Synthesis of extended conjugated indolyl chalcones as potent anti-breast cancer, anti-inflammatory and antioxidant agents

Article abstract of DOI:10.1016/j.bmcl.2017.02.052

In the present investigation, synthesis of a series of extended conjugated δ-chloro-α-cyano substituted indolyl chalcones (5a–p) was accomplished by reacting 3-cyanoacetylindole 2 with 3-chloro-3-phenyl-propenal 4 in the presence of piperidine. The struct

Full text of DOI:10.1016/j.bmcl.2017.02.052

Accepted Manuscript
Synthesis of extended conjugated indolyl chalconesas potent anti-breast cancer,
anti-inflammatory and antioxidant agents
Pravin S. Bhale, Hemant V. Chavan, Sakharam B. Dongare, Sadanand N.
Shringare, Yoginath B. Mule, Samadhan S. Nagane, Babasaheb P. Bandgar
PII:
S0960-894X(17)30184-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.052
BMCL 24724
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
26 November 2016
19 February 2017
21 February 2017
Please cite this article as: Bhale, P.S., Chavan, H.V., Dongare, S.B., Shringare, S.N., Mule, Y.B., Nagane, S.S.,
Bandgar, B.P., Synthesis of extended conjugated indolyl chalconesas potent anti-breast cancer, anti-inflammatory
and antioxidant agents, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2
017.02.052
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Synthesis of extended conjugated indolyl chalconesas potent anti-
breast cancer, anti-inflammatory and antioxidant agents
a,b
Pravin S. Bhale, Hemant V. Chavan, Sakharam B. Dongare, Sadanand N.
c,*
a
a
Shringare, Yoginath B. Mule, Samadhan S. Nagane, Babasaheb P. Bandgar *
a
d
a,
a
Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur
University, Solapur-413 255, Maharashtra, India.
b
Department of Chemistry, Yashwantrao Chavan Mahavidyalaya, Tuljapur, Dist-
Osmanabad-413 601, Maharashtra, India.
c
Department of Chemistry, A.S.P. College, Devrukh, Dist-Ratnagiri-415 804, Maharashtra,
India.
d
Polymer and Advanced Materials Laboratory, National Chemical Laboratory, Pune-
4
11008, Maharashtra, India.
Address for correspondence: Email-hemantchavan.sus@rediffmail.com
------------------------------------------------------------------------------------------------
ABSTRACT
In the present investigation, synthesis of a series of extended conjugated δ-chloro-α-cyano
substituted indolyl chalcones (5a-p) was accomplished by reacting 3-cyanoacetylindole 2
with 3-chloro-3-phenyl-propenal 4 in the presence of piperidine. The structural
interpretations of newly synthesized compounds were based on chemical and spectroscopic
evidences. Anti-tumor evaluation of the synthesized compounds in vitro against MCF-7
(
breast carcinoma) cell line revealed that they possess high anti-tumor activities. Among
them, compound 5e and 5a demonstrated excellent activity against breast carcinoma (GI50
.1 and 4 µM respectively) as good as adriamycin (GI50 <0.1µM). The compounds were also
<
0
screened against the normal Vero monkey cell line, which showed moderate selectivity
against inhibition of cancer cells. The effect of extended conjugation on activity authenticated
by comparing activity profile of compound 5a, 5i and 5m with their simple analogues.
Among the synthesized compounds, 5i and 5l were found to be active anti-inflammatory
agents in addition to having noteworthy antioxidant potential. These results suggest the
possible use of these compounds for the design and development of novel anti-breast cancer
agents.
Key words: Indole chalcones, extended conjugation, cytotoxicity, anti-inflammatory,
antioxidant

