2-Phenylindole derivatives as anticancer agents: synthesis and screening against murine melanoma, human lung and breast cancer cell lines

Article abstract of DOI:10.1080/00397911.2019.1620282

Indole derivatives have attractive anticancer properties and may be a future hope for better anticancer drug(s) of low toxicity and high potency. In this paper, syntheses of 2-phenylindole derivatives have been described via Fischer indole synthesis through a one-pot solvent-free method. The synthesized compounds were screened for anticancer potential in vitro against murine melanoma (B16F10), human lung cancer (A549), and human breast cancer (MDA-MB-231) cell lines. The results highlighted that 2-phenylindole derivatives are also promising anticancer agents in case of melanoma and lung cancer along with the breast cancer. Molecular docking analyses with possible targets for melanoma (NEDD4-1) and lung cancer (EGFR) were also performed to understand specific interactions of 2-phenylindole derivatives with the amino acid residues of the receptors.

Full text of DOI:10.1080/00397911.2019.1620282

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
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ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
2-Phenylindole derivatives as anticancer
agents: synthesis and screening against murine
melanoma, human lung and breast cancer cell
lines
Ruchi Gaikwad, Yamini Bobde, Routholla Ganesh, Tarun Patel, Anju Rathore,
Balaram Ghosh, Kalpataru Das & Shovanlal Gayen
To cite this article: Ruchi Gaikwad, Yamini Bobde, Routholla Ganesh, Tarun Patel, Anju Rathore,
Balaram Ghosh, Kalpataru Das & Shovanlal Gayen (2019): 2-Phenylindole derivatives as
anticancer agents: synthesis and screening against murine melanoma, human lung and breast
cancer cell lines, Synthetic Communications, DOI: 10.1080/00397911.2019.1620282
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https://doi.org/10.1080/00397911.2019.1620282
2-Phenylindole derivatives as anticancer agents: synthesis
and screening against murine melanoma, human lung and
breast cancer cell lines
Ruchi Gaikwada, Yamini Bobdeb, Routholla Ganeshb, Tarun Patela, Anju Rathorea,
Balaram Ghoshb , Kalpataru Dasc, and Shovanlal Gayena
aLaboratory of Drug Design and Discovery, Department of Pharmaceutical Sciences, Dr. H. S. Gour
b
University, Sagar, India; Department of Pharmacy, BITS-Pilani, Hyderabad Campus, Hyderabad, India;
cAdvance Organic Synthesis Laboratory, Department of Chemistry, Dr. H. S. Gour University, Sagar, India
ABSTRACT
ARTICLE HISTORY
Received 6 February 2019
Indole derivatives have attractive anticancer properties and may be a
future hope for better anticancer drug(s) of low toxicity and high
potency. In this paper, syntheses of 2-phenylindole derivatives have
been described via Fischer indole synthesis through a one-pot solv-
ent-free method. The synthesized compounds were screened for
anticancer potential in vitro against murine melanoma (B16F10),
human lung cancer (A549), and human breast cancer (MDA-MB-231)
cell lines. The results highlighted that 2-phenylindole derivatives are
also promising anticancer agents in case of melanoma and lung can-
cer along with the breast cancer. Molecular docking analyses with
possible targets for melanoma (NEDD4-1) and lung cancer (EGFR)
were also performed to understand specific interactions of 2-phenyl-
indole derivatives with the amino acid residues of the receptors.
KEYWORDS
A549; B16F10; anticancer
activity; Fischer indole
synthesis; MDA-MB-231;
molecular docking; 2-
phenylindole
GRAPHICAL ABSTRACT
Introduction
The synthesis of indole and its derivatives is an interesting research field in different
drug discovery programs as it is one of the most common (ninth position of the top
25) nitrogen based scaffold for the preparation of FDA approved drugs in different dis-
eases.[1] The indole heterocyclic ring is found in many naturally occurring compounds
CONTACT Shovanlal Gayen
of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, Madhya Pradesh 470003, India; Kalpataru Das
kalpatarud@gmail.com Advance Organic Synthesis Laboratory, Department of Chemistry, Dr. H. S. Gour University,
shovanlal.gayen@gmal.com
Laboratory of Drug Design and Discovery, Department
Sagar, Madhya Pradesh 470003, India.
Supplemental data for this article can be accessed on the publisher’s website.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsyc.
ß 2019 Taylor & Francis Group, LLC

