Exploration of novel series of chalcone-phosphonates derivatives as

Anticancer Agents: Synthesis, spectral characterization, in vitro anti-

proliferative evaluation, molecular docking and in silico ADMET prediction
Shweta Mishra (  rxshweta10@gmail.com )
SGT College of Pharmacy, Shree Guru Gobind Singh Tricentenary University
Ekta Verma
Agarwal Drugs Pvt. Limited
Biplab Debnath
Bharat Technology, Department of Pharmaceutical Chemistry, Banitabla, Uluberia, Howrah-West Bengal, India, 711316
Amit Chawla
Maa Saraswati Institute of Pharmaceutical Sciences
Prasanna Shama Khandige
NITTE, NGSM Institute of Pharmaceutical Sciences
Bhagawati Saxena
Nirma University
Nityananda Sahoo
Centurion University of Technology and Management
Pardeep Jana
Birla Institute of Technology

Research Article

Keywords: Chalcones, phosphonates, anti-proliferative, hybrid synthesis, anhydrous Mg(ClO4)2

Posted Date: February 1st, 2024

DOI: https://doi.org/10.21203/rs.3.rs-3894516/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Additional Declarations: No competing interests reported.

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Abstract
Based on the wide range of pharmacological aspects related to organophosphates, a novel type of compound, containing chalcones-phosphonates derivative
was synthesized by the reaction of dialkyl phosphite and substituted chalcones using anhydrous Mg(ClO4)2 at 80 °C under solvent-free conditions. All the
obtained structures were confirmed by IR, 1H NMR 13C NMR, and HRMS techniques. The synthesized compounds were tested in vitro against a panel of three
human cancer cell lines against MCF7, HeLa, and A549 cell lines. As compared to the reference drug Doxorubicin (IC50 = 4.17, 3.31, 6.61 µM against MCF7,
HeLa, and A549 cell lines, respectively), most of the synthesized derivatives exhibited moderate to good antiproliferative activity. The structure-activity
relationship of synthesized compounds is discussed. To speculate on the mechanism of anticancer activity, a molecular docking study was carried out. The
molecular docking investigation indicates that all of the synthesized derivatives have good binding ability in the active site of the Vaccinia H1-related (VHR)
phosphatase (PDB: 3F81), PI3- kinase (PDB: 3R7Q), androgen receptor (PDB: 3V49) and VEGFR2 kinase (PDB: 3VHE). Furthermore, all compounds were
screened for in silico drug-likeness, and all were found to have drug-like properties, following the Lipinski rule of 5, with no PAINS alarms. Thus, the in vitro
inhibitory activity and in silico molecular studies confirmed the potency of the chalcone-phosphonate derivatives for anti-proliferative activity.

1. INTRODUCTION
According to the World Health Organization (WHO) report, cancer is a huge problem in the world. Cancer is a leading cause of death worldwide, accounting for
nearly 10 million deaths in 2020, or nearly one in six deaths.1 Based on the information received from the International Union Against Cancer2, the number of
cancer patients is expected to rise by 70% by 2030. Presently, about 140–150 types of FDA-approved anticancer drugs are on the market globally.3
Traditionally chemotherapy, radiation, surgery, molecular targeted therapy, and immunotherapy are commonly used for the treatment of different types of
cancer. However, these treatments may show low specificity, efficacy, resistance, and more side effects.4,5 In this scenario, there is a demand for effective and
more affordable treatments to address this predicament and hopefully overcome it. The poor efficacy, resistance, and considerable toxicity of existing drugs
have made it indispensable to research and develop an accessible approach and to design new anticancer drug molecules.

Chalcones are polyphenolic derivatives of natural products that have attracted the continuing attention of researchers for new drug developments.6 It serves
as the fascinating precursor for the synthesis of a variety of heterocyclic compounds.7,8 It is found in several naturally occurring and synthetic derivatives for
antioxidant9, antituberculosis10,11 antimalarial12, antiviral13, antifungal14, antiallergic15, antibacterial16, anti-inflammation17, and insulin memetic in 3T3 L1
adipocytes.18 Apart from this, it also shows anticancer activity19,20,21 by acting as different mechanisms like inhibition of angiogenesis, inhibition of tubulin
assembly, induction of apoptosis, anti-estrogenic activity, and a combination of these mechanisms.22

Organophosphates are a group of chemical agents that have high bioactivity. Phosphonates are structurally similar to phosphonate esters and carboxylic
acid. They function as enzymatic inhibitors by competing with structural analogs. They target essential metabolic enzymes, such as bisphosphonate, which
acts as an anticancer agent by inhibiting isoprenoid biosynthesis. On the other hand, some studies counseled that organic phosphonates may influence
cancer progression through the inhibition of farnesyl protein transferase (FPT).23 It has emerged as a novel target for anti-cancer agents due to its role in the
posttranslational modification of the Ras oncogene24, or purine nucleoside phosphorylase (PNP) as PNP inhibitors are potentially applicable in the
management of certain hematologic malignancies.25

Based on these biological activities of chalcones and phosphonates, a hybrid pharmacophore approach has been used to design the novel chalcones-
phosphonates molecules. The current research endeavor is supported by several studies that have documented the anticancer action of phosphonate
derivatives.26, 27, 28 Herein, a library of novel phosphonates derivatives bearing substituted chalcones fragments was synthesized from dialkyl phosphonates
and substituted chalcones. The cytotoxic activity of the synthesized analogues was screened against three human cancer cell lines HeLa, MCF-7 (breast
cancer adenocarcinoma), and A549 (adenocarcinoma human alveolar basal epithelial cells) for 48 h as in vitro. Moreover, in vitro haemolytic assays of all the
synthesized derivatives are an indispensable means for evaluating in vivo toxicity to host cells. Structure-activity relationship (SAR) studies were conducted on
the variation in the alkyl phosphonates and the variation of substituents on the phenyl ring (electron-donating groups, electron-withdrawing groups). A
molecular docking investigation was carried out in order to provide evidence for the hypothesized mechanism of anticancer action. Later, in silico ADMET
studies of molecules were carried out on all the compounds. Hence to explore such a privileged chalcone scaffold, in the current study we have designed the
phosphonate-containing chalcone analogs with the aim of the potent cytotoxic activity.

2. Materials and Methods

2.1 Chemistry
The synthesis of all the designed compounds was performed as per Scheme 1. Compounds were routinely checked for their purity on the silica gel GF-254 and
visualized under UV at 254 nm wavelength and chromatographic purification was performed by column chromatography on silica gel (60–120 mesh). The
melting point of synthesized derivatives was determined using the electro-thermal capillary melting point apparatus.

