Novel 1-methoxyindole- and 2-alkoxyindole-based chalcones: design, synthesis, characterization, antiproliferative activity and DNA, BSA binding interactions

Article abstract of DOI:10.1007/s00044-020-02690-6

Indole-based chalcones have been identified as interesting compounds with anticancer properties. In the present study, we report the synthesis and evaluation of new 1-methoxyindole and 2-alkoxyindole chalcone hybrids as antiproliferative agents active aga

Full text of DOI:10.1007/s00044-020-02690-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:897–912
https://doi.org/10.1007/s00044-020-02690-6
ORIGINAL RESEARCH
Novel 1-methoxyindole- and 2-alkoxyindole-based chalcones:
design, synthesis, characterization, antiproliferative activity and
DNA, BSA binding interactions
1
Peter Takáč2 Danica Sabolová3 Mária Vilková4 Matej Baláž5 Tibor Béres6 Ján Mojžiš7
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Zuzana Kudličková
Received: 7 October 2020 / Accepted: 7 December 2020 / Published online: 16 January 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Indole-based chalcones have been identified as interesting compounds with anticancer properties. In the present study, we
report the synthesis and evaluation of new 1-methoxyindole and 2-alkoxyindole chalcone hybrids as antiproliferative agents
active against colorectal carcinoma cell line. Among the 19 investigated molecules, four inhibit the proliferation of colorectal
cancer cells HCT-116 with IC50 values <8 µM and display low cytotoxicity to fibroblast cell line 3T3. The UV–visible, CD
and fluorescence competitive displacement assays with ethidium bromide and Hoechst 33258 performed with two active
chalcones demonstrated that investigated chalcones interact with calf thymus (CT) DNA through the groove binding mode.
Likewise, the quenching interaction of chalcones with bovine serum albumin (BSA) was studied in vitro under optimal
physiological condition (pH = 7.4). The Stern–Volmer constant for chalcone-BSA system was found in the range of 105 M−1
.
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Keywords Chalcones 1-methoxyindole Nucleophilic substitution Antiproliferative activity DNA and BSA binding
activities
Introduction
Chalcone scaffold [(E)-1,3-diphenylprop-2-en-1-one)] retains
an attention among the medicinal chemists due to its wide
variety of pharmacological activities, namely cancer-
preventive and neuroprotective effects, as well as anti-
inflammatory, antibacterial, antiviral, antidiabetic, antioxidant,
antimalarial and other biological activities [1–3]. Several
chalcones have shown significant anticancer activity mediated
through inhibitory effect against various targets [4].
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02690-6) contains supplementary
material, which is available to authorized users.
* Zuzana Kudličková
zuzana.kudlickova@uvlf.sk
1
Department of Chemistry, Biochemistry and Biophysics,
University of Veterinary Medicine and Pharmacy, Košice, Slovak
The indole moiety is a component of biomolecules and
many natural bioactive substances. It is frequently used
structure in an anticancer drug design [5]. Considering
interesting biological properties of indole-based compounds
and their derivatives, several types of hybrid indole-
chalcone compounds have been prepared. The indole ske-
leton may be located at the 1 or 3 position of the prop-2-en-
1-one structure, most commonly attached via indole carbon
3 or 5. Many indole-derived chalcones have shown anti-
tumor activity. For instance, 1-(3,4,5-trimethoxyphenyl)-3-
(1-indol-3-yl)-chalcones 1a,b were identified as selective
and potent anticancer agents against pancreatic carcinoma
PaCa-2 (Fig. 1) [6]. The cytotoxic indole-chalcones 2-4
were synthesized and docking studies that support the
Republic
2
Department of Pharmacology and Toxicology, University of
Veterinary Medicine and Pharmacy, Košice, Slovak Republic
3
Department of Biochemistry, Institute of Chemistry, Faculty of
Science, P. J. Šafárik University, Košice, Slovak Republic
4
Laboratory of NMR, Institute of Chemistry, Faculty of Science,
P. J. Šafárik University, Košice, Slovak Republic
5
Department of Mechanochemistry, Institute of Geotechnics,
Slovak Academy of Sciences, Košice, Slovak Republic
6
Central Laboratories and Research Support, Centre of the Region
Haná for Biotechnological and Agricultural Research, Faculty of
Science, Palacký University, Olomouc, Czech Republic
7
Department of Pharmacology, Faculty of Medicine, P. J. Šafárik
University, Košice, Slovak Republic

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Medicinal Chemistry Research (2021) 30:897–912
Fig. 1 Structure of biologically active chalcones with indol skeleton and 1-methoxybrassinin
potential of these compounds to bind to the colchicine site
of tubulin and inhibit tubulin polymerization were reported
[7–9]. In addition, in vitro and in vivo experiments suggest
that 2 (JAI-51) can easily cross the brain-blood barrier and
therefore the molecule is a potential candidate for a new
treatment of the central nervous system tumors [9].
Indole phytoalexins are a specific group of natural
compounds produced by worldwide-cultivated plants of the
Cruciferae family [10]. Besides their antimicrobial activity
against different plants’ pathogenic species, cruciferous
phytoalexins have shown anticancer activities against var-
ious human cell lines [11]. Nearly half of indole phytoa-
lexins contain 1-methoxyindole skeleton. Among them, 1-
methoxybrassinin (5) showed the most effective cytotoxic
and apoptosis-inducing activity against human Jurkat cell
lines [12]. The study of the antiproliferative activity of
brassinin and its twenty derivatives on human cancer cell
lines revealed that 5 effectively inhibits cell proliferation
and induces apoptosis in colorectal cancer cells. 1-
Methoxybrassinin (5) also has potential as a radio- or che-
mosensing agent [13].
Scheme 1 Synthesis of key synthetic intermediates (i) Na2WO4.2
H2O, H2O2; (ii) K2CO3, DMS; (iii) POCl3, DMF; (iv) ClCH2COCl; (v)
AIBN, (n-Bu)3SnH
hybrid chalcones with 1-methoxy- or 2-alkoxy- indole
moiety and methoxy- or halogen-substituted phenyl ring.
Chemistry
Many natural chalcones contain methoxy and hydroxyl
groups on the phenyl core. The presence of these groups
significantly affects their biological properties and are
important structural elements in antioxidant activities of
chalcones [14]. The importance of the presence of methoxy
groups on the phenyl nucleus was highlighted and investi-
gated in the study of the antitumor activity of indole
hybrid chalcones [15, 16]. To our best knowledge, there is
only one publication of synthesis of chalcones with a
1-methoxyindole skeleton from 3-formyl-2-substituted
1-methoxyindole-5,6-dicarbonitriles [17].
We have synthesized two sets of chalcones with 1-
methoxyindole skeleton in position 1 or 3 of prop-2-en-1-
one. Third set includes 1-(4-fluorophenyl)-3-(2-alkylox-
yindol)prop-2-en-3-ones. Acid or basic catalyzed
Claisen–Schmidt condensation was a key step of the
synthesis. In the first step, aldehyde 8, and ketone 10,
were synthetized from indoline (6, Scheme 1). Oxidation
of indoline (6) using Soimei-Wolframan method and
subsequent methylation with dimethylsulphate according
to the literature [18] resulted in production of 1-
metoxyindole (7). 1-Methoxyindole-3-carboxaldehyde
(8) was prepared by the Vilsmeier–Haack reaction [19]
In the present study, we report the synthesis, character-
ization and evaluation of biological activity of 19 new

Medicinal Chemistry Research (2021) 30:897–912
899
Scheme 2 Preparation of
chalcones: Type A Reagents and
conditions: (i) 50% aq. KOH,
EtOH or iso-ProOH, r.t.; (ii)
SOCl2, dry EtOH, r.t
Scheme 3 Preparation of
chalcones: Type B Reagents and
conditions: (i) 50% aq. KOH,
EtOH or iso-ProOH, r.t.; (ii)
SOCl2, dry EtOH, r.t.; (iii)
piperidine, EtOH, 50 °C
and 3-acetyl-1-methoxyindole (10) [20] in two steps from
the 1-methoxyindole (7).
Interestingly, during the basic catalyzed condensation
(with 50% aq. KOH in EtOH) we have observed the for-
mation of a by-product in a significant concentration.
Analysis of the by-product of reaction with acetophenone
14 h showed that the nucleophilic substitution at the
2-position of the indole with simultaneous liberation of
1-methoxy group leading to the formation of 16b takes
place together with condensation reaction. Indole is a well-
known electron-rich hetero-aromatic structure and electro-
philic substitution reactions of indoles are well studied.
Until the discovery of 1-hydroxyindole chemistry by Somei
[24] the nucleophilic substitution reaction was not common
in the indole chemistry. Since then, it was demonstrated that
hydroxy or methoxy group at the indol nitrogen can behave
as a good leaving group when electron-withdrawing group
is present in the indole skeleton [25]. It was concluded that
1-methoxyindoles having electron-withdrawing group such
as acetyl 10 and formyl 8 at the 3-position readily undergo
nucleophilic substitution reactions regioselectively at the
position 2 with simultaneous release of 1-methoxy group
[20, 26].
General method for preparation of chalcones involves
acid or base catalyzed Claisen–Schmidt condensation [2].
The synthesis of indole hybrid chalcones was described
by Claisen–Schmidt condensation in the presence of
LiOH hydrate in EtOH at r.t [8], the 10% aq. NaOH
[6, 21], the 50% aq. KOH [15, 16] under refluxing con-
ditions in EtOH or MeOH. The first set of chalcones A
was prepared by the condensation of 3-acetyl-1-
methoxyindole (10) with appropriate aldehydes 11a-d
and 8 (Scheme 2) using the 50% aq. KOH in EtOH. The
synthesis of unsubstituted and N-methylated indole
hybride chalcones is usually performed under the reflux
[15, 16], however, in our case this led to the increased
amount of by-products. Therefore, it was advisable to
carry out the reaction at r.t. resulting in the lower presence
of by-products. In the cases where basic catalysis resulted
in the low yield of the desired compound 12a, 12b, 13, an
in situ generated HCl by acid catalytic system of SOCl2 in
anhydrous EtOH [22] was applied.
For the synthesis of 3-(1-methoxyindol-3-yl)propenones
(set B), the same reaction conditions as for the previous set
were used, starting from 1-methoxyindole-3-carboxaldehyde
(8, Scheme 3) and derivatives of acetophenones 14a-h. In
the case of 2-hydroxy-6-methoxyacetophenone 14c, the
application of piperidine-mediated synthesis [23] prevents
cyclisation of 2-hydroxy functional group to the corre-
sponding flavanones that may occur under catalysis with
stronger bases.
These findings, as well as the excellent antiproliferative
potency of the 4-fluoro derivative 15 h, led us to the pre-
paration of the last series C (Scheme 4). We examined
condensations of aldehyde 8 with acetophenone 14 h in
alcohols with different alkyl chains under basic conditions.
The condensation and co-nucleophilic substitution reaction
products 3-(2-alkoxyindol-3-yl)-1-(4-fluorophenyl)prop-2-
en-4-ones 16a–f were obtained in 26–32% yield. The last
two 4-fluorophenyl chalcones 18a and 18b (Scheme 5),

