Green Synthesis, Multitargeted Molecular Docking and ADMET Studies of Chalcones Based Scaffold as Anti-Breast Cancer Agents

 

Jainey P. James*, Pramatha, Mariyam Jouhara, Zakiya Fathima C, Rupal Ria D’Souza

Nitte (Deemed to be University), NGSM Institute of Pharmaceutical Sciences, NGSMIPS, Department of Pharmaceutical Chemistry, Paneer, Deralakatte, Mangalore - 575 018, Karnataka, India.

 

ABSTRACT:

Green synthesis of chalcones is a new alternative to traditional methods, which is eco-friendly and require no solvents. The chalcones exert their anticancer action by the different mechanisms by acting through various targets. The study aimed to synthesise chalcones by green chemistry approach using grinding technique and check their molecular interactions and pharmacokinetic profile by in silico studies. Their anti-breast cancer action was analysed by MTT assay against human breast cancer cells. ADMET and physicochemical properties emphasised the drug-likeness and bioavailability of the synthesised chalcones. All the twelve synthesised chalcones interacted well with the three cancer targets (3ERT, 4OAR and 4WKQ). Among them, the top chalcones were PR2 and PR3, excellently interacting with the targets, which are following the in vitro studies. PR2 and PR3 have obtained good cytotoxic action against human breast cancer cells. Based on these results, it is concluded that the synthesised chalcones can be utilised as leads as anti-breast cancer agents, which can be verified by in vivo studies as future studies.

 

 

 

INTRODUCTION: 

With computational software¹, the pharmacokinetic profile of a molecule can be predicted before synthesis, which can help in predicting toxicity studies These studies also offer the advantage of simulating a physiological model required or even individual receptors if necessary². Thus reduce the need for animal testing and culturing tissue samples which is a tedious process.

 

In cancer³, since there are multiple mechanisms through which remission can be achieved, computational software can be used to see the best fit achieved by a molecule to a receptor. The human estrogen receptor⁴, progesterone receptor⁵ and endothelial growth factor receptor kinase, EGRF kinase⁶ were some of the breast cancer targets.

 

 

Green synthesis is an emerging area, where nontoxic, safe reagents which are eco-friendly and safe are used⁷. The compounds were synthesised using green synthesis without using solvents or harmful reagents⁸,⁹. The conventional synthesis of chalcones involves numerous steps. In contrast, the green synthesis involved the Claisen-Schmidt condensation of methyl ketones and aromatic aldehydes and grinding at room temperature using sodium hydroxide as a catalyst. The time for the reaction completion is 24 hours, but this can be reduced by using microwave-assisted radiation, which completes the reaction in less than 4 hours¹⁰.

 

Chalcone is a term that is given to all compounds having a 1,3 diphenyl-2-propen-1-one structure and is the well-known intermediate in the preparation of various heterocycles^11-13. Chalcones are naturally occurring compounds with a wide range of biological       activities^14-15, such as antimicrobial^16-17, anti-inflammatory, analgesic, antioxidant¹⁸ and anticancer property¹⁸. Recently, it was discovered that the acetylenic derivatives of chalcones had antimalarial and antitubercular activity¹⁹. Chalcones exert their anticancer activity by multiple mechanisms, including mitosis inhibition²⁰, triggering the mitochondrial apoptosis pathway by releasing cytochrome c, the activation of caspases 9 and 3, angiogenesis inhibition, and they exert this effect in the M or G2 phase. The available literature on the anticancer activity of chalcones shows that structural manipulation such as the replacement of aryl rings or hybridisation with other moieties increases anticancer activities^21-22.

 

Based on the above data, it was proposed to prepare chalcones by green synthesis and their molecular interactions, pharmacokinetic profile were tested in silico^23-25. Also, their anti-breast cancer action was confirmed by in vitro studies.

 

MATERIALS AND METHODS:

Most of the chemicals were purchased from Sigma Aldrich, and further purification was not required. Melting points was determined by the capillary method and were uncorrected. Shimadzu Perkin Ekmer 8201 Pc IR Spectrometer used in recording IR spectra (KBr pellets), and frequencies are expressed in cm^-1. Bruker Avance II, 400 NMR spectrometer, recorded NMR spectra and Shimadzu LCMS 8030, Japan Mass spectrometer recorded mass spectra.

