Synthesis and biological evaluation of morpholines linked coumarin–triazole hybrids as anticancer agents

Article abstract of DOI:10.1111/cbdd.13578

A series of novel morpholines linked coumarin–triazole hybrids (6a–6v) has been synthesized and evaluated for their anti-proliferative potential on a panel of five human cancer cell lines, namely bone (MG-63), lung (A549), breast (MDA-MB-231), colon (HCT-15) and liver (HepG2), using MTT assay. Among all, the compound 6n {7-((1-(2,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl) methoxy)-4-((2,6-dimethylmorpholino) methyl)-2H-chromen-2-one} showed significant growth inhibition against MG-63 cells with an IC₅₀ value of 0.80 ± 0.22 μM. Further, induction of apoptosis by 6n of MG-63 cells confirmed as a result of morphological changes, the sub-G1 phase arrest, increased percentage of apoptotic cells, and decrease in mitochondrial membrane potential and increase in reactive oxygen species levels. The in vitro Gal-1 expression in cell culture supernatant of MG-63 cells treated with compound 6n showed dose-dependent reduction. The binding constant (K_a) of 6n with Gal-1 was calculated from the intercept value which was observed as 3.0 × 10⁵ M^?1 by fluorescence spectroscopy. Surface plasmon resonance showed that 6n binds to Gal-1 with binding constant (K_a) of 1.29E+04 1/Ms and equilibrium constant KD value of 7.54E?07 M, respectively. Molecular docking studies revealed the binding interactions of 6n with Gal-1.

Full text of DOI:10.1111/cbdd.13578

DR MALLIKA ALVALA (Orcid ID : 0000-0003-3770-4725)
Article type : Research Article
Synthesis and Biological evaluation of morpholines linked coumarin-triazole hybrids as
anticancer agents
Nerella Sridhar Goud a, Venkatesh Pooladanda b, S. Mahammad Ghouse a, Jakkula Pranayd ,
Santhosh Gatreddid , Insaf A. Qureshid Ravi Alvala c, Chandraiah Godugu b*, Mallika Alvala
a*
a. Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
b. Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
c. G. Pulla reddy College of Pharmacy, Hyderabad, India
d Department of Biotechnology & Bioinformatics, School of Life Sciences, University of
Hyderabad, Hyderabad-500046, India.
*Corresponding Author:
Dr. Mallika Alvala
Department of Medicinal Chemistry,
National Institute of Pharmaceutical Education and Research (NIPER),
Hyderabad 500 037,
India
Tel: 9441117803
Email: mallikaalvala@yahoo.in
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13578
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Abstract:
A series of novel morpholines linked coumarin-triazole hybrids (6a-6v) has been synthesized
and evaluated for their anti-proliferative potential on a panel of five human cancer cell lines
namely, bone (MG-63), lung (A549), breast (MDA-MB-231), colon (HCT-15) and liver
(HepG2), using MTT assay. Among all, the compound 6n {7-((1-(2,4-dichlorobenzyl)-1H-
1,2,3-triazol-4-yl) methoxy)-4-((2,6-di methylmorpholino) methyl)-2H-chromen-2-one}
showed significant growth inhibition against MG-63 cells with an IC50 value of 0.80 ± 0.22
µM. Further, induction of apoptosis by 6n of MG-63 cells confirmed as a result of
morphological changes, the sub G1 phase arrest, increased percentage of apoptotic cells, and
decrease in mitochondrial membrane potential and increase in ROS levels. The in-vitro Gal-1
expression in cell culture supernatant of MG-63 cells treated with compound 6n, showed
dose dependent reduction. The binding constant (Ka) of 6n with Gal-1 was calculated from
the intercept value which was observed as 3.0 x 105 M-1 by Fluorescence spectroscopy.
Surface Plasmon Resonance (SPR) showed that 6n binds to Gal-1 with binding constant (Ka)
of 1.29E+04 1/Ms and equilibrium constant KD value of 7.54E-07 M respectively. Molecular
docking studies revealed the binding interactions of 6n with Gal-1.
Keywords: morpholines, coumarin-triazoles, Galectin-1, binding constant, apoptosis.