General procedure for the synthesis of phosphonate derivatives of chalcones (3a-r)

A mixture of substituted chalcone (1 mmol, 1eq), and dialkylphosphite (1 mmol, 1eq) stirred in the presence of 10 mol% Mg(ClO4)2 under neat condition at
room temperature for a specified time. Column chromatography was performed to obtain the pure product (3a-r) eluting with ethyl acetate in hexane.

2.2 Characterization of synthesized compounds

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Proton nuclear magnetic resonance experiments and carbon-13 nuclear magnetic resonance experiments were recorded on Bruker Advance DX 400 MHz
spectrometer. Chemical shifts were reported in parts per million relative to tetramethylsilane as an internal standard. The 1H NMR spectra were referenced with
respect to the residual CHCl3 proton of the solvent CDCl3 at 7.27 ppm. Coupling constants were reported in hertz (Hz). 13C NMR spectra were fully decoupled
and were referenced to the middle peak of the solvent at 77.00 ppm. Splitting patterns were designated as s, singlet; d, doublet; dd, doublet of doublet; m,
multiplet. HRMS was done on the maXis model and mass recorded in ESI mode. Infra-red spectra were recorded on Nicolet Impact 410 FTIR spectrometer
either as neat samples or using KBr for preparing pellets for solid samples.

2.3 In-vitro cytotoxic activities

The rate of cell survival under the action of test substances was evaluated by an improved MTT assay protocol. In brief, HeLa, A549, and MCF-12 cell lines
were seeded at a density of 5000 or 2500 cells/well in 96-well plates. After 24 hours, cells were exposed to increasing concentrations of the test compounds in
five doses (1 µM, 2µM, 5 µM, 10 µM, and 20 µM). Incubation was terminated after the addition of 50µl of 5 mg/mL solution of MTT solution ((3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Cayman’s MTT proliferation assay kit, 100009365) in phosphate buffer saline. Then incubated for 4
hours, the formed formazan precipitate was dissolved in DMSO, and absorbance was measured at 570 nm and 650nm microplate reader. The data obtained
are expressed as the IC50 values in duplicate. The percentage of viable cells was calculated using the following formula:

\raisebox1ex$[Absrobance (test) − Absorbance(negativecontrol )])$/\raisebox−1ex$[Absorbance (positivecontrol) − Absor

2.4 In vitro Haemolytic activity

The Haemolytic activities of the newly synthesized compounds were assayed on human erythrocyte suspension. Erythrocyte suspension was prepared by
centrifuging 10 mL of whole rat blood with an isotonic buffer for 10 mins at 3000 rpm at 20ºC, followed by washing three times with phosphate-buffered
saline (PBS, pH 7.4). The supernatant was separated and packed cells were suspended with an equal volume of normal saline and recentrifuged. The whole
process was repeated until a clear supernatant was obtained. From the subsequent, 10% erythrocyte suspension was prepared. To 1mL of PBS solution, 2 mL
of saline solution, and 2mL sample solution (1 µM, 2 µM, 5 µM, 10 µM, and 20 µM), and 0.5 mL of test solution were added and incubated for 1h at 37ºC.
Then centrifuged for 10 min at 2,000 rpm at 20ºC. From the supernatant fluid, about 150 µL was transferred to a tube, and the absorbance was measured
spectrophotometrically at 560 nm. The percentage of haemolysis was calculated using the following formula:

% Hemolysis =
\raisebox1ex$[Absrobance (test) − Absorbance(negativecontrol )])$/\raisebox−1ex$[Absorbance (positivecontrol) − Absor

2.5 Molecular docking

Molecular docking study of synthesized compounds was performed using AutoDockVina 4.2 program27, while AutoDockTools-1.5.6 program was used to add
hydrogens, compute Gasteiger charges, merge nonpolar hydrogens, choose torsions, and generate the pdbqt files. BIOVIA Discovery Studio 2017, Visualizer
tool was used for viewing, sharing, analyzing protein, and modeling data. VegaZZ 3.2.0 port 1-WS, a molecular modeling suite was used to perform several
features of docking analysis. PyMOL (Schrodinger, LLC) was employed to visualize the docking pose.

2.6 In silico ADMET

All the synthesized compounds were analyzed by using in silico approaches. For these calculations, all the structures were drawn in ChemBiodraw Ultra 14.0
to collect isomeric SMILES or Mol files format. For absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the synthesized compounds were
predicted by using SwissADME and pkCSM free online tools. With these isomeric SMILES, ADMET properties were obtained.

3. Results and discussion

3.1 Chemistry
The synthetic route of the compounds is outlined in Scheme 1. In continuation of our previous work in the synthesis of phosphonate derivatives of chalcones
via the reaction of chalcones analogs with dialkyl phosphite using anhydrous magnesium perchlorate.29 The product formed by the Phospha-Michael addition
reaction. The reactivity of substituted chalcones has been studied toward different dialkyl phosphonates namely, dimethyl phosphite, diethyl phosphite,
diisopropyl phosphite, and dibutyl phosphite to give phosphonate derivatives of substituted chalcones (Table 1). The phospha-michael addition reaction
between phosphorus nucleophile to a Michael acceptor (substituted chalcones) was performed under a simple and efficient methodology, with the advantages
of one-pot reaction, mild reaction condition, solventless, greener approach, and only catalytic amount of anhydrous Mg(ClO4)2 is required, with excellent yield
in short reaction time. The reactivity of substituted chalcones 1 encouraged us to a robust approach for the synthesis of diverse phosphonate derivatives of
substituted chalcones with different dialkyl phosphite.

In this reaction, the synthesis of dialkyl 1,3-diphenylallylphosphonate derivatives proceeded via the coordination of Mg+2 with the carbonyl oxygen atom and
hydroxyl/alkoxyl group of the dialkyl phosphite. In the next step, intermediates I are formed as a result of the electrostatic attraction between the electrophilic
carbonyl carbon atom and the lone pair of electrons, which further undergoes rearrangement to form intermediate II. Furthermore, Mg(ClO4)2 and γ-oxo
phosphonates were released at the end of the process of proton exchange.

