Synthesis of 3,3-diindolyl oxyindoles efficiently catalysed by FeCl 3 and their in vitro evaluation for anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2010.06.152

A simple and highly efficient method has been developed for the synthesis of 3,3-diindolyl oxyindoles by the reaction of indoles with isatin or 5-fluoro isatin using a catalytic amount (5 mol %) of FeCl₃ at room temperature in a short reaction time in high yields. All these compounds were evaluated against a panel of five human cancer lines and most of them showed potent cytotoxicity. Compound 4b showed IC₅₀ of 4.7 and 5 μM against SK-N-SH and DU-145 cell lines, respectively, whereas 4c, 4d, 4f and 4k showed IC₅₀ of 2.2, 1.2, 3.6 and 3.6 μM, respectively, against DU-145 cell line. Interestingly, some of the compounds are selectively potent in prostate cancer (DU-145) with IC₅₀ values of 1.2-19.6 μM.

Full text of DOI:10.1016/j.bmcl.2010.06.152

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5229–5231
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 3,3-diindolyl oxyindoles efficiently catalysed by FeCl3 and
their in vitro evaluation for anticancer activity
*
Ahmed Kamal , Y. V. V. Srikanth, M. Naseer A. Khan, Thokhir Basha Shaik, Md. Ashraf
Division of Organic Chemistry, Chemical Biology Laboratory, Indian Institute of Chemical Technology, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and highly efficient method has been developed for the synthesis of 3,3-diindolyl oxyindoles by
the reaction of indoles with isatin or 5-fluoro isatin using a catalytic amount (5 mol %) of FeCl3 at room
temperature in a short reaction time in high yields. All these compounds were evaluated against a panel
of five human cancer lines and most of them showed potent cytotoxicity. Compound 4b showed IC50 of
Received 6 April 2010
Revised 9 June 2010
Accepted 30 June 2010
Available online 29 July 2010
4.7 and 5
of 2.2, 1.2, 3.6 and 3.6
are selectively potent in prostate cancer (DU-145) with IC50 values of 1.2–19.6
l
M against SK-N-SH and DU-145 cell lines, respectively, whereas 4c, 4d, 4f and 4k showed IC50
M, respectively, against DU-145 cell line. Interestingly, some of the compounds
M.
l
Keywords:
Anticancer activity
FeCl3
l
Ó 2010 Elsevier Ltd. All rights reserved.
Indoles
Isatin
Oxyindoles
Cancer has become the second cause of mortality in the world
and the development of potent and specific anticancer agents is ur-
gently needed because of the problems like severe toxicity as well
as resistance with the existing drugs. Anticancer agents exert their
biological effect usually by targeting various intracellular targets;
therefore, current research mainly focuses on therapeutic targets
involved in cell proliferation. However, identification of the exact
target for particular class of compounds for their anticancer activ-
ity is one of the challenges to improve efficacy. Indole derivatives
have been found to exhibit anticancer activity by interacting with
different intracellular targets. Naturally occurring and synthetic
indolocarbazoles indicated inhibitory activity against cyclin-
dependent kinases (CDKs) and antiproliferative activities in a vari-
ety of cell lines.1,2 Recently, bis(indolyl)maleimides (1, Fig. 1) have
been identified as NAD+-dependent histone deacetylases (HDAC)
inhibitors.3 Most recently, 3-arylidineindoline-2-one analogues
(2), containing an oxyindole scaffold exhibited potential HDAC
inhibitory activity.4 Furthermore, oxyindole hybrids (3) showed
micromolar activity against lung cancer cells by partial depletion
of intracellular Ca2+ stores and phosphorylation in a growth inhibi-