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R. GAIKWAD ET AL.
as well as in pharmaceutical agents in the field of antiinflammatory, antidepression,
antihypertensive, antiosteoporosis, antimicrobial, anticancer agents, etc.[2–5] The interest
to develop indole derivatives as anticancer agents was initiated long back during 1950s
at the time of introduction of indole-based vinca alkaloids namely vinchristine and vin-
blastin as anticancer drugs. After that indole-based compounds have attracted a huge
attention to the synthetic chemists to develop different anticancer drugs as represented
in Figure 1.[6–10]
Among the various indole derivatives aryl indole, specifically, 2-phenylindole is an effect-
ive scaffold for anticancer activity primarily against breast cancer.[11–14] It was reported that
2-phenylindole derivatives can bind with the estrogen receptor as well as microtubule
receptors in case of estrogen-sensitive breast cancer and metastatic breast cancer, respect-
ively.[15–17] In particular, 2-phenylindole derivatives are not evaluated much against murine
melanoma and human lung cancer cell lines despite several reports published that the other
indole derivatives are effective against melanoma and lung cancer.[18–22] Moreover, 2-phe-
nylindole derivatives can bind to the colchicine binding site of tubulin and can be effective
in multidrug resistance in cancer.[23,24] The importance of 2 phenylindole as anticancer
agents motivated us to study the synthetic routes. The syntheses of 2-phenylindole moiety
(4) have been reported via different methods, namely Fischer indole synthesis, Bischler
indole method, etc.[25] The synthesis of (4) via the rearrangement of (3), has been one of
the most common synthetic methods due to its versatility and broad applicability as per
the reports for the synthesis of a number of biologically active natural and unnatural prod-
ucts.[25–27] Conventionally, the synthesis of 2-phenyl indole derivatives 4 have been
reported in a two-step process via Fischer indole synthesis, The Fischer indole method
involved the intramolecular rearrangement of (3) in the presence of Lewis or Brønsted acid
providing different indole derivatives (Scheme 1). Several Lewis acids including BF3, ZnCl2,
AlCl3, and Pd-based, ionic liquids,[28–32] etc. were reported to catalyze the rearrangement
[28,29]
of (3). Among these catalysts, ZnCl2
was widely used as one of the efficient catalyst
for Fischer indole synthesis (3 ! 4). However, the development of one-pot synthesis of 4
is highly desirable from commercially available starting materials, aryl hydrazine (1) and
aryl ketone (2) to avoid the isolation of unstable intermediate 3.
In continuation of our research towards the development of 2-phenylindole derivatives
as cytotoxic agents [16,17,33], we envisage that the Fischer indole method could be an effi-
cient method for the synthesis of such indole derivatives. In the present communication,
we report a simple, one-pot, solvent-free, method for the synthesis of 2-phenylindole
derivatives 4 using anhydrous ZnCl2 as a catalyst via Fischer indole method. The synthe-
sized 2-phenylindole derivatives were screened against murine melanoma (B16F10),
human lung cancer (A549) including human breast cancer (MDA-MB-231) cell lines for
anticancer activity. Finally, ligand-receptor interactions were mapped using molecular
docking to understand the binding mode of this type of compounds with the receptors.
Results and discussion
To validate our hypothesis for one-pot method for the synthesis of 2-phenylindole
derivatives 4 under solvent-free condition, we began our study to optimize the reaction
using commercially available acetophenone and phenyl hydrazine under various reaction

