Synthesis, 18F-radiolabeling and apoptosis inducing studies of novel 4, 7-disubstituted coumarins

Article abstract of DOI:10.1016/j.bioorg.2020.103663

In present study, a new series of 4, 7-disubstituted coumarin derivatives (7a-y) have been synthesized as galectin-1 targeting apoptosis inducing agents and evaluated for their in vitro cytotoxic potentials against a panel of selected human cancer cell lines namely, Brest (MCF7), Ovarian (SKOV3), Prostate (PC-3 & DU145) and normal embryonic kidney (HEK293T) cells, using MTT assay. Most of the compounds exhibited potent growth inhibitory action against the treated cancer cell lines with an IC₅₀ range of 10–30 μM. Compound 7q exhibited a significant growth inhibition against prostate cancer (PC-3 & DU145) cell lines with an IC₅₀ value of 7.45 ± 0.03 μM, 8.95 ± 0.17 μM respectively. Further, the target compound 7q was radiolabeled with fluorine-18 [¹⁸F] to be used as a novel PET radiotracer for imaging of tumors via targeting galectin-1, using appropriate reaction conditions in the GE Tracer-lab FX2N synthesis module. The purification of the [¹⁸F] radiolabeled compound [¹⁸F]-7q was successfully achieved with 60% ethanol. The radiochemical purity was>85% and residual solvent limits of DMF was 65 ± 3 ppm as analysed by HPLC, TLC & GC analytical methods. The apoptosis studies confirm the inhibition of cell proliferation with morphological changes like cell shrinkage, blebbing and cell wall deformation, increasing the ROS levels, and loss of mitochondrial membrane potential by Acridine orange/Ethidium bromide staining, Hoechst-33342 staining, H₂DCFDA staining, annexin V-FITC/PI, and JC-1 staining methods. In flow cytometric analysis, 7q selectively arrested the sub-G1 phase of the cell cycle in a dose-dependent manner. In Gal-1 ELISA studies, compound 7q efficiently reduced the levels of Gal-1 protein in dose-dependent manner with an IC₅₀ value of 100 μM. The binding constant (K_a) of 7q with Gal-1 was observed as 1.3 × 10⁴ M^?1 by fluorescence spectroscopy. The molecular docking studies clearly showed possible interactions and the pharmacokinetic (ADMET) properties of compound 7q with Gal-1. Hence, the novel 4, 7-disubstituted coumarins could be a potential cytotoxic and PET imaging agents via Gal-1.

Full text of DOI:10.1016/j.bioorg.2020.103663

Journal Pre-proofs
Synthesis, 18F-Radiolabeling and Apoptosis Inducing Studies of Novel 4, 7-
Disubstituted Coumarins
Nerella Sridhar Goud, Venkata Krishna Kanth, Jakkula Pranay, Ravi Alvala,
Insaf A. Qureshi, Pardeep Kumar, Rose Dawn Bharath, Chandana Nagaraj,
Suresh Yerramasetti, Manika Pal-Bhadra, Mallika Alvala
PII:
S0045-2068(20)30035-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.103663
YBIOO 103663
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
6 January 2020
23 January 2020
11 February 2020
Please cite this article as: N. Sridhar Goud, V. Krishna Kanth, J. Pranay, R. Alvala, I.A. Qureshi, P. Kumar, R.
Dawn Bharath, C. Nagaraj, S. Yerramasetti, M. Pal-Bhadra, M. Alvala, Synthesis, 18F-Radiolabeling and
Apoptosis Inducing Studies of Novel 4, 7-Disubstituted Coumarins, Bioorganic Chemistry (2020), doi: https://
doi.org/10.1016/j.bioorg.2020.103663
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Synthesis, 18F-Radiolabeling and Apoptosis Inducing Studies
of Novel 4, 7-Disubstituted Coumarins
Nerella Sridhar Goud a, b, Venkata Krishna Kanth c, Jakkula Pranay e, Ravi Alvala d, Insaf A.
Qureshi e, Pardeep Kumar b, Rose Dawn Bharath b, Chandana Nagaraj b, Suresh Yerramasetti
c, Manika Pal-Bhadra c, Mallika Alvala a*
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India.
b Department of Neuroimaging and Interventional Radiology (NI & IR), National Institute of
Mental Health and Neuro Sciences (NIMHANS), Bangalore 560 029, India.
c Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad
500 007, India.
d G. Pulla Reddy College of Pharmacy, Hyderabad 500 028, India.
e Department of Biotechnology & Bioinformatics, School of Life Sciences, University of
Hyderabad, Hyderabad 500 046, India.
*Corresponding Address:
Dr. Mallika Alvala (PhD)
Faculty of Medicinal Chemistry
NIPER-Hyderabad
Email: mallikaalvala@yahoo.in

Abstract:
In present study, a new series of 4, 7-disubstituted coumarin derivatives (7a-y) have been
synthesized as galectin-1 targeting apoptosis inducing agents and evaluated for their in vitro
cytotoxic potentials against a panel of selected human cancer cell lines namely, Brest
(MCF7), Ovarian (SKOV3), Prostate (PC-3 & DU145) and normal embryonic kidney
(HEK293T) cells, using MTT assay. Most of the compounds exhibited potent growth
inhibitory action against the treated cancer cell lines with an IC50 range of 10-30 µM.
Compound 7q exhibited a significant growth inhibition against prostate cancer (PC-3 &
DU145) cell lines with an IC50 value of 7.45±0.03 µM, 8.95±0.17 µM respectively. Further,
the target compound 7q was radiolabeled with fluorine-18 [18F] to be used as a novel PET
radiotracer for imaging of tumors via targeting galectin-1, using appropriate reaction
conditions in the GE Tracer-lab FX2N synthesis module. The purification of the [18F]
radiolabeled compound [18F]-7q was successfully achieved with 60% ethanol. The
radiochemical purity was greater than 85 % and residual solvent limits of DMF was 65  3
ppm as analysed by HPLC, TLC & GC analytical methods. The apoptosis studies confirm the
inhibition of cell proliferation with morphological changes like cell shrinkage, blebbing and
cell wall deformation, increasing the ROS levels, and loss of mitochondrial membrane
potential by Acridine orange/Ethidium bromide staining, Hoechst-33342 staining, H2DCFDA
staining, annexin V-FITC/PI, and JC-1 staining methods. In flow cytometric analysis, 7q
selectively arrested the sub-G1 phase of the cell cycle in a dose-dependent manner. In Gal-1
ELISA studies, compound 7q efficiently reduced the levels of Gal-1 protein in dose-
dependent manner with an IC50 value of 100 µM. The binding constant (Ka) of 7q with Gal-1
was observed as 1.3 x 104 M-1 by fluorescence spectroscopy. The molecular docking studies
clearly showed possible interactions and the pharmacokinetic (ADMET) properties of
compound 7q with Gal-1. Hence, the novel 4, 7-disubstituted coumarins could be a potential
cytotoxic and PET imaging agents via Gal-1.
Key Words: 4-substituted coumarins, fluorine-18, positron emission tomography, galectin-1,
cancer, apoptosis.
Research Highlights:
 The 7q is a novel anticancer agent with an IC50 of 7.45±0.03 µM mediated by Gal-1
in PC-3 cells.

 Identified novel 7q molecule which induces apoptosis via Gal-1 reduction in PC-3
cells.
 The [18F]-7q radiotracer is an ideal PET imaging agent due to its optimal half-life of
110 min.
 The radiochemical identity and purity were confirmed as per cGMP guidelines for
clinical use.
1. Introduction
Cancer is one of the life-threatening diseases in the world and characterized by uncontrolled
cell division. According to the World Health Organization (WHO), it is estimated to have a
rise of over 13 million by 2030 [1, 2]. Drug-induced toxicity, poor selectivity, and tolerance
of the current chemotherapeutic agents necessitate the development of new anticancer agents
with different mechanisms. The critical cellular process which plays an important role in
tissue growth maintenance and homeostasis is apoptosis [3]. Many studies have suggested
that the tumor cell death induction through apoptosis reserves a physiological advantage in
cancer therapy [4]. Human Galectin-1 (Gal-1) gained interest due to its key role in biological
events like angiogenesis, metastasis, apoptosis, and invasion of tumor cell [5]. Gal-1 is a
protein of galectin family binds to cell-surface and extracellular matrix glycan and affects
cellular functions [6]. The interaction of gal-1 with glycans plays a major role in cancer
biology and tumor immunity. Gal-1 is lower expressed in normal cells and blood, but it is
overexpressed in neoplastic diseases [7]. The Gal-1 has been reported to overexpress in many
cancer types viz. prostate, breast, lung, melanoma and renal, etc. [8, 9]. Gal-1 is considered as
a biomarker in diagnosis, prognosis and treatment condition of various cancer & neurological
conditions. Hence, the Gal-1 is regarded as a molecular target to develop newer therapeutic
agents with increased safety and efficacy for cancer therapy.
The diagnosis with target based positron emission tomography (PET) imaging agent is a
powerful tool for early detection and diagnosis of the condition to the next treatment level.
Therefore, it is necessary to focus on radiochemical synthesis and quality control studies
based on the importance of PET imaging modality for diagnosis [10]. The 18F labeled
radiotracers are emerging as potential weapons for the diagnosis, prognosis and therapy
monitoring of various neurological and tumor-associated conditions through PET imaging
quantitative modality [11]. 18F isotope is highly feasible in the radiolabelling of bioactive
molecules for PET imaging since it has positron-emitting property and favourable half-life of

