Design, synthesis and computational studies involving Indole-Coumarin hybrids as galectin-1 inhibitors

Article abstract of DOI:10.1007/s11696-021-01534-w

In continuation of our quest to develop non-carbohydrate galectin-1 inhibitors, we have designed and synthesized 20 indole-coumarin hybrids linked via chalcone. Compounds 6i and 7e were found to decrease galectin-1 levels significantly in galectin-1 enzyme assay at 20?μM concentration. Binding affinity studies carried out by fluorescence spectroscopy revealed that 6i binds to galectin-1 with a binding constant (K_a) value of 5.4 × 10⁵?M^?1 while 7e was found to have a slightly higher affinity than 6i with K_a of 6.6 × 10⁵?M^?1. Molecular docking was carried out to ascertain the interaction between ligand and protein. To further gain structural insights into the binding of the compounds, 30?ns molecular dynamic simulations were carried out. The studies revealed that compound 7e was stable within the subsite C of galectin carbohydrate recognition domain while 6i fluctuated throughout the simulation. In addition, 7e maintained continuous interaction with Trp68 and His52, the two key amino acid residues are responsible for recognition of ligands within the active site. Furthermore, 7e displayed H-bond interactions with highly conserved amino acids within galectin-1 CRD, i.e., Arg48, Asn61 and Glu71. Free energy of binding evaluated by MM-GBSA calculations was also in accordance with experimental data. 7e was calculated to have binding energy of ??53.40?kcal/mole while 6i was found to have a value of ??45.63?kcal/mole. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11696-021-01534-w

Chemical Papers (2021) 75:2791–2805
https://doi.org/10.1007/s11696-021-01534-w
ORIGINAL PAPER
Design, synthesis and computational studies involving
Indole‑Coumarin hybrids as galectin‑1 inhibitors
Aaftaab Sethi1 · K. Sasikala1 · Pranay Jakkula2 · Divya Gadde3 · Swetha Sanam1 · Insaf A. Qureshi2 · Venu Talla3 ·
Mallika Alvala1,4
Received: 19 November 2020 / Accepted: 21 January 2021 / Published online: 2 February 2021
© Institute of Chemistry, Slovak Academy of Sciences 2021
Abstract
In continuation of our quest to develop non-carbohydrate galectin-1 inhibitors, we have designed and synthesized 20 indole-
coumarin hybrids linked via chalcone. Compounds 6i and 7e were found to decrease galectin-1 levels signifcantly in galec-
tin-1 enzyme assay at 20 µM concentration. Binding afnity studies carried out by fuorescence spectroscopy revealed that 6i
binds to galectin-1 with a binding constant (Ka) value of 5.4×105 M−1 while 7e was found to have a slightly higher afnity
than 6i with Ka of 6.6×105 M−1. Molecular docking was carried out to ascertain the interaction between ligand and protein.
To further gain structural insights into the binding of the compounds, 30 ns molecular dynamic simulations were carried
out. The studies revealed that compound 7e was stable within the subsite C of galectin carbohydrate recognition domain
while 6i fuctuated throughout the simulation. In addition, 7e maintained continuous interaction with Trp68 and His52, the
two key amino acid residues are responsible for recognition of ligands within the active site. Furthermore, 7e displayed
H-bond interactions with highly conserved amino acids within galectin-1 CRD, i.e., Arg48, Asn61 and Glu71. Free energy
of binding evaluated by MM-GBSA calculations was also in accordance with experimental data. 7e was calculated to have
binding energy of − 53.40 kcal/mole while 6i was found to have a value of − 45.63 kcal/mole.
Graphical abstract
Keywords Galectins · Galectin-1 · Gal-1 inhibitors · Non-carbohydrate inhibitors · Indole-coumarin hybrids · Chalcones
* Mallika Alvala
mallikaalvala@yahoo.in
Extended author information available on the last page of the article
Vol.:(0123456789)
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pathological conditions related to neurological diseases
(Wada et al. 2003), HIV-1 viral infection (Mercier et al.
2008) tumor progression (prostate, thyroid, bladder and
ovary), carcinomas, astrocytoma and melanoma (Rabi-
novich 2005). Gal-1 is also a diagnostic tumor marker
(Thijssen et al. 2015). Thus, due to its ubiquitous expres-
sion and wide range of activities, Gal-1 has emerged as a
potential therapeutic target for the treatment of a variety
of disease conditions like neurodegeneration, infamma-
tion, cancer and viral infection (Sundblad et al. 2017; Sethi
et al. 2020).
Introduction
Galectins are proteins that belong to the family of lec-
tins and bind specially to the β-galactoside containing
glycans. They are characterized by the presence of con-
served amino acid sequence motifs (130–140 amino acids)
in their carbohydrate-binding sites known as carbohydrate
recognition domain (CRD) (Barondes et al. 1994). In
total, 15 galectins have been reported till date and they
are classifed into three types, namely, prototype galec-
tins (galectin-1, -2, -5, -7, -10, -11, -13, -14, and -15),
tandem-repeat galectins (galectin-4, -6, -8, -9, and -12)
and chimera galectin (galectin-3) based on their structural
organization (Fig. 1) (Lefer et al. 2002).
Majority of Gal-1 inhibitors reported till date are carbo-
hydrates or carbohydrate derivatives. However, this class of
inhibitors has certain drawbacks. The synthesis and charac-
terization of carbohydrate-based inhibitors is challenging,
expensive and requires extensive protecting group altera-
tion (Boltje et al. 2009). Moreover, they are unable to cross
the cell membrane and aren’t selective. Similarly, peptide-
based inhibitors of Gal-1 also face challenges such as high
cost and poor bioavailability (Blanchard et al. 2016). One
excellent substitute to these class of compounds is hetero-
cyclic molecules as Gal-1 inhibitors. The heterocyclic com-
pounds’ properties can be easily tinkered with, as structural
Galectin-1 (Gal-1) is a highly conserved protein with
a single CRD composed of 135 amino acids. Its expres-
sion is ubiquitous inside our body in normal tissues and
diseased ones (Hughes 1999; Cooper and Barondes 1990).
It regulates cell growth, cell development, cell diferen-
tiation, cell adhesion, signaling and immune system (Liu
et al. 2002; Patterson et al. 2004; He and Baum 2006).
The altered Gal-1 expression is associated with various
Fig. 1 Representative structures
of diferent isoforms of Galectin
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modifcations on these systems are rather easy (Gomtsyan
2012). The literature involved regarding synthetic methods
for developing these molecules is large and well established.
Very recently, there have been a few reports of heterocy-
clic molecules as galectin inhibitors by our research group
and others (Goud et al. 2019a, b, 2020; Gabr et al. 2020).
In continuation of our pursuit to develop non-carbohydrate
Gal-1 inhibitors, herein, we report synthesis of 20 indole-
coumarin hybrids as Gal-1 inhibitors. There was extensive
literature supporting the rationale to choose these hybrids.