1
Cancer is one of the leading causes of death in the society. Cancer is a multifactor
2
disease with superfluous and robust biological network. It is a family of disease, which
despite five decades of intense research still affects 1 in 3 people during the course of their
3
live. Breast cancer is the most common invasive cancer in female worldwide. It accounts for
1
6% of all female cancers and 22.9% of invasive cancers in women, 18.2% of all cancer
deaths worldwide including both in males and females are from breast cancer. Breast cancer
4
rates are much higher in developed nations compared to developing ones. Various kinds of
treatments are available for breast cancer, such as chemotherapy, radiotherapy and hormone
5
therapy. Recent treatment strategies for cancer have limited efficacy. It may require
treatment with compounds that could target multiple intracellular compounds. Nowaday,
cancer therapy interfering with a single biological molecule or pathway has been successfully
6
utilized for the treatment in clinics.
When a cell turns cancerous they develop into tumors, which produce various pro-
inflammatory and inflammatory cytokines and chemokines that attract leukocytes to the site
of growth. The leukocyte in turn produce and assorted array of cytokines, cytotoxic mediators
including reactive oxygen species, serine and cystine proteases, matrix metallo proteinases
(
MMPs) and soluble mediators of cell killing such as tumor necrosis factor-alpha (TNF-α),
interleukins and interferons (IFNs). All these result in inflammation. The leukocyte then tries
to kill the tumor cells to regulate the inflammation. When they are not successful it results in
chronic inflammation and progression of tumors. The development of tumors thus depends
on the balance between pro and anti-inflammatory cytokines. Taking into consideration the
importance of inflammation in cancer development, it can be possible to minimize the risk of
cancer development by using anti-inflammatory agents.
Chalconesconsist of a 1,3-diphenyl-2-propen-1-one framework have received a great
deal of attention due to their relatively simple structures and ample variety of
pharmacological activities including anticancer, anti-inflammatory, antioxidant,
antimicrobial, analgesic, antipyretic, antimalarial, antileishmanicidaland anti-allergic
7
activities. These activities are largely attributed due to the unsaturated ketone moiety.
8
Moreover, introduction of massive substituents on two aryl rings also a subject of interest
because it leads to useful structure-activity relationship (SAR) conclusions and thus helps to
9
synthesize pharmacologically active chalcones. The literature survey revealed that very little
attempts were made towards the synthesis of diversely substituted extended conjugated
chalconesand to verify effects on the biological activity. Extended conjugation and a high
degree of electrophilicity associated with the scaffold play a vital role in the activity of

1
0
suchclass of compounds. The complete delocalization of π-electrons on both the benzene
rings makes these scaffolds more susceptible in undergoing electron transfer reactions that
may be attributed to their superior antioxidant activity. Recently, our research group reported
chloro substituted extended conjugated chalcones as potent anticancer, anti-inflammatory and
1
1
antimicrobial agents.
The indole skeleton is one of the most attractive frameworks with a wide range of
1
biological and pharmacological activities. This physiologically important nucleus is
2
1
abundantly found in therapeutic agents as well as in natural products. Many indole
3
derivatives reported as potent breast cancer agents, such as aplysinopsin analogs, which are
1
indole-derived marine natural products and indole-3-carbinols. Martel-Frachet and
4
15
coworkers reported mono- and bis-indolylchalcones as potent cytotoxic agents against
1
6
bladder carcinoma. Lawrence et al. have reported a series of α-substituted chalconesposses
1
promising antiproliferative activity against different cancer cell lines. Tarleton et al.
7
reported (z)-2-(3,4-dichlorophenyl)-3-(4-nitrophenyl)acrylonitrile shows significant
1
8
cytotoxicity against human breast cancer cell line, MCF-7 (GI50=0.127±0.043µM). More
recently, α-cyano substituted indolylchalcones have found to possess potent anticancer
1
9
potential.
Based upon these observations and in continuation of our ongoing research
2
programmeon the developmentof novel anticancerand anti-inflammatory agents, herein we
0
synthesizeda diverse library of extended conjugated δ-chloro-α-
cyanosubstitutedindolylchalcones as potent anti breast cancer, anti-inflammatory and
antioxidant agents.
In the present study, synthesis of desired extended conjugated δ-chloro-α-cyano
substituted indolylchalcones (5a-p) achieved by the Knoevenagel condensation of 3-
cyanoacetylindoles 2 with substituted 3-chloro-3-phenyl-propenals 4 in the presence of
2
1
piperidine in ethanol (Scheme 1). The starting compounds for the synthesis of title
compounds, namely 3-cyanoacetylindoles2 synthesized in good yields from the reaction of
2
substituted indoles 1 with cyanoacetic acid in presence of acetic anhydride. On the other
2
hand, 3-chloro-3-phenyl-propenals 4 were synthesized using the method described in the
2
literature with minor modifications. The products obtained were purified by column
3
chromatography using silica gel mesh size, 100–200 and elution with 10% ethyl acetate in
1 13
hexane. The structures of target molecules were analyzed by IR, HNMR, CNMR and
HRMS.