110 min unlike other isotopes [12]. The USFDA approved a biologically active molecule
with 18F (Fluorodeoxyglucose-FDG) has gained more significance in the diagnosis and
progression of various tumor cells & neurological conditions using PET [13]. The 18F
radiotracers provide PET images of cellular uptake, transport, neurotransmission and,
metabolic processes on the molecular level in the body. which also act as powerful weapons
to identify new drug targets through pharmacodynamics and pharmacokinetic (ADME)
properties of drugs in-vivo [14, 15]. Ewelina Kluza et al, have reported that the synergistic
targeting of both αvβ3 and galectin-1 improves the specificity of fluorescent liposomes to
tumor endothelium in vivo, which can be easily accessed to Magnetic Resonance Imaging
(MRI) modality for high-resolution tumor images [16].
Coumarin (2H-chromen-2-one), neoflavonoid group of plant secondary metabolites, and
consists of fused benzene with 2-pyrone ring. A wide variety of natural and synthetic
coumarin derivatives with diverse pharmacological activities viz. anticoagulant, antibacterial,
antioxidant, anti-HIV, antitubercular, antihyperglycemic, anticonvulsant, anti-hypertensive,
anti-inflammatory, and anticancer have been reported [17, 18]. Coumarin derivatives are
widely explored for anticancer activities as they possess limited side effects and multidrug
reversal activity. It is proved that the substitution at C-3, C-4 & C-7 positions of coumarin
exhibits tremendous anticancer activities, whereas at C-5, C-6 & C-8 positions showed less
anticancer potentials against a wide range of cancer cell lines [19]. Coumarin derivatives with
C-4 substitution target essential pathways to treat cancer such as HSP90 inhibition, 17βHSD3
inhibition, protein kinase inhibition, aromatase inhibition, TNF-α inhibition, cell cycle arrest,
apoptosis induction, DNA intercalation and microtubulin inhibition [20]. Recently, our
research group has reported a new series of heterocyclic imines linked coumarin-thiazole
hybrids as anticancer agents (1) by targeting Gal-1 with an IC50 of 300 µM [21]. Dong Cao et
al, have reported 4-substituted coumarin derivatives (II), as potential anticancer agents with
an IC50 value ranges from 7-47 nM [22]. Rajput et al., have reported coumarin based
glycoside derivatives as galectin inhibitors. The coumarylmethyl galactoside (II) derivative
showed greater affinity towards various selected galectins [23]. Huiping Zhao et al, have
reported optimization of the novobiocin Scaffold to produce anti-proliferative agents and
developed a new series of novobiocin analogues (IV) as selective Hsp90 inhibitors through
various structural modifications in the sugar moiety by sugar mimics, and piperidine, an aza
sugar mimics incur anticancer activity in nM range against a panel of cancer cell lines (Fig.
1) [24].

<insert Figure 1 here>
The diagnosis with target based PET radiotracer is a powerful tool for early detection, and
understand the disease condition. Hence, overexpression of Gal-1 at different stages in
multiple cancer cells & importance of PET imaging modality for diagnosis, have triggered
our interest to develop novel 4, 7-disubstituted coumarin derivatives. Therefore, the present
18
study aims in synthesis, F-radiolabeling of novel 4, 7-disubstituted coumarins and in-vitro
biological evaluation against a panel of cancer cells with selective galectin-1 inhibition.
2.0. Results and Discussion
2.1. Chemistry
The target coumarin analogues (7a-y) were synthesized by refluxing various aromatic and
aliphatic halides with different cyclic saturated rings conjugated coumarins (4a, 4b, 5a, 5b,
6a) as showed in Scheme 1. Initially, the 4-(chloromethyl)-7-hydroxy-2H-chromen-2-one (3)
was synthesized by pechmenn condensation of resorcinol (1) and ethyl-4-chloroacetoacetate
(2) in the presence of sulphuric acid [25]. In the next step, the 7-hydroxy-4-(substituted
morpholinomethyl)-2H-chromen-2-ones
(4a,
4b),
7-hydroxy-4-(substituted
piperidinomethyl)-2H-chromen-2-ones (5a, 5b) and 4-((3,4-dihydroisoquinolin-2(1H)-yl)
methyl)-7-hydroxy-2H-chromen-2-one (6a) were synthesized by N-alkylation [26] of
substituted morpholines, piperidines and tetrahydroisoquinoline with compound (3). Finally,
the O-alkylation of compounds (4a, 4b, 5a, 5b, 6a) with various aromatic and aliphatic
halides, in the presence of potassium carbonate furnishing the target compounds 7a-y with
moderate to good yields.
<Insert scheme 1 here>
All synthesized compounds (7a-y) were characterized by analytical techniques viz. HRMS,
13
1
1H and C NMR Spectroscopy. The H NMR spectrum of 7a showed characteristic protons
of O- and N-attached methylene groups of morpholine at δ 2.59 and at δ 3.76. The methylene
group protons on the C-4 position of coumarin appeared at δ 3.64 and remaining all protons
13
appeared in the range of δ 5.25-8.50. In the C-NMR spectrum of 7a, the carbonyl and
methylene carbon on the C-4 position of coumarin appeared at δ 161.15 and δ 30.97. The
remaining carbons appeared in the range of δ 53.80-160.54. A similar fashion was observed
1
13
in the rest of all other compounds H NMR and C NMR spectrum (7a-y). In addition, the

compounds 7f-j showed characteristic singlet for dimethyl protons of dimethylmorpholine at
δ 1.16 and the compounds 7k-o, exhibited a sharp singlet of methyl protons of methyl
piperidine at δ 1.42. The ester group of piperidine ester derivatives 7p-t showed a triplet at δ
1.26 and quartet at δ 4.14. The HRMS (ESI) of 7a-y showed corresponding [M+H] + peaks
based on their molecular weights.
2.2. Radiochemistry
The [18F] fluoride is produced via [18O (p, n)18F] nuclear reaction in the cyclotron. Around
350  30 mCi of [18F] radionuclide was transferred from cyclotron to the GE tracer lab FX2N
synthesis module and further, it was trapped in Quaternary Ammonium Anion exchange
(QMA) cartridge and eluted by kryptofix (K222) solution from QMA and eluted into the
reactor (Fig. 2). The [18F]-cryptand complex was dried and radiolabeling of [18F]-7q was
done by aromatic nucleophilic substitution reaction (SNAr) [27], by reacting the precursor
molecule (7q) with [18F]-cryptand (K2.2.2) complex in DMF (Scheme-2). The yield of the
[18F]-7q was 5  2% after purification through tC18 cartridge. In the purification process,
initially the reaction mixture was washed out with water and 20% ethanol, and the maximum
elution was achieved with 60% ethanol. The radiochemical purity was evaluated by High
Pressure Liquid Chromatography (HPLC) analysis [28], and, the usual retention time for free
[18F] radionuclide is at 4.3  0.7 min and found the retention time of [18F]-7q compound at
16.80  0.2 min (Fig. 3A). The HPLC retention time for cold target compounds was found at
16.88  0.2 min (Fig. 3B). The matching of retention time of cold target compound with
[18F]-7q compound confirmed the radiolabeling of the precursor molecule (7q) and proves
that the target compound is our radiolabeled molecule of interest.
<Insert scheme 2 here>
<insert Figure 2 here>
The retention factor was 0.6 for [18F]-7q compound on the TLC study. The radiochemical
purity for the [18F]-7q was around > 85 % as measured by HPLC. The pH of the preparation
was 5.5  0.5 as evaluated by pH paper. The levels of the solvent Dimethylformamide (DMF)
was evaluated in the final preparation using Gas Chromatography (GC) analysis [29]. The GC
analysis showed DMF level was 65  3 ppm (Fig. 4). As per the Food Drug Administration
(FDA) Q3C(R6) guidelines, the DMF is considered as a class 2-solvent and the acceptable
limit is 880 ppm. The levels of the DMF in the final product were in the acceptable limit. The

ethanol content is high as it is an eluting solution. The United States Food and Drug
Administration (USFDA) and European Medicines Agency have found the higher amounts of
ethanol (> 0.5%, the limit for class 3 residual solvents) in radiopharmaceuticals may be
acceptable provided they are synthesized and manufactured under good manufacturing
practice [30]. Our study has shown the successful radiolabeling of the 7q compound with
[18F] radionuclide which can target the Gal-1 protein on the overexpressed cells. The novel 4,
7-disubstituted coumarins could be a potential cytotoxic, but the quantity of molecule used
for PET imaging is in lower nanogram concentration. The final quantity in the radiolabeled
formulation is in nanomoles, therefore, impact of any side effects is very rare. Target based
PET diagnosis is the primary choice to know the disease progression and prescribe better
medication to the patient, hence we have explored the feasibility of radiolabeling of 7q with
18F radionuclide, and succeeded in achieving good results. There are many drugs like
paclitaxel, gefitinib, erlotinib, doxorubicin has been labeled with 18F, and used as PET
imaging agents. These drugs have their own potential side effects but still using as PET
tracers in nanomoles [31-34].
<insert Figure 3A & 3B here>
<insert Figure 4 here>
2.3 Biological evaluation:
2.3.1 Cell proliferation and cytotoxicity analysis:
To evaluate the cell proliferative and cytotoxicity of the novel coumarin analogues (7a-y),
Sulforhodamine B (SRB) cell proliferation assay against a panel of human cancer cell lines
namely, Brest (MCF7), Ovarian (SKOV3), Prostate (PC-3 & DU145) and normal embryonic
kidney (HEK293T) cells, was performed. Briefly, cells were seeded in 96 well plates with a
density of 1*104 cells/well and incubated with target compounds (7a-y) for a period of 72 h
and fixed with 10% Trichloroacetic acid [35]. Plates were stained with a 0.057% SRB stain
solution and eluted with 10mM Tris having pH 10.5. The elute was measured at 510nm with a
spectrophotometer. Inhibitory concentrations of the compounds (7a-y) were calculated with
the sigmoidal curve method and analyzed (Table 1). Overall 7q showed a prominent
inhibition of growth at 7.45±0.03 µM concentration in prostate cancer cell line PC-3. All the
experiments were performed in triplicates and Doxorubicin was employed as a positive
control. In Structural activity relationship (SAR), the morpholine linked coumarin derivatives
(7a-e) exhibited moderate cytotoxicity, whereas the dimethyl morpholine linked coumarin