Coumarin scafold has been utilized previously on many
occasions as Gal-1 inhibitor. Rajput et al. have reported use
of coumarin on two separate occasions to develop Gal-1
inhibitors. I and II with Kd value of 1900 and 16 µM,
respectively (Rajput et al. 2014, 2016). Goud et al. (2019a,
b; 2020) reported a coumarin thiazole derivative as Gal-1
inhibitor (III) with a binding constant (Ka) of 1.9×107 M−1
and in another article reported a 4,7-disubstituted coumarin
(IV) with Ka 1.3×104 M−1. The same group also reported
1-benzyl-1H-benzimidazoles (V) as Gal-1 inhibitor with
Ka = 1.2 × 104 M−1 (Goud et al. 2019a, b). Indole is yet
another decorated scafold in medicinal chemistry. It is the
9th most common nitrogen heterocycle found in US FDA-
approved drugs (Vitaku et al. 2014). Biological importance
of indoles is vast and ranges from anti-microbial, anti-tumor,
anti-infammatory, anti-oxidant, anti-viral, anti-cancer and
anti-parkinsonian among others (Sravanthi and Manju 2016).
But naturally, indoles have also found their way as galectin
inhibitors. Tejler et al. (2005) reported anomeric aldoxime
derivatives of β-galactose as Gal-3 inhibitors. They found
that three of the best inhibitors consisted of a bicyclic aro-
matic moiety in form of naphthalene, quinoline and indole.
Among them, indole derivative was found to be the most
potent inhibitor (VI) with Kd of 180 µM. The same group
in another subsequent study reported inhibitor VII with a
Kd value of 46 µM toward Gal-3 (Tejler et al. 2009). Con-
sequently, along these lines, we decided to design indole-
coumarin hybrids as potential Gal-1 inhibitor. The galectin
CRD is considerably big and thus we wanted an appropri-
ately sized linker which in itself is biologically active and
does not adapt many conformations. Michael acceptors con-
taining an electrophile are considered biologically active as
such motifs can react with biological molecules and bind
with them (Zhuang et al. 2017). Keeping this in mind, chal-
cone was chosen for the role of the linker. Chalcones possess
biological potency for a wide variety of diseases. Chalcones
like metochalcone (VIII) and sofalcone (IX) have also found
their way into clinical practice (Gomes et al. 2017). Kurt
et al. (2020) designed coumarin-chalcone derivatives (X)
among which one of them was reported to have better anti-
cancer activity on H4IIE cancer cell line when compared to
the reference drug Sorafenib. The summary of the rationale
of designed molecules is illustrated in Fig. 2.
Results and discussion
Chemistry
The target compounds 6a–n and 7a–f were synthesized in
3 steps (Scheme 1). Initially, Knoevenagel condensation
between aldehydic functionality of salicylaldehyde (1) and
active methylene of ethylacetoacetate (2) in presence of
piperidine followed by cyclization gave us 3-acetyl coumarin
(3) in high yields. Next, we made use of Claisen–Schmidt
condensation between (3) and substituted indole carbalde-
hyde (4) to give us chalcone (5) in good yield. Finally,
we carried out a simple nucleophilic substitution reaction
between alkyl/aryl halides and (5) which gave us the tar-
get compound N-substituted indole-coumarin hybrids in
moderate-to-excellent yields. All the newly synthesized
compounds 6a–n and 7a–f (Fig. 3) were characterized by
HRMS, 1H, and 13C NMR spectroscopy. The alkene protons
part of the chalcone consistently showed a coupling constant
1
of > 15 Hz in H NMR spectrum of the compounds con-
frming that the E(trans) isomer was formed, which is also
the predominant stereoisomer (Gomes et al. 2017). The 1H
NMR spectrum of the compounds containing CH2 benzylic
protons displayed a sharp singlet between δ5–6. The 13C
NMR spectrum displayed the characteristic peak of carbonyl
carbon of chalcone at>δ180. The benzylic carbon was also
observed in 13C NMR spectrum of the compounds in the
range of δ40–60 as a singlet except for compounds wherein
a fuoro group at the ortho position is present. In those cases,
the benzylic proton was observed as a doublet. The HRMS
(ESI) of compounds showed corresponding [M+H]+ peaks
based on their molecular weights.
1.1 Biological activity (enzymatic assay
and fuorescence studies).
Initially, a preliminary investigation was done using an
enzyme assay to ascertain the ability of the synthesized
molecules 6a–n and 7a–f to reduce the Gal-1 expression.
BT-474 cells were grown in a 12-well plate and treated
with 20 µM concentration of compounds for 24 h. Then
the supernatant was collected as Gal-1 is an extracellular
secreted protein. Equal amounts of supernatant are subjected
to Quantikine Enzyme-Linked Immunoassay (ELISA) as per
manufacturer’s protocol (DGAL 10, R&D systems). The
supernatant was diluted twofold with calibrator diluent and
incubated with human Gal-1-coated plates for 2 h at room
temperature on a horizontal orbital microplate shaker and
washed to remove any unbound protein. Gal-1-specifc poly-
clonal antibody conjugated to horseradish peroxidase was
added and incubated for 2 h on a shaker. It was then washed
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Fig. 2 Illustration highlighting the rationale behind designed molecules
and incubated with substrate solution for 30 min before ter-
minating the reaction with 50 µl of stop solution. Amount of
protein expression was detected at 450 nm using a UV spec-
trophotometer. The percentage reduction of Gal-1 expression
is summarized in Table 1. It was found that among all the
compounds synthesized compound 6i and 7e signifcantly
reduced the Gal-1 expression. These observations are in
accordance with the trend observed for Gal-1 inhibitors
wherein a substitution at 3rd position of the benzyl group at
the far end by fuoro or chloro substituent leads to enhanced
activity as observed in the case of TD-139 and several other
inhibitors (Goud et al. 2019a, b).
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Scheme 1 Synthesis of indole-
coumarin hybrids (6a–n, 7a–f)
Fig. 3 Structure of the synthe-
sized compounds
Encouraged by these results, we set out to determine
the binding afnity of compounds 6i and 7e using fuo-
rescence measurements. The maximum emission spectra
of Gal-1 were found at 343 nm, and there was a decrease
in fuorescence intensity with increasing concentration of
the compounds (Figs. 4, 5). The plot of log[6i] and log[7e]
versus log(Fo − F)/F had given a linear relationship, and
the number of binding sites was calculated from the slope,
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Table 1 The reduction in Gal-1
expression for synthesized
compounds
1.1 Molecular docking, molecular dynamics, MM/
GBSA and ADMET studies
Compound code
Reduction
(%) 20 µM
6a
6b
6c
6d
6e
6f
6g
6h
6i
<20
<20
<20
<20
<20
<20
<20
<20
38.68
<20
<20
<20
<20
<20
<20
<20
<20
<20
81.13
<20
To understand the mode of interaction of compound 6i
and 7e at the Gal-1 CRD, the two compounds were sub-
jected to molecular docking. The docking was carried out
using Glide module of Schrödinger suite 2020–2. The 3D
crystal co-ordinates of human Gal-1 were retrieved from
protein data bank (PDB ID: 4Y24). It was found that the
coumarin moiety in compound 7e was showing multiple
pi-pi stacking interactions with Trp68 residue of Gal-1
protein. In addition, it displayed a H-bond interaction with
Asn61 residue at a distance of 1.88 Å. On the other hand,
compound 6i displayed 2 H-bond interaction with Asn61
residue. One is shown by carbonyl of chalcone at a dis-
tance of 2.35 Å and the other by carbonyl of coumarin at
a distance of 2.41 Å. However, the coumarin moiety in 6i
was tilted slightly far away from the indole ring of Trp68
due to which it was unable to show the pi-pi stacking
interaction (Fig. 6). Interestingly, it has been previously
reported that carbohydrate galectin recognition is charac-
terized by a stacking interaction between the sugar ring
and an aromatic residue of Trp68 (Meynier et al. 2009).