tion assay.5 In addition, bis-indoles and a number of indole deriv-
atives are reported to display potent anticancer activity.6a–g
On the basis of previous studies3–6 on oxyindoles and bisindolyl
maleimides it was speculated that combining the structural char-
acteristics of both these moieties could produce considerable
enhancement in the anticancer activity of such compounds. This
prompted us to design molecules wherein the two indolyl moieties
are linked to the same carbon atom of an indoline-2-one. Keeping
this objective in mind bis-indole derivatives, namely 3,3-diindolyl
oxyindoles have been prepared and evaluated for anticancer activ-
ity. Further in continuation to our previous studies in search of po-
tent molecules that exhibited anticancer activity,7 we have
H
N
O
O
R
HN
O
N
N
R
R
R'
1
2
R
HN
R1
HN
O
O
R
R
R''
N
X
N
X
4a-p
R'
3
* Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
Figure 1. Chemical structures of anticancer indole derivatives.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.152

5230
A. Kamal et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5229–5231
Table 1
synthesized 3,3-diindolyl oxyindoles employing FeCl3 as the cata-
lyst. Various synthetic methods for the preparation of 3,3-diindolyl
oxyindoles have been reported in the literature.8a–f However, the
present method improved the yields using a new catalyst and
the procedure is simple as well as convenient. In recent years iro-
n(III) chloride is found to be a powerful Lewis acid catalyst in var-
ious organic transformations9a–i under mild reaction conditions,
moreover iron salts are cheaper with low toxicity, eco-friendly
and hence this is considered as a practical catalyst in a number
of organic reactions. As a part of our research programme on eval-
uation of biological activity for different heterocyclic scaffolds, we
herein report a new and efficient method to prepare oxyindoles10
under mild conditions and evaluated them for their anticancer
potential.
We first attempted the reaction of isatin with indole and reac-
tion was carried out using 5 mol % anhydrous FeCl3 in acetonitrile
for <1 h at room temperature. Later considering the encouraging
results we expanded our attention towards a various indoles and
isatins to produce 3,3-diindolyl oxyindole derivatives in high
yields. The reaction pathway probably involves the activation of
carbonyl group of isatin moiety as well as indole ring by Fe(III),
and this is similar to that has been reported earlier.8c Both electron
donating and electron withdrawing substituents on indoles, re-
acted effectively with isatin under the same reaction conditions
(Scheme 1, Table 1). Whereas, electron deficient substituents on in-
dole moiety (4g and 4o) required longer reaction times to afford
corresponding oxyindoles with lower yields than those of their
electron-rich counterparts (4b–d, 4j and 4k). However, halogen
substituents on indole and isatin did not show much effect on
the reaction times and yields of the desired products. Further, N-
methyl indole also reacted with isatin and 5-fluoroisatin efficiently
to furnish corresponding 3,3-diindolyl oxyindoles (4h and 4p) in
high yields.
FeCl3 catalysed synthesis of 3,3-diindolyl oxyindolesa (4a–p)
Compd
R
R1
X
Time (min)
Yieldb (%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
H
H
H
H
H
H
H
H
H
F
F
F
F
F
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
CH3
15
10
10
10
30
15
15
20
10
10
10
30
10
50
60
20
93
82
95
92
93
89
81
90
82
80
78
85
94
92
84
92
4-OCH3
5-OCH3
6-OCH3
5-Cl
5-Br
5-NO2
H
H
4-OCH3
5-OCH3
6-OCH3
5-Cl
5-Br
5-NO2
H
F
F
F
a
All the reactions were performed using 5 mol % catalyst.
Yields refer to isolated products.