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Medicinal Chemistry Research (2021) 30:897–912
Scheme 4 Preparation of
chalcones: Type C Reagents and
conditions: (i) 50% aq. KOH,
ROH, r.t
Scheme 5 Preparation of
chalcones 18a,b Reagents and
conditions: 18a [6], for 18b (i)
50% aq. KOH, ROH, r.t., 68%
previously synthesized by [6] supplemented the C series
with indole nitrogen substituted derivatives.
chalcones in comparison with reference drug cisplatin are
listed in Table 1. The data are presented as the mean SD
of three independent experiments and IC50 values less than
μM of Cisplatin are typed in bold.
All compounds were isolated, purified, and characterized
by means of 1H and 13C NMR and HR-MS (Figs. S1–S63 in
supplementary). The vinylic protons were seen as doublets at
6.99–8.02 ppm for H-2 and at 7.48–8.25 ppm for H-3. The
obtained chalcones were assigned as (E)-stereoisomers based
on the observed coupling constants between vinylic hydro-
The highest efficacy was demonstrated by newly syn-
thesized chalcones against leukemic Jurkat cell, which are
rapidly proliferating and more sensitive cells. The IC50
values (excluding three compounds) ranged from 3.9 to
15 μM. In contrast, the lowest sensitivity to test compounds
was found for the mammary adenocarcinoma cell line
MDA-MB-231. In the first tested series, the presence of
three methoxy groups at 2,4,6-positions (compound 12b)
resulted in the best activity with IC50 < 7 µM on HCT116,
MCF-7 and Jurkat cell lines. This finding is consistent with
the data reported for its 1-methylindole analog, one of the
three most active compounds against bladder cancer cells
in the Martel-Frachet study [15]. The regioisomeric analog
15d showed lower activity on all cell lines than 12b, as
well as the regioisomer in the mentioned study. This
indicates the need for a suitable choice of linker to the
propenone chain, which will ensure a suitable electronic
distribution throughout the molecule. The 3,5-dimethoxy
and 3,4,5-trimethoxy substituted chalcones 12a and 12c
from the first series showed very good specificity toward
HCT116 cells. However, the regioisomer, 3,4,5-trimethoxy
substituted chalcone 15e of the second series, exhibits high
toxicity against both cancer and non-cancerous 3T3 cell
lines. In addition to the impact of methoxy groups, we
investigated the effect of the introduction of halogens
on the phenyl moiety on the biological efficiency. The
4-bromo derivatives 12d and 15f displayed no significant
activity, except the one against Jurkat cells. The chalcone
15g with 2-fluorophenyl ring has displayed significant
cytotoxicity against Caco-2 (IC50 8.5 µM), but also con-
siderable toxicity against non-cancer line 3T3. In contrast
1
gens in the range from 15.3 to 15.8 Hz in the H NMR
spectrum. The signal of carbonyl carbon was between δ 182.3
and 194.6 ppm in 13C NMR spectrum. It was found the C=O
stretching vibrations for the enones ranged from 1610 to
1653 cm−1 in IR spectrum.
The presence of a methoxy group on the indole nitrogen
significantly increases the reactivity of the Claisen–Schmidt
condensation starting materials (8, 10), increases the solu-
bility of the products and especially allows the derivatiza-
tion of chalcones in position 2 of the indole skeleton due to
nucleophilic substitution under basic conditions.
Antiproliferative activity
Screening for potential antiproliferative activity of the
synthesized hybrid chalcones was performed on the A549
(human alveolar adenocarcinoma), Caco-2 (human epithe-
lial colorectal adenocarcinoma), HCT116 (human colorectal
carcinoma), HeLa (human cervical adenocarcinoma), MCF-
7 (human breast adenocarcinoma), MDA-MB-231 (human
mammary gland adenocarcinoma), and Jurkat (human acute
T-lymphoblastic leukemia) cell lines. In order to investigate
the selective cytotoxicity against cancer cells over normal
cells, all compounds were further tested using the normal
fibroblast cell line 3T3 (mouse embryo fibroblast). The
determined IC50 values of synthesized indole hybrid

Medicinal Chemistry Research (2021) 30:897–912
901
Table 1 IC50 (μM) SDa of tested compounds in different cell lines after 72 h incubation
Compounds Cell line, IC50 (µM) SDa
R
A549
Caco-2
HCT116 HeLa
MCF-7
MDA-MB-231 Jurkat
3T3
cLogPb
12a
3,5-di-MeO
55.3 8.4 62.3 12.5 5.5 2.2 25.4 8.2 30.9 4.3 >100
15.2 3.7 43.4 5.3 3.43
12b
12c
2,4,6-tri-MeO 11.2 5.9 40.3 12.2 5.0 1.2 18.3 5.1 6.3 1.2
3,4,5-tri-MeO 60.2 10.3 35.2 10.1 5.4 1.6 42.2 4.2 >100
>100
>100
>100
6.5 0.6 >100
>100 >100
3.43
2.73
4.29
3.23
3.65
3.67
3.83
3.63
12d
4-Br
>100
>100
63.5 7.9 >100
>100
12.3 1.8 >100
8.2 0.6 >100
13
>100
70.4 15.4 45.7 5.3 40.2 5.1 39.5 5.1 >100
15ac
15bc
15cc
15dc
15e
4-MeO
>100
>100
>100
50.0
5
>100
>100
>100
>100
2,6-di-MeO
80 5.7
33.8 7.3 15.1 0.8 50.0 4.3 >100
53.0 10.4
10.4 3.3 >100
7.1 1.5 >100
35.2 2.8 >100
2-OH-6-MeO >100
2,4,6-tri-MeO >100
3,4,5-tri-MeO 5.9 2.2
80.6 10.1 42.9 5.4 35.5 1.9 36.4 4.6 >100
>100
10.4 4.2 45.6 5.7 46.3 2.3 >100
5.7 0.2 7.6 0.1 33.4 6.2 25.3 5.6
5.2 0.8
5.5 0.7 7.2 1.8 2.93
8.7 1.6 72.3 8.3 4.36
5.9 0.9 23.4 1.7 3.19
15f
4-Br
2-F
>100
53.9 8.6 8.5 0.9
65.6 4.8 62.6 10.6 5.8 1.2 59.9 12.1 72.3 8.7 >100
32.9 8.7 >100 30.2 5.9 22.2 7.6 28.2 6.7 38.8 6.8
40.6 8.6 35.1 5.4 7.5 1.7 28.1 5.7 60.5 9.6 >100
69.8 0.2 27.1 1.1 70.7 10.4 59.0 13.4 >100
15g
15h
16a
37.9 8.9 19.2 2.9 20.0 8.0 21.1 3.9
4-F
10.1 2.2 >100
4.7 0.7 29.3 3.4 3.89
4.6 0.6 >100 4.42
3.64
OMe
OEt
16b
16c
O-nPr
O-isoPr
O-nBu
O-isoBu
H
>100
32.7 8.6 26.5 4.6 28.3 5.6 50.4 8.9 34.7 9.3
5.4 0.1 36.7 7.4 4.95
3.9 0.9 5.4 3.4 4.73
16d
16e
5.5 0.1
>100
5.4 0.2
>100
4.4 1.7 6.4 1.3
5.2 2.0
>100
10.3 1.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
8.6 0.9 >100
5.48
5.35
3.83
4.30
–
16f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
18a
>100
>100
>100
>100
18b
Cisplatin
Me
27.1 3.5 8.5 1.3
9.5 0.2
>100
>100
15.2 0.3 15.3 0.5 13.1 0.2 15.6 0.3 17.5 0.5
16.2 0.6 20.9 0.3
aStandard deviation
bLogarithm of calculated octanol-water partition (calculated from ChemBioDrawUltra 12.0.2)
cAntiproliferative data previously published in [46]
4-fluorophenyl derivative 15h has shown very high and
selective activity against HCT116 (IC50 5.8 µM). The
knowledge of the rapidly expanding role of fluorine in
the design and development of new drugs has led us to the
synthesis and examination of a third group of chalcones
having a substituted indole skeleton and 4-fluorophenyl
ring. In recent years, the number of fluorine-containing
drugs represents a one-third of all approved small molecule
drugs [27]. Fluorine incorporation into the structure of a
drug may dramatically change its physiochemical proper-
ties such as lipophilicity, solubility, metabolic stability, and
partition coefficient [28]. The fluoro-substituted chalcone
derivatives with excellent antiproliferative activity were
synthesized by Burmaoglu [29]. Roman et al. [30] identi-
fied 4-fluoro-3,4,5-trimethoxychalcone as a new anti-
invasive agent with cytotoxic effects. The first member of
the third series, isostere 16a having methoxy group at
position 2 of indole skeleton has a moderate activity and
increased toxicity against non-cancer 3T3 cells. The 2-
ethoxyindol-3-yl derivative 16b has selective activity on
the same cancer cell lines HCT116 as lead compound 15h
with IC50 7.5 µM. The iso-propyl chain has caused high
toxicity to both cancer and non-cancer cells with IC50
below 10.3 µM. In contrast, the lipophilic (cLog P > 5) and
bulky butoxy group caused a decrease in biological activ-
ity. For biological studies, we prepared and investigated
4-fluorophenyl derivatives both without a substituent on
the indole nitrogen 18a and with the methyl group on the
indole nitrogen 18b, previously described by Kumar et al.
[6]. Unsubstituted chalcone 18a showed no activity, while
N-methylated chalcone 18b showed selective activity
against Caco-2 cell lines to the same extent as 15g deri-
vative and moderate activity against A-549 cells. It can be
stated that the presence of methoxy group on the indole
nitrogen has a positive effect on the antiproliferative
activity of hybrid indole chalcones.
DNA binding activities
DNA is one of the central components of cellular machinery
and storage unit of genetic information. It plays a key role in