Trp68 a conserved residue within the galectin family and
is a key amino acid involved in recognition of galactose
(Di-Lella et al. 2009). Thus, it was gratifying to fnd that
both the compounds ft well into the Gal-1 CRD and 7e
displayed the important Trp68 pi-pi stacking interaction.
The respective docking scores of 6i and 7e were found to
be − 4.195 and − 4.135.
6j
6k
6l
6m
6n
7a
7b
7c
7d
7e
7f
which inferred interaction of protein and compound in
1:1 ratio. The binding constant (Ka) was calculated from
the intercept value which was observed as 5.4× 105 M−1
for 6i and 6.6 × 105 M−1 for 7e which corroborated well
with the ELISA studies. The intensity of the fuorescence
was quenched upon increase of ligand concentration, and
the bimolar quenching constant (Kq) was calculated to be
6.6×1012 M−1 s−1 for 6i and 5.9×1012 M−1 s−1 for 7e which
is larger than difusion control limit suggesting an interac-
tion of protein and ligand as well as the mode of quenching
to be static (Lakowicz 2013).
Merely relying on the docking score is not suitable as
the protein is considered to be rigid in most of the soft-
ware, while the system in which protein and ligand coexist
is dynamic (Sethi et al. 2019). Thus, to further understand
the mode of binding, compounds 6i and 7e were subjected
to molecular dynamics (MD) simulation of 30 ns. The MD
Fig. 4 Fluorescence binding study of compound 6i with Gal-1
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Fig. 5 Fluorescence binding study of compound 7e with Gal-1
Fig. 6. 3D interaction diagram of Gal-1 protein with a 6i (cyan),
H-bond interactions (yellow) can be observed between carbonyl
oxygen of chalcone and coumarin with Asn61 residue. b 7e (cyan),
H-bond interaction (yellow) can be observed between carbonyl oxy-
gen of coumarin and Asn61 residue in addition to hydrophobic inter-
action (blue) with Trp68
was performed using Desmond Module of Schrödinger
suite 2020–2.
Figure 8 demonstrates the type of protein–ligand contacts
exhibited by the complexes employed in MD simulation.
It was found that 6i displayed a H-bond interaction with
Arg48 and His52. Also, it displayed hydrophobic interaction
with Val31, His44, Arg48, His52 and Trp68. Although the
interaction with Trp68 wasn’t intense, compound 7e dis-
played H-bond interactions Asn61 and water bridge-medi-
ated H-bonding with Glu71. Additionally, it exhibited a very
strong hydrophobic interaction with Trp68 and relatively
mild ones with Arg48 and His52. Thus, it is clear that com-
pound 7e was able to maintain its interactions with Asn61
and Trp68 during the course of the simulation. The simu-
lation also demonstrated the importance of pi-pi stacking
To understand the conformational change in the protein
and ligand from its native structure the root mean square
deviation (RMSD) values were analyzed (Fig. 7). Confgu-
ration RMSD for 6i complex was frst found to increase and
then converge after 15 ns during equilibration phase while
for 7e complex it was found to increase as well but converge
after 12 ns. Ligand 7e fuctuated initially but was found to
stabilize after 15 ns while ligand 6i fuctuated throughout
the simulation and only seemed to stabilize at the end of
the simulation. Protein–ligand interactions of both the com-
plexes were examined during the course of MD simulation.
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found to be non-mutagenic; however, certain degree of
hepatotoxicity was predicted for 7e as well.
Conclusion
In conclusion, we have synthesized a series of 20 novel
indole-coumarin hybrids (6a-n, 7a–f) and character-
1
ized by spectral techniques viz. H-NMR, 13C-NMR and
HRMS. Initially, all the compounds were tested for Gal-1
inhibition using enzymatic assay. The two most promis-
ing molecules (6i and 7e) were further studied by fuo-
rescence spectroscopy to determine their binding values.
To understand their mode of binding, the two compounds
were subjected to extensive computational studies. It was
concluded that both these compounds interacted with
Trp68 in Gal-1 CRD which led to their recognition inside
the active site in addition to displaying H-bond network
with multiple amino acids present within the Gal-1 CRD.
MD studies further validated the interactions with Gal-1
residues within the CRD. In-silico toxicity profling was
carried out for both the molecules to identify any potential
toxic efects. The molecules were found to have suitable
ADME properties and were found to be non-mutagenic.
However, they have been predicted to be hepatotoxic. This
needs to be kept in mind while considering similar mol-
ecules as clinical candidates.
Fig. 7 Average RMSD of protein (Orange) and ligand (Blue) for
Gal-1 ligand complexes a 6i and b 7e during molecular dynamics
simulation
interaction with Trp68. Compound 7e stabilized early while
6i fuctuated throughout most of the simulation. The free
energy of binding was calculated for the docked complexes
of 6i and 7e using the MM/GBSA method incorporated in
Prime module of Schrödinger suite 2020–2. The free energy
of binding was determined to be − 53.40 kcal/mole for 7e
while for 6i it was found to be − 45.63 kcal/mole. These
results are in accordance with the experimental and MD
simulation data.
Experimental section
1.1 Chemistry
All the described starting materials, reagents and solvents in
the current study were purchased from respective commer-
cial suppliers. Analytical thin-layer chromatography (TLC)
was performed on MERCK pre-coated silica gel 60-F254
aluminum plates. Visualization on TLC plates was achieved
via UV light or in a few cases by iodine chamber. All melt-
ing points were recorded on Stuart® SMP30 melting point
apparatus. Column chromatography was performed using
silica gel (60–120 mesh) and was eluted with ethyl acetate-
hexane mixture. NMR spectra were recorded on Bruker 500
(500 MHz for 1 H NMR and 125 MHz for 13C NMR) using
DMSO as solvent. Chemical shift was reported in parts
per million (ppm) with respect to internal standard Tetra
Methyl Silane (TMS). Coupling constants were quoted in
Hertz (Hz). High-resolution mass spectra (HRMS) were
obtained on Agilent Q-TOF-Mass Spectrometer 6540-UHD
LC/HRMS operating at 70 eV using direct inlet.
The absorption, distribution, metabolism, excretion and
toxicity (ADMET) data were evaluated for compounds 6i
and 7e using pkCSM web server (Pires et al. 2015). 6i
was found to have a high intestinal absorption and high
Caco-2 permeability and had a blood brain barrier (BBB)
permeability of 0.236. This signifes its inability to cross
the BBB. The efect of cytochrome P450 metabolism for
CYP3A4 was positive while for CYP2D6 was found to be
negative. The clearance value was found to be 0.897, and
the compound was found to be non-mutagenic; however,
certain degree of hepatotoxicity was predicted. 7e was
found to have a high intestinal absorption and high Caco-2
permeability too and had a blood brain barrier (BBB) per-
meability of 0.123. CYP2D6 inhibition was found to be
negative while CYP3A4 was positive. The compound was
1.1 Synthetic protocol.
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Fig. 8 Bar-chart representation
of protein–ligand contacts of
Gal-1 ligand complexes a 6i and
b 7e during molecular dynamics
simulation
Synthesis of 3‑acetyl coumarin (3)
reaction was continued at 70 °C for 2–3 h. The reaction pro-
gress was monitored by TLC. After completion of the reac-
tion, the reaction mixture was added into a beaker containing
ice and stirred vigorously. The precipitate thus obtained was
then fltered under vacuum and recrystallized in absolute
ethanol to yield compounds 5a or 5b.