O
CN
R1
R1
i
R2
R2
N
H
N
H
R1
O
Cl
1
2
iii
R
3
R = H, Br, OCH3
1
R = H, CH3
2
CN
HN
R4
R2
O
Cl
R5
R3
R4
R3
CHO
5a-p
ii
R4
R5
4
R5
3
R = H, OCH3
3
R = H, OCH3
4
R = OCH , Br
5
3
Scheme 1: Synthesis of extended conjugated indolyl chalcones
Reagents and conditions: i) CNCH COOH, (CH CO) O, Reflux ii) DMF-POCl
Piperidine, Ethanol, Reflux 1-4 h
2
3
2
3
iii)
All the synthesized extended indolylchalcones (5a-p) were evaluated for their in vitro
cytotoxic potencies in human breast cancer cell line MCF-7 and normal Vero monkey cell
2
4,25
line by employing the sulforhodamine B (SRB) assay method.
Adriamycin is one of the
most effective anticancer agents used as reference drug. Three parameters such as GI50, TGI
and LC50 were determined during the screening process and the results summarized in Table
1
.
The GI50 values (growth inhibitory activity) refer to the drug concentration that
produces a 50% reduction of cellular growth compared with the untreated control cells. The
TGI (cytostatic activity) and LC50 (cytotoxic activity) values refer to the drug concentration
required for total growth inhibition and killing 50% of the cells, respectively. GI50 values
used to classify a compound’s activity as follows: inactive, >100 µM; moderate, between >10
and <100 µM; and active, <10 µM.
It is worth mentioning that most of the compounds were significantly cytotoxic
against MCF-7 compared to the standard drug adriamycin, with the concentration of the drug
that produced 50% inhibition of cell growth (GI50). Compound 5e and 5a exhibited potent
activity (GI50 = <0.1 and 4 µM respectively) against the MCF-7 cell line which was almost as
good as that of adriamycin (GI50 = <0.1 µM). Compounds 5f, 5i, 5j, 5m, 5b, 5n and 5c also
exhibited good cytotoxicity (GI50 = 29.64 - 47.30 µM). On the other hand, all other
compounds showedweak cytotoxicity (GI50 = 56.74 - 100 µM) against MCF-7 cell line.

A comparison of the TGI concentrations of the compounds with adriamycin was also
done. Most of the compounds were inactive and a few exhibited weak activity against the
MCF-7 cell line. Compounds 5h (TGI = 73.75 µM), 5d (TGI = 81.5 µM), 5e (TGI = 83.7
µM), 5f (TGI = 92.97 µM), 5a (TGI = 94.3 µM) and 5i (TGI = 96.5) exhibited weak activity
against MCF-7 cell line. All the other compounds found inactive (TGI >100 µM) as compared to
standard drug adriamycin.
The LC50 concentrations of the compounds were compared to that of adriamycin to get an
indication of the cytotoxic effects of these compounds against the MCF-7 cell line. The
compounds were inactive (LC50 >100 µM) like adriamycin (LC50 = 97.1 µM) against the MCF-
7
cell line.
Structure activity relationship (SAR) study revealed that introduction of methoxy
groups at 3,4,5-positions of benzene ring increase in activity was observed, whereas, decrease
in activity was observed with bromo substitution.The presence of methyl group at 2-position
of indole also enhanced activity to greater extent. Compound 5e acquiring methyl group at 2-
position of indole ring exhibited potent activity (GI50 = <0.1 µM, TGI = 83.7 µM) against
MCF-7 cell line.
To confirm the effect of extended conjugation on cytotoxic potential, we have
prepared three simple α-cyano substituted indolyl chalcone analogues of compound 5a, 5i
and 5m by reacting suitably substituted 3-cyanoacetylindole with 3,4,5-
trimethoxybenzaldehyde by refluxing in ethanol in the presence of piperidine. These simple
chalcone analogues were also subjected for cytotoxicity evaluation against MCF-7 cancer cell
line. The comparison of GI50 values for 5a, 5i and 5m and their simple chalcone analogues
reveals that presence of extended conjugation greatly increases the cytotoxic potential of
compound 5a, 5i and 5m (Figure 1). Significant increase in activity of compound 5e (Indole
ring bearing 2-methyl group) compared to compound 5a (absence of methyl group)
authenticates the presence of methyl group at 2-position of indole moiety is essential for
activity (Figure 1).