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Medicinal Chemistry Research (2021) 30:897–912
the studied compounds observed on gradual addition of CT
DNA to its fixed concentration (Fig. 2A, B), was attributed
to interaction of its π* molecular orbitals with the π-orbitals
of the DNA base pairs, resulting in a decreased π–π*
transition probability and hence hypochromism [33].
Fluorescence binding experiments
Fluorescence displacement titration is based on the
quenching of fluorescence using dyes as reporter molecules.
The extent of fluorescence decrease is directly related to the
binding of the ligand to CT DNA. The displacement assay
was carried out with a well-known minor groove binder
Hoechst 33258 (HO, Fig. S64 in supplementary) and the
intercalator Ethidium bromide (EB, Fig. S65 in supple-
mentary), using protocols reported in the literature [32, 33].
In the case of EB we have noted only negligible changes
in fluorescence spectra after the addition of the chalcones to
DNA-EB complex. This implies that the tested substances
are not intercalators. Subsequently we did the minor groove
displacement assay with HO. The addition of the studied
chalcones caused a significant decrease in the intensities of
the emission maxima at 465 nm, suggesting a noticeable
interaction of the drugs with Hoechst 33258 (Fig. 3).
Linear quenching plots were used to estimate the values
of the Stern–Volmer constants (KSV), which are 1.71 × 104
M−1 for 12b and 1.54 × 104 M−1 for 15h (Table 2, Figs.
S66, S67 in supplementary). Fluorescence binding experi-
ments confirmed that examined chalcones preferred minor
groove binding mode. The analogous fluorescence
quenching profile was obtained for S009-131 coumarin-
chalcone hybrid reported in literature [34].
Plasma protein binding is an important factor to under-
stand the pharmacokinetic and pharmacodynamic properties
of drugs, as it strongly influences drug distribution and
determines the free fraction, which is available to the target
[35]. Fluorescence methods have been widely used also to
investigate the interaction between ligands and plasma
proteins and can give information about the quenching
mechanism, binding constants, and binding sites [36]. We
have utilized this technique to study the interaction between
12b, 15h and bovine serum albumin (BSA). The fluores-
cence spectra of BSA at increasing concentrations of 12b
(A) and 15h (B) are shown in Fig. 4.
Fig. 2 Absorption spectra of compound 12b (A), 15h (B) (10.4 µM) in
0.01 M Tris buffer (pH 7.3, 24 °C) with increasing concentration of CT
DNA. The uppermost black line corresponds to the spectrum of the
free chalcone
replication, transcription, protein-coding, and cell integrity
[31]. In order to validate the specific binding mode of the
new bioactive compounds, the chalcones with significant
activity against HCT116 cells 12b and 15h, were selected
for further biophysical spectroscopic studies.
UV–vis DNA binding studies
The binding of the chalcones to DNA helix is often mon-
itored through spectral changes in the absorbance and
wavelength shifting [32]. To clarify the interaction between
the chalcones and calf thymus DNA (CT DNA), the
absorption spectra of 12b and 15h in the absence and pre-
sence of DNA are illustrated in Fig. 2. Absorption titration
experiments of studied chalcones in Tris buffer were per-
formed using fixed compound concentration to which
increments of the CT DNA stock solution were added. With
increasing CT DNA concentration, for 12b, the hypochro-
mism of the band at 378 nm reaches 38.2% with a blue shift
of 6 nm. The absorption peak for 15h at 361 nm showed
only slight blue shift (1 nm) and substantial hypochromic
effect (about 39.1%) upon addition of CT DNA (Table 2).
The hypochromism or decrease in absorption intensity of
We have observed that the fluorescence intensity of BSA
decreased regularly with an increased concentration of
chalcones 12b and 15h. The Ksv constants for chalcone-
BSA systems show values in the order of 105 M−1 (Table 2,
Figs. S68, S69 in supplementary), which suggest the sig-
nificant affinity of investigated drugs to important plasma
protein. The slightly higher value of the Ksv constant was
found for the chalcone 12b, which has three methoxy
substituents in its structure.

Medicinal Chemistry Research (2021) 30:897–912
903
Table 2 Spectral and binding
Compound
[M−1
]
Ksv
[M−1
]
HO
BSA
λmax [nm]
λmax [nm]
Hypochromism [%]
Ksv
characteristics of chalcones
12b and 15h
12b
378
361
6
1
38.2
39.1
1.71 × 104
1.54 × 104
2.55 × 105
2.19 × 105
15h
Fig. 3 Fluorescence emission spectra of HO bound to CT DNA in the
presence of 12b (A) and 15h (B) in 0.01 M Tris buffer (pH 7.3), λex =
343 nm, 24 °C). The uppermost line corresponds to the spectrum
before addition of chalcones
Fig. 4 Fluorescence quenching spectra of BSA–chalcone system with
increased concentration of 12b (A) and 15h (B) in PBS buffer pH 7.3,
λex = 280 nm at 24 °C. The uppermost black line corresponds to the
spectrum of the free BSA
any small molecule and can therefore be exploited to study
the interaction of small molecules with DNA [41]. Simple
groove binding and electrostatic interactions of drugs lead
either to no changes or to marginal perturbations in the two
CD bands of B-DNA. On the other hand, when classical
intercalators (e.g., methylene blue or ethidium bromide) are
bound to DNA, the positive band shows an increase in
molar elipticity at 275 nm and the negative band shows a
decrease in intensity at 246 nm with a slight red shift of the
band maximum [42]. Figure 5 shows the CD spectra of CT
DNA in the absence and presence of the studied chalcones.
Both the positive (275 nm) and negative (246 nm) bands
were minimally decreased in intensity with slight red-shift
after the addition of the compounds 12b and 15h. Further-
more, the incubation of DNA with tested compounds caused
perturbation of the negative band, which is indicative of the
Circular dichroism
In addition, the interaction of chalcones with CT DNA was
examined using circular dichroism (CD) measurements. CD
measures the difference in the absorption of right and left
circularly polarized light. It is a sensitive method for
monitoring conformational changes of DNA [37, 38].
Transitions in the DNA bases give rise to a CD spectrum in
the 200–350 nm region due to the chiral sugar at N1 or N9
and due to the helical arrangement of the bases [39]. The
CD spectrum of free CT DNA in UV region (Fig. 5) has a
positive band with maximum at 275 nm attributable to base
stacking and a negative band with maximum at 246 nm due
to helicity, which is characteristic for right-handed B-form
of DNA [40]. These peaks are sensitive to the binding of

904
Medicinal Chemistry Research (2021) 30:897–912
Experimental procedures
Chemistry
All reagents used in the synthesis were obtained commercially
and were used without further purification, unless otherwise
specified. The reactions were monitored by thin-layer chro-
matography (TLC) using TLC-sheets ALUGRAM-SIL G/
UV254 (Macherey Nagel, Germany). Purification by flash
chromatography was performed using Silica gel 60 Å
(0.0040–0.063 mm, Merck, Germany) with the indicated
eluent. Melting points of all the synthesized derivatives were
determined by open-capillary tube method on digital melting
point apparatus (Electrothermal). NMR spectra were recorded
at r.t. on a VNMRS spectrometer (Varian) operating at
Fig. 5 CD spectra of CT DNA (4.4 × 10−4 M) in the absence (black
line) and presence 12b (green dashed line) and 1h (red dotted line) in
0.01 M Tris buffer (pH = 7.3, 24 °C)
unwinding of the DNA helix. We have noticed only minimal
changes in CD spectra. This fact provides further evidence
of groove binding nature of investigated chalcones.
1
600 MHz for H and 150 MHz for 13C at 299.15 K and on
Varian Mercury Plus (Varian) 400 MHz operating at 400 MHz
1
for H and 100 MHz for 13C. Chemical shifts (δ in ppm) are
given from internal solvent, DMSO-d6 or CDCl3. The infrared
spectra were recorded in the range 4000–500 cm−1 on
IRAffinity-1 FTIR Spectrophotometer (Shimadzu) using the
KBr method or on FTIR Spectrometer Tensor 27 (Bruker
Optik GmbH, Germany). High-resolution mass spectra were
determined using a the Synapt G2-Si Mass Spectrometer
(Waters, Manchester, UK), according the procedure described
in literature [43] with the following modifications: the samples
were dissolved in chloroform or MeOH (1 mg mL−1) and
diluted 1000-fold. The atmospheric solid analysis probe was
dipped into the sample solution, placed into the ion source and
analyzed in full scan mode. The probe was kept at a constant
temperature of 450 °C for 2 min.
Conclusion
In summary, two series of chalcones containing 1-
methoxyindole unit were synthesized and characterized.
The nucleophilic substitution reaction on the 1-
methoxyindole nucleus accompanying the condensation
reaction was used to prepare the third series of compounds:
3-(2-alkoxyindol-3-yl)-1-(4-fluorophenyl)prop-2-en-1-ones.
The obtained results of antiproliferative activity confirmed
the assumption of suitability of preparation of 1-
methoxyindole hybrid chalcones as antiproliferative
agents. Most of the tested compounds showed activity
against human leukemic T cell lymphoma Jurkat with IC50
below 15 µM. Compounds 12b, 12c, 15h, 16b displayed
most prominent antiproliferative activity against human
colon cancer cell line HCT116 with IC50 < 8 µM and weak
toxicity against a mouse embryo fibroblast cell line 3T3
(IC50 > 100 µM). In addition, chalcone 12b was also effec-
tive against other cancer cell lines MCF-7 and Jurkat with
IC50 < 7 µM and A549 with IC50 = 11.2 µM. Of the series
tested, the chalcones 12b and 15h provided the best balance
between high antiproliferative potency and low cytotoxicity
and were selected for biochemical spectroscopic studies.
The present study also demonstrated binding interaction of
CT DNA and BSA with novel chalcone derivatives using a
variety of spectroscopic methods. The results obtained from
fluorescence titration experiments suggest that the investi-
gated chalcones interact with CT DNA through minor
groove binding mode and that these drugs could strongly
quench the intrinsic fluorescence of BSA (Ksv constants are
in the range from 2.19 × 105 M−1 to 2.54 × 105 M−1). The
results of this study suggest that the 1-methoxyindolyl
chalcone hybrids are potential candidates for future devel-
opment of therapeutic agents against colon cancer.
General procedure for 1-(1-metoxyindol-3-yl)prop-2-
en-1-ones (Set A)
(A) Base-catalyzed Claisen–Schmidt condensation: To a
stirred solution of 3-acetyl-1-methoxyindole (10, 0.2 mmol)
in EtOH or iso-PrOH (2 ml) was added KOH (0.2 ml,
50% solution in H2O) and benzaldehyde derivative 11
(0.2 mmol). The mixture was stirred at r.t. After completion
of the reaction, reaction mixture was acidified with 1 M HCl
to pH 4, extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4 and evaporated. The
product was purified by column chromathography on SiO2
and then crystalized to yield the chalcones.
(B) Acid-catalyzed Claisen–Schmidt condensation: To a
stirred solution of the 3-acetyl-1-methoxyindole (10,
0.2 mmol) and substituted benzaldehyde (11, 0.2 mmol) in
anhydrous EtOH (2 ml) thionyl chloride (0.4 mmol) was
added. The solution turned deep red immediately. After
completion of the reaction, reaction mixture was poured
into H2O, extracted with EtOAc, organic layer washed with
brine and after drying over Na2SO4 evaporated. Chalcone