3.2 Spectral characterization

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The spectral properties of the newly synthesized compounds agree with their proposed structures. The IR spectra analysis of compound 3a displayed
stretching band of C-P bonds at ≃ 1023 cm-1 and lacked the stretching band for the C=C bond (of α, β-unsaturated carbonyl group). The typical characteristic
absorbance peaks of aromatic rings can be seen at around 1651 cm-1 due to the stretching of phenyl C = C in plane. Likewise, 1H and 13C NMR also confirmed
the chemical structure of represented compound 3a, the spectra are shown in Fig. 1 (A) and Fig. 1 (B) and the assignment of each peak is marked. The total
number of aromatic protons in compound 3a is calculated to be 10. All the protons of aromatic regions appeared in the range of 7.82 – 7.11 δ ppm. The
chemical shift of proton in the CH, which is attached to the phosphonate group, was observed to be between 4.01 and 3.88 δ ppm. The appearance of
carbonyl group (C=O) was confirmed by 13C NMR, as shown at 196.11 ppm in the spectrum. The peak at 39.11 ppm and 31.11 ppm have appeared for carbon
of CH2 and CH respectively which confirm the formation of phosphonate bond. The methoxy CH3 carbon signals have appeared at 54.32 ppm. The aromatic
carbon has appeared in the range of 136.40-126.34 ppm. The masses of the newly synthesised compound were confirmed by the high-resolution mass
spectra (HRMS). The HRMS spectra of compound 3a, which shows a mass peak at m/z 341.0931, due to the presence of [M+Na]+ (with 4 decimals), also
confirmed the synthesis of the target compound.

3.3 In-vitro antiproliferative activities and SAR discussion

The in vitro antiproliferative activity of all the new synthesized compounds were investigated against three different human cancer cell line (MCF7, HeLa
and A549) using MTT assay30, in comparison with doxorubicin as a positive control. The observed IC50 values of all compounds are showed in Table 1.

The general structure of target compounds was varied by alkyl phosphite and substituents on Michael adduct. Through an overall analysis of the in vitro
antiproliferative activities, it could be concluded that most of the compounds (3a-r) exhibited the significant activity against MCF7 and HeLa comparing to
doxorubicin, while they demonstrated minimal activities against A549 cell lines. About MCF7, some of the compounds including 3f, 3l, 3m, 30, 3p, and 3q
showed better potency to doxorubicin, with lowest IC50 values. In addition, higher antiproliferative activities were observed for compounds 3l, 3m, and 3p
against HeLa cells compared to the reference drug. It should be highlighted that three compounds with chloro and fluro substations (3l and 3m) and one with
dibenzylideneacetone (3p) were shown to be the most active against two of the evaluated cell lines (Figure 2).

Table 1: In vitro anti-proliferative screening of synthesized compounds

Taking the results of antiproliferative activity into consideration, structure–activity relationships (SAR) studies have been conducted. It is interesting to note
that the presence of an electron-withdrawing group (F or Cl) on the phenyl ring (3k, 3l, or 3m) had a favourable impact on the antiproliferative activity when
tested on MCF7 and HeLa cell lines. Moreover, the presence of diethyl phosphonate group on the β position improved the cytotoxicity on both cell lines. For
example, compounds 3e, 3f, 3g, 3k, 3l, 3m, 3p having diethyl phosphonate substitution showed better IC50 values. Furthermore, dibenzylideneacetone
(compounds 3o, 3p, 3q, 3r) was introduced in place of benzylacetophenone, resulting in moderate to significant activity MCF7 and HeLa IC50 values that were
more potent than the standard drugs. According to these results, compound 3e and 3f were the most active against cervical cancer cell line (HeLa) and breast
cancer cell line (MCF7), respectively. All the compounds were found to be weaker activity against lung cancer cell line (A549). With the aforementioned results
into consideration, it is significant to highlight that these newly developed derivatives were able to achieve the optimum anticancer activity against the tested
cancer cell lines.

The most effective approach for chemotherapy involves maximum cytotoxicity towards cancer cells and minimal damage to normal cells. The selectivity
index (SI) measures how harmful a chemical is to tumour cells when compared to normal cells. Consequently, the cytotoxicity of the most active compounds
was evaluated against normal Vero cell line. The selected potent compounds were evaluated for their selectivity toward the MCF7 and HeLa cancer cells using
the selectivity of doxorubicin for reference. The SI was determined by dividing the IC50 values against normal cell lines and the cancer cell lines as presented in
Table 2 and Figure 3. The high SI values indicate a selectivity towards cancer cells, whilst values below 2 indicate a lack of selectivity. Except for compound
3o, all of the tested compounds shown SI values greater than 2 against the MCF-7 and HeLa cell lines. As a result, these compounds can be considered
selective against both the MCF-7 and the HeLa cell lines. The selectivity index study showed that compound 3l exhibited a high SI value of 11.83 and 10.27
respectively against MCF7 and HeLa cell lines. It has been concluded that, compound shows low toxicity against the normal cell line, but great selectivity
towards malignant cell lines, suggesting the safety of the synthetic compounds

3.4 In vitro Haemolytic activity

Haemolysis testing is the preliminary method to determine the hemocompatibility properties and the cytotoxicity of any new synthesized compounds31.
Haemolysis assessment is an acceptable in vitro screening tool for gauging probable in vivo toxicity to the host cells. The haemolytic activity in terms of
HD 50 haemolytic dose (μg/mL) of the synthesized compounds 3a-3r are shown in Figure 4. Standard drug doxorubicin had HD 50 value at 3.89 μg/mL. In
contrast to the reference standard, the most of the synthesized compounds exhibit relatively low in vitro cytotoxicity to human erythrocytes. Compound 3d, 3n,
3q and 3d moderately showed haemolytic toxicity. Hence haemolytic assay anticipated that all the synthesized compounds were biocompatible with
erythrocyte thereby evidencing the lack of haemolytic toxicity of anticancer drugs.

3.5 Molecular Docking study

In above study, three foremost (cervical, breast, and lung) cancer cell lines have been selected to identify the anti-proliferative potency of new chalcone-
phosphonate derivatives and these derivatives exhibited potent anti-proliferative activity against two cancer cell lines. In order to find out the anti-proliferative
activity in different targets, molecular docking study was performed. All the compounds were docked into the active site of Vaccinia H1-related (VHR)
phosphatase (PDB: 3F81)32, PI3- kinase (PDB: 3R7Q) 33, androgen receptor (PDB: 3V49) 34 and VEGFR2 kinase (PDB: 3VHE) 35. The binding energy computed

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for co-crystalized ligand was -5.1 kcal/mol, -10.2 kcal/mol, -11.4 kcal/mol, and -12.6 kcal/mol respectively for 3F81, 3R7Q, 3V49, and 3VHE. The binding score
of all synthesized compounds are shown in Table 3 and binding energy overview presented in Figure 5.