Introduction
Cancer is one of dreadful diseases in the world and characterized by uncontrolled cell growth
(Rashid, 2012). According to World Health Organization (WHO), it is estimated to have a
rise in the disease to over 13 million by 2030 (Bray, 2005). Despite, many anticancer agents
are available but the drug induced toxicity, poor selectivity and tolerance, indicate the
necessity to develop new potential anticancer agents.
Galectin-1 (Gal-1) a 14KDa laminin protein is considered to be a molecular target in the
treatment of numerous cancers due to its fundamental role in tumour growth and in the
multiple processes of metastasis, angiogenesis, apoptosis and invasion (Astorgues, 2014).
The apoptosis is the critical cellular process, plays an important role in the cell growth
maintenance and homeostasis. The changes in the regulation of apoptotic pathway have been
indicated in numerous diseases including cancer (Hanahan, 2011). Therefore, ideology
towards development of novel anticancer agents that would target Gal-1 protein and induce
apoptosis will be more effective strategy in cancer treatment.
Coumarin (2H-chromen-2-one), a fused heterocyclic ring system consisting of benzene and
2-pyrone ring belongs to the family of neo flavonoids of plant secondary metabolites known
for its wide variety of biological applications (Gormley, 1996; Fylaktakidou, 2004; Yuce,
2011; Manvar, 2011; Kostova, 2011;Hassan, 2016; Sashidhara, 2011;Thakur, 2015).
Recently, Vishal K. Rajput et al, have reported a selective galactose based coumarin derived
Galectin inhibitors and showed that the coumarin-arginine interactions are essential for
Galectin inhibition through molecular modelling studies (Rajput, 2016).
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1,2,3-triazole, a class of five membered aza-heterocycles, known to interact with different
biomolecular targets [14] and found in many bioactive molecules (Kolb, 2003; Lal, 2016;
Kharb, 2011; Kumar, 2014; Kadaba, 1988; Patp, 2012; Giffin, 2008; Buckle, 1983; Ma,
2014) ;Sheng, 2015). Johan Tejler et al, have reported multivalent lactose derivatives with
triazole moiety for selective Gal-1 inhibition (Tejler, 2006). Ring systems such as
morpholine, dimethyl morpholine, piperidine, piperazine etc. are reported to exhibit
significant cytotoxicity against various cancer cells when incorporated (Taylor, 2014).
1,2,3-Triazole linked (arylamidomethyl)-dihydrofurocoumarin hybrids I as phosphodiesterase
inhibitors, have been reported by A.V. Lipeeva et al. for selective cytotoxicity (Lipeeva,
2015). Linking of coumarin with triazoles has been known for selective cytotoxicity such as
Triazoles linked with 6- hydroxycoumarin II, chalcone-coumarin hybrid with a triazole linker
III and Coumarin-1,2,3-triazole-dithiocarbamate hybrid IV against a panel of cancer cell
lines [(Shakeel, 2014; Pingaew, 2014;Ye, 2014)] (Figure 1).
Guided by these literature investigations, and in our continuous interest to develop potent
cytotoxic molecules, we have synthesized a series of morpholines linked coumarin-triazole
hybrids and evaluated for their in vitro cytotoxicity against a panel of cancer cells are
discussed herein.
Results and Discussion
Chemistry
The target compounds 7-((1-substitutedbenzyl-1H-1,2,3-triazol-4-yl) methoxy)-4-(substituted
morpholino methyl)-2H chromen-2-one derivatives (6a-6v) were synthesized by utilizing
Huisgen 1,3-dipolar cycloaddition reaction of terminal alkyne of coumarin linked
morpholiness (5a, 5b) with various benzyl azides in the presence of copper(I)-catalyst
(Wang, 2017) (Scheme 1). Initially, the 4-(Chloromethyl)-7-hydroxy-2H-chromen-2-one (3)
was prepared by Pechmenn condensation of resorcinol (1) with ethyl-4-chloroacetoacetate (2)
in the presence of sulphuric acid (Benci, 2012). Further, Compound (3) was treated with
substituted morpholines derivatives and offered morpholines linked coumarin intermediates
4a and 4b, which are further involved in the O-alkylation process with propargyl bromide in
the presence of Potassium carbonate resulted morpholines linked coumarin with terminal
alkyne derivatives 5a and 5b (Khachatryan, 2015). Finally, copper(I)-catalyzed 1,3-dipolar
cycloaddition of morpholines linked coumarin with terminal alkyne derivatives (5a, 5b) and
various benzyl azides furnished the target compounds 6a-6v with moderate to good yields.