Table 1.In vitro anticancer screening of extended conjugated indolyl chalcones (5a-p) against
a
human breast cancer cell line MCF-7.
R1
O
Cl
R3
CN
HN
R4
R2
R5
MCF-7
Vero (Normal)
Compound
b
c
d
R
1
R
2
R
3
R
4
R
5
LC50
TGI
GI50
LC50
TGI
GI50
5
5
a
H
H
H
H
OCH
OCH
H
3
OCH
OCH
OCH
Br
3
3
3
OCH
H
3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
97.1
94.3
>100
>100
81.5
4.0
54.2
NT
35.9
16.2
NT
NT
NT
12.4
11.8
NT
NT
7.6
8.2
NT
NT
9.5
NT
NT
NT
19
b
3
44.20
47.30
72.50
<0.1
NT
NT
5
c
H
H
H
NT
5
d
H
H
H
H
NT
NT
5
e
H
CH
CH
CH
CH
H
3
3
3
3
OCH
OCH
H
3
OCH
OCH
OCH
Br
3
3
3
OCH
3
3
3
83.7
68.3
65.9
NT
42.6
41.1
NT
5
f
H
3
H
92.97
>100
73.75
96.5
29.64
56.74
73.91
30.84
31.90
80.30
>100
39.20
44.90
87.10
79.86
<0.1
5
5
g
H
H
h
H
H
H
NT
NT
5
5
i
Br
OCH
OCH
H
3
OCH
OCH
OCH
Br
3
3
3
OCH
H
41.5
51.8
NT
22.1
38.4
NT
j
Br
H
3
>100
>100
>100
>100
>100
>100
>100
11.0
5
k
Br
H
H
5
l
Br
H
H
H
NT
NT
5m
OCH
OCH
OCH
OCH
3
3
3
3
H
OCH
OCH
H
3
OCH
OCH
OCH
Br
3
3
3
OCH
H
45.2
NT
12.1
NT
5
n
o
p
H
3
5
H
H
NT
NT
5
H
H
H
NT
NT
Adriamycin
52.6
>10
a
b
Concentrations in µM; Concentration of drug resulting in a 50% reduction in the measured protein at the end
c
of the drug treatment as compared to that at the beginning) calculated from [(Ti - Tz)/Tz]x100 = -50; Drug
d
concentration resulting in total growth inhibition (TGI) will calculated from Ti = Tz; Growth inhibition of 50%
(GI50) calculated from [(Ti - Tz)/(C - Tz)] x 100 = 50; NT = Not.tested.

O
Cl
O
OCH3
OCH3
OCH3
OCH3
CN
CN
HN
HN
HN
HN
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
5
5
5
a GI50 = 4 mM
GI50 = 91.7 mM
Br
Br
O
Cl
O
OCH3
OCH3
OCH3
OCH3
CN
CN
HN
i GI50 = 30.84 mM
GI50 = 63.1 mM
H CO
3
H CO
3
O
Cl
O
OCH3
OCH3
OCH3
OCH3
CN
CN
HN
m GI50 = 39.20 mM
GI50 = 64.6 mM
O
Cl
OCH3
OCH3
CN
HN
OCH3
5e GI50 = < 0.1 mM
Figure 1. Effect of extended conjugation on cytotoxicity potential of compound 5a, 5i and
5
m
Most of the reported drugs affect the normal cell growth, which serves to be a major
disadvantage in the progress of anticancer drug development. Therefore, we have ensured the
selectivity of some active compounds by in vitro screening against the normal Vero Monkey
cell line. This cellular level normal screening results help to reveal the safety profile of active
compounds. This cytotoxicity study revealed that the GI50 values of compound 5a, 5e, 5f, 5i,
5
j and 5m are 16.2, 12.4, 11.8, 7.6, 8.2 and 9.5 µM respectively (Table 1). These results
illustrate that the synthesized methyl substituted extended conjugated indolylchalcones5a and
e may have drug like properties at cellular level. This new lead compounds also show
moderate selectivity against cancer lines over normal cell line.
5