Medicinal Chemistry Research (2021) 30:897–912
905
was separated by column chromathography on SiO2 and
then crystalized.
7.50–7.48 (m, 1H, H-7′), 7.38 (td, 1H, J 7.2, 1.4 Hz, H-6′),
7.35 (td, 1H, J 7.2, 1.3 Hz, H-5′), 7.22 (d, 1H, J 15.5 Hz, H-
2), 6.87 (s, 2H, H-2″, H-6″), 4.21 (s, 3H, N-OCH3), 3.94 (s,
6H, OCH3), 3.90 (s, 3H, OCH3). 13C (CDCl3, 100 MHz):
184.2 (C, C-1), 153.6 (2 C, C-3″,5″), 141.9 (CH, C-3),
140.2 (C, C-4″), 132.6 (C, C-7′a), 130.9 (C, C-1″), 128.4
(CH, C-2′), 124.5 (CH, C-6′), 123.4 (CH, C-5′), 123.3 (CH,
C-4′), 123.2 (C, C-3′a), 123.0 (CH, C-2), 113.9 (C, C-3′),
108.6 (CH, C-7′), 105.6 (2CH, C-2″,6″), 66.9 (CH3, N-
OCH3), 61.2 (CH3, OCH3), 56.4 (2CH3, OCH3). IR (KBr)
νmax 3127, 2945, 1638, 1582, 1506, 1450, 1420, 1339,
1268, 1243, 1125, 829, 756 cm−1. HRMS: m/z [M + H]+:
368.1498 for C21H22NO5 (calcd. 368.1498).
(2E)-3-(3″,5″-dimethoxyphenyl)-1-(1′-methoxy-1′H-
indol-3′-yl)prop-2-en-1-one (12a)
Procedure B: 22 h; 52%. Rf = 0.43 (hexane/EtOAc 2:1);
1
light-yellow crystals; mp 141–142 °C (CH2Cl2/hexane). H
(CDCl3, 400 MHz): 8.53–8.51 (m, 1H, H-4′), 8.06 (s, 1H,
H-2′), 7.73 (d, 1H, J 15.6 Hz, H-3), 7.50–7.49 (m, 1H, H-
7′), 7.38 (td, 1H, J 7.2, 1.4 Hz, H-6′), 7.34 (td, 1H, J 7.2,
1.3 Hz, H-5′), 7.29 (d, 1H, J 15.5 Hz, H-2), 6.79 (d, 2H,
J 2.3 Hz, H-2″,H-6″), 6.52 (t, 1H, J 2.2 Hz, H-4″), 4.21 (s,
3H, N-OCH3), 3.85 (s, 6H, OCH3). 13C (CDCl3, 100 MHz):
184.2 (C, C-1), 161.2 (C, C-3″, C-5″), 141.7 (CH, C-3),
137.3 (C, C-1″), 132.6 (C, C-7′a), 128.5 (CH, C-2′), 124.4
(CH, C-6′), 124.2 (CH, C-5′), 123.4 (CH, C-2), 123.3 (CH,
C-4′), 123.3 (C, C-3a′), 113.9 (CH, C-3′), 108.6 (CH, C-7′),
106.3 (2CH, C-2″,6″), 102.3 (CH, C-4″), 66.9 (CH3, N-
OCH3), 55.6 (CH3, OCH3). IR (KBr) νmax 3139, 2942,
2833, 1648, 1596, 1507, 1455, 1379, 1331, 1240, 1205,
1160, 1070, 972, 823, 740 cm−1. HRMS: m/z [M + H]+:
338.1395 for C20H20NO4 (calcd. 338.1392).
(2E)-3-(4″-bromophenyl)-1-(1′-methoxy-1′H-indol-3′-
yl)prop-2-en-1-one (12d)
Procedure A: iso-PrOH; 7 h; 58%. Rf = 0.649 (hexane/acetone
1:1); light-yellow crystals; mp 158–159 °C (CH2Cl2/hexane).
1H (CDCl3, 400 MHz): 8.53–8.50 (m, 1H, H-4′), 8.06 (s, 1H,
H-2′), 7.75 (d, 1H, J 15.6 Hz, H-3), 7.55 (d, 2H, J 8.6, H-3″,
H-5″), 7.51 (d, 2H, J 8.6, Hz, H-2″, H-6″), 7.50-4.48 (m, 1H,
H-7′), 7.38 (1H, td, J 7.2, 1.5 Hz, H-6′), 7.35 (1H, td, J 7.2,
1.4 Hz, H-5′), 7.32 (d, 1H, J 15.6 Hz, H-2), 4.21 (s, 3H,
OCH3). 13C (CDCl3, 100 MHz): 183.9 (C, C-1), 140.3 (CH,
C-3), 134.3 (C, C-1″), 132.6 (C, C-7′a), 132.3 (2CH, C-3″,5″),
129.7 (2CH, C-2″,6″), 128.5 (CH, C-2′), 124.5 (CH, C-6’),
124.3 (C, C-4″), 124.2 (CH, C-2), 123.5 (CH, C-5’), 123.3
(CH, C-4′), 123.2 (C, C-3′a), 113.9 (C, C-3′), 108.7 (CH, C-
7′), 67.0 (CH3, OCH3). IR (KBr) νmax 3046, 2982, 2942,
1653, 1595, 1516, 1487, 1401, 1380, 1330, 1059, 1006, 943,
819, 731 cm−1. HRMS: m/z [M + H]+: 356.0287 for
(2E)-1-(1′-methoxy-1′H-indol-3′-yl)-3-(2″,4″,6″-
trimethoxyphenyl)prop-2-en-1-one (12b)
Procedure A: EtOH; 24 h; 16%. Procedure B: 10 min;
63%. Rf 0.32 (hexane/EtOAc 3:2); light-yellow crystals; mp
172–173 °C (CH2Cl2/hexane). 1H (CDCl3, 400 MHz,):
8.55–8.53 (m, 1H, H-4′), 8.25 (d, 1H, J 15.8 Hz, H-3), 7.99
(s, 1H, H-2′), 7.72 (d, 1H, J 15.8 Hz, H-2), 7.48-7.46 (m
1H, H-7′), 7.34 (td, 1H, J 7.3, 1.22 Hz, H-6′), 7.31 (td, 1H, J
7.3, 1.4 Hz, H-5′), 6.16 (s, 2H, H-3″, H-5″), 4.18 (3H, s, N-
OCH3), 3.93 (6H, s, OCH3), 3.86 (s, 3H, OCH3). 13C
(CDCl3, 100 MHz): 186.5 (C, C-1), 162.7 (C, C-4″), 161.6
(2 C, C-2″,C-6″), 132.9 (CH, C-3), 132.5 (C, C-7′a), 128.1
(CH, C-2′), 123.9 (CH, C-6′), 123.9 (CH, C-2), 123.3 (C, 3′
a), 123.3 (CH, C-4′), 122.9 (CH, C-5′), 114.5 (C, C-3′),
108.5 (CH, C-7′), 106.8 (C, C-1″), 90.8 (2CH, C-3″, C-5″),
66.8 (CH3, N-OCH3), 56.0 (2CH3, OCH3), 55.5 (CH3,
OCH3). IR (KBr) νmax 2942, 2836, 1638, 1602, 1571, 1508,
C18H15NO2Br calcd. 356.0286).
(
(2E)-1,3-bis(1-methoxy-1H-indol-3-yl)prop-2-en-1-
one (13)
Procedure B: 1-Methoxyindol-3-carbaldehyde (0.2 mmol)
was used as the aldehyde; 4 h; 52%. Rf 0.20 (hexane/acet-
one 4:1); yellow solid; mp 75–76 °C. 1H (CDCl3,
400 MHz): 8.57–8.55 (m, 1H, H-4′), 8.08 (s, 1H, H-2′),
8.03 (d, 1H, J 15.6 Hz, H-3), 8.03–8.00 (m, 1H, H-4″), 7.67
(s, 1H, H-2″), 7.51–7.48 (m, 2H, H-7″, H-7′), 7.38 (d, 1H, J
15.4 Hz, H-2), 7.39–7.29 (m, 4H, H-5′, H-6′, H-5″, H-6″),
4.22 (s, 3H, OCH3), 4.16 (s, 3H, OCH3). 13C (CDCl3,
100 MHz): 184.8 (C, C-1), 134.7 (CH, C-3), 133.1 (C, C-7″
a), 132.6 (C, C-7′a), 127.9 (CH, C-2′), 126.3 (CH, C-2″),
124.2 (CH, C-6′), 123.7 (CH, C-6″), 123.3 (CH, C-4′),
123.2 (C, C-3′a), 123.1 (CH, C-5′) 122.6 (C, C-3″a), 121.9
(CH, C-5″), 120.8 (CH, C-4″), 120.1 (CH, C-2), 114.2 (C,
C-3′), 109.7 (C, C-3″), 109.1 (CH, C-7″), 108.6 (CH, C-7′),
66.9 (CH3, OCH3), 66.6 (CH3, OCH3). IR: HRMS: m/z
[M + H]+: 347.1398 for C21H19N2O3 (calcd. 347.1396).
1460, 1376, 1327, 1212, 1155, 1121, 1064, 977, 743 cm−1
.
HRMS: m/z [M + H]+: 368.1498 for C21H22NO5 (calcd.
368.1498).
(2E)-1-(1′-methoxy-1′H-indol-3′-yl)-3-(3″,4″,5″-
trimethoxyphenyl)prop-2-en-1-one (12c)
Procedure A: iso-PrOH; 24 h; 60%. Procedure B: 48 h;
42%. Rf = 0.53 (hexane/EtOAc 1:1); mp 196–197 °C
(CH2Cl2/hexane). 1H (CDCl3, 400 MHz): 8.54–8.51 (m,
1H, H-4′), 8.07 (s, 1H, H-2′), 7.74 (d, 1H, J 15.5 Hz, H-3),