1
All synthesized compounds (6a-6v) were characterized by spectral techniques viz. H-NMR,
13C NMR and HRMS. The 1H NMR spectrum of 6a showed characteristic protons of O- and
N-attached methylene groups of morpholine ring at δ 3.60 and 2.45 ppm respectively. All the
remaining protons appeared in the range of δ 3.70-8.50 ppm. 13C NMR spectrum showed the
characteristic carbonyl and N-attached methylene carbons of morpholine at δ 161.3 and δ
53.3 ppm respectively, and all the remaining carbons appeared in the range of δ 53.7-160.7.
The similar pattern was observed for rest of the compounds (6a-6v).
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Scheme 1: Synthesis of morpholines linked coumarin-triazole hybrids (6a-6v).
Biological evaluation
In vitro anti proliferative activity
All the synthesized morpholines linked coumarin-triazole derivatives (6a-6v) were screened
for their in vitro cytotoxicity against a panel of selected human cancer cell lines viz. bone
(MG-63), lung (A549), breast (MDA-MB-231), colon (HCT-15) and liver (HepG2) and
normal embryonic fibroblast cells (NIH-3T3) using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay (Sharma, 2015). The IC50 (µM) values of
compounds 6a-6v along with standard, Cisplatin are displayed in Table-1. Among the all the
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derivatives, the compound 6n has displayed significant cell cytotoxicity with an IC50 value of
0.80 ± 0.22 µM, 2.97 ± 0.04 µM, 4.05 ± 2.28 µM, 3.93 ± 2.56 µM and 7.19 ± 1.12 µM
against MG-63, A549, HCT-15, MDA-MB-231 and HepG2 cell lines respectively and no
cytotoxicity against NIH-3T3 normal fibroblast cells The compounds 6e, 6i, 6l, 6p and 6u
have displayed moderate cytotoxicity with IC50 values in the range of 1.0 to 10 µM.
In Structural activity relationship (SAR), the coumarin scaffold with morpholines and triazole
ring (6a-6j), attached with benzyl group bearing an electron-withdrawing groups especially
halogens exhibited good cytotoxic activity in compounds viz. 6e (R2= 4-Br), 6i (R2= 4-F)
and 6k (R2= 3-Cl). The presence of an electron-releasing group in 6f (3- OCH3), 6g (4-
OCH3) and 6j (3,5-di CH3) showed moderate to less cytotoxicity. In contrast, other electron-
withdrawing groups in 6b (4–NO2), 6c (-2,4 dichloro), 6d (4-CN) and unsubstituted
derivatives 6a and 6h exhibited less to no cytotoxicity. The coumarin scaffold with dimethyl
morpholines and triazole ring (6k-6v), attached with benzyl group bearing an electron-
withdrawing groups like halogens displayed significant cytotoxic activity in compounds viz.
6n (R2= 2,4-dichloro) and 6p (R2= 4- Br). The presence of an electron-releasing group in 6q
(3-OCH3) and 6u (3,5-di CH3) showed less to good cytotoxicity.
Whereas unsubstituted derivative 6l showed moderate activity as compared to 6s. In contrast,
other electron-withdrawing groups in 6m (4–NO2), 6o (4-CN), 6r (3,5-Difluoro), 6s (4-F) and
6v (3-Cl) exhibited less to no cytotoxicity. Hence, we concluded that dimethylmorpholine
derivatives with an electron-withdrawing group viz. 6n and 6p are more cytotoxic than simple
morpholine derivatives like 6e, 6i and 6k.