 

Green Synthesis of Chalcones:

The compounds were synthesised by the Claisen-Schmidt condensation reaction of an aromatic aldehyde and ketone in the presence of 30% NaOH as a catalyst. This mixture was grinded for 10 minutes and the resultant mixture was then poured into ice and acidified using conc. HCl to precipitate the crude product, which was later isolated by filtration²⁶ (Figure 1).

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(4-bromophenyl)prop-2-en-1-one (PR1):

Brown Crystals (EtOH); m.p. 195–197°C; Yield 84%; IR (KBr) cm⁻¹: 3043 (aromatic C–H), 1699 (α,β unsaturated ketone), 1556 (C=C str), 667 (C-Br); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.76 (1H, d, J = 16.2 Hz), 7.83 (1H, d, J = 16.2 Hz), 7.02-8.56 (9H, m, Ar-H); Mass (m/z): (M⁺) 228

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(4-chlorophenyl)prop-2-en-1-one (PR2):

Yellow Crystals (EtOH); m.p. 261–263°C; Yield 80%; IR (KBr) cm⁻¹: 3012 (aromatic C–H), 1713 (α,β unsaturated ketone), 1545 (C=C str), 817 (C-Cl); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.71 (1H, d, J = 16.2 Hz), 7.01 (1H, d, J = 16.2 Hz), 7.23-8.45 (9H, m, Ar-H); Mass (m/z): (M⁺) 298

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(4-fluorophenyl)prop-2-en-1-one (PR3):

Cream Crystals (EtOH); m.p. 232–234°C; Yield 79%; IR (KBr) cm⁻¹: 3034 (aromatic C–H), 1703 (α,β unsaturated ketone), 1543 (C=C str), 1407 (C-F); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.73 (1H, d, J = 16.2 Hz), 7.15 (1H, d, J = 16.2 Hz), 7.15-8.12 (9H, m, Ar-H); Mass (m/z): (M⁺) 282

 

 

Figure 1. Scheme

 

(E)-1-(4-aminophenyl)-3-(benzo[b]thiophen-2-yl)prop-2-en-1-one (PR4):

Yellow Crystals (EtOH) m.p. 207–209°C; Yield 66%; IR (KBr) cm⁻¹: 3028 (aromatic C–H), 1697 (α,β unsaturated ketone), 1;567 (C=C str); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.76 (1H, d, J = 16.2 Hz), 7.45 (1H, d, J = 16.2 Hz), 7.12-7.89 (9H, m, Ar-H); Mass (m/z): (M⁺) 279.36

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(4-hydroxyphenyl)prop-2-en-1-one (PR5):

Brown Crystals (EtOH); m.p. 223–225°C; Yield 71%; IR (KBr) cm⁻¹: 3077 (aromatic C–H), 1699 (α,β unsaturated ketone), 1559 (C=C str); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.34 (1H, d, J = 16.2 Hz), 7.67 (1H, d, J = 16.2 Hz), 7.15-7.99 (9H, m, Ar-H); Mass (m/z): (M⁺) 280.34

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one (PR6):

Yellow Crystals (EtOH); m.p. 277–279°C; Yield 56%; IR (KBr) cm⁻¹: 3089 (aromatic C–H), 1685 (α,β unsaturated ketone), 1578 (C=C str); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.45 (1H, d, J = 16.2 Hz), 7.17 (1H, d, J = 16.2 Hz), 7.32-7.91 (9H, m, Ar-H); Mass (m/z): (M⁺) 309.34

 

(E)-3-(benzo[b]thiophen-2-yl)-1-p-tolylprop-2-en-1-one (PR7):

Cream Crystals (EtOH); m.p.207-209°C; Yield 62%; IR (KBr) cm⁻¹: 3084 (aromatic C–H), 1686 (α,β unsaturated ketone), 1521 (C=C str); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.67 (1H, d, J = 16.2 Hz), 7.98 (1H, d, J = 16.2 Hz), 7.31-7.90 (9H, m, Ar-H); Mass (m/z): (M⁺) 278.37

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(4-methoxyphenyl)prop-2-en-1-one (PR8)