derivatives (7f-j) and methyl piperidine linked coumarin derivatives (7k-0) showed good
cytotoxicity. The piperidine ester linked coumarin derivatives (7p-t) exhibited a significant
cytotoxicity and isoquinoline linked coumarin derivatives (7u-y) exhibited less cytotoxicity.
Hence, we have concluded that piperidine ester derivatives (7p-t) are promising cytotoxic
agents.
<Insert Table 1 here>
2.3.2 Effect of compound 7q on Gal-1 protein levels:
Targeting Gal-1 is considered as one of the emerging approaches for the treatment of cancer,
as it plays a vital role in tumor development and metastasis. It is evident from the literature
that Gal-1 expression is associated with numerous tumor types [36]. Compound 7q was
examined for its effect on Gal-1 levels. PC-3 cells were grown in 60mm cell culture dishes,
treated with 7q 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 [37].
Equal amounts of supernatant were subjected to quantitative enzyme immunoassay as per
manufacture’s protocol [DGAL10, R&D Systems, USA]. In details, 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 30minutes before terminating the reaction with 50µl
of the stop solution. The amount of protein expression was detected at 450 nm using UV-
spectrophotometer. Compound 7q effectively reduced the levels of Gal-1 in dose-dependent
manner at the concentrations of 10, 30, 100 and 300 µM (Fig. 5, Table 2) and the IC50 value
found to be 100µM stipulating that the molecular target of these coumarin analogues to be
Gal-1.
<Insert Figure 5 here>
<Insert Table 2 here>
2.3.3. Fluorescence studies:
In fluorescence studies, there a decrease in fluorescence intensity with increasing
concentration of the compound, and maximum emission spectra of Gal-1 was at 343 nm (Fig.
6A). The plot of log[7q] versus log(Fo-F)/F had given a linear relationship (Fig. 6B) and the
number of binding sites was calculated from the slope, which was found to be 0.89 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 1.3 x 104 M-1. The intensity of the

fluorescence was quenched upon the increase of ligand concentration and the bimolar
quenching constant (Kq) was calculated to be 3.8 x 1012 M-1 s-1 which is larger than diffusion
control limit [38] suggesting interaction of protein and ligand as well as the mode of
quenching to be static.
<Insert Figure 6A & 6B here>
2.3.4 Effect on the Cell cycle distribution:
The 7q that demonstrated the strong cytotoxic effect on PC-3 cells was further studied for cell
cycle analysis using intercalating fluorescent dye, Propidium Iodide (PI) which can be
visualized with the aid of fluorescent flow cytometry. PC-3 cells were seeded in 60mm dishes
at a density of 5x105 cells/plate and treated with 7q at 5, 7.5, 10µM concentrations for 48 h
followed by overnight fixation with 70% ethanol. Cells were further stained with PI and
analyzed with the aid of BD Accuri™ C6 flow cytometry for the distribution of cell cycle
phases (Fig. 7A). Cells demonstrated an increase in the percentage of the sub-G1 phase of cell
cycle upon treatment with 7q in a concentration-dependent manner with 13.36% at 10µM
(Fig. 7B). Fluorescent based cell cycle analysis clearly demonstrated an arrest at the sub-G1
phase of cell cycle post-treatment in PC-3 cells.
<Insert Figure 7A & 7B here>
2.3.5 Annexin-V/Propidium iodide dual staining assay:
To establish the extent of cells undergoing apoptosis, Annexin-V/Propidium Iodide staining
was employed. Annexin-V is a marker of early apoptotic cells that binds specifically to the
protein of phosphatidylserine PS [39]. Cells were grown in 60mm dishes and treated with 7q
for 48h. Thereafter, the cells were trypsinized, stained with Annexin-V/Propidium iodide and
analyzed with fluorescent based flow cytometry. Annexin-V/propidium iodide staining reveals
the distribution of cells between live, necrotic, early and late apoptotic phases. Cells treated
with 7q for 48 h demonstrated an increased cell population in the apoptotic phase in a
concentration dependent manner (Fig. 8). At 10µM concentration, approximately 28% of the
cells underwent apoptosis.
<Insert Figure 8 here>
2.3.6 Acridine orange/ethidium bromide (AO/EB) staining:
Acridine orange (AO) is a DNA intercalating dye that binds to both live and dead cells,
whereas Ethidium bromide (EB) stains only nonviable cells, i.e., cells which have lost the cell

membrane integrity [40]. The green vs red fluorescence clearly demonstrates the extent of
cells undergoing apoptosis. Cells were grown on coverslips for 48 h post-treatment with 7q,
stained with AO/EB dual stain and imaged with a fluorescent inverted microscope at 10X
magnification. PC-3 cells treated with 7q initiated apoptosis at 5 µM concentration treatment,
with a gradual increase in a concentration-dependent manner i.e., cells at 10 µM showed a
clear induction of apoptosis (Fig. 9).
<Insert Figure 9 here>
2.3.7 Hoechst-33342 nuclear morphology staining:
Cells undergoing apoptosis or necrosis, develop a condensed or fragmented DNA indicating
an ideal mark to distinguish viable and non-viable cells [41]. Hoechst 33342 is a cell-
permeable fluorescent benzimidazole dye that stains DNA by binding to the minor groove. To
observe morphological changes and to confirm above-mentioned results cells were grown in
coverslips and Hoechst staining was performed post-treatment with 7q for 48 h. Results
revealed a clear condensation of DNA as marked by arrows in Fig. 10. This clearly
demonstrates altered nuclear architecture due to the treatment with 7q in PC-3 cells.
<Insert Figure 10 here>
2.3.8 Effect on the Mitochondrial membrane potential:
The loss of mitochondrial membrane potential distinctly indicates cell apoptosis [42]. As
induction of apoptosis by the treatment of 7q was evidently established we wanted to see the
effect on mitochondrial membrane potential. The dysfunction of mitochondria has been long
associated with early induction apoptosis and measurement of the depolarization of
mitochondria reveals the extent of the induction. JC-1, a carbocyanine cationic dye that
accumulates in the mitochondria in a potential-dependent manner i.e., JC-1 forms J-aggregates
(red fluorescence) in energized mitochondria whereas loss of membrane potential leads to the
formation of JC-1 monomers (green fluorescence) [43]. JC-1 forms J-aggregates in turn
emitting red fluorescence in untreated control PC-3 cells, whereas depolarized mitochondria
due to the 7q treatment for 48 h emit green fluorescence i.e., the formation of J-monomers
(Fig. 11A). Analysis with fluorescent-based flow cytometry revealed that there was increased
depolarization of mitochondrial membrane potential upon 7q treatment (fig. 11B). This data
establishes the role of 7q in the induction of apoptosis through the loss of mitochondrial
membrane potential.

<Insert Figure 11A & 11B here>
2.3.9 Estimation of ROS levels by DCFDA fluorescence assay:
Reactive oxygen species (ROS) play a seemingly vital role in physiological as well as
pathological conditions including apoptosis [44]. As 7q has the ability to induce apoptosis,
levels of ROS were studied. To evaluate the levels of ROS, the reduced fluorescein,
H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) was utilized. H2DCFDA is a non-
fluorescent dye, which emits fluorescence upon the breakage of the acetate moieties due to the
presence of intracellular esterases. Treatment of the PC-3 cells with 7q showed a clear
elevation in ROS levels as visualized by the emission of green fluorescence by the H2DCFDA
dye (Fig. 12).
<Insert Figure 12 here>
2.4 Molecular docking Studies:
The interactions of Gal-1 with the compound 7q were studied by molecular
docking calculations using the Glide docking module of Schrodinger suite [45]. The
3D crystal co-ordinates of human Gal-1 were retrieved from the protein data bank
(PDB ID: 4Y24). The 2D, 3D ligand interaction, and electronic surface interaction diagrams
of 7q was shown in Figure 13A, 13B & 13C. Hydrogen bonding interaction was found at a
distance of 3.06Å between NH of Glu71 acting as a donor (atom no. 1518) and C=O of ester
moiety of ethyl isonipecotate moiety acting as an acceptor (atom no. 2031). Another
hydrogen bond was found at a distance of 2.07Å between OH of Ser29 as a donor (atom no.
1229) and the oxygen of nitro on phenyl acting as an acceptor (atom no. 2040). Further, π- π
stacking between the indole ring of Trp68 and coumarin ring and the π-cation stacking
interaction between the nitrogen of Arg48(atom no. 354) and the coumarin ring seems to
stabilize the docking pose of the 7q molecule. A set of pharmacokinetic Absorption,
Distribution, Metabolism, Elimination (ADMET) related properties were calculated using
qikprop program [46]. The physicochemical properties of 7q matched with the prescribed
ranges as represented in Table 3. Additionally, the molecular weight (mol. Wt.),
hydrogen bond donors (donor HB), hydrogen bond acceptors (accept HB), partition
coefficient (QPlogPo/w), exhibited acceptable values that followed Lipinski rule of five.