Apoptosis studies
Morphological observations using phase contrast microscopy
Apoptosis or programmed cell death is recognized by distinctive pattern of morphological,
biochemical and molecular changes occurring in a cell (Kalle, 2010). Compound 6n treated
cells (0.5, 1 and 2.5µM) showed prominent morphological changes like cell shrinkage with
rounding of cells and formation of membrane blebs as evidenced by phase contrast
microscopy studies and proportional to the concentration (Figure 2). However, these
distinctive morphological features are absent in the controlled cells.
Acridine orange/ethidium bromide (AO/EB) staining
Acridine orange/ethidium bromide (AO/EB) staining assay was used to differentiate live,
apoptotic and necrotic cells (Sharma, 2016). AO/EB staining method recognize the live cells
from dead cells, since AO can permeate the intact cell membrane and stain the nuclei green,
whereas EB can only stain the nucleus of cells that have lost membrane integrity in orange. It
can be inferred from Figure 3 that the control cells displayed normal morphology and
appeared in green colour. Fluorescence microscopic images of MG-63 cells treated with 0.5
µM concentration of compound 6n clearly showed early stage apoptosis. The clear
morphological changes such as cell shrinkage, membrane blebbing, chromatin condensation
and apoptotic body formation were observed at 1 µM and 2.5 µM respectively, hence these
results suggesting that 6n induced apoptosis in MG-63 cancer cells in a dose dependent
manner.
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DAPI nucleic acid staining
DAPI (4',6-diamidino-2-phenylindole), a blue fluorescent dye aids in the visualization of
chromatin condensation or nuclear damage and distinguishes live cells from apoptotic cells
by staining the condensed nuclei of apoptotic cells was considered of interest to detect the
induction of apoptosis by 6n in MG-63 cells (Tarnowski, 1991). The results from Figure 4
demonstrated that the nuclear structure of untreated cells was intact, whereas cells treated
with 6n displayed condensed, horse-shoe shaped or fragmented nuclei in cells.
Cell cycle analysis
Further to understand the mechanism of inhibition of cell proliferation of MG-63 cells by 6n,
cell death was quantified in terms of percentage of apoptotic cells using FACS Verse TM
flow analyser (Ravi, 2011). Propidium iodide staining of cells treated with 6n at different
concentrations showed dose dependent increase in apoptotic cells (Figure 5). Untreated
control cells exposed to DMSO-d6 showed 3.01% cells in sub G1 phase, whereas compound
6n treatment resulted in increased sub G1 population to 6.38% at 0.5 µM, 7.31% at 1 µM and
12.84% at 2.5 µM concentration in 24 h. These results clearly demonstrate that 6n induce
apoptosis by sub G1 phase arrest of cell cycle.
Mitochondrial membrane potential
The loss of mitochondrial membrane potential is known to be hallmark for apoptosis (Singh,
2011). Mitochondrial depolarization occurring in the early stages of apoptosis in cells treated
with 6n was investigated by JC1 (a carbocyanine cationic dye) staining. It exhibits potential-
dependent accumulation in mitochondria, indicated by a fluorescence emission shift from red
to green. The enhanced ROS levels induce the loss of mitochondrial membrane potential
which further activates the apoptotic signalling and leads to cell death. The polarised
mitochondria show red colour due to the formation of J-aggregates, whereas the depolarised
mitochondria exhibit green colour due to J-monomers. Figure 6A shows increase population
of P2 (green) mitochondrial depolarised cells in concentration dependent manner in contrast
to untreated (Figure 6A). The loss of mitochondrial membrane potential increased in a dose
dependent manner with compound 6n (Figure 6B). Thus, compound 6n induced apoptosis by
inducing the mitochondrial membrane potential loss.
AnnexinV/Propidium iodide dual staining assay
To quantify the percentage of apoptosis induced by compound 6n, the Annexin V/Propidium
iodide dual staining assay (Praveen Kumar, 2016) was carried out. The Annexin V-Alexa
Flour 488/PI dual staining assay facilitates the detection of live cells (LL; AV-/PI-), early
apoptotic cells (LR; AV+/PI-), late apoptotic cells (UR; AV+/PI+) and necrotic cells (UL;
AV-/PI+). As shown in Figure 7, the compound 6n increased the percentage of late apoptotic
cells from 6.19% (control) to 21.19% at 1 µM and 27.03% at 2.5 µM respectively which
clearly indicates that the compound 6n induces apoptosis in MG-63 cells in a dose dependent
manner.