A mixture of salicylaldehyde (1 equiv.), ethyl acetoacetate
(1 equiv.) and a few drops of piperidine were stirred at
room temperature in neat conditions. Precipitation occurred
almost immediately, and the reaction progress was moni-
tored by TLC. After completion of the reaction, the reac-
tion mixture was added into a beaker containing ice and
stirred for 15 min. The precipitate thus obtained was then
fltered and dried under vacuum. The obtained compound
was recrystallized in absolute ethanol to yield compound 3.
General procedure for synthesis of N‑substituted
and unsubstituted indole‑coumarin hybrids (6a–n, 7a–f)
Compound 5a–b (1 equiv.), obtained from above protocol,
was added together with 1 equiv. of potassium carbonate in
acetonitrile. Further, respective benzyl bromide or alkyl hal-
ides were added with constant stirring at room temperature.
After completion of addition, the reaction was maintained
at 70 °C for 3–4 h and the reaction progress was monitored
by TLC. On completion, work-up was carried out or the ace-
tonitrile was directly evaporated under reduced pressure to
obtain the crude compound. The compounds were subjected
Synthesis of (E)‑3‑(3‑(1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one and methoxy derivative
(5a–b)
Compound 3 (1 equiv.) was added to absolute ethanol with
stirring, followed by the addition of simple indole-3-car-
boxaldehyde or methoxy-substituted derivative (1 equiv.).
A catalytic amount of piperidine was then added, and the
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to column chromatography on silica gel to obtain the pure
products.
1.1.1 (E)‑3‑(3‑(1‑benzyl‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6d)
Yellow solid; 89% yield; mp: 192–194 °C; FT-IR (cm−1):
1
4.3. Spectral data
2920, 1715, 1652, 1600; H NMR (500 MHz, DMSO-
d6) δ 8.64 (d, J = 18.0 Hz, 1H), 8.27 (d, J = 20.0 Hz,
1H), 8.07–7.99 (m, 2H), 7.96 (dd, J = 7.7, 1.3 Hz, 1H),
7.77–7.72 (m, 1H), 7.71–7.66 (m, 1H), 7.62–7.58 (m, 1H),
7.51 (d, J=7.3 Hz, 1H), 7.43 (t, J=11.2, 3.9 Hz, 1H), 7.34
(dd, J = 9.3, 5.5 Hz, 2H), 7.32–7.24 (m, 5H), 5.51 (s, 2H);
13C NMR (125 MHz, DMSO-d6) δ 186.67, 159.26, 154.82,
146.65, 139.30, 137.92, 137.57, 137.17, 134.31, 130.74,
129.17, 128.16, 127.69, 126.53, 126.17, 125.32, 123.57,
122.18, 120.92, 119.56, 119.05, 116.59, 112.96, 111.97,
50.02; HRMS (ESI): m/z calcd for C27H19NO3 405.1365;
found 406.1428 [M + H]+.
1.1.1 (E)‑3‑(3‑(1H‑indol‑3‑yl)acryloyl)‑2H‑chromen‑2‑one
(6a)
Yellow solid; 90% yield; mp: 200–202 °C; FT-IR (cm−1):
3261, 1698, 1645, 1602; 1H NMR (500 MHz, DMSO-d6) δ
11.96 (s, 1H), 8.66 (s, 1H), 8.10–8.03 (m, 2H), 8.01–7.94 (m,
2H), 7.75 (t, J=7.3 Hz, 1H), 7.69 (d, J=15.8 Hz, 1H), 7.51
(dd, J=11.6, 4.9 Hz, 2H), 7.44 (t, J=7.5 Hz, 1H), 7.29–7.22
(m, 2H); 13C NMR (125 MHz, DMSO-d6) δ 186.60, 159.27,
154.81, 146.62, 140.18, 138.18, 134.85, 134.29, 130.74,
126.59, 125.36, 125.32, 123.39, 121.83, 120.73, 119.07,
118.99, 116.58, 113.44, 113.13; HRMS (ESI): m/z calcd for
C20H13NO3 315.0895; found 316.0980 [M+H]+.
1.1.1 (E)‑3‑(3‑(1‑(4‑bromobenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6e)
1.1.1 (E)‑3‑(3‑(1‑methyl‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6b)
Yellow solid; 86% yield; mp: 203–205 °C; FT-IR (cm−1):
3044, 1717, 1650, 1602; 1H NMR (500 MHz, DMSO-d6)
δ 8.66 (s, 1H), 8.25 (s, 1H), 8.01 (dt, J = 30.6, 11.4 Hz,
3H), 7.75 (dd, J = 14.8, 7.1 Hz, 1H), 7.71–7.63 (m, 1H),
7.59–7.56 (m, 1H), 7.52 (dd, J=19.2, 8.0 Hz, 3H), 7.43 (t,
J=7.3 Hz, 1H), 7.29–7.19 (m, 4H), 5.50 (s, 2H); 13C NMR
(125 MHz, DMSO-d6) δ 186.72, 159.23, 154.82, 146.65,
139.18, 137.82, 137.02, 137.01, 134.34, 132.08, 130.76,
129.92, 126.54, 126.19, 125.34, 123.65, 122.25, 121.31,
120.94, 119.72, 119.04, 116.60, 113.06, 111.91, 49.36;
HRMS (ESI): m/z calcd for C27H18BrNO3 483.0470; found
486.0624 [M + H + 2]+.
Yellow solid; 92% yield; mp: 210–212 °C; FT-IR (cm−1):
1
3044, 1727, 1652, 1605; H NMR (500 MHz, DMSO-
d6) δ 8.66 (s, 1H), 8.07 (s, 1H), 8.01 (t, J = 11.2 Hz, 2H),
7.96 (dd, J = 7.7, 1.4 Hz, 1H), 7.77 – 7.73 (m, 1H), 7.69
(d, J = 15.7 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.50 (d,
J=8.3 Hz, 1H), 7.45–7.42 (m, 1H), 7.31 (ddd, J=14.9, 13.9,
6.9 Hz, 2H), 3.87 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
δ 186.46, 159.26, 154.82, 146.65, 139.39, 138.66, 137.94,
134.31, 130.75, 126.56, 125.88, 125.32, 123.44, 122.09,
120.76, 119.06, 118.95, 116.58, 112.40, 111.53, 33.55;
HRMS (ESI): m/z calcd for C21H15NO3 329.1052; found
330.1152 [M+H]+.
1.1.1 (E)‑3‑(3‑(1‑(3‑chlorobenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6f)
1.1.1 (E)‑3‑(3‑(1‑ethyl‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6c)
Yellow solid; 70% yield; mp: 206–208 °C; FT-IR (cm−1):
1
3044, 1717, 1650, 1602; H NMR (500 MHz, DMSO-
d6) δ 8.67 (s, 1H), 8.27 (s, 1H), 8.15–7.99 (m, 2H), 7.96
(d, J = 7.6 Hz, 1H), 7.76 (dd, J = 14.7, 7.0 Hz, 1H), 7.70
(d, J = 15.9 Hz, 1H), 7.62 (dd, J = 9.3, 2.9 Hz, 1H), 7.51
(dt, J = 14.7, 7.4 Hz, 3H), 7.43 (dd, J = 13.8, 6.3 Hz, 1H),
7.33–7.24 (m, 4H), 5.57 (s, 2H); 13C NMR (125 MHz,
DMSO-d6) δ 186.76, 159.25, 154.83, 146.67, 140.36,
139.18, 137.82, 137.05, 134.35, 131.41, 131.09, 130.76,
130.45, 126.77, 126.54, 126.14, 125.35, 123.72, 122.37,
122.30, 120.97, 119.79, 119.05, 116.61, 113.11, 111.89,
49.24; HRMS (ESI): m/z calcd for C27H18ClNO3 450.0975;
found 451.1292 [M + H]+.