Denaturation of proteins is a well-documented cause of inflammation. In the present
study, the in vitro anti-inflammatory effect of synthesized compounds was evaluated against
2
6
denaturation of egg albumin and results are summarized in Table 3. Compound 5i and 5l
showed significant inhibition (90.82%) compared to the Diclofenac sodium, a standard anti-
inflammation drug (90.21%) at 1mM concentration. All the other compounds were also found
to be effective in inhibiting heat induced albumin denaturation (78.23-88.07%) except
compound 5d and 5j.
Table 3. Effect of extended conjugated chalcones on heat induced protein denaturation
Entry
% inhibition (1 mM)
86.23
5a
5
b
85.32
5
c
80.23
5
d
38.53
5
e
80.73
5
f
79.81
5g
83.10
5
h
85.32
5
i
90.82
5
j
21.10
5k
78.23
5
l
90.82
5
m
84.40
5
n
87.15
5o
79.53
5
p
88.07
Diclofenac
sodium
9
0.21
Free radicals especially the reactive oxygen species (ROS) are proposed to be the key
players in the pathophysiological mechanisms associated with various inflammatory
2
disorders. These radicals reacts indiscriminately with almost every type of biomolecules
7
2
8
found in living cell and may deviate the cells from its normal physiological functions.
Antioxidants are the compounds capable of scavenging the free radicals; for this antioxidant
2
9
therapy is one of the recent options. Taking into the account of multifactorial character of

oxidative stress; involved in many pathological states, the entire series of extended
indolylchalcones (5a-p) were evaluated for their direct scavenging activity against a variety
3
0
of reactive oxygen and nitrogen species such as 2,2-diphenyl-2-picrylhydrazyl (DPPH),
3
nitric oxide (NO) andsuperoxide (SOR).
1
32,33
Free radical scavenging activity was measured
in terms of percent inhibition and results are presented in Table 4. All the synthesized
compounds have shown good to excellent scavenging activity against DPPH, NO and SOR
radicals (Fig. 2). Most of the synthesized compounds possesses excellent NO radical
scavenging activity. Compounds 5h, 5c, 5d, 5p, 5f, 5b, 5e, 5g and 5a have shown excellent
NO radical scavenging activity (78.33-73.33%), whereas, all other compounds showed good
activity (68.33-63.66%). The compounds 5m and 5p showed good DPPH free radical
scavenging activity (40–46%). All other compounds showed moderate DPPH radical
scavenging activity (11-34%). Compounds 5l, 5j and 5h were found to possess significant
SOR scavenging activity (83.63-69.09%) as compared to standard ascorbic acid (AA)
(
74.07%). All other compounds were moderate SOR scavengers (66.37–50%).
Table 4. In vitro anti-oxidant activity of extended conjugated indolyl chalcones (5a-p)
%
inhibition at 1mM
NO
Entry
DPPH
21.05
15.78
23.36
18.42
14.47
22.36
34.17
15.78
26.31
11.84
15.60
15.78
46.05
34.21
16.70
40.78
44.18
SOR
59.09
63.63
61.32
65.45
58.18
66.36
63.07
69.09
50.00
80.90
62.17
83.63
50.00
58.18
60.72
54.54
74.07
5
5
a
73.33
75.00
76.88
76.66
74.16
75.83
74.00
78.33
36.66
57.50
56.18
67.50
68.33
74.16
41.63
76.66
42.63
b
5
c
5
d
5
e
f
5
5
5
g
h
5
i
5
j
5
k
5
l
5
m
5
n
o
p
5
5
AA
Standard: AA = Ascorbic acid; data represent mean of two replicates.