906
Medicinal Chemistry Research (2021) 30:897–912
General procedure for 3-(1-metoxyindol-3-yl)prop-2-
en-1-ones (Set B)
(CDCl3, 400 MHz): 7.89 (d, 1H, J 8.0 Hz, H-4′), 7.54 (s,
1H, H-2′), 7.48 (d, 1H, J 16.1 Hz, H-3), 7.45 (d, 1H,
J 8.0 Hz, H-7′), 7.33 (t, 1H, J 8.4 Hz, H-4″), 7.32 (ddd, 1H,
J 8.0, 7.1, 1.1 Hz, H-6′), 7.24 (ddd, 1H, J 8.0, 7.1, 1.1 Hz,
H-5′), 6.99 (d, 1H, J 16.1 Hz, H-2), 6.63 (d, 2H, J 8.4, H-3″,
H-5″), 4.11 (s, 3H, N-OCH3), 3.79 (s, 6H OCH3). 13C
(CDCl3, 100 MHz): 182.3 (C, C-1), 157.7 (2 C, C-2″, C-6″),
138.6 (CH, C-3), 133.1 (C, C-7′a), 130.5 (CH, C-4″), 126.6
(CH, C-2′), 125.6 (CH, C-2), 123.8 (CH, C-6′), 122.6 (C, C-
3′a), 122.1 (CH, C-5′), 121.0 (CH, C-4′), 119.1 (C, C-1″),
109.4 (C, C-3′), 109.0 (CH, C-7′), 104.3 (2CH, C-3″, C-5″),
66.6 (CH3, N-OCH3), 56.2 (2CH3, OCH3). IR νmax 3088,
2949, 2842, 1610, 1589, 1472, 1433, 1240, 1103, 1059,
953, 741 cm−1. HRMS: m/z [M + H]+: 338.1393 for
C20H20NO4 (calcd. 338.1392).
(A) Base-catalyzed Claisen–Schmidt condensation: To a
stirred solution of substituted acetophenone 14 (0.2 mmol)
in EtOH or iso-PrOH (2 mL) KOH (0.2 mL, 50% solution
in H2O) and 1-methoxyindol-3-carbaldehyde (8, 0.2 mmol)
were added. The mixture was stirred at r.t. After completion
of the reaction, reaction mixture was acidified with 1 M HCl
to pH 4, extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4 and evaporated. The
product was purified by column chromathography on SiO2
and then crystalized to yield the chalcones.
(B) Acid-catalyzed Claisen-Smidt condensation: To a
stirred solution of substituted acetophenone 14 (0.2 mmol)
and 1-methoxyindol-3-carbaldehyde (8, 0.2 mmol) in
anhydrous EtOH (2 mL) with molecule sieves (3 Å) thionyl
chloride (0.4 mmol) was added. The solution turned deep
red immediately. After completion of the reaction, reaction
mixture was poured into water, extracted with EtOAc, the
organic layer was washed with brine and after drying over
anhydrous Na2SO4, filtered and evaporated. Product was
separated by column chromathography on SiO2 and then
crystalized.
(2E)-1-(2″-Hydroxy-6″-methoxyphenyl)-3-(1′-
methoxy-1′H-indol-3′-yl)prop-2-en-1-one (15c)
To a solution of 1-methoxyindol-3-carboxaldehyde (8;
0.070 g; 0.4 mmol) and 2-hydroxy-6-methoxyacetofenone
(14c; 0.066 g; 0.4 mmol) in EtOH (2 ml) was added
dropwise piperidine (0.04 ml; 0.4 mmol). Reaction mix-
ture was heated at 50 °C for 21 h. After evaporation of
solvent product was isolated by column chromathography
on SiO2 (eluent hexane/EtOAc 2:1) with the yield 28%.
Rf 0.66 (hexane/EtOAc 2:1); colourless powder; mp
118–121 °C (EtOAc /hexane). 1H (CDCl3, 400 MHz):
13.58 (s, 1H, OH), 8.08 (d, 1H, J 15.6 Hz, H-3), 8.02 (d,
1H, J 15.6 Hz, H-2), 8.01 (d, 1H, J 8.1 Hz, H-4′), 7.66 (s,
1H, H-2′), 7.51 (d, 1H, J 8.1 Hz, H-7′), 7.37 (ddd, 1H,
J 8.1, 7.1, 1.1 Hz, H-6′), 7.35 (t, 1H, J 8.2 Hz, H-4″), 7.31
(ddd, 1H, J 8.1, 7.2, 1.1 Hz, H-5′), 6.63 (dd, 1H, J 8.4,
0.9 Hz, H-3″), 6.45 (dd, 1H, J 8.3, J 0.8 Hz, H-5″), 4.16
(s, 3H, N-OCH3), 4.01 (s, 3H, OCH3). 13C (CDCl3,
100 MHz): 194.1 (C, C-1), 165.2 (C, C-2″), 161.0 (C, C-
6″), 137.1 (CH, C-3), 135.4 (CH, C-4″), 133.3 (C, C-7′a),
127.7 (CH, C-2′), 123.9 (CH, C-6′), 123.8 (CH, C-2),
122.5 (C, C-3′a), 122.2 (CH, C-5′), 121.0 (CH, C-4′),
112.2 (C, C-1″), 111.2 (CH, C-3″), 110.3 (C, C-3′), 109.2
(CH, C-7′), 101.7 (CH, C-5″), 66.7 (CH3, N-OCH3), 56.1
(CH3, OCH3). IR νmax 3103, 2947, 1622, 1577, 1548,
1456, 1371, 1234, 1085, 1041, 858, 735 cm−1. HRMS: m/
z [M + H]+: 324.1238 for C19H18NO4 (calcd.324.1236).
(2E)-3-(1′-methoxy-1′H-indol-3′-yl)-1-(4″-
methoxyphenyl)prop-2-en-1-one (15a)
Procedure B: There was used
2
eq. of 4-
methoxyacetophenone because of close Rf values of pro-
duct and unreacted aldehyde 6; 40 min; 65%. Rf 0.41
(hexane/EtOAc 2:1); light-yellow crystals; mp 94–96 °C
(CH2Cl2/hexane). 1H (CDCl3, 400 MHz): 8.07 (d, 2H, J 8.9,
H-2″, H-6″), 8.02 (d, 1H, J 15.5 Hz, H-3), 8.00 (dd, 1H, J
7.8, 1.0 Hz, H-4′), 7.68 (s, 1H, H-2′), 7.56 (d, 1H, J 15.5 Hz,
H-2), 7.50 (dd, 1H, J 7.9, 1.0 Hz, H-7′), 7.36 (ddd, 1H, J 8.1,
7.3, 1.1 Hz, H-6′), 7.31 (ddd, 1H, J 8.1, 7.1, 1.2 Hz, H-5′),
7.00 (d, 2H, J 8.8 Hz, H-3″, 5″), 4.16 (s, 3H, N-OCH3), 3.90
(s, 3H, OCH3). 13C (CDCl3, 100 MHz): 189.0 (C, C-1),
163.2 (C, C-4″), 137.2 (CH, C-3), 133.1 (C, C-7′a), 131.8
(C, C-1″), 130.7 (2CH, C-2″, C-6″), 126.9 (CH, C-2′), 123.8
(CH, C-6′), 122.6 (C, C-3′a), 122.1 (CH, C-5′), 120.9 (CH,
C-4′), 118.1 (CH, C-2), 113.9 (2CH, C-3″, C-5″), 109.8 (C,
C-3′), 109.1 (CH, C-7′), 66.6 (CH3, N-OCH3), 55.6 (CH3,
OCH3). IR νmax 3103, 3005, 2935, 1652, 1584, 1564, 1506,
1365, 1277, 1167, 978, 948, 820, 737 cm−1. HRMS: m/z [M
+ H]+: 308.1289 for C19H18NO3 (calcd. 308.1287).
(2E)-3-(1′-Methoxy-1′H-indol-3′-yl)-1-(2″,4″,6″-
trimethoxyphenyl)prop-2-en-1-one (15d)
(2E)-1-(2″,6″-Dimethoxyphenyl)-3-(1′-methoxy-1′H-
indol-3′-yl)prop-2-en-1-one (15b)
Procedure B: 6 h; 33%. Rf 0.42 (hexane/acetone 2:1); red
crystals; mp 47–49 °C (acetone/hexane). 1H (CDCl3,
600 MHz): 7.89 (d, 1H, J 8.0 Hz, H-4′), 7.54 (s, 1H, H-2′),
7.53 (d, 1H, J 16.2 Hz, H-3), 7.45 (d, 1H, J 8.1 Hz, H-7′),
7.32 (t, 1H, J 7.6 Hz, H-6′), 7.23 (t, 1H, J 7.2 Hz, H-5′),
Procedure A: EtOH; 14 h; 34%. Rf 0.21 (hexane/acetone
1
2:1); yellow crystals; mp 146–148 °C (EtOAc/hexane). H