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Measurement of ROS levels by DCFDA
The dissipation of mitochondrial membrane potential is caused by high levels of reactive
oxygen species (ROS), which further leads to cause intrinsic apoptosis. The effect of 6n on
cellular ROS levels in MG-63 cells was studied by cell permeant fluorogenic dye DCFDA
(2,7-dichloro fluorescein diacetate) (Eruslanov, 2010). Treatment with 6n for 48 h caused a
significant increase of ROS in MG-63 cells in dose dependent manner (0.5, 1 and 2.5 µM) as
shown in Figure 8.
Effect of compound 6n on Gal-1 protein levels:
Inhibition of human Gal-1 is considered as one of the potential therapeutic approaches for the
treatment of cancer, as it plays a major role in tumour development and metastasis by
modulating various biological functions viz. apoptosis, angiogenesis, migration, cell immune
escape (Rabinovich, 2005). Effect of 6n on Gal-1 levels during apoptosis induction was
studied using Gal-1 quantitative enzyme immunoassay. Briefly, MG-63 cells were grown in
60mm cell culture dishes, treated with 6n for 48 h and the supernatant was collected. As Gal-
1 is an extracellular secreted protein, its expression can be quantified from the cellular
external environment (Wu, 2018). Equal amounts of supernatant were subjected to
quantitative enzyme immunoassay as per manufacture’s protocol [DGAL10, R&D Systems,
USA]. The supernatant was incubated with human Gal-1 coated plates and washed to remove
unbound protein. Furthermore, an enzyme linked antibody was added to the reaction, specific
to human Gal-1 and incubated with substrate solution for 30 minutes before terminating the
reaction with 50µL of the stop solution. Amount of protein expression was detected with the
aid of a UV-spectrophotometer. Compound 6n effectively reduced the levels of Gal-1 protein
in dose dependent manner at the concentrations of 10, 30, 100 and 300 µM (Figure 9, Table
2).
Fluorescence Measurements for Gal-1 binding
The maximum emission spectra of Gal-1 was at 343 nm and there was decrease in
fluorescence intensity with increasing concentration of the compound (Figure 10A). The plot
of log[6n] versus log(Fo-F)/F had given a linear relationship (Figure 10B) and the number of
binding sites were calculated from the slope, which were found to be 1.13 and infers
interaction of protein and compound in 1:1 ratio. The binding constant (Ka) was calculated
from the intercept value which was observed as 3.0 x 105 M-1. The intensity of the
fluorescence was quenched upon increase of ligand concentration and the bimolar quenching
constant (Kq) was calculated to be 8.2 x 1012 M-1 s-1 which is larger than diffusion control
limit (Lakowicz, 2006) suggesting interaction of protein and ligand as well as the mode of
quenching to be static.
Surface Plasmon Resonance (SPR) studies
The Gal-1/6n interaction was analyzed through SPR using immobilized Gal-1 (ligand, 8070
RU) and compound 6n at various concentrations. Sensorgram was measured for each of
concentration of compound 6n (Figure 11) and fitted using 1:1 interaction steady state
affinity model. SPR showed that 6n binds to Gal-1 with binding constant (Ka) of 1.29E+04
1/Ms and equilibrium constant KD value of 7.54E-07 M respectively, using Biacore T200
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Evaluation software version 2.0. SPR analysis has shown interaction of Gal-1/6n, which is in
corroboration with florescence studies.
Molecular docking
The interactions of Gal-1 with the compound 6n was studied using molecular docking
calculations using the Glide module of Schrödinger suite 2014-3 (Schrödinger suite, 2014).
The 3D crystal co-ordinates of human Gal-1 were retrieved from protein data bank (PDB ID:
4Y24). The docking results showed that the compound 6n well accommodated inside the
binding site of the Gal-1. The compound 6n has shown three hydrogen bonding interactions
with the essential residues of the binding site like Ser29, Asn33 and Arg48 additionally, the
π-π stacking interactions with His44, His52 and Trp68 adds to the stability of the docked
pose of compound 6n. Figure 12, illustrate the interactions of compound 6n with the binding
site of Gal-1. It is notable from the interaction diagram that the coumarin and the triazole
rings are making the key binding interactions with the binding site residues.