<Insert Figure 13A, 13B & 13C here>
<Insert Table 3 here>
3.0 Conclusion:
A series of novel 4, 7-disubstituted coumarin derivatives were synthesized (7a-y) and
cytotoxic activity was evaluated against a panel of cell lines. Amongst all the synthesized
conjugates, 7q exhibited potential anti-proliferative against prostate cancer cell line, PC-3 at a
concentration of 7.45±0.03 µM. Cell cycle distribution analysis revealed an arrest at the sub-
G1 phase, following treatment with conjugate 7q. Annexin-V staining revealed an increasing
population of cells undergoing apoptosis in a concentration-dependent manner by treatment
with conjugate 7q in PC-3 cells. This was supported by both, Acridine Orange/Ethidium
Bromide and Hoechst 33342 nuclear staining. Loss of mitochondrial membrane potential and
increased reactive oxygen species, as established by the JC-1 and H2DCFDA staining,
respectively suggest a possible mechanism of action of the conjugate 7q in inducing
apoptosis. In addition, compound 7q efficiently reduced the levels of Gal-1 protein in dose-
dependent manner confirmed by ELISA. we have explored the feasibility of radiolabeling
18
with F radionuclide as to show that this cytotoxic molecule 7q can also be used as a PET
tracer in low concentration. Our studies show the potential of novel 4, 7-disubstituted
coumarin derivatives in the development of a novel class of small molecules against
oncogenesis.
4.0 Methods and Materials:
4.1 Chemistry
All the starting materials, specific reagents and solvents were purchased from commercial
suppliers and utilized without further purification. 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 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
13
1H-NMR and 125 MHz for C NMR) using CDCl3 and DMSO 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.
4.1.1. Synthetic procedures and spectral data
4.1.1.1. Synthesis of 4-(chloromethyl)-7-hydroxy-2H-chromen-2-one (3)
A 50 mL round bottom flask was charged with resorcinol (1 equi) and ethyl-4-
chloroacetoacetate (1.1 equi), make it into a clear solution than the solution was added drop
wise to an externally cooled conc. H2SO4 (10 mL) contains in a 50 mL beaker at 10 °C and
the reaction mixture was then stirred at room temperature for 20 min. After confirmation by
TLC, the mixture was poured into ice water contained in a beaker, the precipitate compound
3 obtained was collected by suction filtration and washed with cold water, dried and
1
recrystallized from ethanol. white solid, yield 91%; mp 186-188 °C; H NMR (500 MHz,
DMSO) δ 10.66 (s, 1H), 7.68 (d, J = 7.8 Hz, 1H), 6.90 – 6.71 (m, 2H), 6.42 (s, 1H), 4.96 (s,
2H). 13C NMR (126 MHz, DMSO) δ 161.94, 160.61, 155.78, 151.45, 127.01, 113.55, 111.54,
109.83, 103.00, 41.83. HRMS (ESI): m/z calcd for C10H7ClO3, 210.0084, found 211.0157
[M+H] +
4.1.1.2. Synthesis of 7-hydroxy-4-(substituted morpholinomethyl)-2H-chromen-2-ones (4a,
4b)
To a solution of 4-(chloromethyl)-7-hydroxy-2H-chromen-2-one (1 mmol) (3) in freshly
distilled acetone (5 mL), the substituted morpholines (1 mmol) and anhydrous K2CO3 (2.5
mmol) were added and the reaction mixture was stirred under reflux for 4-5 hr. After
confirmation by TLC, solvent in the reaction mixture was removed by evaporation and left
out crude solid residue was extracted with ethyl acetate (3x20 ml), dried over Na2SO4. The
combined organic layer was concentrated in vacuo and the residues were purified by column
chromatography on silica gel.
4.1.1.2.1. 7-hydroxy-4-(morpholinomethyl)-2H-chromen-2-one (4a)
1
white solid, yield 86%; mp 193-195 °C; H NMR (500 MHz, DMSO) δ 10.56 (s, 1H), 7.80
(d, J = 6.9 Hz, 1H), 6.84 (dd, J = 8.7, 2.4 Hz, 1H), 6.76 (d, J = 2.4 Hz, 1H), 6.29 (s, 1H), 3.66
(s, 2H), 3.66 – 3.62 (t, J = 5.9 Hz, 4H), 2.51 (s, 4H). 13C NMR (126 MHz, DMSO) δ 161.52,
160.82, 155.69, 152.86, 127.21, 113.24, 111.35, 110.67, 102.65, 66.69, 58.76, 53.83. HRMS
(ESI): m/z calcd for C14H15NO4, 261.1001, found 262.1075 [M+H] +
4.1.1.2.2. 4-((2,6-dimethylmorpholino) methyl)-7-hydroxy-2H-chromen-2-one (4b)

1
Off white solid, yield 84%; mp 216-218 °C; H NMR (500 MHz, DMSO) δ 10.51 (s, 1H),
7.75 (d, J = 8.8 Hz, 1H), 6.78 (dd, J = 8.7, 2.4 Hz, 1H), 6.70 (d, J = 2.4 Hz, 1H), 6.23 (s, 1H),
3.59 – 3.54 (m, 4H), 2.76 (d, J = 10.3 Hz, 2H), 1.75 (t, J = 10.7 Hz, 2H), 1.04 (d, J = 6.3 Hz,
6H). 13C NMR (126 MHz, DMSO) δ 161.51, 160.84, 155.70, 152.92, 127.28, 113.23, 111.38,
110.77, 102.64, 71.50, 59.51, 58.44, 19.36. HRMS (ESI): m/z calcd for C16H19NO4,
289.1314, found 290.1396 [M+H] +
4.1.1.3. Synthesis of 7-hydroxy-4-(substituted piperidinomethyl)-2H-chromen-2-ones (5a,
5b)
To a solution of 4-(chloromethyl)-7-hydroxy-2H-chromen-2-one (1 mmol) (3) in freshly
distilled acetone (5 mL), the substituted piperidines (1 mmol) and anhydrous K2CO3 (2.5
mmol) were and the reaction mixture was stirred under reflux for 5-6 hr. After confirmation
by TLC, solvent in the reaction mixture was removed by evaporation left a crude solid
residue that was extracted with ethyl acetate (3x20 ml) and dried over Na2SO4. The combined
organic layer was concentrated in vacuo and the residues were purified by column
chromatography on silica gel.
4.1.1.3.1. 7-hydroxy-4-((4-methylpiperidin-1-yl) methyl)-2H-chromen-2-one (5a)
1
white solid, yield 83%; mp 144-146 °C; H NMR (500 MHz, DMSO) δ 10.50 (s, 1H), 7.75
(d, J = 8.7 Hz, 1H), 6.78 (dd, J = 8.7, 2.1 Hz, 1H), 6.71 (d, J = 2.1 Hz, 1H), 6.20 (s, 1H), 3.58
(s, 2H), 2.83 (d, J = 11.2 Hz, 2H), 2.02 (dd, J = 19.6, 8.9 Hz, 2H), 1.58 (d, J = 12.1 Hz, 2H),
13
1.40-1.32 (m, 1H), 1.19 – 1.11 (m, 2H), 0.89 (d, J = 6.5 Hz, 3H). C NMR (125 MHz,
DMSO) δ 161.55, 160.93, 155.67, 153.80, 127.14, 113.23, 111.42, 110.22, 102.64, 58.88,
54.09, 34.48, 30.57, 22.20. HRMS (ESI): m/z calcd for C16H19NO3, 273.1365, found
274.1439 [M+H] +
4.1.1.3.2. ethyl 1-((7-hydroxy-2-oxo-2H-chromen-4-yl) methyl) piperidine-4-carboxylate
(5b)
1
white solid, yield 80%; mp 162-164 °C; H NMR (500 MHz, DMSO) δ 7.76 (d, J = 8.8 Hz,
1H), 6.79 (dd, J = 8.7, 2.3 Hz, 1H), 6.71 (d, J = 2.3 Hz, 1H), 6.21 (s, 1H), 4.07 (q, J = 7.1 Hz,
2H), 3.60 (s, 2H), 2.82 (d, J = 11.5 Hz, 2H), 2.32 (s, 1H), 2.13 (dd, J = 11.2, 9.3 Hz, 2H),
13
1.85 – 1.78 (m, 2H), 1.63 – 1.55 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). C NMR (125 MHz,
DMSO) δ 174.75, 161.55, 160.89, 155.67, 153.50, 127.18, 113.23, 111.37, 110.35, 102.64,
60.25, 58.68, 52.97, 40.49, 28.48, 14.55. HRMS (ESI): m/z calcd for C18H21NO5, 331.1420,
found 332.1496 [M+H] +