Medicinal Chemistry Research (2021) 30:897–912
907
6.99 (d, 1H, J 16.0 Hz, H-2), 6.18 (s, 2H, H-3″, H-5″), 4.11
(s, 3H, N-OCH3), 3.87 (s, 3H, OCH3), 3.77 (s, 6H, OCH3).
13C (CDCl3, 150 MHz): 194.6 (C, C-1), 162.2 (C, C-4″),
158.9 (2 C, C-2″, C-6″), 137.8 (CH, C-3), 133.0 (C, C-7′a),
126.4 (CH, C-2′), 126.0 (CH, C-2), 123.8 (CH, C-6′), 122.6
(C, C-3′a), 122.0 (CH, C-5′), 120.9 (CH, C-4′), 112.3 (C, C-
1″), 109.5 (C, C-3′), 108.9 (CH, C-7′), 90.9 (2CH, C-3″, C-
5″), 66.6 (CH3, N-OCH3), 56.1 (2CH3, OCH3), 55.6 (CH3,
OCH3). IR νmax 2927, 2853, 1599, 1585, 1454, 1414, 1226,
1203, 1155, 1122, 1026, 740 cm−1. HRMS: m/z [M + H]+:
368.1496 for C21H22NO5 (calcd. 368.1498).
HRMS: m/z [M + H]+: 356.0284 for C18H15NO2Br
(calcd. 356.0286).
(2E)-1-(2″-Fluorophenyl)-3-(1′-methoxy-1′H-indol-3′-
yl)prop-2-en-1-one (15g)
Procedure A: iso-PrOH; 1 h; 50%. Rf 0.48 (hexane/acetone
1
2:1); yellow crystals; mp 110–112 °C (CH2Cl2/hexane). H
(DMSO-d6, 600 MHz): 8.47 (s, 1H, H-2′), 7.98 (d, 1H, J 8.0,
H-4′), 7.84 (dd, 1H, J 15.7, 0.9 Hz, H-3), 7.76 (dd, 1H, J 7.6,
1.7 Hz, H-6″), 7.66-7.62 (m, 1H, H-4″), 7.58 d (d, 1H, J 8.1,
H-7′), 7.35 – 7.40 (m, 3H, H-6′, H-3″,H-5″), 7.33 (dd, 1H, J
15.8, 2.2 Hz, H-2), 7.29 (ddd, 1H, J 8.1, 7.2, 1.0 Hz, H-5′),
4.15 (s, 3H, OCH3). 13C (DMSO-d6,151 MHz): 188.3 (C, d,
JCF.2.0 Hz, C-1), 160.1 (CH, JCF.250.1 Hz, C-2″), 138.5
(CH, C-3), 133.6 (C, d, JCF.8.7 Hz, C-4″), 132.5 (C, C-7′a),
130.4 (CH, d, JCF.2.9 Hz, C-6″), 129.8 (CH, C-2′), 127.5 (C,
d, JCF.13.9 Hz, C-1″), 124.8 (CH, d, JCF.3.3 Hz, C-5″), 123.8
(CH, C-6′), 122.2 (CH, C-5′), 121.7 (C, C-3′a), 120.9 (CH, d,
JCF.4.7, C-2), 120.4 (CH, C-4′), 116.5 (CH, d, JCF.22.6 Hz,
C-3″), 109.2 (CH, C-7′), 108.2 (C, C-3′), 66.7 (CH3, OCH3).
IR (KBr) νmax 3098, 1629, 1613, 1607, 1570, 1505, 1448,
1376, 1313, 1292, 1244, 1234, 1108, 1081, 976, 951, 768,
741 cm−1. HRMS: m/z [M + H]+: 296.1088 for C18H15NO2F
(calcd. 296.1087).
(2E)-3-(1′-Methoxy-1′H-indol-3′-yl)-1-(3″,4″,5″-
trimethoxyphenyl)prop-2-en-1-one (15e)
Procedure A: iso-PrOH; 45 min; 26%. Procedure B: 3 h;
27%. Rf 0.3 (hexane/acetone 2:1); yellow crystals; mp
120–122 °C (CH2Cl2/hexane). 1H (CDCl3, 400 MHz): 8.02
(d, 1H, J 15.5 Hz, H-3), 7.97 (dt, 1H, J 7.8, 0.9 Hz, H-4′),
7.71 (s, 1H, H-2′), 7.51 (dd, 1H, J 8.1, 1.0 Hz, H-7′), 7.47
(d, 1H, J 15.5 Hz, H-2), 7.37 (ddd, 1H, J 8.1, 7.3, 1.2 Hz,
H-6′), 7.31 (ddd, J 7.9, 7.2, 1.2 Hz, H-5′), 7.30 (s, 2H, H-
2″, H-6″), 4.17 (s, 3H, N-OCH3), 3.97 (s, 6H, OCH3), 3.94
(s, 3H, OCH3). 13C (CDCl3, 100 MHz): 189.6 (C, C-1),
153.3 (2 C, C-3″, C-5″), 142.2 (C, C-4″), 138.0 (CH, C-3),
134.5 (C, C-1″), 133.1 (C, C-7′a), 127.0 (CH, C-2′), 124.0
(CH, C-6′), 122.7 (C, C-3′a), 122.2 (CH, C-5′), 120.8 (CH,
C-4′), 118.2 (CH, C-2), 109.7 (C, C-3′), 109.2 (CH, C-7′),
106.1 (2CH, C-2″, C-6″), 66.7 (CH3, N-OCH3), 61.1 (CH3,
OCH3), 56.6 (2CH3, OCH3). IR (KBr) νmax 3215, 2991,
2938, 1646, 1588, 1559, 1503, 1466, 1455, 1415, 1343,
1275, 1234, 1160, 1130, 810, 732 cm−1. HRMS: m/z [M +
H]+: 368.1500 for C21H22NO5 (calcd.368.1498).
(2E)-1-(4″-Fluorophenyl)-3-(1′-methoxy-1′H-indol-3’-
yl)prop-2-en-1-one (15h)
Procedure A: iso-PrOH; 1.5 h; 37%. Procedure B: 4 h; 52%.
Rf 0.58 (hexane/acetone 2:1); yellow crystals; mp 78–80 °C
(CH2Cl2/hexane). 1H (CDCl3, 600 MHz): 8.08 (dd, J 8.8,
5.4 Hz, H-2″, H-6″), 8.03 (d, 1H, J 15.5 Hz, H-3), 7.98 (dt, 1H,
J 8.0, 1.1 Hz, H-4′), 7.69 (s, 1H, H-2′), 7.51 (d, 1H, J 15.5 Hz,
H-2), 7,51 (dt, 1H, J 8.2, 1.1 Hz, H-7′), 7.37 (ddd, 1H, J 8.2,
7.1, 1.1 Hz, H-6′), 7.32 (ddd, 1H, J 8.1, 7.2, 1.1 Hz, H-5′), 7.18
(t, 2H, J 8.6 Hz, H-3″, H-5″), 4.17 (s, 3H, OCH3). 13C (CDCl3,
150 MHz): 189.0 (C, C-1), 165.5 (C, d, JCF.253.4 Hz, C-4″),
138.3 (CH, C-3), 135.4 (C, d, JCF.3.0 Hz, C-1″), 133.2 (C, C-7′
a), 131.0 (2CH, d, JCF.9.1 Hz, C-2″, C-6″), 127.3 (CH, C-2′),
124.0 (CH, C-6′), 122.6 (C, C-3′a), 122.3 (CH, C-5′), 120.9
(CH, C-4′), 117.9 (CH, C-2), 115.7 (2CH, d, JCF.21.8 Hz, C-
3″, C-5″), 109.7 (C, C-3′), 109.2 (CH, C-7′), 66.7 (CH3,
OCH3). IR (KBr) νmax 3094, 1652, 1601, 1584, 1506, 1375,
1280, 1211, 1158, 957, 809, 725 cm−1. HRMS: m/z [M + H]+:
296.1085 for C18H15NO2F (calcd. 296.1087).
(2E)-1-(4″-Bromophenyl)-3-(1’-methoxy-1’H-indol-3’-
yl)prop-2-en-1-one (15f)
Procedure A: iso-PrOH; 1 h; 27%. Procedure B: 5 h;
35%. Rf 0.58 (hexane/acetone 2:1); yellow crystals; mp
105–107 °C (CH2Cl2/hexane). 1H (CDCl3, 400 MHz):
8.03 (d, 1H, J 15.5 Hz, H-3), 7.97 (d, 1H, J 7.9 Hz, H-4′),
7.91 (d, 2H, J 8.5 Hz, H-2″, H-6″), 7.70 (s, 1H, H-2′),
7.65 (d, 2H, J 8.6 Hz, H-3″, H-5″), 7,50 (d, 1H, J 8.1 Hz,
H-7′), 7.47 (d, 1H, J 15.5 Hz, H-2), 7.37 (ddd, 1H, J 8.1,
7.2, 0.9 Hz, H-6′), 7.32 (ddd, 1H, J 7.9, 7.1, 1.0 Hz, H-
5′), 4.16 (s, 3H, OCH3). 13C (CDCl3, 101 MHz): 189.5
(C, C-1), 138.7 (CH, C-3), 137.8 (C, C-1″), 133.2 (C, C-
7′a), 131.9 (2CH, C-3″, C-5″), 130.0 (2CH, C-2″, C-6″),
127.4 (CH, C-2′), 127.3 (C, C-4″), 124.0 (CH, C-6′),
122.6 (C, C-3′a), 122.3 (CH, C-5′), 120.9 (CH, C-4′),
117.6 (CH, C-2), 109.7 (C, C-3′), 109.2 (CH, C-7′), 66.6
(CH3, N-OCH3). IR νmax 3099, 2947, 1647, 1591, 1581,
1560, 1375, 1278, 1244, 1007, 950, 804, 727 cm−1
General procedure for 3-(2′-alkoxy-1’H-indol-3′-yl)-
1-(4″-fluorophenyl)prop-2-en-1-ones
To a stirred solution of 4-fluoroacetophenone (14h,
0.5 mmol) in alcohol (5 ml) was added 50% KOH in H2O
(0.5 ml from a) and 1-methoxyindol-3-carbaldehyde