Yellow solid; 90% yield; mp: 202–204 °C; FT-IR (cm−1):
2979, 1722, 1648, 1600; 1H NMR (500 MHz, DMSO-d6) δ
8.66 (s, 1H), 8.14 (s, 1H), 8.01 (t, J=12.1 Hz, 2H), 7.96 (dd,
J=7.8, 1.6 Hz, 1H), 7.77–7.72 (m, 1H), 7.67 (d, J=15.7 Hz,
1H), 7.63 (dd, J=7.6, 1.3 Hz, 1H), 7.50 (d, J=8.2 Hz, 1H),
7.44 (td, J=7.6, 1.2 Hz, 1H), 7.30 (dddd, J=14.8, 8.6, 7.2,
1.3 Hz, 2H), 4.36–4.19 (m, 2H), 1.42 (t, J = 7.2 Hz, 3H);
13C NMR (125 MHz, DMSO-d6) δ 186.56, 159.25, 154.81,
146.59, 139.51, 137.70, 136.41, 134.29, 130.73, 126.60,
126.07, 125.32, 123.41, 122.07, 120.89, 119.06, 118.99,
116.59, 112.59, 111.54, 41.39, 15.54; HRMS (ESI): m/z
calcd for C22H17NO3 343.1208; found 344.1295 [M+H]+.
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2801
1.1.1 (E)‑3‑(3‑(1‑(3‑methoxybenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6g)
1.1.1 (E)‑3‑(3‑(1‑(2‑fuorobenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6j)
Yellow solid; 94% yield; mp: 213–215 °C; FT-IR (cm−1):
Yellow solid; 86% yield; mp: 214–216 °C; FT-IR (cm−1):
3051, 1718, 1654, 1603; 1H NMR (500 MHz, DMSO-d6) δ
8.66 (d, J=4.4 Hz, 1H), 8.23 (d, J=44.2 Hz, 1H), 8.04 (d,
J=16.3 Hz, 2H), 7.95 (d, J=6.8 Hz, 1H), 7.71 (dd, J=16.6,
8.4 Hz, 2H), 7.62 (d, J = 5.2 Hz, 1H), 7.49 (d, J = 7.9 Hz,
1H), 7.45–7.41 (m, 1H), 7.38 (d, J = 6.7 Hz, 1H), 7.29
(s, 2H), 7.25–7.19 (m, 1H), 7.15 (d, J = 8.8 Hz, 1H), 7.10
(d, J = 6.1 Hz, 1H), 5.55 (d, J = 14.8 Hz, 2H); 13C NMR
(125 MHz, DMSO-d6) δ 186.78, 186.72, 159.25, 154.83,
146.68, 146.63, 139.25, 139.19, 137.85, 137.09, 134.33,
131.22, 130.75, 130.26, 126.55, 126.53, 126.03, 125.33,
123.75, 123.68, 122.26, 120.96, 119.78, 119.74, 119.06,
116.60, 114.68, 113.08, 111.90, 111.69, 49.41, 44.22;
HRMS (ESI): m/z calcd for C27H18FN2O3 423.1271; found
424.1348 [M+H]+.
1
2912, 1714, 1651, 1601; H NMR (500 MHz, DMSO-
d6) δ 8.66 (s, 1H), 8.25 (s, 1H), 8.06–7.94 (m, 3H), 7.75 (t,
J=7.7 Hz, 1H), 7.68 (d, J=15.8 Hz, 1H), 7.63–7.58 (m, 1H),
7.50 (d, J=8.3 Hz, 1H), 7.44 (t, J=7.4 Hz, 1H), 7.29–7.21 (m,
3H), 6.90–6.79 (m, 3H), 5.47 (s, 2H), 3.72 (s, 3H); 13C NMR
(125 MHz, DMSO-d6) δ 186.71, 159.93, 159.25, 154.82,
146.63, 139.32, 139.09, 137.95, 137.21, 134.32, 130.75,
130.32, 126.57, 126.13, 125.33, 123.58, 122.19, 120.91,
119.78, 119.56, 119.06, 116.60, 113.79, 113.25, 112.93,
111.99, 55.53, 49.95; HRMS (ESI): m/z calcd for C28H21NO4
435.1471; found 436.1526 [M+H]+.
1.1.1 (E)‑3‑(3‑(1‑(2‑nitrobenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6h)
Yellow solid; 82% yield; mp: 200–202 °C; FT-IR (cm−1):
2923, 1720, 1644, 1604; 1H NMR (500 MHz, DMSO-d6) δ
8.67 (s, 1H), 8.22–8.15 (m, 2H), 8.09–8.02 (m, 2H), 7.97 (d,
J=7.7 Hz, 1H), 7.74 (dd, J=15.8, 9.7 Hz, 2H), 7.68–7.55 (m,
2H), 7.52 (dd, J=17.3, 8.1 Hz, 2H), 7.44 (t, J=7.4 Hz, 1H),
7.29 (dt, J=15.0, 7.3 Hz, 2H), 6.59 (d, J=7.6 Hz, 1H), 5.93
(s, 2H); 13C NMR (125 MHz, DMSO-d6) δ 186.78, 159.27,
154.85, 147.70, 146.74, 139.02, 138.19, 137.35, 134.91,
134.37, 133.41, 130.78, 129.36, 128.30, 126.52, 126.05,
125.68, 125.35, 123.88, 122.44, 121.01, 120.11, 119.06,
116.61, 113.46, 111.96, 47.68; HRMS (ESI): m/z calcd for
C27H18FN2O3 450.1216; found 451.1375 [M+H]+.
1.1.1 (E)‑3‑(3‑(1‑(3,5‑difuorobenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6k)
Yellow solid; 85% yield; mp: 207–209 °C; FT-IR (cm−1):
3046, 1717, 1649, 1601; 1H NMR (500 MHz, DMSO-d6) δ
8.67 (s, 1H), 8.28 (s, 1H), 8.03 (dd, J=12.3, 6.4 Hz, 2H),
7.96 (dd, J = 7.7, 1.2 Hz, 1H), 7.77–7.69 (m, 2H), 7.63
(dd, J=6.0, 2.9 Hz, 1H), 7.50 (d, J=8.3 Hz, 1H), 7.44 (t,
J=7.4 Hz, 1H), 7.33–7.27 (m, 2H), 7.17 (tt, J=9.3, 2.2 Hz,
1H), 7.02 (d, J = 6.2 Hz, 2H), 5.54 (s, 2H); 13C NMR
(125 MHz, DMSO-d6) δ 186.72, 163.98, 163.88, 162.02,
161.91, 159.25, 154.83, 146.73, 142.09, 139.07, 137.78,
136.98, 134.35, 130.77, 126.49, 126.14, 125.33, 123.78,
122.35, 120.98, 119.90, 119.04, 116.60, 113.22, 111.82,
111.13, 110.93, 103.95, 103.75, 103.54, 49.10; HRMS
(ESI): m/z calcd for C27H17F2NO3 441.1176; found 442.1261
[M+H]+.