4.1.1.4. Synthesis of 4-((3,4-dihydroisoquinolin-2(1H)-yl) methyl)-7-hydroxy-2H-chromen-
2-one (6a)
To a solution of 4-(chloromethyl)-7-hydroxy-2H-chromen-2-one (1 mmol) (3) in freshly
distilled acetone (5 mL), the tetrahydroisoquinoline (1 mmol) and anhydrous K2CO3 (2.5
mmol) were and the reaction mixture was stirred under reflux for 3-4 hr and remaining
procedure is as similar as the synthesis of 4a, 4b compounds. Brown solid, yield 84%; mp
171-173 °C; 1H NMR (500 MHz, DMSO) δ 7.83 (d, J = 8.8 Hz, 1H), 7.20-7.10 (m, 4H), 6.85
– 6.78 (m, 1H), 6.75 (d, J = 6.3 Hz, 1H), 6.30 (s, 1H), 3.65 (s, 2H), 3.02 (t, J = 5.7 Hz, 1H),
2.88 – 2.75 (m, 5H). 13C NMR (125 MHz, DMSO) δ 162.24, 160.97, 155.83, 153.34,
134.96, 134.38, 129.40, 128.93, 127.24, 126.52, 125.97, 113.48, 111.07, 110.31, 102.70,
58.43, 55.95, 51.03, 29.13. HRMS (ESI): m/z calcd for C19H17NO3, 307.1208, found
308.1287 [M+H] +
4.1.2. General procedure for the synthesis of final compounds 7a-y.
To the individual solution of compounds 4a, 4b, 5a, 5b and 6a (1 equi) in freshly distilled
acetone (10 mL), added K2CO3 (2.5 equi) as base and various alkyl halides and aryl halides
(1.1 equi) were added and stirred under reflux for 2-3 h. The solvent was evaporated
completely and the obtained residue was extracted with ethyl acetate (3x20 ml) and dried
over Na2SO4. The combined organic layer was concentrated in vacuo and the residues were
purified by column chromatography on silica gel, offered the final target compounds 7a-y
with moderate to good yields.
4.1.3 Spectral data for final compounds 7a-7y
4.1.3.1 4-(morpholinomethyl)-7-((4-nitrobenzyl)oxy)-2H-chromen-2-one (7a)
Off white solid, yield 85%; mp 174-176 °C; 1H NMR (500 MHz, CDCl3) δ 8.30 – 8.25 (m,
2H), 7.81 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 9.2, 4.6 Hz, 1H), 6.88 (d, J
= 2.5 Hz, 1H), 6.42 (s, 1H), 5.25 (s, 2H), 3.76 (s, 4H), 3.64 (s, 2H), 2.59 (s, 4H). 13C NMR
(125 MHz, DMSO+ CDCl3) δ 161.15, 160.54, 159.57, 155.49, 147.56, 144.18, 128.31,
126.80, 123.84, 112.83, 112.47, 112.32, 102.08, 68.99, 66.44, 53.64, 30.97. HRMS (ESI):
m/z calcd for C21H20N2O6, 396.1321, found 397.1396 [M+H] +
4.1.3.2 2-((4-(morpholinomethyl)-2-oxo-2H-chromen-7-yl) oxy)acetonitrile (7b)
Off white solid, yield 84%; mp 164-166 °C; 1H NMR (500 MHz, CDCl3) δ 7.86 (d, J = 8.6
Hz, 1H), 6.98 – 6.91 (m, 2H), 6.44 (s, 1H), 4.85 (s, 2H), 3.74 (t, J = 9.1 Hz, 4H), 3.60 (s, 2H),

2.54 (t, J = 8.7 Hz, 4H). 13C NMR (125 MHz, CDCl3) δ 160.67, 158.89, 155.36, 151.22,
126.66, 114.32, 114.22, 113.51, 111.99, 102.45, 66.89, 59.61, 53.80, 53.49. HRMS (ESI):
m/z calcd for C16H16N2O4, 300.1110, found 301.1394 [M+H] +
4.1.3.3 4-(morpholinomethyl)-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (7c)
Brown solid, yield 82%; mp 117-119 °C; 1H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 8.8 Hz,
1H), 6.99 – 6.87 (m, 2H), 6.40 (s, 1H), 4.77 (d, J = 2.3 Hz, 2H), 3.73 (t, J = 8.8 Hz, 4H), 3.60
(s, 2H), 2.58 (t, J = 2.3 Hz, 1H), 2.55 (t, J = 3.8 Hz, 4H). 13C NMR (125 MHz, CDCl3) δ
161.19, 160.34, 155.42, 151.53, 125.97, 113.05, 112.74, 112.47, 102.12, 77.42, 76.53, 66.90,
59.53, 56.17, 53.82. HRMS (ESI): m/z calcd for C17H17NO4, 299.1158, found 300.1235
[M+H] +
4.1.3.4 7-((4-bromobenzyl)oxy)-4-(morpholinomethyl)-2H-chromen-2-one (7d)
White solid, yield 83%; mp 184-186 °C; 1H NMR (500 MHz, CDCl3) δ 7.76 (d, J = 8.9 Hz,
1H), 7.55 – 7.50 (m, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.93 – 6.85 (m, 2H), 6.39 (s, 1H), 5.08 (s,
2H), 3.74 (s, 4H), 3.60 (s, 2H), 2.55 (s, 4H). 13C NMR (125 MHz, CDCl3) δ 161.37, 161.03,
155.58, 134.85, 131.93, 129.11, 126.00, 122.37, 112.86, 112.71, 112.32, 101.94, 69.69,
66.83, 59.56, 53.80. HRMS (ESI): m/z calcd for C21H20BrNO4, 429.0576, found 430.0652
[M+H] +
4.1.3.5 7-(allyloxy)-4-(morpholinomethyl)-2H-chromen-2-one (7e)
Off white solid, yield 84%; mp 106-108 °C; 1H NMR (500 MHz, CDCl3) δ 7.75 (d, J = 8.8
Hz, 1H), 6.93 – 6.81 (m, 2H), 6.40 (s, 1H), 6.05 (s, 1H), 5.45 (dd, J = 17.3 Hz, 1H), 5.35 (dd,
J = 16.7 Hz, 1H), 4.61 (d, J = 5.3 Hz, 2H), 3.76 (s, 4H), 3.62 (s, 2H), 2.57 (s, 4H). 13C NMR
(125 MHz, CDCl3) δ 161.70, 161.12, 155.62, 132.14, 125.87, 118.58, 112.91, 112.35,
101.84, 69.27, 66.63, 58.95, 53.70. HRMS (ESI): m/z calcd for C17H19NO4, 301.1314, found
302.1392 [M+H] +
4.1.3.6 4-((2,6-dimethylmorpholino)methyl)-7-((4-nitrobenzyl)oxy)-2H-chrom en-2-one (7f)
White solid, yield 86%; mp 195-197 °C; 1H NMR (500 MHz, CDCl3) δ 8.34 – 8.22 (m, 2H),
7.80 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.00 – 6.81 (m, 2H), 6.40 (s, 1H), 5.24 (s,
2H), 3.71 (s, 2H), 3.58 (s, 2H), 2.76 (s, 2H), 1.90 (s, 2H), 1.16 (d, J = 6.3 Hz, 6H). 13C NMR
(125 MHz, CDCl3) δ 163.36, 160.81, 155.58, 147.85, 143.12, 127.73, 126.31, 124.00,
112.95, 112.78, 112.63, 101.99, 69.06, 59.51, 59.24, 30.93, 18.98. HRMS (ESI): m/z calcd
for C23H24N2O6, 424.1634, found 425.1715 [M+H] +