908
Medicinal Chemistry Research (2021) 30:897–912
(8, 0.5 mmol). The reaction mixture was stirred at r.t.
After completion of the reaction, reaction mixture was
cooled to 0–10 °C, acidified with 1 M HCl to pH 4. Then
the precipitated product was filtered, washed with H2O,
dried and crystalized. When the product was not pre-
cipitated, the mixture was extracted with EtOAc and the
extract was washed with brine. After drying over anhy-
drous Na2SO4 filtrate was evaporated to dryness and
product was purified by column chromathography on
SiO2 and then crystalized to yield the chalcones.
(2E)-1-(4″-Fluorophenyl)-3-(2′-propoxy-1′H-indol-3′-
yl)prop-2-en-1-one (16c)
Reaction time: 25 h; yield: 32%, Rf 0.32 (hexane/acetone
1
2:1); orange crystals; mp 177–179 °C (acetone/hexane). H
(DMSO-d6, 400 MHz): 8.13 (dd, 2H, J 8.4, 5.8 Hz, H-2″,
H-6″), 8.03 (d, 1H, J 15.2 Hz, H-3), 7.85 (d, 1H, J 7.8 Hz,
H-4′), 7.34 (t, J 8.8 Hz, 2H, H-3″, H-5″), 7.33 (d, 1H, J
15.3 Hz, H-2), 7,32 (d, 1H, J 7.8 Hz, H-7′), 7.17 (t, 1H, J
7.5 Hz, H-6′), 7.11 (t, 1H, J 7.5 Hz, H-5′), 4.37 (t, 2H, J
6.5 Hz, OCH2CH2CH3), 1.86 (sextet, 2H, J 6.9 Hz,
OCH2CH2CH3), 1.05 (t, 3H, J 7.4 Hz, OCH2CH2CH3). 13C
(DMSO-d6, 100 MHz): 186.8 (C, C-1), 164.4 (C, d,
JCF.251.8 Hz, C-4″), 157.1 (C, C-2′), 136.5 (CH, C-3),
135.7 (C, d, JCF.2.8 Hz, C-1″), 132,4 (C, C-7′a), 130.6
(2CH, d, JCF.9.1 Hz, C-2″, C-6″), 125.1 (C, C-3′a), 121.7
(CH, C-5′), 121.2 (CH, C-6′), 119.1 (CH, C-4′), 115.5 (CH,
d, JCF.21.8 Hz, C-3″,C-5″), 111.5 (CH, C-7′), 110.8 (CH, C-
2), 94.3 (C, C-3′), 73.0 (CH2, OCH2CH2CH3), 22.2 (CH2,
OCH2CH2CH3), 10.2 (CH3, OCH2CH2CH3). IR (KBr) νmax
3071, 2974, 1624, 1591, 1570, 1506, 1489, 1337, 1285,
1217, 1153, 1044, 1011, 817, 744 cm−1. HRMS: m/z [M +
H]+: 324.1403 for C20H19NO2F (calcd. 324.1400).
(2E)-1-(4″-Fluorophenyl)-3-(2′-methoxy-1′H-indol-3′-
yl)prop-2-en-1-one (16a)
Reaction time: 5 days; yield: 32%. Rf 0.43 (hexane/acetone
1
3:2); orange crystals; mp 177–179 °C (acetone/hexane). H
(DMSO-d6, 600 MHz): 12.11 (s, NH), 8.14 (dd, 2H, J 8.8,
5.6 Hz, H-2″, H-6″), 8.02 (d, 1H, J 15.2 Hz, H-3), 7.86 (d,
1H, J 7.8 Hz, H-4′), 7,34 (d, 1H, J 7.7 Hz, H-7′), 7.34 (t, J
8.8 Hz, 2H, H-3″, H-5″), 7.33 (d, 1H, J 15.1 Hz, H-2), 7.19
(td, 1H, J 7.7, 1.1 Hz, H-5′), 7.13 (td, 1H, J 7.8, 1.1 Hz, H-
6′), 4.17 (s, 3H, OCH3). 13C (DMSO-d6, 150 MHz): 186.7
(C, C-1), 164.3 (C, d, JCF.249.8 Hz, C-4″), 157.5 (C, C-2′),
136.2 (CH, C-3), 135.6 (C, d, JCF.2.7 Hz, C-1″), 131.9 (C,
C-7′a), 130.6 (2CH, d, JCF.9.1 Hz, C-2″, C-6″), 125.1 (C, C-
3′a), 121.7 (CH, C-5′), 121.0 (CH, C-6′), 119.0 (CH, C-4′),
115.4 (2CH, d, JCF 21.6 Hz, C-3″, C-5″), 111.4 (CH, C-7′),
110.9 (CH, C-2), 93.7 (C, C-3′), 58.8 (CH3, OCH3). IR
(KBr) νmax 3215, 2937, 1638, 1595, 1560, 1533, 1506,
1489, 1344, 1219, 1065, 1017, 814, 737 cm−1. HRMS: m/z
[M + H]+: 296.1086 for C18H15NO2F (calcd. 296.1087).
(2E)-1-(4″-Fluorophenyl)-3-(2′-isopropoxy-1′H-indol-
3′-yl)prop-2-en-1-one (16d)
Reaction time: 25 h; yield: 26%. Rf 0.35 (hexane/acetone
1
2:1); orange crystals; mp 167–169 °C (acetone/hexane). H
(CDCl3, 600 MHz): 8.82 (s, NH), 8.19 (d, 1H, J 15.4 Hz, H-
3), 8.08 (dd, 2H, J 8.6, 5.6 Hz, H-2″, H-6″), 7.81 (d, 1H, J
7.9 Hz, H-4′), 7.44 (d, 1H, J 15.4 Hz, H-2), 7.29 (d, 1H,
J 8.0 Hz, H-7′), 7.25 (t, 1H, J 7.7 Hz, H-6′), 7.18 (t, 1H, J
7.6 Hz, H-5′), 7.17 (t, 2H, J 8.7 Hz, H-3″, H-5″), 4.76 (t,
1H, J 6.1 Hz, OCH(CH3)2), 1.43 (d, 6H, J 6.1 Hz, OCH
(CH3)2). 13C (CDCl3, 150 MHz): 188.8 (C, C-1), 165.3 (C,
d, JCF.252.7 Hz, C-4″), 155.1 (C, C-2′), 137.7 (CH, C-3),
135.7 (C, d, JCF.2.8 Hz, C-1″), 131.9 (C, C-7′a), 130.8 (CH,
d, JCF.9.0 Hz, C-2″, C-6″), 125.6 (C, C-3′a), 122.3 (2CH, C-
5′, C-6′), 119.7 (CH, C-4′), 115.6 (2CH, d, JCF.21.7 Hz,
C-3″, C-5″), 114.0 (CH, C-2), 111.3 (CH, C-7′), 98.5 (C, C-
3′), 77.6 (CH, OCH(CH3)2), 22.6 (2xCH3, OCH(CH3)2). IR
(KBr) νmax 3210, 2978, 2926, 1630, 1597, 1522, 1506,
1481, 1339, 1277, 1217, 1153, 1101, 1051, 978, 812,
(2E)-3-(2′-ethoxy-1′H-indol-3′-yl)-1-(4″-fluorophenyl)
prop-2-en-1-one (16b)
Reaction time: 22 h; yield: 30%. Rf 0.34 (hexane/acetone 2:1);
orange crystals; mp 174–175 °C (acetone/hexane). 1H
(DMSO-d6, 600 MHz): 12.06 (s, NH), 8.14 (dd, 2H, J 8.8,
5.6 Hz, H-2″, H-6″), 8.03 (d, 1H, J 15.2 Hz, H-3), 7.87 (d, 1H,
J 7.8 Hz, H-4′), 7.35 (d, 1H, J 15.3 Hz, H-2), 7.35 (t, J 8.7 Hz,
2H, H-3″, H-5″), 7,33 (d, 1H, J 7.7 Hz, H-7′), 7.18 (td, 1H, J
7.6, 1.1 Hz, H-6′), 7.13 (td, 1H, J 7.8, 1.1 Hz, H-5′), 4.47 (q,
2H, J 7.0 Hz, OCH2CH3), 1.47 (t, 3H, OCH2CH3). 13C
(DMSO-d6, 150 MHz): 186.7 (C, C-1), 164.3 (C, d,
JCF.250 Hz, C-4″), 156.8 (C, C-2′), 136.4 (CH, C-3), 135.6
(C, d, JCF.2.7 Hz, C-1″), 132 (C, C-7′a), 130.6 (2CH, d,
JCF.9.1 Hz, C-2″, C-6″), 124.9 (C, C-3′a), 121.7 (CH, C-5′),
121.1 (CH, C-6′), 119.1 (CH, C-4′), 115.5 (2CH, d,
JCF.21.6 Hz, C-3″, C-5″), 111.3 (CH, C-7′), 110.8 (CH, C-2),
94.2 (C, C-3′), 67.6 (CH2, OCH2CH3), 14.7 (CH3,
OCH2CH3). IR (KBr) νmax 3079, 2981, 1627, 1601, 1560,
1507, 1481, 1339, 1220, 1160, 1055, 815, 736 cm−1. HR-MS:
m/z [M + H]+: 310.1243 for C19H17NO2F (calcd. 310.1243).
739 cm−1
.
HRMS: m/z [M + H]+: 324.1399 for
C20H19NO2F (calcd. 324.1400).
(2E)-3-(2′-butoxy-1′H-indol-3′-yl)-1-(4″-fluorophenyl)
prop-2-en-1-one (16e)
Reaction time: 29 h; yield: 31%. Rf 0.35 (hexane/EtOAc
2:1); red crystals; mp 151–153 °C (acetone/hexane). 1H
(DMSO-d6, 600 MHz): 8.13 (dd, 2H, J 8.8, 5.6 Hz, H-2″,