90
80
70
60
50
40
30
20
10
DPPH
NO
SOR
0
5a
5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p AA
Compound
Figure 2. Antioxidant activity profile of synthesized compounds
In summary, we rationally designed and synthesized a series of extended conjugated
δ-chloro-α-cyano substituted indolyl chalcone derivatives and in vitro evaluated them for
potential cytotoxic activity against breast carcinoma (MCF-7) Most of the compounds under
investigation exhibited significant antitumor activities. Among them, compound 5e and 5a
show higher activity against breast carcinoma as good as adriamycin. These results further
support its safety margin by studying the activity on normal Vero monkey cell line. In
general, the extended conjugation and substitution of alkyl group at 2-position of indole leads
to increase the cytotoxic activity. Anti-inflammatory and Antioxidant potential of synthesized
compounds were also evaluated. All the compounds found to possess marked anti-
inflammatory potential by effectively inhibiting the heat induced albumin denaturation. Most
of the compounds exhibited significant NO and SOR scavenging activity, whereas moderate
DPPH radical scavenging activity. The present investigation has thus provides impetus for
the design and development of more potent chalcone derivatives as anticancer leads.
Acknowledgements
Authors express deep thank to Tata Memorial Centre, Advanced Centre for Treatment
Research and Education in Cancer (ACTREC), Kharghar, Navi Mumbai-410210 for
conducting the in vitro anticancer screening and Dr. Sandip Patil, Birnale College of
Pharmacy, Sangli, India for anti-oxidant and anti-inflammatory activity determinations.

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Chem., 2010, 45, 2629–2633 (b)Bandgar BP, Gawande SS, Bioorg. Med. Chem.2010,
18, 2060-2065.
2. R. J. Sundberg, Indoles, Academic Press: London, (1996). (b) J. A. Joule, K. Mills,
Heterocyclic Chemistry; Blackwell Science: Oxford (2000)

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3. J. A. Joule, In Science of Synthesis, Thomas, E. J., Ed., Thieme: Stuttgart, Vol. 10 (2000).
b) G. W. Gribble, J. Chem. Soc., Perkin Trans., 2000, 1, 1045-1075. (c) S.Hibino, T.
(
Choshi, Nat. Prod. Rep., 2002, 19, 148-180 (c) J. A. Joule, K. Mills, G. F.
Smith,Heterocyclic Chemistry; Stanley Thornes Ltd.:Cheltenham, (1995). (d) T. L.
Gilchrist, Heterocyclic Chemistry, Addison-WesleyLongman Limited: Singapore, (1997).
(
e) T. L. Gilchrist, J. Chem. Soc., Perkin Trans.2001, 1,2491-2515
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4. (a) Hollenbeak, K. H;. Schmitz, F., J. Lloydia, 1997, 40, 479; (b) Kondo, K.; Nishi, J.;
Ishibashi, M.; Kobayashi, J., J. Nat. Prod., 1994, 57, 1008; (c) Bialonska, D.; Zjawiony,
J. K., Marine Drugs, 2009, 7, 166.
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5. Brew, C. T.; Aronchik, I.; Kosco, K.; McCammon, J.; Bjeldanes, L. F.; Firestone, G. L.,
Int J. Cancer, 2009, 124, 2294.
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6143.
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8. Tarleton, M.; Robertson, M. J.; Gilbert, J.; Sakoff, J.A.; McCluskey, A.; Med. Chem.
Commun., 2011, 2, 31.
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2013, 21, 333; (b) Kumar, D.; Kumar, N. M.; Tantak, M. P.; Ogura, M.; Kusaka, E.; Ito,
T., Bioorg. Med. Chem. Lett., 2014, 24, 5170.
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0. (a)Masand, V. H.; Mahajan, D. T.; Hadda, T. B.; Jawarkar, R. D.; Chavan, H.; Bandgar,
B. P.; Chauhan, H., Med. Chem. Res., 2014, 23, 417; (b) Bandgar, B.P.; Patil, S.A.; Totre,
J. V.; Korbad, B. L.; Gacche, R. N.; Hote, B. S.; Jalde, S. S.; Chavan, H. V., Bioorg. Med.
Chem. Lett. 2010, 20, 2292; (c) Bandgar, B.P.; Adsul, L.K.; Lonikar, S.V.; Chavan, H.V.;
Shringare, S.N.; Patil,S.A.; Jalde, S.S.; Koti, B.A.; Dhole, N.A.; Gacche, R.N.; Shirfule,
A., J. Enz. Inhib. Med. Chem.,2013, 28, 593; (d) Bandgar, B.P.; Jalde, S.S.; Korbad, B.L.;
Patil, S.A.; Chavan, H.V.; Kinkar, S.N.; Adsul, L.K.; Shringare, S.N.; Nile S.H., J. Enz.
Inhib. Med. Chem.,2012, 27, 267; (e) Chavan, H.V.; Adsul, L.K.; Kotmale, A.S.;
Dhakane, V.D.; Thakare, V.N.; Bandgar, B.P., J. Enz. Inhib. Med. Chem., 2015, 30, 22;
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f) Chavan, H.V.; Bandgar, B.P., ACS Sustainable Chem. Eng. 2013, 1, 929; (g) Jalde,
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in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 2014, 44, 623.