4.1.3.7 2-((4-((2,6-dimethylmorpholino) methyl)-2-oxo-2H-chromen-7-yl) oxy) acetonitrile
(7g)
Off white solid, yield 83%; mp 146-148 °C; 1H NMR (500 MHz, CDCl3) δ 7.89 – 7.83 (m,
1H), 6.94 (dd, J = 7.3, 2.4 Hz, 2H), 6.43 (s, 1H), 4.86 (s, 2H), 3.70 (m, 2H), 3.57 (s, 2H), 2.73
(d, J = 10.4 Hz, 2H), 1.89 (t, J = 10.7 Hz, 2H), 1.16 (d, J = 6.3 Hz, 6H). 13C NMR (125
MHz, CDCl3) δ 160.74, 158.89, 155.34, 151.43, 126.72, 114.34, 114.27, 113.48, 112.02,
102.41, 71.66, 59.52, 59.22, 53.51, 30.93, 19.01. HRMS (ESI): m/z calcd for C18H20N2O4,
328.1423, found 329.1502 [M+H] +
4.1.3.8 4-((2,6-dimethylmorpholino) methyl)-7-(prop-2-yn-1-yloxy)-2H-chrom en-2-one (7h)
Off white solid, yield 82%; mp 112-114 °C; 1H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 7.2
Hz,1H), 6.98 – 6.87 (m, 2H), 6.39 (s, 1H), 4.77 (d, J = 2.2 Hz, 2H), 3.75 – 3.65 (m, 2H), 3.54
(d, J = 18.9 Hz, 2H), 2.74 (d, J = 10.6 Hz, 2H), 2.58 (t, J = 2.3 Hz, 1H), 1.88 (t, J = 10.6 Hz,
2H), 1.16 (d, J = 6.3 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 161.26, 160.32, 155.42,
151.74, 126.04, 113.10, 112.73, 112.47, 102.09, 77.43, 76.52, 71.68, 59.57, 59.16, 56.16,
19.02. HRMS (ESI): m/z calcd for C19H21NO4, 327.1471, found 328.1548 [M+H] +
4.1.3.9 4-(((4-((2,6-dimethylmorpholino) methyl)-2-oxo-2H-chromen-7-yl)oxy) methyl)
benzonitrile (7i)
white solid, yield 83%; mp 180-182 °C; 1H NMR (500 MHz, CDCl3) δ 7.78 (d, J = 8.8 Hz,
1H), 7.71 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 6.94 – 6.84 (m, 2H), 6.39 (s, 1H), 5.19
(s, 2H), 3.71 (s, 2H), 3.57 (s, 2H), 2.75 (d, J = 10.1 Hz, 2H), 1.89 (t, J = 9.6 Hz, 2H), 1.16 (d,
J = 6.3 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 155.58, 141.17, 132.58, 127.65, 126.26,
118.47, 112.80, 112.23, 101.99, 71.31, 69.30, 59.04, 30.93, 18.96. HRMS (ESI): m/z calcd
for C24H24N2O4, 404.1736, found 405.1811 [M+H] +
4.1.3.10 7-(allyloxy)-4-((2,6-dimethylmorpholino) methyl)-2H-chromen-2-one (7j)
Off white solid, yield 82%; mp 108-110 °C; 1H NMR (500 MHz, CDCl3) δ 7.75 (d, J = 9.6
Hz, 1H), 6.91 – 6.78 (m, 2H), 6.37 (s, 1H), 6.05 (m, 1H), 5.45 (dd, J = 17.3, 1.3 Hz, 1H), 5.35
(dd, J = 10.5, 1.2 Hz, 1H), 4.61 (d, J = 5.3 Hz, 2H), 3.75 – 3.65 (m, 2H), 3.54 (s, 2H), 2.74 (d,
J = 10.7 Hz, 2H), 1.88 (t, J = 10.6 Hz, 2H), 1.16 (d, J = 6.3 Hz, 6H). 13C NMR (125 MHz,
CDCl3) δ 161.62, 161.31, 155.60, 132.16, 125.91, 118.56, 112.86, 112.43, 101.77, 71.52,
69.25, 59.41, 58.92, 30.93, 18.99. HRMS (ESI): m/z calcd for C19H23NO4, 329.1627, found
330.1704 [M+H] +

4.1.3.11 4-((4-methylpiperidin-1-yl) methyl)-7-((4-nitrobenzyl) oxy)-2H-chrom en-2-one
(7k)
Off white solid, yield 86%; mp 198-200 °C; 1H NMR (500 MHz, CDCl3) δ 8.32 – 8.23 (m,
2H), 7.86 (d, J = 8.2 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.7 Hz, 1H), 6.87 (d, J =
2.5 Hz, 1H), 6.43 (s, 1H), 5.24 (s, 2H), 3.65 (s, 2H), 2.95 (s, 2H), 2.17 (s, 2H), 1.65 (s, 2H),
1.42 (s, 3H), 0.95 (d, J = 5.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 161.16, 160.82,
155.51, 147.84, 144.01, 143.23, 127.71, 126.39, 123.99, 113.35, 112.62, 101.91, 69.02,
54.33, 33.81, 30.43, 21.71. HRMS (ESI): m/z calcd for C23H24N2O5, 408.1685, found
409.1763 [M+H] +
4.1.3.12 2-((4-((4-methylpiperidin-1-yl) methyl)-2-oxo-2H-chromen-7-yl) oxy) acetonitrile
(7l)
Off white solid, yield 83%; mp 152-154 °C; 1H NMR (500 MHz, CDCl3) δ 7.92 (d, J = 8.6
Hz, 1H), 6.98 – 6.90 (m, 2H), 6.46 (s, 1H), 4.85 (s, 2H), 3.63 (s, 2H), 2.95 (s, 2H), 2.24 –
2.05 (s, 2H), 1.64 (s, 2H), 1.48 – 1.22 (m, 3H), 0.94 (d, J = 6.2 Hz, 3H). 13C NMR (125
MHz, CDCl3) δ 160.87, 158.81, 155.28, 126.83, 114.55, 114.31, 113.35, 111.88, 102.39,
59.52, 54.34, 53.51, 34.17, 30.47, 21.75. HRMS (ESI): m/z calcd for C18H20N2O3, 312.1474,
found 313.1550 [M+H] +
4.1.3.13
4-(((4-((4-methylpiperidin-1-yl)
methyl)-2-oxo-2H-chromen-7-yl)oxy)
methyl)benzonitrile (7m)
1
Off white solid, yield 84%; mp 177-179 °C; H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 8.7
Hz, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 6.95 – 6.82 (m, 2H), 6.40 (s, 1H),
5.19 (s, 2H), 3.59 (s, 2H), 2.90 (s, 2H), 2.14 (d, J = 28.0 Hz, 2H), 1.63 (s, 2H), 1.45 – 1.26
(m, 3H), 0.94 (d, J = 6.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 160.99, 155.52, 141.25,
132.58, 127.65, 126.39, 118.50, 113.00, 112.69, 112.19, 101.94, 69.28, 58.95, 54.26, 33.81,
30.27, 21.60. HRMS (ESI): m/z calcd for C24H24N2O3, 388.1787, found 389.1864 [M+H] +
4.1.3.14 7-(allyloxy)-4-((4-methylpiperidin-1-yl) methyl)-2H-chromen-2-one (7n)
Brown solid, yield 82%; mp 107-109 °C; 1H NMR (500 MHz, CDCl3) δ 7.71 (d, J = 8.8 Hz,
1H), 6.83 – 6.71 (m, 2H), 6.31 (s, 1H), 5.98 (m, 1H), 5.37 (dd, J = 17.3, 1.4 Hz, 1H), 5.27
(dd, J = 10.5, 1.2 Hz, 1H), 4.53 (d, J = 5.3 Hz, 2H), 3.53 (s, 2H), 2.84 (s, 2H), 2.08 (s, 2H),
1.55 (s, 2H), 1.37 – 1.20 (m, 3H), 0.87 (d, J = 6.3 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ
161.52, 161.43, 155.54, 132.22, 125.98, 118.51, 112.76, 112.65, 101.75, 69.22, 59.23, 54.30,

33.99, 30.40, 21.70. HRMS (ESI): m/z calcd for C19H23NO3, 313.1678, found 314.1753
[M+H] +
4.1.3.15 7-((4-bromobenzyl)oxy)-4-((4-methylpiperidin-1-yl)methyl)-2H-chrom en-2-one
(7o)
white solid, yield 84%; mp 149-151 °C; 1H NMR (500 MHz, CDCl3) δ 7.81 (d, J = 8.8 Hz,
1H), 7.56 – 7.50 (m, 2H), 7.30 (d, J = 9.8 Hz, 2H), 6.94 – 6.83 (m, 2H), 6.40 (s, 1H), 5.08 (s,
2H), 3.63 (s, 2H), 2.93 (s, 2H), 2.16 (s, 2H), 1.64 (s, 2H), 1.33 (m, 3H), 0.94 (d, J = 5.9 Hz,
3H). 13C NMR (125 MHz, CDCl3) δ 161.41, 161.15, 155.53, 134.85, 131.92, 131.92,
129.12, 126.19, 122.36, 112.88, 102.01, 69.69, 54.22, 33.88, 30.25, 21.59. HRMS (ESI): m/z
calcd for C23H24BrNO3, 442.3530, found 444.0997 [M+2] +
4.1.3.16 ethyl 1-((7-(benzyloxy)-2-oxo-2H-chromen-4-yl)methyl)piperidine-4-carboxylate
(7p)
Brown solid, yield 84%; mp 130-132 °C; 1H NMR (500 MHz, CDCl3) δ 8.28 – 8.21 (m,
2H), 7.84 (d, J = 8.8 Hz, 1H), 7.67 – 7.52 (m, 3H), 6.96 – 6.82 (m, 2H), 6.38 (s, 1H), 5.24 (s,
2H), 4.14 (q, J = 7.1 Hz, 2H), 3.60 (s, 2H), 2.97 – 2.79 (m, 2H), 2.34 (s, 1H), 2.24 – 2.14 (m,
2H), 1.93 (s, 2H), 1.82 (s, 2H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ
160.81, 155.54, 147.83, 143.15, 129.93, 127.73, 126.46, 126.20, 124.05, 124.00, 112.83,
112.74, 102.07, 69.05, 60.60, 52.93, 30.92, 29.69, 14.20. HRMS (ESI): m/z calcd for
C25H27NO5, 421.1889, found 422.1966 [M+H] +
4.1.3.17 ethyl 1-((7-((4-nitrobenzyl) oxy)-2-oxo-2H-chromen-4-yl)methyl) piperidine-4-
carboxylate (7q)
Brown solid, yield 86%; mp 135-137 °C; 1H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 8.8 Hz,
1H), 7.42 (m, 3H), 7.35 (m, 1H), 6.93 – 6.87 (m, 2H), 6.34 (s, 1H), 5.13 (s, 2H), 4.14 (q, J =
7.1 Hz, 2H), 3.56 (s, 2H), 2.87 (d, J = 11.4 Hz, 2H), 2.31 (m, 1H), 2.20 – 2.11 (m, 2H), 1.94 –
1.86 (m, 2H), 1.82 – 1.74 (m, 2H), 1.25 (t, J = 6.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ
174.98, 161.59, 161.48, 155.55, 152.38, 135.88, 128.76, 128.36, 127.51, 126.06, 112.84,
111.92, 101.87, 70.45, 60.41, 53.30, 40.85, 30.92, 28.27, 14.22. HRMS (ESI): m/z calcd for
C25H26N2O7, 466.1740, found 467.1818 [M+H] +
4.1.3.18 ethyl 1-((7-((4-bromobenzyl) oxy)-2-oxo-2H-chromen-4-yl) methyl) piperidine-4-
carboxylate (7r)