1.1.1 (E)‑3‑(3‑(1‑(4‑fuorobenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6i)
Yellow solid; 84% yield; mp: 218–220 °C; FT-IR (cm−1):
1
3044, 1717, 1650, 1602; H NMR (500 MHz, DMSO-d6)
δ 8.66 (s, 1H), 8.25 (s, 1H), 8.06 – 7.99 (m, 2H), 7.96 (dd,
J=7.8, 1.7 Hz, 1H), 7.77 – 7.73 (m, 1H), 7.69 (d, J=15.7 Hz,
1H), 7.64 – 7.60 (m, 1H), 7.50 (d, J=8.4 Hz, 1H), 7.44 (t,
J=7.5 Hz, 1H), 7.38 – 7.33 (m, 2H), 7.28 (dt, J=10.4, 3.5 Hz,
2H), 7.20 – 7.15 (m, 2H), 5.50 (s, 2H); 13C NMR (125 MHz,
DMSO-d6) δ 186.72, 163.05, 161.11, 159.25, 154.83, 146.64,
139.24, 137.81, 136.98, 134.32, 133.79, 133.77, 130.75,
129.94, 129.87, 126.56, 126.19, 125.33, 123.61, 122.22,
120.93, 119.65, 119.06, 116.60, 116.07, 115.90, 113.01,
111.94, 49.24; HRMS (ESI): m/z calcd for C27H18FNO3
423.1271; found 424.1348 [M+H]+.
1.1.1 (E)‑3‑(3‑(1‑(2,5‑difuorobenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6l)
Yellow solid; 92% yield; mp: 211–213 °C; FT-IR (cm−1):
1
3046, 1717, 1649, 1601; H NMR (500 MHz, DMSO-d6)
δ 8.65 (s, 1H), 8.19 (s, 1H), 8.07–7.99 (m, 2H), 7.96 (d,
J=7.5 Hz, 1H), 7.75 (t, J=7.6 Hz, 1H), 7.69 (d, J=15.8 Hz,
1H), 7.64 (d, J=7.6 Hz, 1H), 7.50 (d, J=8.3 Hz, 1H), 7.43
(t, J = 7.3 Hz, 1H), 7.31 (dt, J = 13.9, 6.8 Hz, 3H), 7.23
(d, J = 7.8 Hz, 1H), 7.11 (s, 1H), 5.56 (s, 2H); 13C NMR
(125 MHz, DMSO-d6) δ 186.82, 159.24, 157.71, 157.59,
154.83, 146.66, 139.16, 137.80, 136.95, 134.34, 130.75,
129.97, 126.54, 126.01, 125.34, 123.77, 122.34, 120.99,
119.94, 119.05, 117.93, 117.87, 117.75, 117.67, 117.16,
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Chemical Papers (2021) 75:2791–2805
117.09, 116.97, 116.89, 116.66, 116.60, 113.20, 111.63,
44.06, 4.15; HRMS (ESI): m/z calcd for C27H17F2NO3
441.1176; found 442.1250 [M+H]+.
1.1.1 (E)‑3‑(3‑(5‑methoxy‑1‑methyl‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (7b)
orange solid; 90% yield; mp: 195–197 °C; FT-IR (cm−1):
3196, 2988, 1715, 1641, 1601; 1H NMR (500 MHz, DMSO-
d6) δ 8.69 (s, 1H), 7.99 (dd, J=24.3, 9.4 Hz, 2H), 7.91 (m,
1H), 7.73 (d, J=15.4 Hz, 1H), 7.69 (m, 1H) 7.49 (m, 3H),
7.44–7.41 (m, 1H), 6.96 (d, J = 7.9 Hz, 1H), 3.86 (s, 3H),
3.83 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 185.96,
159.39, 155.91, 154.86, 146.85, 139.45, 137.95, 134.35,
133.69, 130.79, 126.64, 126.40, 125.33, 119.08, 118.39,
116.57, 112.77, 112.34, 112.19, 103.32, 55.98, 33.72;
HRMS (ESI): m/z calcd for C22H17NO4 359.1158; found
360.1277 [M+H]+.
1.1.1 (E)‑3‑(3‑(1‑(3,5‑dimethylbenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6m)
Yellow solid; 84% yield; mp: 210–212 °C; FT-IR (cm−1):
1
2913, 1714, 1654, 1603; H NMR (500 MHz, DMSO-d6)
δ 8.66 (s, 1H), 8.22 (s, 1H), 8.06–7.93 (m, 3H), 7.79–7.72
(m, 1H), 7.68 (d, J=15.7 Hz, 1H), 7.59 (d, J=8.7 Hz, 1H),
7.51 (t, J=7.4 Hz, 1H), 7.47–7.41 (m, 1H), 7.29–7.26 (m,
2H), 6.91 (s, 3H), 5.41 (s, 2H), 2.21 (s, 6H); 13C NMR
(125 MHz, DMSO-d6) δ 186.73, 159.26, 154.82, 146.60,
139.43, 138.25, 137.96, 137.38, 137.28, 134.32, 130.74,
129.64, 126.59, 126.11, 125.49, 125.34, 123.57, 122.17,
120.91, 119.49, 119.07, 116.60, 112.83, 111.98, 50.03,
21.35; HRMS (ESI): m/z calcd for C29H23NO3 433.1678;
found 434.1766 [M+H]+.
1.1.1 (E)‑3‑(3‑(1‑ethyl‑5‑methoxy‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (7c)
yellow solid; 92% yield; mp: 192–194 °C; FT-IR (cm−1):
3192, 2980, 1712, 1644, 1601; 1H NMR (500 MHz, DMSO-
d6) δ 8.68 (d, J=4.3 Hz, 1H), 8.08 (d, J=4.3 Hz, 1H), 8.02
(dd, J = 15.6, 4.5 Hz, 1H), 7.98–7.95 (m, 1H), 7.73 (dt,
J = 16.0, 6.3 Hz, 2H), 7.57–7.48 (m, 3H), 7.47–7.40 (m,
1H), 6.96 (d, J=10.1 Hz, 1H), 4.28–4.19 (m, 2H), 3.87 (d,
J=4.6 Hz, 3H), 1.49–1.35 (m, 3H); 13C NMR (125 MHz,
DMSO-d6) δ 186.05, 159.37, 155.85, 154.85, 146.76,
139.55, 136.36, 134.31, 132.68, 130.76, 126.83, 126.43,
125.31, 119.08, 118.44, 116.56, 112.74, 112.38, 112.32,
103.43, 55.97, 41.55, 15.56; HRMS (ESI): m/z calcd for
C23H19NO4 373.1314; found 374.1395 [M+H]+.
1.1.1 (E)‑3‑(3‑(1‑(4‑methylbenzyl)‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (6n)
yellow solid; 89% yield; mp: 200–202 °C; FT-IR (cm−1):
1
2921, 1720, 1650, 1603; H NMR (500 MHz, DMSO-
d6) δ 8.66 (s, 1H), 8.24 (s, 1H), 8.07–7.92 (m, 3H), 7.73
(d, J = 6.7 Hz, 1H), 7.68 (d, J = 15.7 Hz, 1H), 7.59 (d,
J=2.7 Hz, 1H), 7.49 (d, J=7.7 Hz, 1H), 7.43 (s, 1H), 7.26
(s, 2H), 7.19 (d, J=7.0 Hz, 2H), 7.13 (d, J=7.0 Hz, 2H),
5.44 (s, 2H), 2.24 (s, 3H); 13C NMR (125 MHz, DMSO-
d6) 186.71, 159.24, 154.82, 146.58, 139.35, 137.89, 137.42,
137.11, 134.49, 134.30, 130.73, 129.70, 127.76, 126.60,
126.19, 125.33, 123.51, 122.14, 120.88, 119.51, 119.06,
116.60, 112.86, 112.00, 49.86, 21.12; HRMS (ESI): m/z
calcd for C28H21NO3 419.1521; found 420.1595[M+H]+.