The compounds 3f and 3k showed higher binding affinity (ΔGb = -5.7 kcal/mol) with VHR phosphate as compared to the co-crystallized ligand and other
newly synthesized compounds (ΔGb = -5.1 kcal/mol). These compounds may be promising therapeutic candidate for the management of cervical cancer
through VHR inhibition since these compounds are also effective against HeLa cancer cell line. On other hand, the compound 3o (figure 6) revealed high
binding affinity ΔGb = -7.6 kcal/mol, -8.1 kcal/mol, and -8.7 kcal/mol with PI3-Kinase, VEGFR2 kinase and androgen receptor modulators respectively. This
compound may be shown antiproliferative activity through inhibiting the aberrant activation of PI3K/AKT signaling pathway, tumor angiogenesis and AR
expression. However, the binding affinity score of 3o compound is lower than co-crystallized ligand.

3.6 In-silico ADMET studies

Pharmaceutical properties and ADME i.e. absorption, distribution, metabolism, and excretion have a tremendous impact on both drug development and the
success of drug candidates. ADME properties are widely used in drug development to translate the balance of molecular properties, to predict drug likeness36,
37
. In order to assess the molecular properties, all the synthesized compounds 3a-3r were subjected to the amenability to the Lipinski rule. These properties
were computed by SwissADME and pkCSM online tools (Table 4). It has been reported that lipophilicity plays an important role in the passage of compounds
through the high lipid-containing cell. Therefore, cLogP values for all the synthesized compounds are significant, though no direct correlation could be
established between the activity and cLogP data of synthesized compounds of the series. Based on the predicted results in Table 4, all compounds followed
the Lipinski rule and showed no violation of the above criteria. The log P values were within ranges of 2.05- 3.38 and the hydrogen bond acceptor count
between 3-5 and hydrogen bond donor count between 3-4. It is reported that a large number of rotatable bonds (≥10) are associated with poor oral
bioavailability, mostly when related to ≥140Å2 a high topological polar surface area. Results show the number of rotatable bonds in all compounds varied
between 5-7, predicted within the range for oral bioavailability. As a result, these analogs may have the potential to become candidates for new drugs.

The identification of Pan-assay interference compounds (PAINS) is required for the removal of false positives at the beginning of the drug discovery process.
Compounds with PAINS properties are known to interfere with in vitro assay results in a variety of ways. As shown in Table 5, no single analogue was found to
have such fragments.

The bioavailability radar and BOILED EGG model of most potent compounds were predicted using SwissADME online tool, as shown in figure 7. Bioavailability
radar is used to predict rapid assessment of drug likeness parameters such as lipophilicity, size, polarity, solubility, flexibility, and saturation. In the
bioavailability radar, the pink area denoted the optimal range for each physicochemical property. As shown in figure 7, compound 3l, 3m, 3o and 3p fall entirely
or partially in the pink area and to be considered drug-like, as compared to reference drug doxorubicin. In the BOILED EGG representation, the yolk denotes the
points which allow passive blood–brain-barrier permeation, while the white region denotes points for passive gastrointestinal tract absorption. The graph is
plotted against WlogP[logP (n-octanol/water partition coefficient; a method developed by Wildman and Crippen] on ordinate versus TPSA or Topological Polar
Surface Area on abssica.

For molecules that are actively effluxed by P-glycoprotein, the dot should be blue, while for those that are not substrates of P-glycoprotein, the dot should be
red. The results show that compounds 3l and 3m appear in the white ellipse with a red tip, indicating a high likelihood of passive absorption via the
gastrointestinal tract. While blue dots appeared for compound 3o and 3p in the yellow area, they have a high possibility of permeating the BBB to access the
CNS and can be a substrate of the P-gp. Although doxorubucin appears outside the Grey zone, it is not expected to be readily absorbed or to permeate the BBB
(Figure 7)

3.7 In silico toxicological prediction

The preliminary toxicity testing of any new chemical substance can provide valuable

information by increased efficiency in toxicity testing and decreased the animal usage without evaluating the testing system in light of overall risk assessment
and management needs. All new compounds should be tested earlier for biological safety in order to reduce the risk of elimination at the later phase of clinical
development. In the present study, the toxicity risk assessment was carried using pkCSM online tool to predict mutagenicity, hepatotoxicity, skin irritation,
ames toxicity etc38. For the toxicity assessment initially, all the selected potent compounds were assigned SMILES (simplified molecular input line entry
system). With the SMILES, a toxicity profile was obtained. The predicted toxicity data of the compounds are shown in Table 6.

It was found from the obtained results that all the investigated compounds do not showed ames toxicity. None of the compounds exhibited mutagenicity and
skin irritant and it was predicted that the compounds are safe for the human body. Compound 3o showed hepatoxicity and other compounds did not exhibit
hepatotoxicity. They were included in category III acute oral toxicity which suggests that, for oral administration, the compounds are relatively harmless. Acute
fathead minnow toxicity is an important basis of hazard and risk assessment for compounds in the aquatic environment. The predicted minnow toxicity of
most of the compounds except 3p, was found to be <3 which shows less oral toxicity. However, all the compounds showed good to better drug-likeness
features as compared to standard drug doxorubicin. On the whole, the drug-likeness ranged from average to good score.

4. Conclusion
A new class of chalcone-phosphonates derivatives have been designed, synthesized and characterized. All new derivatives evaluated for their anti-proliferative
efficacy on three (MCF7, HeLa and A549) cell lines. The compounds 3e, 3f 3k, 3m-p displayed anti-proliferative activity superior to that of Doxorubicin against
MCF7. The compounds 3b, 3e, 3f, 3l had anti-proliferative activity against HeLa cell line and their efficacy were higher than that of doxorubicin. According to

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docking studies, compounds 3f and 3i showed higher binding affinity (ΔGb = -5.7 kcal/mol) with VHR phosphate as compared to the co-crystallized ligand
(ΔGb = -5.1 kcal/mol) Additionally, the compound 3o had high binding score against PI3-Kinase, VEGFR2 kinase and androgen receptor modulators but their
binding scores were lower than that of co-crystallized ligand. Accordingly, we can conclude that the chalcone-phosphonates derivatives can be good leads for
anticancer treatment. They are effective against cervical and breast cancer and ineffective against lung cancer.