Brown Crystals (EtOH); m.p. 253–257 °C; Yield 86%; IR (KBr) cm⁻¹: 3020 (aromatic C–H), 1693 (α,β unsaturated ketone), 1514 (C=C str), 687 (C-Br); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.52 (1H, d, J = 16.2 Hz), 7.61 (1H, d, J = 16.2 Hz), 7.21-7.91 (9H, m, Ar-H)

Mass (m/z): (M⁺) 294.37

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(pyrimidin-2-yl)prop-2-en-1-one (PR9)

Yellow Crystals (EtOH); m.p.203–205 °C; Yield 66%; IR (KBr) cm⁻¹: 3063 (aromatic C–H), 1709 (α,β unsaturated ketone), 1564 (C=C str); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.67 (1H, d, J = 16.2 Hz), 7.98 (1H, d, J = 16.2 Hz), 7.31-7.90 (9H, m, Ar-H); Mass (m/z): (M⁺) 266.32

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(thiazol-2-yl)prop-2-en-1-one (PR10)

Yellow Crystals (EtOH); m.p.193–195 °C; Yield 61%; IR (KBr) cm⁻¹: 3020 (aromatic C–H), 1693 (α,β unsaturated ketone), 1578 (C=C str); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.81 (1H, d, J = 16.2 Hz), 7.42 (1H, d, J = 16.2 Hz), 7.20-7.83 (9H, m, Ar-H); Mass (m/z): (M⁺) 271.36

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(thiophen-2-yl)prop-2-en-1-one (PR11)

Red Crystals (EtOH); m.p.204–206 °C; Yield 73%; IR (KBr) cm⁻¹: 3055 (aromatic C–H), 1694 (α,β unsaturated ketone), 1566 (C=C str); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.89 (1H, d, J = 16.2 Hz), 7.92 (1H, d, J = 16.2 Hz), 7.23-7.96 (9H, m, Ar-H); Mass (m/z): (M⁺) 270.37

 

(E)-3-(benzo[b]thiophen-2-yl)-1-(5-chlorothiophen-2-yl)prop-2-en-1-one (PR12)

Yellow Crystals (EtOH); m.p.286–288 °C; Yield 72%; IR (KBr) cm⁻¹: 3020 (aromatic C–H), 1688 (α,β unsaturated ketone), 1585 (C=C str); ¹H NMR (400 MHz, DMSO, δ/ppm): 6.81 (1H, d, J = 16.2 Hz), 7.72 (1H, d, J = 16.2 Hz), 7.19-7.90 (9H, m, Ar-H); Mass (m/z): (M⁺) 304.81

 

In silico platform:

All computational analysis was carried on Maestro 12.3 version (LigPrep, Glide XP docking, binding free energy calculations, ADMET, pharmacophore modeling) (Schrödinger 2020-4, LLC, New York). This software package programmed on DELL Inc.27" workstation machine running with Linux–x86_64 as the operating system.

 

 

Molecular docking and binding free energy calculation:

The selected targets for breast cancers are human estrogen receptor (PDB ID:3ERT)²⁷, progesterone receptor (PDB ID:4OAR)²⁸ and endothelial growth factor receptor kinase, EGFR kinase (PDB ID:4WKQ)²⁹. Their crystal structures are downloaded from the protein data bank and minimised by Protein Preparation Wizard, using the OPLS-2005 force field of Schrodinger software. The designed and synthesised chalcones were prepared by the LigPrep application (Schrödinger, 2020-4)³⁰ and were used for docking. The grid was generated by the grid box by applying default parameters. Glide-XP (extra precision) (Schrödinger, 2020-4)^31-34 was used for molecular docking computations. The binding free energy MMGBSA (Molecular Mechanics, Generalized Born Model and Solvent Accessibility) dGbind (kcal/mol), between the receptor and ligands, were calculated by the Prime module (Schrödinger, 2020-4)³⁵.

 

ADMET and Physicochemical Properties:

ADMET and physicochemical properties of ligand molecules were determined by using QikProp of Schrodinger software (Schrödinger 2020-4: QikProp)^36-37. The prepared ligands were incorporated into the QikProp tool and processed. The ADMET features include Caco-2 cell permeability, BBB permeability, percentage human oral absorption and solvent accessible surface area (SASA) and physicochemical properties like molecular weight, log P, donor-HB, and accept-HB analyses Lipinski Rule of five (Lipinski, 2004) were assessed.