b
Table 2
Anticancer potency of compounds 4a–p in selected human cancer cell lines
Compd
IC50 (l
M)a
A549b
SK-N-SHc
MCF-7d
Hep G-2e
DU-145f
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
8.6
15.3
11.2
7.8
8.3
8.0
10.3
10.1
12.7
7.8
10.6
14.4
12.1
10.3
7.7
11.3
4.7
3.9
13.6
9.4
13.2
11.8
5.1
67.3
12.1
7.0
4.1
8.5
49.8
14.1
33.6
45.4
20.4
9.0
8.7
5.0
2.2
1.2
8.1
3.6
8.0
5.6
12.8
9.4
30.2
12.0
14.6
15.8
23.4
12.0
6.6
18.0
36.6
16.0
16.6
12.8
23.8
13
g
—
46.8
g
—
—
g
23.6
7.5
All the synthesized compounds were evaluated for in vitro cyto-
toxicity against a panel of five human cancer cell lines including
lung (A-549), CNS (SK-N-SH), breast (MCF-7), liver (Hep-2) and
g
—
3.6
12.0
24.8
12.8
13.1
19.6
16.3
7.5
prostate (DU-145) by using MTT assay.11 IC50 values (in
lM), which
2.8
6.8
6.5
g
—
is the concentration required to inhibit 50% of cell viability by
the test compounds after exposure to cells, have been deter-
mined. Results in Table 2 indicate that most of the compounds
displayed good anticancer potency against the cell lines tested in
the assay. Compounds 4c, 4d, 4f and 4k showed excellent antican-
17.5
9.2
11
a
b
c
d
e
f
Mean values from three separated experiments.
Lung cancer.
CNS cancer.
Breast cancer.
Liver cancer.
Prostate cancer.
cer potency with IC50 values of 2.2, 1.2, 3.6 and 3.6
tively, against prostate cancer cell line DU-145 cell line in
comparison to structurally related analogues, (GI50 = 1.08–
12.51
M) against DU-145 cell line.5 In most cases, methoxy sub-
lM, respec-
g
Inactive up to concentration 100 lM.
3
l
stituents in the 4-, 5- and 6-position of the indole ring led, for
4b–d, to significant increase in potency compared to other ana-
logues. Compounds possessing methoxy group in the 5- and 6-po-
sition (4c and 4d) showed higher potency in DU-145 with IC50
to reduction in potency to some extent particularly against liver
cancer cell line (Hep G-2) with IC50 values in the range of 16.0–
36.6
and 4o) also exhibited significant activity against DU-145 cell line
with IC50 values of 8.0 and 7.5 M, but found to be inactive against
lM. Compounds with nitro substituents at indole ring (4g
values of 2.2 and 1.2
methoxy group in the 4-position which exhibited IC50 value of
M in the same cell line. On the other hand, introduction of a flu-
lM, respectively, compared to 4b having
l
breast cancer cell line MCF-7. Compound 4n with bromo substitu-
ent at indole ring selectively more active in SK-N-SH cell line with
5
l
oro substituent in the 5-position of the oxyindole ring led, for 4j–l,
HN
R1
O
R1
O
R
FeCl3 (5 mol%)
MeCN, rt
O
+
R
R
N
X
N
H
N
X
N
X
4a-p
Scheme 1. Synthesis of 3,3-diindolyl oxyindoles (4a–p).

A. Kamal et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5229–5231
5231
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Chourasia, M.; Sastry, G. N. Bioorg. Med. Chem. 2009, 17, 1557; (c) Kamal, A.;
Khan, M. N. A.; Srikanth, Y. V. V.; Rajesh, S. V. C. R. N. C. Chem. Biol. Drug Des.
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M. J.; Pushpavalli, S. N. C. V. L.; Ray, P.; Bhadra, M. P. Eur. J. Med. Chem. 2010, 45,
2173.
an IC50 value of 2.8 lM, where as fluorine substituent did not alter
the potency of the compounds. Incorporation of a methyl group in
1-position of indole moiety (4h and 4p) also displayed consider-
able cytotoxicity in all most all cell lines tested.
Notably, some of the compounds displayed marked potency
selectively against prostate cancer cell line and CNS cancer cell line
8. (a) Rad-Moghadam, K.; Sharifi-Kiasaraie, M.; Taheri-Amlashi, H. Tetrahedron
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Mohammadizadeh, M. R.; Karimi, A. R. Catal. Commun. 2006, 7, 752; (f) Paira,
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with IC50 value up to 1.2 and 2.8 lM, respectively. It is expected
that these compounds which resemble with indole derivatives
(1–3), could exhibit anticancer activity by acting as HDAC or CDK
inhibitors. However, further studies by structural modifications
in both isatin and indole moieties to improve the anticancer effi-
cacy is likely to provide an insight in to the mechanism of action
of diindolyl oxyindoles.