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SYNTHETIC COMMUNICATIONSV
3

4
R. GAIKWAD ET AL.
2
R
CH
3
2
R
1
+
Step 1
R
Step 2
H+
NH2
O
1
1
R
2
R
N
R
NH
N
H
N
H
1
4
3
2
Scheme 1. Synthesis of 2-phenylindole via Fischer indole method.
ZnCl2 (2 equiv.)
AcOH (0.1 N)
R1
CH3
180 oC
R1
+
NH2
O
R2
NH
15-20 min.
One-pot
Solvent-free
N
H
R2
1
2
4a
86 % (R1 = H, R2 = H)
Scheme 2. Synthesis of 2-phenylindole via modified Fischer indole method.
conditions (Scheme 2). Initially, we screened various metal salts as catalyst at different
reaction conditions including room temperature (25 ꢀC) and conventional heating. We
performed the optimization of reaction using different Lewis acids catalyst such as
cupric chloride, ferric chloride, nickel chloride, terbium chloride, indium chloride, zinc
chloride, and acetic acid as additives under different reaction conditions. The reaction
was monitored with the help of TLC. When the reaction mixture was heated at 180 ꢀC,
the formation of 2-phenyl indole 4a was observed in the presence of anhydrous zinc
chloride as Lewis acid and other catalysts provided a complex mixture as observed from
1H NMR studies. However, in the presence of zinc chloride, the formation of only phe-
nyl hydrazone 3 (cf. Scheme 1) was observed at room temperature. Thus, zinc chloride
was found to be the best catalyst for the formation of 2-phenyl indole 4a under heating
condition. After the screening of several reaction conditions, we achieved the best reac-
tion condition when the reaction was carried out under solvent-free and one-pot reac-
tion condition using 2 equiv. ZnCl2 and few drops of acetic acid (0.1 N) as additives.
Under this condition, the reaction was completed within 15 min with 86% isolated yield
of 2-phenyl indole 4a (Scheme 2).
Employing the above reaction conditions (Scheme 2), several 2-phenylindole deriva-
tives (4a–4k) were synthesized. The results are summarized in Table 1.
The 11 synthesized compounds (4a–4k) were screened for their anticancer activity
against three different cell lines: murine melanoma (B16F10), human lung cancer
(A549), and human breast cancer cell lines (MDA-MB-231). The viability of the cells
was evaluated in two different doses of the synthesized compounds (100 mM and 10 mM)
in triplicate for 48 h using MTT assay. In the assay, the MTT reagent [3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used in the concentration of
5 mg/mL. The results are plotted in Figure 2. It is observed that the results are promis-
ing for all the eleven tested compounds and therefore, all of them were selected for the
determination of IC50 values i.e. concentration needed to inhibit cancer cell by 50% by
the treatment of synthesized compounds (Figure 3). Interestingly synthesized indole

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5
Table 1. Synthesis of various 2-phenylindole derivatives via the optimized conditions.a
Me
Me
Me
N
H
N
H
N
H
4a (86%)
4b (30%)
4c (40%)
Me
Cl
F
Cl
N
H
N
H
N
H
4d (51%)
4e (61%)
4f (40%)
Me
H3C
OMe
F
N
H
N
N
H
H
4g (47%)
4h (45%)
4i (50%)
Me
NO2
NO2
N
H
N
H
4k (20%)
4j (25%)
aReaction conditions: Unless otherwise mentioned the reaction was carried out with Phenylhydrazine 1 (5.1 mmol), ace-
tophenone 2 (5 mmol), and anhydrous zinc chloride (200 mol %), few drops of acetic acid (0.1 N) under solvent-free
condition as mentioned in Scheme 2. Isolated yields by column chromatography are shown in parenthesis.
derivatives have shown moderate to potent anticancer activities against different cell
lines. Against murine melanoma (B16F10) cell line the synthesized compounds 4a–k
have IC50 in the range of 23.81 lM to 60.11 lM and the highest activity was observed
for compound 4k. The most potent anticancer activity (IC50 ¼ 29.12 lM) was found in
case of compound 4b against human lung cancer cell line (A549). This is might be due
to the electron donating properties of methyl group as another compound 4c which has
also a methyl group at the p-position of phenyl, has IC50 of about 29.39 lM. However,
electron donating methoxy group is not favorable for anticancer activity. This is evident
from anticancer activity of compound 4g. The compounds 4a–4i have shown compara-
tively weak anticancer activities against human breast cancer MDA-MB-231 cell line
than the B16F10 and A549 cell lines. The lowest anticancer activity, IC50 of 92.73 lM
was also found against MDA-MB-231 cell line for compound 4f. However, compounds
4j and 4k have shown better anticancer activity of 16.18 lM and 25.59 lM respectively
against MDA-MB-231 cell line (Figure 3).
Until now most of the 2-phenylindole derivatives were tested against breast can-
cer cell line[6,12,13,21] due to their tubulin targeting property. Indole derivatives are
also proved to be inhibitors of EGFR, nitric oxide synthase and NFkB[12], Pim-1
kinase, Caspase-3, HDACs, DNA topoisomerase, sirtuin, r receptor, etc.[4,9] Thus,
indole-based compounds may have a broad spectrum of anticancer activity and
give useful lead molecule in other forms of cancers. Our results reported above
point out that most of the 2-phenylindole derivatives have better anticancer