1
Off white solid, yield 81%; mp 127-129 °C; H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 8.8
Hz, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H), 6.94 – 6.85 (m, 2H), 6.42 (s, 1H),
5.09 (d, J = 13.3 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.69 (s, 2H), 2.95 (s, 2H), 2.38 (s, 2H),
2.17 (s, 1H), 1.99 (s, 2H), 1.83 (s, 2H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ 161.53, 155.55, 134.82, 131.92, 129.12, 126.20, 122.36, 112.91, 112.66, 102.05,
69.70, 60.59, 52.94, 30.92, 28.14, 14.20. HRMS (ESI): m/z calcd for C25H26N2O7, 500.3890,
found 502.1053 [M+2] +
4.1.3.19 ethyl 1-((7-((4-methoxybenzyl) oxy)-2-oxo-2H-chromen-4-yl) methyl) piperidine-4-
carboxylate (7s)
Brown solid, yield 80%; mp 105-107 °C; 1H NMR (500 MHz, CDCl3) δ 7.76 (d, J = 9.2 Hz,
1H), 7.36 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 6.90 – 6.87 (m, 2H), 6.34 (s, 1H), 5.05
(s, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H), 3.54 (d, J = 19.1 Hz, 2H), 2.88 (d, J = 11.4 Hz,
2H), 2.35 – 2.28 (m, 1H), 2.20 – 2.14 (m, 2H), 1.91 (d, J = 11.2 Hz, 2H), 1.80 (d, J = 17.7,
6.8 Hz, 2H), 1.25 (d, J = 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 174.95, 161.69,
161.50, 159.74, 155.55, 129.33, 127.86, 126.01, 114.18, 113.80, 112.91, 112.61, 111.88,
101.83, 70.28, 60.41, 55.33, 53.27, 40.81, 30.91, 28.24, 14.21. HRMS (ESI): m/z calcd for
C26H29NO6, 451.1995, found 452.2069 [M+H] +
4.1.3.20 ethyl 1-((7-((4-cyanobenzyl) oxy)-2-oxo-2H-chromen-4-yl) methyl) piperidine-4-
carboxylate (7t)
1
white solid, yield 84%; mp 149-151 °C; H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 8.2 Hz,
1H), 7.71 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 6.89 (m, 2H), 6.39 (s, 1H), 5.19 (s,
2H), 4.14 (q, J = 7.1 Hz, 2H), 3.61 (s, 2H), 2.94 (d, J = 22.8 Hz, 2H), 2.34 (s, 1H), 2.17 (s,
2H), 1.93 (s, 2H), 1.83 (s, 2H), 1.26 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ
160.80, 155.51, 147.82, 143.13, 129.90, 127.71, 126.42, 126.22, 124.03, 124.01, 112.80,
112.71, 102.09, 69.04, 60.61, 52.92, 30.94, 29.67, 14.24. HRMS (ESI): m/z calcd for
C26H26N2O5, 446.1842, found 447.1918 [M+H] +
4.1.3.21 4-((3,4-dihydroisoquinolin-2(1H)-yl) methyl)-7-((4-nitrobenzyl) oxy)-2H-chromen-
2-one (7u)
Brown solid, yield 85%; mp 190-192 °C; 1H NMR (500 MHz, CDCl3) δ 8.26 (d, J = 8.7 Hz,
2H), 7.89 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.7 Hz, 2H), 7.16 (m, 3H), 7.02 (d, J = 7.0 Hz,
1H), 6.93 – 6.86 (m, 2H), 6.49 (s, 1H), 5.23 (s, 2H), 3.88 (d, J = 25.2 Hz, 4H), 2.99 (s, 4H).
13C NMR (125 MHz, CDCl3) δ 161.05, 160.95, 155.56, 147.81, 143.21, 133.46, 128.77,

127.72, 126.64, 126.59, 125.99, 123.99, 113.05, 112.72, 102.00, 69.02, 58.60, 55.87, 50.91,
28.62. HRMS (ESI): m/z calcd for C26H22N2O5, 442.1529, found 443.1594 [M+H] +
4.1.3.22 7-((4-bromobenzyl) oxy)-4-((3,4-dihydroisoquinolin-2(1H)-yl) methyl)-2H-
chromen-2-one (7v)
Pale brown solid, yield 80%; mp 168-170 °C; 1H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 8.5
Hz, 1H), 7.51 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.3 Hz, 2H), 7.19 – 7.11 (m, 3H), 7.01 (d, J =
6.7 Hz, 1H), 6.90 – 6.85 (m, 2H), 6.45 (s, 1H), 5.07 (s, 2H), 3.82 (d, J = 20.0 Hz, 4H), 2.98 –
2.88 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 161.37, 161.29, 155.56, 152.13, 134.90,
134.16, 134.03, 131.91, 129.12, 128.74, 126.53, 126.39, 126.36, 125.80, 122.33, 112.87,
112.81, 112.33, 101.88, 69.65, 59.19, 56.25, 51.00, 29.07. HRMS (ESI): m/z calcd for
C26H22BrNO3, 475.0783, found 476.0860 [M+H] +
4.1.3.23 4-((4-((3,4-dihydroisoquinolin-2(1H)-yl) methyl)-2-oxo-2H-chromen-7-yl) oxy)
methyl) benzonitrile (7w)
Brown solid, yield 82%; mp 197-199 °C; 1H NMR (500 MHz, CDCl3) δ 7.89 (d, J = 8.8 Hz,
1H), 7.70 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.22 – 7.14 (m, 3H), 7.02 (d, J = 7.3
Hz, 1H), 6.90 (m, 2H), 6.51 (s, 1H), 5.19 (s, 2H), 3.94 (s, 4H), 3.02 (s, 4H). 13C NMR (125
MHz, CDCl3) δ 161.15, 160.95, 155.54, 141.32, 133.83, 132.60, 132.55, 128.79, 127.67,
127.63, 126.54, 125.94, 118.55, 113.11, 112.73, 112.19, 101.96, 69.30, 58.96, 56.10, 51.00,
28.89. HRMS (ESI): m/z calcd for C27H22N2O3, 422.1630, found 423.1708 [M+H] +
4.1.3.24 2-((4-((3,4-dihydroisoquinolin-2(1H)-yl) methyl)-2-oxo-2H-chromen-7-yl) oxy)
acetonitrile (7x)
Brown solid, yield 81%; mp 135-137 °C; 1H NMR (500 MHz, CDCl3) δ 7.95 (d, J = 8.8 Hz,
1H), 7.18 – 7.11 (m, 3H), 7.02 (d, J = 6.6 Hz, 1H), 6.92 (m, 2H), 6.50 (s, 1H), 4.84 (s, 2H),
3.84 – 3.77 (m, 4H), 2.97 – 2.86 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 160.79, 158.89,
155.36, 151.75, 134.00, 133.94, 128.75, 127.00, 126.53, 126.46, 125.85, 114.42, 114.27,
113.55, 112.00, 102.39, 59.19, 56.20, 53.50, 50.99, 29.02. HRMS (ESI): m/z calcd for
C21H18N2O3, 346.1317, found 347.1393 [M+H] +
4.1.3.25 7-(allyloxy)-4-((3,4-dihydroisoquinolin-2(1H)-yl) methyl)-2H-chromen -2-one (7y)
Pale brown solid, yield 82%; mp 102-104 °C; 1H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 8.7
Hz, 1H), 7.18 (m, 3H), 7.02 (d, J = 7.2 Hz, 1H), 6.85 (m, 2H), 6.49 (s, 1H), 6.04 (m, 1H),
5.44 (dd, J = 17.3, 1.2 Hz, 1H), 5.35 (d, J = 16.3, 1.2 Hz, 1H), 4.60 (d, J = 5.3 Hz, 2H), 3.93