2
1. General procedure for the synthesis of extended conjugated indolylchalcones (5a-p):To a
mixture of 3-(1H-indol-3-yl)-3-oxopropanenitrile2 (1 mmol) in ethanol (15 ml) was
added piperidine (0.3 ml) and stirred for 5 min. Then, added the purified β-chlorovinyl
o
aldehyde 4 (1 mmol) and this mixture was heated to 70 C for 1-4 h. After completion of
reaction (monitored by TLC), reaction mixture was poured over crushed ice and acidified
with acetic acid. The precipitated solid was filtered, washed with water and oven dried. It
was column purified by column chromatography using silica gel mesh size, 100–200 and
elution with 10% ethyl acetate in hexane.
2
2
2
2. Slatt, J.; Romero, I.; Bergman, J. Synthesis,2004,16, 2760-65.
3. Meesala, R.; Nagarajan, R. Tetrahedron Lett.,2006, 47, 7557–7561.
4. Procedure of the SRB-assay: Tumor cells (human breast cancer cell line MCF-7) were
grown in tissue culture flasks in growth medium (RPMI-1640 with 2 mM glutamine, pH
o
.4, 10% fetal calf serum, 100 mg/ml streptomycin, and 100 units/ml penicillin) at 37 C
7
under the atmosphere of 5% CO
The cells at subconfluent stage were harvested from the flask by treatment with trypsin
0.05% trypsin in PBS containing 0.02% EDTA) and placed in growth medium. The cells
2 2
and 95% relative humidity employing a CO incubator.
(
with more than 97% viability (trypan blue exclusion) were used for cytotoxicity studies.
An aliquot of 100 ml of cells were transferred to a well of 96-well tissue culture plate.
o
The cells were allowed to grow for one day at 37 C in a CO
2
incubator as mentioned
above. The test materials at different concentrations were then added to the wells and
cells were further allowed to grow for another 48 h. Suitable blanks and positive controls
were also included. Each test was performed in triplicate. The cell growth was stopped by
o
gently layering of 50 ml of 50% trichloroacetic acid. The plates were incubated at 4 C for
an hour to fix the cells attached to the bottom of the wells. Liquids of all the wells were
gently pipette out and discarded. The plates were washed five times with doubly distilled
water to remove TCA, growth medium, etc and were air-dried. 100 ml of SRB solution
(
0.4% in 1% acetic acid) was added to each well and the plates were incubated at ambient
temperature for half an hour. The unbound SRB was quickly removed by washing the
wells five times with 1% acetic acid. Plates were air dried, tris-buffer (100 ml of 0.01 M,
pH 10.4) was added to all the wells and plates were gently stirred for 5 min on a
mechanical stirrer. The optical density was measured on ELISA reader at 540 nm. The
cell growth at absence of any test material was considered 100% and in turn growth
inhibition was calculated. GI50 values were determined by regression analysis.