5. Spectral data
5.1. Dimethyl 3-oxo-1,3-diphenylpropylphosphonate 3a
Yield: 82% (white solid). mp 112˚C. IR (KBr) ν: 3397.66, 1651.71, 1238.94, 1053.18 cm-1; 1H NMR (400 MHz, CDCl3) (ppm): 7.82–7.80(m, 2H), 7.41–7.29 (5H),
7.19–7.10(m, 3H ), 4.01–3.88 (m, 1H), 3.69–3.47(m, 5H), 3.39–3.35 (m, 3H); 13C NMR (100 MHz, CDCl3): 196.10, 136.97, 133.32, 128.61, 126.30, 54.60, 39.11,
20.87, 16.13; HRMS (ESI) m/z calcd for C17H19O4PNa+ [M + Na]+, 341.0898 ; Found 341.093

5.2 Diethyl 3-oxo-1,3-diphenylpropylphosphonate 3b

Yield: 76% (Brownish oily liquid). IR (KBr) 3058, 2979, 1687, 1250.55, 1053.16 cm-1; 1H NMR (CDCl3, 400 MHz) (ppm): 7.9329–7.9303 (d, 2H), 7.22–7.25 (m,
1H), 7.14 (t, J = 7.48 Hz, 2H), 6.69 (t, J = 7.32 Hz, 1H), 6.58 (d, J = 8.28, 2H), 4.26 (s, 2H), 3.90 (s, 1H); 13C NMR (100 MHz, CDCl3): 168.2, 161.5, 153.5, 137.5,
136.2, 128.5, 126.9, 126.7, 125.3, 124.5, 121.6, 42.9; HRMS (ESI) m/z calcd for C15H12N2OSNa+ [M + Na]+, 347.133; Found 369.1663

5.3 Diisopropyl 3-oxo-1,3-diphenylphosphonate 3c

Yield:73% (brown oily liquid ). IR (KBr): 3058, 2979, 1687, 1232.06, 1023.33 cm-1. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.84 (d, J = 1.04 Hz, 2H), 7.52 (t, J = 6.92
Hz, 1H), 7.46–7.41 ( m, 4H), 7.29–7.25 (m, 2H),7.21–7.17 (m,1H), 4.77–4.65 (m, 1H), 4.46–4.38(m, 1H), 3.94–3.85 (m,1H), 3.64–3.59 (m,1H), 3.80–3.72
(m,1H) 1.22 (d, J = 6.16 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm) 196.73, 136.33, 133.22, 129.51, 128.58, 128.10, 127.13, 71.66, 39.45, 24.26, 23.14. HRMS
(ESI): m/z for C21H27O4PNa+ [M + Na]+, calc.: 397.1598 ; observed: 397.1558.

5.4 Dibutyl 1,3-diphenylallylphosphonate 3d

Yield: 66% (yellowish oily liquid). IR (KBr) 3058, 2979, 1687 1235.15, 1027.36 cm-1; 1H NMR (CDCl3, 400 MHz) (ppm): 7.8524–7.8321 (d, 2H), 7.4396–7.3989
(m, 2H), 7.2566–7.2344 (d, J = 8.908 Hz, 2H), 6.69 (t, J = 7.32 Hz, 2H), 6.58 (d, J = 8.28, 2H), 4.26 (s, 2H), 2.4003 (s, 3H); 13C NMR (100 MHz, CDCl3):195.92,
137.02, 132.94, 129.30, 128.43, 127.06, 133.32, 66.51, 40.09, 32.56, 32.38, 18.69, 13.32; HRMS (ESI) m/z calcd for C21H27O4PNa+ [M + Na]+, 397.1598; Found
397.1558

5.5 Diethyl 3-(4-fluorophenyl)-3-oxo-1-phenylpropylphosphonate 3e

Yield: 64% (yellow semisolid). IR (KBr) ν: 3457.45, 3107.74, 1688.86, 1597.98, 1507.19, 1496.69, 1235.77, 1158.34, 1098.56, 1031.39, 966.74 cm-1; 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.91–7.86(m, 2H), 7,36-7.34(m, 2H), 7.23–7.16(m, 2H), 7.15–7.11(m, 1H), 7.05–6.99(m, 2H), 4,04-3.94(m, 2H), 3.90–3.77(m, 2H),
3.69–3.54 (m, 3H), 1.20 (t, J = 7.04Hz, 3H), 0.99 (t, J = 7.04Hz, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 194.79, 164.57, 135.90, 130.81, 129.26,128.54, 115.84,
63.07, 39.76, 29.69, 16.33. HRMS (ESI) m/z C19H22FO4PNa+[M + Na]+, 387.1098.

5.6 Diethyl 1-(4-fluorophenyl)-3-oxo-3-phenylpropylphosphonate 3f

Yield: 72% (yellow semisolid). IR (KBr) ν: 3062.39, 2871.21, 2345.30 1966.29, 1894.91, 1688.93, 1598.05, 1478.08, 1391.89, 1355.99, 1231.70, 1162.08,
1051.34, 967.52 cm-1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.99–7.95 (m, 2H), 7.58–7.54 (m, 2H), 7.47–7.40 (m, 2H), 6.99 (t, J = 8.52 Hz, 2H), 4.14–4.05 (m,
2H), 4.00-3.89 (m, 2H), 3.82–3.61 (m, 3H), 1.26 (t, J = 7.04Hz, 3H), 1.11 (t, J = 7.04Hz, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 196.34, 163.3, 160.80, 136.45,
133.41, 131.80, 115.54, 6 3.04, 39.20, 29.70, 18.71. HRMS (ESI) m/z C19H22FO4PNa+[M + Na]+, 387.1140.

5.7 Diethyl 3-(4-methoxyphenyl)-3-oxo-1-phenylpropylphosphonate 3g

Yield: 70% (yellow oily liquid). IR (KBr): 3410.74, 2926.64, 2852.55, 1710.85, 1449.54 cm-1; 1H NMR (400 MHz, CDCl3) (ppm): 7.9423–7.9377 (dd, J = 1.84 Hz,
2H), 7.55–7.53 (m, 1H), 7.46–7.42 (m, 2H), 7.37–7.34 (m, 2H), 6.84–6.82 (d, J = Hz, 2H) 4.10–4.06 (m, 2H), 3.95–3.89 (m, 2H), 3.775 (s, 3H), 3.76–3.64 (m,
2H) 1.35–1.26 (m, 3H), 1.13–1.09 (m, 2H), 13C NMR (100 MHz, CDCl3) δ (ppm): 196.72, 158.67, 139.13, 136.65, 133.14, 130.45, 128.78, 114.39, 70.79, 60.27,
39.51, 33.76, 29.63, 23.99; HRMS (ESI) m/z calcd for C20H25O5PNa+ [M + Na]+, 399.1298; Found 399.1258.