(s, 4H), 3.03 (s, 4H). 13C NMR (125 MHz, CDCl3) δ 161.65, 161.25, 155.62, 133.62,
132.19, 128.75, 126.59, 126.17, 125.99, 118.53, 112.85, 112.68, 112.45, 101.83, 69.24,
58.32, 55.87, 50.88, 28.78. HRMS (ESI): m/z calcd for C22H21NO3, 347.1521, found
348.1599 [M+H] +
4.2. Radiochemistry
4.2.1 General Procedure:
All the chemicals and reagents were procured from Loba chemie (Mumbai, INDIA). The
quality control was performed using various instruments like Gas chromatography (Scion 436
GC, Netherlands) having flame ionization detector (FID), Thin layer chromatography (TLC)
scanner (EZ-SCAN, California, USA) with multimode radiation detector (OMNI-RAD,
California. USA), Dose calibrator (Capintec CRC-25PET, New Jersey. USA), High
performance liquid chromatography (HPLC) system (Dionex, ICS- 5000+, +, California.
USA), pH paper (Fisher Scientific, New Hampshire. USA). The [18F] radioisotopes were
produced with 16.5MeV in-house Cyclotron (PETtrace 860, GE Healthcare, USA) by the
18
18
proton bombardment on the enriched O-18 water using O (p, n) F reaction. The proton
bombardment was done with the beam current of range of 30 µA -65 µA for 10 min
depending upon the requirement of [18F]. For the radiolabeling, the produced [18F] was
delivered to semiautomatic synthesizer module (Tracerlab FX2N, GE Healthcare, Chicago.
USA) using the Helium (UHP-5.5) as a carrier gas.
4.2.2 Radio synthesis of target compound
A stock solution for [18F] eluent was prepared by dissolving 60 mg of Kryptofix (K222) and
12 mg of potassium carbonate in 12.0 ml of acetonitrile along with 525 L of the deionized
water. The [18F] was eluted from conditioned QMA cartridge with a mixture of QMA eluent
stock solution (1.5 mL) and acetonitrile (0.5 mL) into the reactor. Initially, complex of [K
2.2.2] K+[18F]- was dried and, the reaction with the precursor (4±0.5 mg,) in 1.5 ml of DMF
(3) was carried out at 160C for 30 min. After completion of the reaction, the reaction
mixture was diluted with acidic water (6 mL, pH=3.0) and passed through tC18 light
cartridge for primary purification. The cartridge was washed with 4 mL of water, and 3 mL of
8% ethanol. The final purification was performed using 3 mL of 20-100% ethanol.
4.2.3.-Quality control parameters

The radiochemical purity of the preparation was estimated by thin-layer chromatography
(TLC) and high-performance liquid chromatography (HPLC).
a) TLC parameters, around 3-5 µL of each sample was spotted on silica gel TLC sheet just
0.5 cm above the bottom and run the TLC using acetonitrile/saline (1:1) the following
suitable solvent system.
a) HPLC parameters
The mobile phase was run in a gradient method for better resolution of peaks on the spectra.
The acetonitrile (ACN) (solvent A) and water (solvent B) are used in different proportions.
The gradient method was started with 5% acetonitrile (0-5 min), 5% to 100% acetonitrile (5-
20 min), then100% acetonitrile (20-25 min) and again at 5% acetonitrile (25-30min). The
stationary phase was the C18 column (250 mm X 4.6 mm, 5 μm), and UV detection
wavelength (λmax) was 280 nm with a flow rate of 1 mL/min.
c) Residual solvent analysis
The residual analysis was performed using gas chromatography. 1.0 L of the sample was
injected into the injector and analysis was started. The column was operated initially at 40°C
for the first 3 min and then rise 50°C/min up to 8 min and the final temperature was set at
240°C and the column was BR-200ms, 0.32 mm ID. The makeup gas consists of Helium (28
mL/min), zero air (300 mL/min) and hydrogen gas (30 mL/min) flow at the rate of 2 mL/min.
4.3 Biology:
4.3.1 Maintenance of cell culture and cell proliferation assay:
All the cell lines were procured from ATCC (American Type Culture Collection, USA). Cells
were grown in DMEM (Dulbecco’s modified Eagle’s medium) with 10% fetal bovine serum.
Cell was maintained in a humidified incubator with 5% CO2 at 37oC. For the anti-
cytotoxicity assay, cells were seeded at a density of 10,000 cells/well and treated with
conjugates for 72 h. Further, cells were fixed with 10% Trichloroacetic acid for 1hour at 4 oC
and washed with running tap water. 100µl of 0.057% of sulphorhodamine B was added per
well and incubated for 30mins and further washed with 1% acetic acid to remove the
unbound dye. To elute the dye, 10mM Tris having pH 10.5 was added and reading was
captured at 510nm with Varioskan Flash spectral scanning multimode reader.
4.3.2 Quantikine ELISA Human Gal-1 Immunoassay

This assay utilizes the quantitative sandwich enzyme immunoassay technique. The assay was
performed as per the protocol of quantitative enzyme immunoassay manufacture’s (R & D
human galectins quantikine ELISA kit, USA).
4.3.3 Fluorescence measurements
In order to perform fluorescence binding studies of Gal-1 with compound 7q, full-length ORF
of Gal-1 was cloned into pET28a expression vector followed by purification using Ni-NTA
affinity chromatography as reported by Hsieh and co-authors, 2015 [38]. Intrinsic
fluorescence measurements were carried out on a Jasco spectrofluorimeter equipped with
Peltier at 25°C. Compound 7q was dissolved in DMSO to prepare a stock of 10 mM and was
used in the range of 0-65 µM. Purified Gal-1with concentration of 13 μM in 10 mM
phosphate buffer (pH 7.5) was excited at 280 nm and the emission was recorded from 300-
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. Buffer correction was made for
each spectrum. Binding constant (Ka) and number of binding sites (n) were determined using
modified Stern-Volmer equation i.e. log(Fo-F)/F = logKa + nlog[Q] where Fo and F is 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.
4.3.4 Cell cycle analysis:
For analysis of cell cycle distribution, cells were seeded in 60mm culture dishes and treated
with conjugate for 48 h. Post-treatment, cells were trypsinized, washed with phosphate buffer
saline (PBS) and fixed with 70% ethanol overnight. On the day of analysis, cells were
washed with PBS and incubated at 37oC for 30 minutes in a staining solution containing
propidium iodide and triton x-100. Post-incubation, cells were washed and analyzed with BD
Accuri C6 flow cytometer. The sample analysis was processed with FCSalyzer software.
4.3.5 Annexin-V Alexa Fluor 488 staining:
To evaluate the extent of apoptosis undergoing cells, Annexin-V Alexa 488/propidium iodide
staining was employed. Staining was carried out as per manufacturer’s protocol (#V13241,
Invitrogen, USA). Briefly, cells were seeded in 60mm culture dishes and treated with
conjugates for 48 h. Post-treatment, cells were stained with 5 µl of Annexin V Alexa Fluor

488 and 1µl of 100µg of Propidium Iodide (PI) for 15 minutes. Cells were then analyzed with
the BD Accuri C6 flow cytometer and FCSalyzer software was used for analysis.
4.3.6 Acridine orange/Ethidium bromide staining:
Acridine orange/Ethidium staining reveals the extent of apoptosis undergoing cells in the
treated versus untreated cells. Cells were seeded on coverslips and incubated for 48 h with the
conjugate. Further, a staining solution containing 100µg/ml of acridine orange and 100µg/ml
of Ethidium bromide was added to cells and incubated in dark at 37oC. Cells were imaged
with a fluorescence inverted microscope (CKX41, Olympus, USA) at 10X magnification.
4.3.7 Hoechst 33342 staining:
To analyze the alterations in the nuclear architecture, PC-3 cells were seeded on cover slips
and treatment with conjugate for 48 h. Cells were washed and fixed with 4% p-formaldehyde
and incubated for 20 minutes. Plates were washed and stained with Hoechst 33342 (2µg/ml)
for 5-10 minutes. Excess stain was washed with PBS and mounted on slides. Slides were
analyzed under a fluorescent inverted microscope (Olympus, USA) at 10X magnification.
4.3.8 JC-1 staining:
For measuring the mitochondrial membrane potential, cells were seeded in 60mm culture
dishes and treated with conjugates for 48 h. Further, cells were harvested with trypsin and
stained with 10µg/ml of JC-1 (#PK-CA 707-7001, PromoKine, Germany) for 10minutes at
37 oC. Post-incubation, red and green fluorescence were measured by BD Accuri C6 flow
cytometer and analysis with FCSalyzer was carried out.
4.3.9 H2DCFDA staining:
For the estimation of reactive oxygen species (ROS), H2DCFDA staining was employed.
Cells seeded in 60mm dishes were treated with conjugates for 48 h. Cells were trypsinized
and stained with carboxy-H2DCFDA for 30 minutes in dark at 37 oC. Images were captured
with an Olympus fluorescence inverted microscope (CKX41) at 10X objective.
4.4 Molecular modeling studies

All computational calculations were performed on an Intel (R) Xenon(R) 2 Duo CPU E7600
@ 3.06GHz processor with the LINUX operating system. The software package used was the
Schrodinger drug discovery consisting of modules for ligand, protein preparation and Glide
for high-throughput virtual screening for docking. Protein preparation wizard was used to
prepare Gal-1 downloaded from PDB (PDB ID: 4Y24), ready for docking i.e. removing
waters, adding missing side chains and energy minimization by an OPLS-2005 force field.
The final compound 7q was sketched and converted to 3D using Ligprep. The Glide XP
docking algorithm was employed using a grid box volume of 10x 10x 10 Å at the center of
TD-139 as standard. A set of ADMET related properties were calculated using qikprop
program i.e., Qikprop, version 6.5, Schrödinger, LLC, New York, NY, 2014.
Acknowledgments
1. Department of Biotechnology (DBT), Govt. of India, New Delhi for financial assistance
(6242-19/RGCB/PMD/DBT/MLKA/2015).
2. National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad for
resources and facilities.
Conflict of Interest
Authors declare no conflicts
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