Conclusion
In summary, we have synthesized a series of novel morpholines linked coumarin-triazole
1
hybrids (6a-6v) and characterized by spectral techniques viz. H-NMR, 13C-NMR and
HRMS. Initially, these compounds were evaluated for anti-proliferative activities on bone
(MG-63), lung (A549), breast (MDA-MB-231), colon (HCT-15), liver (HepG2), using MTT
assay. The compound 6n {7-((1-(2,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl) methoxy)-4-
((2,6-di methylmorpholino) methyl)-2H-chromen-2-one} exhibited significant growth
inhibition against MG-63 cells with an IC50 value of 0.80 ± 0.22 µM and no toxicity against
NIH-3T3 fibroblast cells. Compound 6n induced apoptosis confirmed by significant changes
in cell morphology, growth arrest at sub G1 phase, increased percentage of apoptotic cells,
decrease in mitochondrial membrane potential, increase in ROS levels and Gal-1 inhibition.
Importance of di methyl substituted morpholines is confirmed by docking studies with Gal-1.
Materials and Methods
Chemistry
All the starting materials, reagents and solvents were purchased from commercial suppliers.
Analytical thin layer chromatography (TLC) was performed on MERCK pre-coated silica gel
60-F254 aluminum plates. Visualization of the spots on TLC plates was achieved either by
exposure to iodine vapour and UV light. All melting points were recorded on Stuart® SMP30
melting point apparatus and are uncorrected. Column chromatography was performed using
silica gel (60-120 mesh) and was eluted with ethyl acetate-hexane. NMR spectra were
recorded on Bruker 500 (500 MHz for 1H-NMR and 125 MHz for 13C NMR) using CDCl3
and DMSO-d6 as solvents. 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 Synthetic,
biology procedures and spectral data of synthesized compounds were included in supporting
information.
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Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
Thankful to Department of Biotechnology (DBT), Govt. of India, New Delhi, for the young
investigators in cancer biology start up grant (6242-19/RGCB/PMD/DBT/MLKA/2015).
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Figure 1. Representative examples structures of 1,2,3-triazole-coumarin hybrids (I-IV) and
proposed structure of compounds 6a-6v.
Figure 2. Morphological changes due to induction of apoptosis by 6n were observed by
phase-contrast microscopy. The MG-63 cells lost their characteristics of epithelial cells with
increasing concentrations of 6n
Figure 3. Effect of compound 6n on apoptotic changes determined by AO/EB staining. The
MG-63 cells were treated with compound 6n with the concentration of 0.5, 1 and 2.5 µM.
After 48 h of incubation, cells were stained by AO/EB dual staining. Images were captured at
200X magnification
Figure 4. Nuclear morphology of MG-63 cells treated with 6n: Nuclear morphology of MG-
63 cancer cells visualised after DAPI staining. cells were treated with different concentrations
of 6n compound for 48 h and stained with DAPI. The images were captured with
fluorescence microscope at 200X. The arrows indicate the nuclear condensation and nuclear
fragmentation
Figure 5. Cell cycle analysis: MG-63 Cells were treated with 0.5, 1 and 2.5 µM
concentrations 6n and analysed by propidium iodide (PI) staining after 24 h of incubation.
Figure 6. Flowcytometric analysis of the mitochondrial membrane potential: (A)
mitochondrial membrane depolarisation of MG-63 cells treated with 0.5, 1 and 2.5 µM
concentrations of 6n. P1red) indicates the percentage of polarised cells (live), where is
P2(green) indicates the percentage of depolarised cells (dead). (B) Loss rate of the
mitochondrial membrane potential represented by Graphpad Prism 6.01 software. Data
represent as mean ± SEM (n=3). **p<0.01 and ****p<0.0001 versus control.
Figure 7. Effect of compound 6n on induction of apoptosis in MG-63 cells. After 48 h of
compound 6n (0.5, 1 and 2.5 µM) incubation, cells were stained with Annexin V/PI and
analysed for apoptosis using BD c6 accuri flow cytometer. 30.74% apoptotic cells were
observed at 2.5 µM concentration. The 10,000 cells from each sample were analysed by flow
cytometry.