5.8 Diisopropyl (3-(4-methoxyphenyl)-3-oxo-1-phenylpropyl)phosphonate 3h

Yield: 64% (brownish oily liquid). IR (KBr): 3410.74, 2926.64, 2852.55, 1710.85, 1449.54 cm-1; 1H NMR (400 MHz, CDCl3) (ppm): 7.89–7.87 (m, 2H), 7.47–7.45
(t, J = 8 Hz, 1H), 7.39–7.31 (m, 4H), 6.78–6.76 (d, J = 8 Hz, 2H), 4.65–4.63 (m, 1H) 4.39–4.37 (m, 4H), 3.95–3.89 (m, 2H), 3.775 (s, 3H), 3.76–3.64 (m, 2H),
3.69–3.55 (m, 4H) 1.32–1.29 (m, 2H), 1.26–1.22 (m, 3H), 1.19–1.18 (m, 3H), 13C NMR (100 MHz, CDCl3) δ (ppm): 194.72, 156.67, 136.13, 132.65, 131.11,
130.35, 127.16, 114.39, 72.29, 57.27, 39.51, 33.76, 29.63, 23.99; HRMS (ESI) m/z calcd for C22H29O5PNa+ [M + Na]+, 427.1758; Found 427.2222.

5.9 Diethyl 1-(4-fluorophenyl)-3-(4-methoxyphenyl)-3-oxopropylphosphonate (Table 4,

Entry 9):

Page 6/17
Yield: 72% (yellow oily liquid). IR (KBr) ν: 3431.70, 1686.22, 1232.40, 1027.62 cm-1; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.00-7.96 (m, 2H), 7.37–7.34 (m, 2H),
7.14–7.10 (m, 2H), 6.85(d, J = 8.4Hz, 2H), 4.11–4.07(m, 2H), 3.95–3.85 (m, 2H), 3.78 (s, 3H), 3.76–3.70 (m, 1H), 3.65–3.61 (m,2H), 1.30 (t, J = 7.04, 3H), 1.11 (t,
J = 7.04, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 195.01, 167.02, 158.75, 133.04, 115.7 6, 62.95, 39.09, 37.43, 20.94. HRMS (ESI) m/z Calcd for C20H24FO5P
Na+[M + H]+, 395.1450, Found, 395.1426.

5.10 Diethyl (3-(4-fluorophenyl)-1-(4-methoxyphenyl)-3-oxopropyl)phosphonate 3j

Yield: 52% (yellow oily liquid). IR (KBr) ν: 3431.70, 1686.22, 1232.40, 1027.62 cm− 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.99–7.95 (d, 2H), 7.13–7.08 (t, 2H),
6.84–6.82 (d, J = 8Hz, 2H), 4.14–4.03 (m, 3H), 3.94–3.84 (m, 2H), 3.74 (m, 3H), 3.71–3.57 (m, 3H), 1.31–1.26 (t, J = 17.24 Hz, 3H), 1.12–1.09 (t, J = 14.12 Hz,
3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 195.92, 167.22, 164.78, 158.77, 133.05, 130.87, 115.49, 63.97, 55.17, 39.09, 16.50. HRMS (ESI) m/z Calcd for
C20H24FO5P Na+[M + H]+, 395.1450, Found, 395.1426.

5.11 Diethyl (1-(4-fluorophenyl)-3-oxo-3-(p-tolyl)propyl)phosphonate 3k

Yield: 62% (yellow oily liquid). IR (KBr) ν: 3431.70, 1686.22, 1232.40, 1027.62 cm− 1; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.85–7.83 (d, J = 8.04 Hz, 2H), 7.43–
7.39 (m, 2H), 7.25–7.23 (d, J = 8Hz, 2H), 7.009–6.966 (t, J = 17.24 Hz, 2H), 4.07-4.00 (m, 2H), 3.98–3.89 (m, 2H), 3.82–3.57 (m, 3H), 2.40 (m, 3H), 1.31–1.26 (t,
J = 17.24 Hz, 3H), 1.13–1.09 (t, J = 14.12 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 195.92, 163.22, 160.78, 144.24, 134.05, 130.87, 115.49, 62.97, 39.09,
37.43, 29.68, 16.34. HRMS (ESI) m/z Calcd for C20H24FO5P Na+[M + H]+, 395.1450, Found, 395.1426.

5.12 Diethyl (1-(4-chlorophenyl)-3-(4-fluorophenyl)-3-oxopropyl)phosphonate 3l

Yield: 55% (yellow oily liquid). IR (KBr) ν: 3476.35, 2987.65, 1686.45, 1598.93, 1492.01, 1480.84,1411.72, 1395.76, 1007.10, 816.00, 799.86; 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.97–7.94 (m, 2H), 7.38–7.35 (m, 2H), 7.13–7.08 (m, 2H), 4.14–4.04 (m, 2H), 3.95–3.86 (m, 2H), 3.82–3.54 (m, 3H), 1.26 (t, J = 7.04Hz, 3H),
1.11 (t, J = 7.04Hz, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 194.67, 167.20, 167.20, 164.66, 134.56, 133.24, 132.88, 130.80, 128.72, 115.92, 63.14, 39.20,
29.68, 16.40. HRMS (ESI) m/z calcd; C19H21ClFO4P Na+ [M + Na]+, 421.0798; Found 421.0763

5.13 Diethyl 1-(2-chlorophenyl)-3-(4-fluorophenyl)-3-oxopropylphosphonate 3m

Yield: 52% (yellow oily liquid). IR (KBr): 3459,3067.93, 2983.84, 1690.68, 1055.49, 969.57; 1H NMR (400 MHz, CDCl3) δ 8.00-7.96, (m, 2H), 7.52, (d, 1.72, 1H),
7.41 (d, 7.8, 1H), 7.23–7.09(m, 4H), 4.66–4.57 (m, 1H), 4.19–4.10 (m, 2H), 3.93–3.87 (m, 1H), 3.78–3.71 (m, 3H), 1.31 (t, J = 7.08Hz, 3H), 1.09 (t, J = 7.04Hz,
3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 194.40, 167.03, 164.49, 135.18, 134.04, 132.76, 130.76, 129.67, 129.08, 128.26, 126.89, 62.93, 39.19, 18.02, and
16.42. HRMS (ESI) m/z calcd for C19H21ClFO4P H+[M + H]+, 399.7957; Found 399.0937

5.14 Diethyl 3-oxo-1-phenylbutylphosphonate 3n

Yield: 49% (Brownish oily liquid). IR (KBr); 2987 cm-1, 2428, 1688, 1371 cm− 1; 1H NMR (CDCl3, 400 MHz) (ppm): 7.38–7.33 (m, 2H), 7.28 (t, J = 7.28 Hz, 2H),
7.23–7.19 (m, 1H), 13C NMR (100 MHz, CDCl3): 204.94, 160.78, 144.24, 131.97, 129.32, 128.18, 115.49, 62.97, 39.02, 21.61, 16.34; HRMS (ESI) m/z calcd for
C14H21O4PH+[M + H]+, 284.1207; Found 285.1259

