by slowly adding 25 lL of cold 50% (w/v) TCA (fi-
nal 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. Fifty microliters 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. Un-
bound SRB was removed by five washes with tap water, followed
4.2.14. 4-Bromo-3-(6-methyl-benzothiazol-2-ylimino)-1-
piperidin-1-ylmethyl-1,3-dihydro-indol-2-one (5n)
This compound was obtained as a yellowish orange solid in 52%
yield. Mp 194–196 °C; 1H NMR (200 MHz, CDCl3): d 1.21–1.30 (m,
2H, CH2 piperidinyl), 1.45–1.63 (m, 4H, CH2 piperidinyl), 2.29–2.45
(m, 4H, CH2 piperidinyl), 2.51 (s, 3H, CH3), 5.51–5.54 (m, 2H, CH2),

7592
V. R. Solomon et al. / Bioorg. Med. Chem. 17 (2009) 7585–7592
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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:
ðTi ꢀ TzÞ=ðC ꢀ TzÞ ꢁ 100
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(variable slope) curve which is fitted by GraphPad Prism v.4.03 soft-
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5.4. Flow cytometry
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.3,30 The
ethanol was then removed by centrifugation, and cells were resus-
pended in 1ꢁ PBS solution, followed by centrifuge. The cell pellet
was than stained with PI master mix (100
100
lg/mL RNase A,
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using CXP software.
Acknowledgments
The authors wish to thank G. Ulibarri (Laurentian University)
for letting us use his laboratory equipment in the process of syn-
thesizing compounds. This work was supported by funds from
the Natural Sciences and Engineering Council of Canada (NSERC)
and the Northern Cancer Research Foundation to H.L. V.R.S. is a re-
cipient of a postdoctoral fellowship from the Ontario Ministry of
Research and Innovation.
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