1.1.1 (E)‑3‑(3‑(1‑benzyl‑5‑methoxy‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (7d)
yellow solid; 89% yield; mp: 196–198 °C; FT-IR (cm−1):
3196, 2988, 1723, 1647, 1601; 1H NMR(500 MHz, DMSO-
d6) δ 8.68 (s, 1H), 8.20 (s, 1H), 8.03 (d, J=15.7 Hz, 1H),
7.96 (d, J = 6.8 Hz, 1H), 7.76–7.71 (m, 2H), 7.49 (dd,
J=7.5, 3.2 Hz, 3H), 7.43 (t, J=7.5 Hz, 1H), 7.36–7.32 (m,
2H), 7.27 (t, J=6.9 Hz, 3H), 6.91 (dd, J=8.9, 2.3 Hz, 1H),
5.47 (s, 2H), 3.85 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
δ 186.20, 159.37, 155.89, 154.86, 146.82, 139.33, 137.61,
137.17, 134.35, 132.86, 130.78, 129.15, 128.14, 127.64,
126.96, 126.40, 125.32, 119.07, 119.02, 116.57, 112.91,
112.77, 112.72, 103.42, 55.96, 50.20. HRMS (ESI): m/z
calcd for C28H21NO4 435.1471; found 436.1559 [M+H]+.
1.1.1 (E)‑3‑(3‑(5‑methoxy‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (7a)
yellow solid; 92% yield; mp: 190–192 °C; FT-IR (cm−1):
3196, 2988, 1715, 1641, 1601; 1H NMR (500 MHz, DMSO-
d6) δ 11.85 (s, 1H), 8.69 (s, 1H), 8.08–8.00 (m, 2H), 7.97 (d,
J=7.7 Hz, 1H), 7.77–7.71 (m, 2H), 7.49 (t, J=5.4 Hz, 2H),
7.43 (dd, J = 14.9, 8.1 Hz, 2H), 6.91 (dd, J = 8.8, 2.2 Hz,
1H), 3.86 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 186.14,
159.39, 155.59, 154.84, 146.79, 140.23, 134.92, 134.33,
133.01, 130.76, 126.42, 126.07, 125.32, 119.07, 118.51,
116.56, 113.80, 113.37, 112.83, 103.20, 55.90; HRMS
(ESI): m/z calcd for C21H15NO4 345.1001; found 346.1080
[M+H]+.
1.1.1 (E)‑3‑(3‑(1‑(3‑chlorobenzyl)‑5‑methoxy‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (7e)
yellow solid; 82% yield; mp: 187–189 °C; FT-IR (cm−1):
3192, 2981, 1724, 1646, 1603; 1H NMR (500 MHz, DMSO-
d6) δ 8.69 (s, 1H), 8.22 (s, 1H), 8.03 (d, J=15.7 Hz, 1H),
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2803
7.97 (d, J = 7.8 Hz, 1H), 7.75 (ddd, J = 11.8, 4.5, 2.2 Hz,
2H), 7.51 (dd, J=7.6, 5.2 Hz, 3H), 7.45–7.42 (m, 1H), 7.36
(dd, J = 3.9, 2.1 Hz, 3H), 7.21 (dd, J = 6.5, 1.9 Hz, 1H),
6.93 (dd, J = 8.9, 2.3 Hz, 1H), 5.49 (s, 2H), 3.85 (s, 3H);
13C NMR (125 MHz, DMSO-d6) 186.25, 159.37, 155.96,
154.87, 146.86, 140.17, 139.20, 137.05, 134.37, 133.76,
132.75, 131.10, 130.79, 128.16, 127.51, 126.97, 126.38,
126.33, 125.34, 119.26, 119.07, 116.58, 113.04, 112.89,
112.68, 103.48, 55.98, 49.48; HRMS (ESI): m/z calcd for
C28H20ClNO4 469.1081; found 470.1189 [M+H]+.
4.4.2. Fluorescence measurements
In order to perform fuorescence binding studies of Gal-1
with compounds 6i and 7e, full length ORF of Gal-1 was
cloned into pET28a expression vector followed by purifca-
tion using Ni–NTA afnity chromatograph. Intrinsic fuo-
rescence measurements were carried out on a Jasco spec-
trofuorimeter equipped with peltier at 25 °C. Compounds
6i and 7e were dissolved in DMSO to prepare a stock of
10 mM and were used in the range of 0–65 µM. Purifed
Gal-1 with concentration of 13 μM in 10 mM phosphate
bufer (pH 7.5) was excited at 280 nm, and the emission
was recorded from 300 to 400 nm using a cuvette of 10 mm
path length. Slit width 5 nm was used for excitation and
emission, while scan speed was maintained at 100 nm/
min. Bufer correction was made for each spectrum. Bind-
ing constant (Ka) and number of binding sites (n) were
determined using modifed Stern–Volmer equation, i.e.,
log(Fo − F)/F = logKa + nlog[Q] where Fo and F are the
intensity of the protein in the absence and presence of the
ligand, respectively, whereas n is the number of binding sites
and Q is the ligand concentration.
1.1.1 (E)‑3‑(3‑(1‑(4‑fuorobenzyl)‑5‑methoxy‑1H‑indol‑3‑yl)
acryloyl)‑2H‑chromen‑2‑one (7f)
yellow solid; 92% yield; mp: 190–192 °C; FT-IR (cm−1):
1
3192, 2980, 1723, 1648, 1603; H NMR (500 MHz,
DMSO-d6) δ 8.69 (d, J = 4.8 Hz, 1H), 8.33–8.12 (m, 1H),
8.03 (dd, J = 15.7, 2.3 Hz, 1H), 7.97 (d, J = 7.7 Hz, 1H),
7.74 (dd, J = 15.8, 8.1 Hz, 2H), 7.50 (dd, J = 9.7, 4.4 Hz,
3H), 7.46–7.41 (m, 1H), 7.38 (dd, J = 13.8, 7.3 Hz, 1H),
7.28–7.21 (m, 1H), 7.20–7.06 (m, 2H), 6.97–6.88 (m, 1H),
5.49 (t, J=23.1 Hz, 2H), 3.85 (s, 3H); 13C NMR (125 MHz,
DMSO-d6) 186.27, 159.38, 155.94, 154.86, 146.89, 146.84,
139.27, 139.22, 137.10, 134.36, 132.82, 132.76, 131.26,
131.19, 130.79, 130.63, 130.20, 126.95, 126.83, 126.38,
125.33, 125.27, 124.41, 123.70, 123.35, 119.24, 119.07,
116.58, 112.87, 103.46, 55.97, 49.59, 44.37; HRMS (ESI):
m/z calcd for C28H20FNO4 453.1376; found 454.1532
[M+H]+.