5.15 Dimethyl 3-oxo-1,5-diphenylpennt-4-enylphosphonate 3o

Yield: 56% (yellow oily liquid). IR (KBr): 3444.72, 2985.01, 2089.59, 1659.38, 1613.83 cm− 1; 7.54–7.51 (m, 3H), 7.45–7.35 (m, 5H), 7.33–7.27 (m, 2H), 7.25–
7.23 (m, 1H), 6.70 (d, J = 16.24 Hz, 1H), 3.99–3.90 (m, 1H), 3.87–3.78 (m, 2H), 3.74 (d, J = 10.72Hz, 3H), 3.48 (d, J = 10.52Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
(ppm): 196.34, 163.3, 160.80, 136.45, 133.41, 131.80, 115.54, 6 3.04, 39.20, 29.70, 18.71. HRMS (ESI) m/z calcd for C19H21O4PNa+[M + Na]+, 367.1098 ; Found
367.1080.

5.16 Diethyl 3-oxo-1,5-diphenylpennt-4-enylphosphonate 3p

Yield: 67% (yellow oily liquid). IR (KBr): 3444.72, 2985.01, 2089.59, 1659.38, 1613.83 cm-1; 1H NMR (CDCl3, 400 MHz) (ppm): 7.58–7.45 (m, 3H), 7.44–7.38 (m,
5H), 7.36–7.28 (m, 2H), 7.25–7.21 (m, 1H), 6.68 (d, J = 16.24 Hz, 1H), 4.16–4.04 (m,2H), 3.96–3.85 (m, 2H), 3.77–3.69 (m, 1H), 3.67–3.34 (m, 2H), 1.31 (t, J =
7.04Hz, 3H), 1.09 (t, J = 7.04Hz, 3H) );13C NMR (100 MHz, CDCl3): 195.96, 142.89, 130.35, 129.27, 128.35, 127.12, 126.15, 62.74, 40.16, 38.76; HRMS (ESI) m/z
calcd for C19H21O4P H+[M + H]+, 345.1253 ; Found 345.1270.

5.17 Diisopropyl 3-oxo-1, 5-diphenylpent-4-enylphosphonate 3q

Yield: 59% (yellow oily liquid). IR (KBr) cm-1; 3329.77, 2975.95, 1691.04, 1614.20 cm-1; 1H NMR (CDCl3, 400 MHz) (ppm): 7.52–7.44 (m, 3H), 7.43–7.39 (m,
,2H), 7.38–7.36 (m, 3H), 7.32–7.28 (m, 2H), 7.24–7.19 (m, 1H), 6.67 (d, J = 16.24Hz, 1H), 4.74–4.66 (m, 1H), 4.44–4.36 (m, 1H), 3.85–3.76 (m, 1H), 3.50–3.30
(m, 2H), 1.32 (q, J = 6.18Hz, 6H), 1.24 (d, J = 12.16 Hz, 3H), 0.86 (d, J = 6.2Hz, 3H); 13C NMR (100 MHz, CDCl3): 196.65, 143.08,134.04, 130.58, 129.50, 128.94,
126.05, 70.43, 39.11, 24.30, 23.08. HRMS (ESI) m/z calcd for C23H29O4P H+ [M + Na]+, 423.1870 ; Found 423.1707

5.18 Dibutyl 3-oxo-1,5-diphenyl-4-enylphosphonate 3r

Yield: 52% (Yellow oily liquid). IR (KBr) cm-1; 3456.11, 3060.84, 2960.02, 1691.71, 1668.89, 1023.63; 1H NMR (CDCl3, 400 MHz) (ppm):7.56–7.48 (m, 3H),
7.43–7.36 (m, 5H), 7.31–7.27 (m, 2H), 7.23–7.19 (m, 1H), 6.66 (d, J = 16.24Hz, 1H), 4.10–3.80 (m, 5H), 3.65–3.61 (m, 1H), 3.59–3.34 (m, 1H), 1.73–1.58 (m,
3H), 1.42–1.32 (m, 3H), 1.26–1.17 (m, 2H), 0.95-.89 (m, 3H), 0.82 (t, J = 7.2 Hz, 3H), 13C NMR (100 MHz, CDCl3): 196.21, 143.20, 136.02, 134.28, 128.94,
127.25, 125.95, 66.57, 40.99, 32.40, 24.52, 18.76, 13.62; HRMS (ESI) m/z calcd for C25H33O4PNa+ [M + Na]+ 451.2020; Found 451.1998

Page 7/17
Declarations
Author Contribution
Shweta Mishra- conception or design of the work; synthesis of compounds; manuscript writingEkta Verma- Methodology, reviewed the manuscript.Biplab
Debnath- Formal analysis, reviewed the manuscript.Amit Chawla- Drafting the work or reviewing it critically , reviewed the manuscript.Prasanna Shama
Khandige-interpretation of data for the work, reviewed the manuscript.Bhagawati Saxena- In vitro activity, Methodology, reviewed the manuscript.Nityananda
Sahoo- Softwere analysis, reviewed the manuscript.Pardeep Jana- Molecular docking, reviewed the manuscript.

Competing Interest declaration

The authors declare no conflict of interest, financial or otherwise.

Declaration of Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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Schemes
Schemes 1-2 are available in the Supplementary Files section.

Tables
Table 1 is available in the Supplementary Files section

Table 1: In vitro anti-proliferative screening of synthesized compounds

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 SNo. Compounds MCF7 (µM) HeLa (µM) A549 (µM)

1. 3a 11.80 10.51 40.66

2. 3b 9.23 6.80 38.68

3. 3c 10.85 7.96 41.91

4. 3d 11.37 11.83 46.23

5. 3e 2.94 3.49 >50

6. 3f 2.09 3.12 >50

7. 3g 12.91 8.89 >50

8. 3h 13.32 11.34 >50

9. 3i 8.39 8.57 47.81

10. 3j 8.19 8.31 46.12

11. 3k 6.72 6.17 39.62

12. 3l 1.12 1.29 20.26

13. 3m 1.63 1.44 21.85

14. 3n 3.68 5.89 39.92

15. 3o 1.56 5.10 48.92

16. 3p 1.09 2.40 46.13

17. 3q 2.13 8.55 >50

18. 3r 3.41 9.36 >50

19. Doxorubicin 2.89 2.56 5.13

IC50 value, the concentration of a compound at which 50% growth inhibition is observed

Table 2 Selectivity index of compounds

Compounds Vero Cell line IC50 Selectivity index Selectivity index HeLa Selectivity index A549

MCF7

3l 13.25 11.83 10.27 ND

3m 14.09 8.64 9.78 ND

3o 9.13 5.85 1.79 ND

3p 7.67 7.03 3.19 ND

Doxorubicin 88.34 6.34 7.16 ND

Table 3 Docking score of synthesized compounds

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Sl. No. Compound name 3F81 3QJZ 3R7Q 3V49 3VHE

3a -5.5 -6.7 -7.1 -7.5 -7.2

1.