Medicinal Chemistry Research (2021) 30:897–912
909
H-6″), 8.02 (d, 1H, J 15.2 Hz, H-3), 7.86 (d, 1H, J 7.8 Hz,
H-4′), 7.35 (t, 2H, J 8.8 Hz, H-3″, H-5″), 7.35 (d, 1H,
J 15.3 Hz, H-2), 7.33 (d, 1H, J 7.8 Hz, H-7′), 7.18 (td, 1H,
J 7.6, 1.0 Hz, H-6′), 7.12 (td, 1H, J 7.8, 1.0 Hz, H-5′), 4.42
(t, 2H, J 6.4 Hz, OCH2CH2CH2CH3), 1.83 (tt, 2H, J 7.6,
6.4 Hz, OCH2CH2CH2CH3), 1.52 (qt 2H, J 7.4, 7.6 Hz,
OCH2CH2CH2CH3), 0.98 (t, 3H, J 7.4, OCH2CH2CH2CH3).
13C (DMSO-d6, 150 MHz): 186.8 (C, C-1), 164.4 (C, d,
JCF.250.0 Hz, C-4″), 156.9 (C, C-2′), 136.5 (CH, C-3), 135.7
(C, d, JCF.2.8 Hz, C-1″), 132.2 (C, C-7′a), 130.7 (2CH, d,
JCF.9.2 Hz, C-2″,C-6″), 125.0 (C, C-3′a), 121.7 (CH, C-5′),
121.1 (CH, C-6′), 119.1 (CH, C-4′), 115.5 (2CH, d,
JCF.21.6 Hz, C-3″,C-5″), 111.3 (CH, C-7’), 110.9 (CH, C-2),
94.2 (C, C-3′), 71.4 (CH2, OCH2CH2CH2CH3), 30.7 (CH2,
OCH2CH2CH2CH3), 18.6 (CH2, OCH2CH2CH2CH3), 13.6
(CH3, OCH2CH2CH2CH3). IR (KBr) νmax 3177, 2962, 2873,
1632, 1599, 1526, 1477, 1450, 1377, 1261, 1225, 1155,
827, 741 cm−1. HRMS: m/z [M + H]+: 338.1557 for
C21H21NO2F (calcd. 338.1556).
(m, 1H, H-4′), 8.06 (d, 1H, J 15.4 Hz, H-3), 7.64 (d, 1H, J
15.4 Hz, H-2), 7.50–7.49 (m, 1H, H-7′), 7.38 (t, 2H, J 8.8 Hz,
H-3″, H-5″), 7.25 (ddd, 1H, J 7.6, 7.1, 1.5 Hz, H-6′), 7.23
(ddd, 1H, J 7.5, 7.1, 1.5 Hz, H-5′). 13C (DMSO-d6,
150 MHz): 187.3 (C, C-1), 164.6 (C, d, JCF 250.6 Hz, C-4″),
139.2 (CH, C-3), 137.5 (C, C-7′a), 135.1 (C, d, JCF.2.8 Hz,
C-1″), 133.4 (CH, C-2′), 131.0 (2CH, d, JCF.9.2 Hz, C-2″, C-
6″), 125.1 (C, C-3′a), 122.7 (CH, C-6′), 121.2 (CH, C-5′),
120.4 (CH, C-4′), 115.6 (2CH, d, JCF.21.6 Hz, C-3″, C-5″),
115.0 (CH, C-2), 112.8 (C, C-3′), 112.4 (CH, C-7′). IR (KBr)
ν
max 3257, 1648, 1630, 1596, 1561, 1536, 1432, 1360, 1226,
1046, 817, 747 cm−1. HRMS: m/z [M + H]+: 266.0981 for
C17H13NOF (calcd.266.0981).
(2E)-1-(4″-Fluorophenyl)-3-(1′-metyl-1′H-indol-3′-yl)prop-2-
en-1-one (18b)
Procedure A: EtOH; 8 h; 68%. Rf 0.38 (hexane/ethylacetate
1
2:1); yellow crystals; mp 154–156 °C (acetone/hexane). H
(DMSO-d6, 400 MHz): 8.21 (dd, J 8.8, 5.8 Hz, H-2″, H-6″),
8.12 (d, 1H, J 8.2 Hz, H-4′), 8.11 (s, H-2′), 8.02 (d, 1H,
J 15.4 Hz, H-3), 7.63 (d, 1H, J 15.4 Hz, H-2), 7.57 (d, 1H,
J 7.8 Hz, H-7′), 7.38 (t, 2H, J 8.7 Hz, H-3″, H-5″), 7.32
(ddd, 1H, J 7.6 Hz, H-6′), 7.28 (t, 1H, J 7.6 Hz, H-5′), 3.86
(s, 3H, N-CH3). 13C (DMSO-d6, 100 MHz): 187.2 (C, C-1),
164.6 (C, d, JCF 249.3 Hz, C-4″), 138.6 (CH, C-3), 138.0
(C, C-7′a), 136.8 (CH, C-2′), 135.1 (C, d, JCF.2.8 Hz, C-1″),
131.0 (2CH, d, JCF.9.2 Hz, C-2″, C-6″), 125.6 (C, C-3′a),
122.8 (CH, C-6′), 121.4 (CH, C-5′), 120.6 (CH, C-4′),
115.0 (CH, C-2), 115.6 (2CH, d, JCF.21.6 Hz, C-3″, C-5″),
111.7 (C, C-3′), 110.9 (CH, C-7′), 33.1 (CH3, N-CH3). IR
(KBr) νmax 3094, 1652, 1601, 1584, 1506, 1375, 1280,
1211, 1158, 957, 809, 725 cm−1. HRMS: m/z [M + H]+:
280.1136 for C18H15NOF (calcd. 280.1138).
(2E)-3-(2′-isobutoxy-1′H-indol-3′-yl)-1-(4″-
fluorophenyl)prop-2-en-1-one (16f)
Reaction time: 24 h; yield: 27%. Rf 0.43 (hexane/EtOAc 2:1);
orange crystals; mp 171–172 °C (acetone/hexane). 1H
(DMSO-d6, 600 MHz): 12.08 (s, NH), 8.13 (dd, 2H, J 8.8,
5.5 Hz, H-2″, H-6″), 8.04 (d, 1H, J 15.2 Hz, H-3), 7.86 (d,
1H, J 7.8 Hz, H-4′), 7.35 (t, 2H, J 8.6 Hz, H-3″, H-5″), 7.35
(d, 1H, J 15.3 Hz, H-2), 7.32 (d, 1H, J 7.9 Hz, H-7′), 7.18 (td,
1H, J 7.6, 1.1 Hz, H-6′), 7.12 (t, 1H, J 7.7, 1.0 Hz, H-5′),
4.21 (d, 2H, J 6.6 Hz, OCH2CH(CH3)2), 2.17 (nonet, 1H, J
6.6, Hz, OCH2CH(CH3)2), 1.06 (d 6H, J 6.7 Hz, OCH2CH
(CH3)2). 13C (DMSO-d6, 150 MHz): 186.7 (C, C-1), 164.3
(C, d, JCF.250.0 Hz, C-4″), 156.9 (C, C-2′), 136.3 (CH, C-3),
135.7 (C, d, JCF.2.7 Hz, C-1”), 132.0 (C, C-7′a), 130.6 (2CH,
d, JCF.9.1 Hz, C-2″, C-6″), 125.0 (C, C-3′a), 121.7 (CH, C-
5′), 121.1 (CH, C-6′), 119.0 (CH, C-4′), 115.5 (2CH, d,
JCF.21.6 Hz, C-3″, C-5″), 111.4 (CH, C-7′), 110.9 (CH, C-2),
94.0 (C, C-3′), 77.3 (CH2, OCH2CH(CH3)2), 27.9 (CH,
OCH2CH(CH3)2), 18.7 (2xCH3, OCH2CH(CH3)2). IR (KBr)
Antiproliferative activity
Hela cells (human cervical carcinoma), HCT116 (human
colorectal carcinoma), and Jurkat (human leukemic T cell
lymphoma) were cultured in RPMI 1640 medium (Bio-
sera, Kansas City, MO, United States). MCF-7 (human
breast adenocarcinoma), MDA-MB-231 (human mam-
mary gland adenocarcinoma), A-549 (human lung ade-
nocarcinoma), Caco-2 (cervical adenocarcinoma) and 3T3
(murine fibroblasts) cell lines were maintained in a growth
medium consisting of high glucose Dulbecco’s Modified
Eagle Medium with sodium pyruvate (GE Healthcare,
Piscataway, NJ, United States). The growth medium
was supplemented with a 10% fetal bovine serum, 1X
HyClone™ Antibiotic/Antimycotic Solution (GE Health-
care, Little Chalfont, UK). Cells were cultured in an
atmosphere containing 5% CO2 in humidified air at 37 °C.
ν
max 3167, 2959, 1634, 1599, 1526, 1479, 1373, 1357, 1265,
1224, 1151, 1015, 823, 737 cm−1. HRMS: m/z [M + H]+:
338.1556 for C21H21NO2F (calcd. 338.1556).
Data for 1-(4″-Fluorophenyl)-3-(1′H and 1-metyl-
indol-3′-yl)prop-2-en-1-ones
(2E)-1-(4″-Fluorophenyl)-3-(1′H-indol-3′-yl)prop-2-en-1-one
(18a)
mp 185–188 °C [6], yellow crystals; mp 185–187 °C (acet-
one/hexane). 1H (DMSO-d6, 600 MHz): 11.92 (s, NH), 8.21
(dd, J 8.8, 5.6 Hz, H-2″, H-6″), 8.12 (s, 1H, H-2′), 8.11–8.09

910
Medicinal Chemistry Research (2021) 30:897–912
Cell viability, estimated by trypan exclusion, was >95%
before each experiment.
emission spectra of DNA-HO system were measured in the
range of 360–600 nm using excitation wavelength of
343 nm, with a slit width of 5.5 nm for the excitation and
emission beams. The DNA-HO complex was prepared by
adding 1.2 × 10−6 M of dye and 39.2 × 10−6 M of CT DNA
in 10 mM Tris-HCl buffer (pH 7.3, 24 °C). KSV quenching
constant were determined according to the classical
Stern–Volmer Eq. (1) in literature [44, 45].
The effect of synthetic chalcone derivatives on the via-
bility and proliferation of seven cancer cell lines and one
non-cancer cell line was evaluated using 3-(4,5-dimethyl-
thiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay.
Briefly, 5 × 103 cells were plated per well in 96-well poly-
styrene microplates (SARSTEDT, Nümbrecht, Germany).
Twenty four hours after cell seeding, various concentrations
(100, 50, 10, 5 and 1 μmol/L,) of tested compounds were
added. After 72 h of incubation, 10 μl of MTT (5 mg/ml)
were added in each well and incubated for an additional 4 h
at 37 °C in 5% CO2 during which insoluble formazan was
produced, followed by addition of 100 μL of 10% sodium
dodecyl sulfate in each well and another 12 h were allowed
for the formazan to be dissolved. The absorbance was
measured at 540 nm using the automated Cytation™ 3 Cell
Imaging Multi-Mode Reader (Biotek, Winooski, VT, Uni-
ted States). Three independent experiments were performed
for each test. The measured values were shown as percent
metabolic activity of the cells as compared to unaffected
control, which amounting 100%.
I0=I ¼ 1 þ KSV ½QŠ
ð1Þ
where I0 and I are the fluorescence intensities in the absence
and the presence of the quencher at 470 nm. KSV is the
Stern–Volmer quenching constant and [Q] is the concen-
tration of quencher.
Circular dichroism measurements
The CD spectra of DNA in the presence of chalcones
recorded on a JASCO (J-810) spectropolarimeter in the
wavelength range of 235–320 nm, in 10 mM Tris-HCl
buffer (pH 7.3) at 24 °C. The concentration of CT DNA and
chalcones was 4.4 × 10−4 M and 5.2 × 10−4 M, respectively.
Acknowledgements This research was supported by the Grant Agency
of Ministry of the Education, Science, Research and Sport of the
Slovak Republic [VEGA project no. 2/0044/18, VEGA project no. 1/
0753/17, VEGA project no. 1/0016/18, VEGA project no. 1/0138/20].
The support of Slovak Research and Development Grant Agency
[project APVV-18-0357] is also gratefully acknowledged.
DNA binding activity
Chemicals for biochemical study
For DNA binding experiments Dimethyl Sulfoxide
(DMSO), Calf Thymus DNA (CT DNA), Ethidium Bro-
mide (EB), Hoechst 33 258 (HO) and Tris(hydroxymethyl)
aminomethane (Tris) were purchased from Sigma-Aldrich
Chemie.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
UV–Vis absorption measurements
All the spectra were measured in 0.01 M Tris buffer (pH
7.3) by using a Varian Cary 100 Bio spectrophotometer in
the range of 300–500 nm and recorded at r.t. (24 °C) using
quartz cuvette of 1 cm path length. The concentration of
investigated chalcones was 10.4 × 10−6 M. During the
titration, an aliquot of buffered CT DNA solution was
added to each cuvette (sample and reference) to eliminate
the absorbance of DNA itself. The DNA concentration per
nucleotide was determined by absorption spectroscopy
using the molar absorption coefficient (6600 M−1 cm−1) at
260 nm.
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