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R. GAIKWAD ET AL.
Figure 2. Cell viability assay of the synthesized compounds using two different concentration
(100 mM and 10 mM).
activities in murine melanoma and human lung cancer cell lines than the breast
cancer cell line. Thus, these 2-phenylindoles could be promising anticancer agents
against the murine melanoma and lung cancer cell lines.

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7
Figure 3. The compounds were screened further to find out their IC50 values. Various concentration
(1.5–400 mM) were used treat B16F10, A549, and MDA-MB-231 cell lines for 48 h and cell viability was
measured using MTT assay.
In order to find out how these compounds can bind with the targets in case of mel-
anoma and lung cancer, we have performed molecular docking study with the potent 2-
phenylindole derivative, i.e., compound 4k for melanoma and compound 4b for lung
cancer. For melanoma, NEDD4-1 protein (pdb id: 5c91) as an emerging target and
inhibition of this protein triggers the tumor suppressor to induce an anticancer effect in
melanoma.[34] In case of lung cancer, epidermal growth factor receptor (EGFR) is a

8
R. GAIKWAD ET AL.
Figure 4. Docking interaction of compound 4k in NEDD4-1 receptor and compound 4b in
EGFR receptors.
validated target.[35] EGFR (pdb id: 3w32) is a tyrosine kinase receptor and over expres-
sion of this receptor is linked to non-small-cell lung cancer. The reason behind our
selection of these receptors for docking analysis is that some indole derivatives (see
Supplementary Figure 1) were reported to bind with these receptors.[36–38]
Molecular docking analysis with compound 4k has shown that the compound can nicely
bind with the pocket of NEDD4-1 enzyme. Docking results showed that the compound 4k
can bind better with the target with a binding affinity of ꢁ7.0 kcal/mol than the inbound
indole-based ligand, methyl (2E)-4-f[(5-methoxy-1,2-dimethyl-1H-indol-3-yl)carbonyl]a-
minogbut-2-enoate, with binding affinity of ꢁ5.4 kcal/mol. The 2-phenylindole moiety of
the compound 4k has aromatic p-p stacking interaction with two tyrosine amino acids 605
and 634 and anion-p interaction with the E 554 residue of the receptor. Other residues, A
550, T 551, H 631, C 627, etc. provide important van der Waals interaction with the ligand
4k. In case of EGFR target, the compound 4b can nicely bind with the binding pocket as
shown in Figure 4B. L 777, L 788, V 726, and A 743 showed alkyl-p interaction with 2-phe-
nylindole moiety. Important van der Waals interactions are formed by the residues D 855, T
854, T 790, and L 844. However, in this case, the binding affinity of compound 4b is
ꢁ7.9 kcal/mol lower than the inbound ligand, a pyrimido[4,5-b]azepine derivative (the bind-
ing affinity ꢁ11.9 kcal/mol). In this scenario, we wish to mention that the compound 4b
may have high ligand efficiency as it is very low molecular weight molecule giving huge
opportunity to incorporate multiple substituents both in the indole and phenyl moieties and
thus, may come out as better alternative inhibitor of EGFR in future.
In summary, a solvent-free and one-pot method for synthesis of substituted 2-phenyl-
indole derivatives was developed. The synthesized compounds were screened for anti-
cancer activity against melanoma, human lung cancer, and human breast cancer cell
lines. All the compounds have shown promising anticancer activities in melanoma and
lung cancer along with the breast cancer cell line. This result opens a new avenue in
anticancer research with 2-phenylindole based compounds that may provide good lead
structure for drug discovery in case of melanoma and lung cancer. Finally, molecular
docking analysis was applied in order to find out the binding mode of 2-phenylindole
derivatives with the possible receptors.