2
2
5. Skehan, P.; Strong, R.; Scadiaro, D.; Monks, A.; Mc-Mahon, J.; Vistica, D.; Warren, J.T.;
Bokesch, H.; Kenney, S.; Boyed, M. R. J. Natl. Cancer Inst.,1990, 82, 1107-12.
6. In vitro anti-inflammatory activity by protein denaturation method: The reaction mixture
(
10 ml) consisted of 0.4 ml of egg albumin (from fresh hen’s egg), 5.6 ml of phosphate
buffered saline (PBS, pH 6.4) and 4 ml of synthetic derivative (1 mM). Similar volume of
double-distilled water served as control. Then the mixtures were incubated at (37oC ± 2)
o
in an incubator for 15 min and then heated at 70 C for 5 min. After cooling, their
absorbance was measured at 660 nm by using vehicle as blank. Diclofenac sodium (1
mM) was used as reference drug and treated similarly for the determination of
absorbance. The percentage inhibition of protein denaturation was calculated by using the
following formula, % inhibition = 100 x (Vt / Vc - 1); Where, Vt = absorbance of test
sample, Vc = absorbance of control.
2
7. Trouba, K. J.; Hamadeh, H. K.; Amin, R. P.; Germolec, D. R. Antioxid. Redox Signal,
2002, 4, 665-73.
2
2
8. Barry, H.; John, M. C. G. Biochem. J., 1984, 219, 1-14.
9. Velavan, S.; Naghlendran, K.; Mahesh, R.; HazeenaB.V. PHCOGMAG, 1998. ISSN:
0973–1296.
3
0. DPPH radical scavenging activity: The ability of compounds 5a-p to scavenge DPPH
2
radical was assessed using reportedmethod with modification. Briefly, 1 ml of
4
synthesized compounds as 1 mM was mixed with 3.0 ml DPPH (0.5 mmol/L in
methanol), the resultant absorbance was recorded at 517 nm after 30 min incubation at
o
7 C. The percentage of scavenging activity was derived using the following formula,
3
Percentage inhibition (%) = [(Acontrol– Asample) / Acontrol] x 100; Where, Acontrol is
absorbance of DPPH; Asample is absorbance of reaction mixture (DPPH with Sample).
3
1. NO radical scavenging activity: NO radical scavenging activity of compounds 5a-p was
2
carried out as per the method. NO radicals were generated from sodium nitroprusside
4
solution. One ml of 10 mM sodium nitroprusside was mixed with 1 ml of 1 mM synthetic
o
compounds in phosphate buffer (0.2 M pH 7.4). The mixture was incubated at 25 C for
150 min. After incubation the reaction mixture mixed with 1.0 ml of pre-prepared Griess
reagent (1% sulphanilamide, 0.1% napthylethylenediamine dichloride and 2% phosphoric
acid). The absorbance was measured at 546 nm and percentage of inhibition was

calculated using the same formula as above. The decreasing absorbance indicates a high
nitric oxide scavenging activity.
3
2. Superoxide radical (SOR) scavenging assay: The reaction mixture consisting of 1ml of
nitro blue tetrazolium (NBT) solution (156 mM NBT in phosphate buffer, pH 7.4), 1 ml
NADH solution (468 mM NADH in phosphate buffer, pH 7.4), and 1ml of synthetic
compound (1mM) solution was mixed. The reaction was started by adding 1 ml of
phenazine methosulfate (PMS) solution (60 mM PMS in phosphate buffer, pH 7.4) to the
o
mixture. The reaction mixture was incubated at 25 C for 5 min and the absorbance was
measured at 560 nm against blank sample and compared with standards and percentage of
inhibition was calculated using the same formula as above. Decreased absorbance of
reaction mixture indicated increased SOR scavenging activity.
3
3. Kumar, R. S.; Sivakumar, T.; Sundaram, R. S.; Sivakumar, P.; Nethaji, R.; Gupta, M.;
Mazumdar, U. K. Iranian J. Pharmacol Therap., 2006, 5, 35-41.

Graphical Abstract
Synthesis of extended conjugated indolyl chalcones as potent anti-
breast cancer, anti-inflammatory and antioxidant agents
a,b
Pravin S. Bhale, Hemant V. Chavan, Sakharam B. Dongare, Sadanand N.
c,*
a
a
a
d
a,
Shringare, Yoginath B. Mule, Samadhan S. Nagane, Babasaheb P. Bandgar *
Extended conjugation increases activity
O
O
Cl
OCH3
OCH3
Methoxy
groups
CN
CN
HN
HN
OCH3 increases
activity
R
OCH3
R
OCH3
OCH3
alkyl substitution
enhances activity
R = H; (MCF-7; GI50 = 91.7 µM)
5
5
a R = H; (GI = 4 µM)
50
e R = CH ; (GI = <0.1 µM)
MCF-7
3 50