Computational studies
Molecular docking
Compounds 6i and 7e were sketched by using the
2D-sketcher in Maestro (Schrodinger Suite 2020–2). These
two compounds were then prepared using the “LigPrep”
module by generating low-energy ionization and tautomeric
states with a pH of 7.4 using OPLS-2005 force feld for
minimization. The molecular docking studies for the pre-
pared compounds were performed using the Glide module in
Maestro Schrodinger Suite 2020–2. The three-dimensional
crystal structure of the target protein Gal-1 with the PDB ID:
4Y24 was retrieved from Protein Data Bank, PDB. First, the
retrieved protein was prepared using the “Protein prepara-
tion wizard.” The protein was imported and pre-processed
to assign the bond orders and add hydrogen. All the water
molecules were deleted followed by optimization and mini-
mization by using OPLS-2005 force feld. Next, a grid was
generated around the co-crystal of the prepared protein by
“Receptor grid generation.” The grid generation was per-
formed to defne the active site of the protein. The region
was enclosed in a box size of 10×10×10 Å. The prepared
compounds were then docked at the active site using Glide
XP fexible ligand docking. Van der Waals radius was main-
tained < 0.8. The scoring function and the protein–ligand
interactions were further analyzed.
4.4. Biology
4.4.1. Quantikine ELISA human Gal‑1 immunoassay
BT-474 cells were grown in 12-wellplate and treated with
20 µM concentration of compounds for 24 h. Then the super-
natant was collected as Gal-1 is an extracellular secreted
protein. Equal amounts of supernatant are subjected to
Quantikine Enzyme-Linked Immunoassay (ELISA) as per
manufacturer’s protocol (DGAL 10, R&D systems). The
supernatant was diluted twofold with calibrator diluent and
incubated with human Gal-1-coated plates for 2 h at room
temperature on a horizontal orbital microplate shaker and
washed to remove any unbound protein. Gal-1-specifc poly-
clonal antibody conjugated to horseradish peroxidase was
added and incubated for 2 h on shaker. It was then washed
and incubated with substrate solution for 30 min before ter-
minating the reaction with 50 µl of stop solution. Amount
of protein expression was detected at 450 nm using UV
spectrophotometer.
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2804
Chemical Papers (2021) 75:2791–2805
Di-Lella S, Ma L, Diaz Ricci JC, Rabinovich GA, Asher SA, Álvarez
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4.5.2. Molecular dynamics simulation and MM‑GBSA
calculations
In order to more precisely evaluate the protein and the ligand
interactions, MD simulations of 30 ns were performed for
6i and 7e using Desmond module of Schrödinger suite
(2020–2). Aqueous biological system was built by using
OPLS_2005 force field, and TIP3P model was used to
simulate the water molecules. Orthorhombic periodic
boundary conditions were set up to specify the shape and
size of the repeating unit bufered at 10 Å distances. The
physiological pH was neutralized by adding 0.15 M NaCl.
300 K temperature and 1.01325 bar pressure was main-
tained by using Nose–Hoover temperature coupling and
Martina–Tobias–Klein method for the constant pressure,
respectively. Reversible reference system propagation algo-
rithm (REPSA), a time stepping algorithm, was used for
near non-bonded (2 fs), far non-bonded (6 fs) and bonded
interactions (2 fs). In the end, molecular dynamics simula-
tions (30 ns) were performed for the two ligands.
Gabr M, Rehman AU, Chen HF (2020) A quinoline-pyrazole scaf-
fold as a novel ligand of galectin-3 and suppressor of trem2
signaling. ACS Med Chem Lett. https://doi.org/10.1021/acsme
dchemlett.0c00330
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PV, Andrade CH, Neves BJ (2017) Chalcone derivatives: prom-
ising starting points for drug design. Molecules 22(8):1210.
https://doi.org/10.3390/molecules22081210
Gomtsyan A (2012) Heterocycles in drugs and drug discovery. Chem
Heterocycl Comp 48(1):7–10. https://doi.org/10.1007/s1059
3-012-0960-z
Goud NS, Ghouse MS, Vishnu J, Pranay J, Alvala R, Talla V,
Qureshi IA, Alvala M (2019a) Synthesis and biological evalu-
ation of novel heterocyclic imines linked coumarin-thiazole
hybrids as anticancer agents. Curr Med Chem: Anti-Cancer
Agents 19(4):557–566. https://doi.org/10.2174/1871520619
666190207140120
Goud NS, Ghouse SM, Vishnu J, Komal D, Talla V, Alvala R, Pra-
nay J, Kumar J, Qureshi IA, Alvala M (2019b) Synthesis of
1-benzyl-1H-benzimidazoles as galectin-1 mediated anticancer
agents. Bioorg Chem 89:103016. https://doi.org/10.1016/j.bioor
g.2019.103016
The MM/GBSA method (Schrödinger Release 2020–2:
Prime, Schrödinger, LLC, New York, NY, 2020–2) was
used to calculate the binding free energy (ΔGbind), for recep-
tor–binder complex systems.
Goud NS, Makani VK, Pranay J, Alvala R, Qureshi IA, Kumar P,
Bharath RD, Nagaraj C, Yerramsetty S, Pal-Bhadra M, Alvala
M (2020) Synthesis, 18F-radiolabeling and apoptosis induc-
ing studies of novel 4,7-disubstituted coumarins. Bioorg Chem
97:103663. https://doi.org/10.1016/j.bioorg.2020.103663
He J, Baum LG (2006) Galectin interactions with extracellular matrix
and efects on cellular function. Methods Enzymol 417:247–256.
https://doi.org/10.1016/S0076-6879(06)17017-2
Supplementary Information The online version supplementary mate-
rial available at https://doi.org/10.1007/s11696-021-01534-w.
Hughes RC (1999) Secretion of the galectin family of mammalian car-
bohydrate-binding proteins. Biochim Biophys Acta 1473(1):172–
185. https://doi.org/10.1016/S0304-4165(99)00177-4
Kurt BZ, Kandas NO, Dag A, Sonmez F, Kucukislamoglu M (2020)
Synthesis and biological evaluation of novel coumarin-chalcone
derivatives containing urea moiety as potential anticancer agents.
Arab J Chem 13(1):1120–1129. https://doi.org/10.1016/j.arabj
c.2017.10.001
Acknowledgements Authors are thankful to DoP, Ministry of Chemi-
cals and Fertilizers, Govt. of India, New Delhi, for the award of NIPER
fellowship and supporting the work.
Funding The research carried out received no external funding.
Compliance with ethical standards
Lakowicz JR (2013) Principles of fuorescence spectroscopy. Springer,
New York
Conflict of interest The authors declare that they have no confict of
Lefer H, Carlsson S, Hedlund M, Qian Y, Poirier F (2002) Intro-
duction to galectins. Glycoconj J 19(7–9):433–440. https://doi.
org/10.1023/B:GLYC.0000014072.34840.04
interest.
Liu FT, Patterson RJ, Wang JL (2002) Intracellular functions of galec-
tins. Biochim Biophys Acta 1572(2–3):263–273. https://doi.
org/10.1016/S0304-4165(02)00313-6
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Authors and Afliations
Aaftaab Sethi1 · K. Sasikala1 · Pranay Jakkula2 · Divya Gadde3 · Swetha Sanam1 · Insaf A. Qureshi2 · Venu Talla3 ·
Mallika Alvala1,4
1
3
Department of Medicinal Chemistry, National Institute
of Pharmaceutical Education and Research (NIPER),
Hyderabad 500037, India
Department of Pharmacology and Toxicology, National
Institute of Pharmaceutical Education and Research
(NIPER), Hyderabad 500037, India
2
4
Department of Biotechnology and Bioinformatics,
School of Life Sciences, University of Hyderabad,
Hyderabad 500046, India
MARS Training Academy, Hyderabad, India
1 3