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9
Experimental
General procedure for the synthesis of 2-phenylindole (4a)
Phenylhydrazine 1a (5.1 mmol) and acetophenone 2a (5 mmol) and anhydrous zinc
chloride (200 mol %) were mixed together using pestle in mortar and few drops of
acetic acid (0.1 N) was added drop-wise with continuous mixing at room temperature
for 10 min. The mixture was transferred into a round-bottomed flask fitted with a reflux
condenser attached with CaCl2 guard tube. Then the mixture was heated slowly up to
the temperature of 180 ꢀC. The reaction was monitored by TLC using 10% ethyl acetate
in n-hexane and the reaction was observed to be completed within 15 min. The mixture
was cooled to room temperature and diluted with 5 mL dichloromethane and 5 mL
water. The dichloromethane layer was separated and aqueous layer extracted repeatedly
with dichloromethane (3 ꢂ 5 mL). The combined organic layer was dried over anhyd-
rous Na2SO4, filtered, and solvent was evaporated under reduced pressure. The crude
product was purified using flash column chromatography over silica gel using 6% ethyl
acetate in hexane as eluent to afford the pure product 2-phenylindole 4a in 86% yield
1
as light yellow solid (Table 1, entry 1). MP 194 ꢀC; H-NMR (500 MHz, CDCl3) ꢀ: 8.33
(brs, 1H), 7.67–7.65 (m, 3H), 7.46–7.39 (m, 3H), 7.35–7.13 (m, 3H), 6.84 (s, 1H); 13C
NMR (125 MHz, CDCl3) ꢀ 137.99, 136.91, 132.45, 129.35, 129.14, 127.83, 125.27, 122.47,
120.79, 120.49, 111.03, 100.08; IR (mmax: 3428.01, 3053.42, 1524.95, 1345.96, 1181.15,
739.99, 685.12 cmꢁ1): HRMS (EIþ): m/z calculated for C14H11N [M þ H]þ: 194.0970,
found 194.0964.
2-P-tolyl-1H-indole (4b)
1
Light pale yellow powder (30% yield), MP 165 ꢀC; H NMR (400 MHz, CDCl3) ꢀ 8.27
(s, 1H), 7.61 (d, J ¼ 7.8 Hz, 1H), 7.55 (d, 2H), 7.37 (d, 1H), 7.24 (s, 1H), 7.22 (s, 1H),
7.15 (dd, 2H), 6.77 (s, 1H), 2.38 (s, 3H).13C NMR (101 MHz, CDCl3) ꢀ 138.09, 137.67,
136.73, 129.74, 129.60, 129.37, 125.10, 122.14, 120.54, 120.22, 110.84, 99.42, 21.26; IR
(mmax: 3614.49, 3433.52, 2980.78, 1435.23, 1229.68, 786.09 cmꢁ1): HRMS (EIþ): m/z cal-
culated for C15H13N [M]þ 207.1048, found 207.0994.
General method for screening for anticancer activity of synthesized indole
derivatives using MTT assay
The newly synthesized compounds (4a–k) of the series were screened for anticancer
activity using murine melanoma cell lines (B16F10), human lung (A549) and breast can-
cer cell lines (MDA-MB-231) by following standard MTT assay procedure.[33,38]
Acknowledgments
The authors thank Sophisticated Instrumentation Center, Dr. Harisingh Gour University, Sagar,
for recording NMR spectral data and HRMS of our compounds.

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R. GAIKWAD ET AL.
Funding
SG and KD are thankful to the University Grants Commission (UGC), New Delhi for Startup
grants No. F.30-106/2015-BSR and No. F.30-56/2014 BSR, respectively.
ORCID
Balaram Ghosh
Shovanlal Gayen
http://orcid.org/0000-0002-3425-2439
http://orcid.org/0000-0002-3367-578X
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