Figure 8. Measurement of ROS. (A) MG-63 cells were treated with 6n (0.5, 1 and 2.5 µM)
for 48 h and DCF fluorescent images were captured at 200X magnification. (B) Relative DCF
fluorescence intensity was measured and represented as bar chart. Data represent as mean ±
SEM (n=3). *p<0.05 and **p<0.01 versus control.
Figure 9: (A) Gal-1 protein levels in MG-63 cells with or without 6n at 10, 30, 100 and 300
µM concentration. (B) Human Gal-1 Standard Curve. (Data represent as Mean ± SEM,
***p<0.001, 6n vs. control
Figure 10: Fluorescence binding study of compound 6n with the Gal-1. (A) Fluorescence
spectra of Gal-1 in the increasing concentrations of ligand from 0 to 65 μM (from top to
bottom) at pH 7.5. The protein was excited at 280 nm and emission spectra was collected in
the range of 300–400 nm. (B) Modified Stern–Volmer plot of Gal-1 by compound 6n used
for the calculation of binding affinity.
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Figure 11. Sensorgram of SPR for Gal-1 binding to compound 6n. The y-axis represents the
amount of bound analyte in terms of RU, while x-axis shows time after injection (seconds).
The colored lines indicate different concentrations of compound 6n.
Figure 12: 3D ligand interaction diagram of target compound 6n (A) and co-crystal (TD-
139) (B) with the binding domain of Gal-1: 6n making interactions with Ser29, Asn33,
Arg48, His44 and His52 residues of Gal-1(PDB ID: 4Y24).
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MDA-MB-
231e
Compound
MG-63b
A549c
HCT-15d
HepG2f
NIH-3T3g
6a
>30
>30
>30
>30
>30
-
-
-
-
-
-
-
-
-
-
-
-
6b
6c
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
6d
6e
>30
>30
>30
>30
>30
3.77 ± 0.17
>30
7.82 ± 2.38
>30
>30
>30
>30
6f
>30
>30
>30
6g
6h
6i
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
6.55 ± 1.02
19.47 ± 1.40
>30
7.55 ± 0.97
8.30 ± 0.51
9.00 ± 0.38
>30
2.54 ± 0.23
22.88 ± 2.72
28.23 ± 1.45
>30
6j
11.96 ± 4.92 >30
6k
6l
>30
27.08 ± 5.07 22.97 ± 4.61
9.45 ± 1.31
>30
4.78 ± 1.08
>30
>30
>30
6m
6n
6o
6p
6q
6r
>30
>30
>30
0.80 ± 0.22
>30
2.97 ± 0.04
>30
4.05 ± 2.28
>30
3.93 ± 2.56
>30
7.19 ± 1.12
>30
49.07 ± 0.09
-
-
-
-
-
-
-
-
-
1.72 ± 0.09
>30
9.97 ± 3.57
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
6s
>30
>30
>30
>30
>30
6t
>30
>30
>30
>30
>30
6u
6v
Cisplatin
1.89 ± 0.05
>30
10.93 ± 0.65 >30
>30
24.15 ± 2.14 19.45 ± 1.75 16.97 ± 2.45
>30
>30
19.50 ± 1.02 >30
14.25 ± 2.52
25.73 ± 1.41
17.42 ± 0.77
Table 1. IC50 (µM) valuesa for the derivatives 6a-6v determined by MTT assay.
a50% Inhibitory concentration after 48 h of drug treatment. b Human osteosarcoma. c Human
lung cancer. d Human colon cancer. e Human breast cancer. f Human liver cancer. g Mouse
embryonic fibroblast cells.
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Table 2. Amount of Gal-1 protein in control and compound 6n (10, 30, 100 and 300 μM)
treated MG-63 cells.
Concentration Absorbance at 450 nm
Gal-1 concentration(µg/mL)
Control
0.6074
0.6060
0.5493
0.4143
0.2786
4.03
4.02
3.49
2.24
0.97
6n (10 µM)
6n (30 µM)
6n (100 µM)
6n (300 µM)
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