3b -5.4 -6.6 -6.5 -7.0 -7.2

2.

3c -5.5 -6.1 -6.8 -6.7 -7.6

3.

3d -5.2 -6.5 -6.9 -6.5 -6.9

4.

3e -5.6 -5.9 -6.9 -7.2 -7.4

5.

3f -5.7 -6.0 -6.6 -7.4 -7.5

6.

3h -5.5 -5.9 -6.8 -6.6 -7.7

7.

3g -5.2 -6.0 -6.4 -7.2 -7.3

8.

3i -5.3 -5.8 -7.4 -7.1 -7.6

9.

3j -5.6 -6.3 -7.1 -6.9 -8.2

10.

3k -5.7 -6.3 -6.7 -7.5 -7.8

11.

3l -5.5 -6.1 -6.7 -6.8 -7.8

12.

3m -4.9 -5.8 -5.7 -6.5 -6.0

13.

3n -4.9 -5.6 -6.2 -6.5 -5.9

14.

3o -5.5 -7.4 -7.6 -8.1 -8.7

1.

3p -5.5 -5.6 -7.6 -7.1 -8.2

16.

3q -5.1 -6.2 -7.0 -6.6 -8.1

17.

3r -4.9 -6.1 -7.1 -6.6 -7.8

18.

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 Co-Crystalized ligand -5.1 -8.5 -10.2 -11.4 -12.6
19.

Table 4: Physicochemical properties and Drug likeness prediction using SwissADME

Lipinski’s Parameters

Entry nViol cLogP Mol. Wt. HBA HBD RB tPSA

3a 0 2.92 318.30 4 0 7 62.41

3b 0 3.62 346.36 4 0 9 62.41

3c 0 4.17 374.41 4 0 9 62.41

3d 0 4.95 402.46 4 0 13 62.41

3e 0 3.91 364.35 5 0 9 62.41

3f 0 3.96 364.35 5 0 9 62.41

3g 0 3.62 376.38 5 0 10 71.64

3h 0 3.92 394.37 4 0 10 71.64

3i 0 3.27 378.37 5 0 9 62.41

3j 0 3.45 398.79 5 0 9 62.41

3k 0 3.44 398.79 5 0 9 62.41

3l 0 2.49 284.29 4 0 8 62.41

3m 0 3.30 344.34 4 0 8 62.41

3n 0 3.91 372.39 4 0 10 62.41

30 0 4.54 400.45 4 0 10 62.41

3p 1 5.33 428.50 4 0 14 62.41

3q 0 4.21 404.44 5 0 10 71.64

3r 0 4.47 422.43 6 0 10 71.64

Doxorubucin 3 2.58 543.52 12 6 5 206.07

Table 5 Pharmacokinetic properties and medicinal chemistry parameters predicted using SwissADME and pkCSM online tool

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 ADME properties

Compound code % GI absorption BBB Score BBB permeant Caco2 permeability CYP2D6 substrate Total Clearance
PAINS# VDss

Alert

3a 0 93.344 -0.038 Yes 1.676 -0.382 Yes 0.435

3b 0 93.46 0.282 Yes 1.872 -0.477 Yes 0.516

3c 0 93.981 -0.252 No 1.24 -0.758 Yes 0.359

3d 0 92.079 0.174 No 1.154 -0.287 Yes 0.642

3e 0 93.591 0.006 No 1.692 -0.63 Yes 0.254

3f 0 93.257 -0.245 No 1.687 -0.649 Yes 0.372

3g 0 95.038 -0.146 No 1.574 -0.55 Yes 0.436

3h 0 94.835 -0.673 No 1.766 -0.671 Yes 0.292

3i 0 93.966 -0.261 No 1.678 -0.528 Yes 0.314

3j 0 92.096 -0.036 No 1.677 -0.579 Yes 0.126

3k 0 92.096 -0.036 No 1.677 -0.579 Yes 0.126

3l 0 93.862 0.023 Yes 1.704 -0.152 No 0.49

3m 0 94.094 0.305 Yes 1.9 -0.588 Yes 0.304

3n 0 92.991 0.273 Yes 1.202 -0.349 Yes 0.399

30 0 93.506 -0.259 No 1.299 -0.681 Yes 0.243

3p 0 91.61 0.166 No 1.213 -0.185 Yes 0.528

3q 0 94.78 -0.667 No 1.174 -0.847 Yes 0.29

3r 0 94.713 -1.057 No 1.214 -0.981 Yes 0.144

Doxorubicin 1 62.10 -1.358 No 0.341 1.712 No 0.985

Table 6 In silico predicted toxicological parameters using pkCSM online tool

Parameters 3l 3m 3o 3p Doxorubicin

AMES toxicity No No No No No

Max. tolerated dose (human) 1.004 1.001 0.707 0.785 0.019

hERG I inhibitor No No No No No

hERG II inhibitor No No No No Yes

Oral Rat Acute Toxicity (LD50) 2.587 2.629 2.155 2.268 2.411

Oral Rat Chronic Toxicity (LOAEL) 1.21 1.219 1.355 1.454 3.544

Hepatotoxicity No No Yes No Yes

Skin Sensitisation No No No No No

T.Pyriformis toxicity 0.781 0.823 0.744 0.937 0.285

Minnow toxicity -1.244 -1.538 -2.939 -3.518 4.594

Figures

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Figure 1

(A) Assigned 1H and 13C NMR peak of compound 3a (B) Spectral characterization (1H and 13C NMR spectra) of compound 3a

Figure 2

Comparison of antiproliferative activity of synthesized compounds

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Figure 3

Comparison of selectivity index (SI) values of the selective tested compounds

Figure 4

Comparison of HD 50 values of the all the tested compounds

Figure 5

Binding energy overview of synthesized compounds

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Figure 6

2D and 3D pose of compound 3o

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Figure 7

Bioavailability radar and BOILED EGG model representation of most potent compounds (denoted as Molecule 1 and represented by a blue/red dot in the
figure).

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.

Supportinginformation.docx
GraphicalAbstract.png
Scheme1.png
Scheme2.png
Table1.docx

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