In conclusion, we have developed a simple and highly efficient
method for the conversion of 3,3-diindolyl oxyindoles from indole
and isatin using 5 mol % of FeCl3 in high yields. The advantages of
this method over previous reports include its simplicity, clean
reactions, high yields, shorter reaction times and use of inexpen-
sive catalyst. These compounds were screened for their anticancer
potency and compounds 4b, 4c, 4f and 4k have exhibited potential
anticancer potency thereby suggesting that these scaffolds could
be further developed as possible anticancer agents by the struc-
tural modification in both indole and oxyindole moieties for
improving the anticancer efficacy.
10. General: To a mixture of isatin (1 mmol) and indole (2 mmol), FeCl3 (5 mol %)
was added. The reaction mixture was stirred at room temperature for the
appropriate time (Table 1). After completion of the reaction as monitored by
TLC the solvent was removed under vacuum and quenching with a saturated
solution of NaHCO3 and the products were extracted into ethyl acetate
(3 Â 30 mL). The combined organic layers were washed with water, dried over
anhydrous Na2SO4 and concentrated in vacuo. Purification by column
chromatography using hexane/ethyl acetate (9:1) furnished the
corresponding 3,3-diindolyl oxyindoles. 4d: White solid, mp 242–243 °C; 1H
NMR (300 MHz, DMSO-d6): d 10.7 (br s, 2H, NH), 10.5 (br s, 1H, NH), 7.25 (d,
J = 8.3 Hz, 3H, Ar–H), 7.20 (d, J = 7.6 Hz, 1H, Ar–H), 7.01–6.89 (m, 2H, Ar–H),
6.84 (d, J = 3.0 Hz, 2H, Ar–H), 6.73–6.64 (m, 4H, Ar–H), 3.52 (s, 6H, OCH3). 13C
NMR (75 MHz, DMSO-d6): 178.3, 152.4, 141.4, 134.6, 132.2, 127.8, 126.4, 125.2,
124.9, 121.5, 113.6, 112.1, 110.4, 109.4, 103.4, 55.1, 52.6; LRMS (ESI, m/z) 424
Acknowledgement
The author Y.V.V.S. and M.A. thanks UGC, New Delhi, for the
award of fellowships.
References and notes
m
(M+1)+; IR (KBr) ( max/cmÀ1): 3382 (NH), 1686 (C@O), 1481, 1213 (C–O), 804,
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755.
4o: Pale yellow solid, mp 333–334 °C; 1H NMR (300 MHz, DMSO-d6): d 11.8 (br
s, 2H, NH), 11.0 (br s, 1H, NH), 8.23 (s, 2H, Ar–H), 7.97 (d, J = 9.8 Hz, 2H, Ar–H),
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m
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11. Cell proliferation assay using MTT:
This assay is a quantitative colorimetric method for determination of cell
survival and proliferation.12 The assessed parameter is the metabolic activity of
viable cells. Metabolically active cells reduce pale yellow tetrazolium salt
(MTT) to
a dark blue water-insoluble formazan, which can be directly
quantified after solubilisation with DMSO. The absorbance of the formazan
directly correlates with the number of viable cells. The cells were plated in 96-
well plates at a density of 2.0 Â 104 in 200
lL of medium per well of 96-well
plate. Cultures were incubated with different concentrations of test material
and incubated for 48 h. The medium was replaced with fresh medium
containing
diphenyltetrazolium bromide (MTT) for 2–3 h. The supernatant was
aspirated and MTT-formazon crystals dissolved in 100 DMSO; OD
100
lg/mL
of
3-(4,5-dimethylthiazol-2-yl)-2,5-
l
L
measured at k 540 nm (reference wavelength, k 620 nm) on ELISA reader cell
viability% was calculated by comparing the absorbance of treated versus
untreated cells.
12. (a) Slater, T. Biochem. Biophys. Acta 1963, 77, 383; (b) van de Loosdrecht, A. A. J.
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