 

In Vitro anticancer study by MTT assay:

We procured (MCF-7) (Human breast cancer cells) cell culture from National Centre for Cell Sciences (NCCS), Pune, India. Twelve compounds were incubated with different concentrations (25, 50, 100, 200 µM) to screen the cytotoxic activity of the compounds against human breast cancer cells (MCF-7). The cell viability was then determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after 24 h of incubation. Per cent inhibition was calculated from the absorbance as % growth inhibition^38-39.

 

RESULTS AND DISCUSSION:

Chemistry:

Twelve chalcone derivatives have been synthesised through Claisen-Schmidt condensation reaction by grinding technique in the presence of NaOH.  IR, NMR and mass spectroscopic techniques were used to confirm the structures.

 

Molecular docking:

The twelve chalcone derivatives were docked with 3ERT, 4OAR and 4WKQ (Tables 1-4 and Figures 2a,b,c).

 

Table 1: Docking scores and binding free energies of chalcones with protein 3ERT

S. No.	Chalcones	G Score	MMGBSA dG Bind
1.	PR2	-7.198	-63.55
2.	PR1	-7.026	-73.1
3.	PR3	-6.924	-70.62
4.	PR5	-6.831	-72.1
5.	PR7	-6.635	-58.72
6.	PR6	-6.507	-70.52
7.	PR4	-6.377	-75.4
8.	PR8	-6.017	-69.98
9.	PR12	-5.573	-73.09
10.	PR11	-5.42	-69.46
11.	PR9	-4.475	-58.58
12.	PR10	-4.324	-67.32
13.	Hydroxy tamoxifen	-10.152	-83.22

 

Table 2. Docking scores and binding free energies of chalcones with protein 4OAR

S. No.	Chalcones	G score	MMGBSA dG bind
1.	PR3	-6.683	-68.21
2.	PR2	-6.658	-71.96
3.	PR6	-6.531	-58.31
4.	PR1	-6.482	-72.56
5.	PR9	-6.467	-59.1
6.	PR5	-6.368	-64.49
7.	PR10	-6.115	-66.5
8.	PR12	-6.114	-83.13
9.	PR11	-6.109	-78.54
10.	PR8	-6.014	-70.16
11.	PR4	-6.003	-69.06
12.	PR7	-5.462	-50.49
13.	Ulipristal acetate	-6.398	-97.23

 

Table 3. Docking scores and binding free energies of chalcones with protein 4WKQ

S. No.	Chalcones	G Score	MMGBSA dG Bind
1.	PR2	-7.799	-69.13
2.	PR3	-7.74	-64.03
3.	PR1	-7.729	-68.87
4.	PR4	-7.576	-67.7
5.	PR12	-7.408	-72.27
6.	PR11	-7.363	-67
7.	PR6	-7.229	-66.57
8.	PR8	-6.578	-70.57
9.	PR5	-6.541	-64.91
10.	PR10	-6.094	-69.44
11.	PR9	-5.633	-61.26
12.	PR7	-5.542	-67.67
13.	Gefitinib	-8.806	-95.15

 

 

 

Table 4: Docking interactions of chalcones with target proteins 3ERT, 4OAR and 4WKQ

S. No	Chal cones	Protein ID	Hydrophobic Interactions	Polar Interactions	Hydrogen Bonding	Pi-Pi stacking
1.	PR1	3ERT	Phe 404, Met 421, Ile 424, Met 388, Leu 391, Leu 387, Leu 384, Trp 383, Leu 525 Met 528, Cys 530, Ala 350, Leu 346, Met 343, Leu 428	Thr 347
		4OAR	Cys 891, Tyr 890, Leu 797, Leu 887, Met 756, Met 759 Met 801, Val 760, Leu 763,Leu 721, Phe 778, Leu 718, Leu 715	Thr 894, Asn 719, Gln 725		Phe 778
		4WKQ	Leu 718, Met 793,Leu 792, Leu 788,Phe 856, Met 766, Leu 777, Ala 743, Leu 844, Val 726	Gln 791, Thr 790, Thr 854	Met 793
2.	PR2	3ERT	Phe 404, Met 421, Ile 424, Me, 388, Leu 391, Leu 387, Leu 384, Trp 383, Leu 525 Met 528, Cys 530, Ala 350, Leu 346, Met 343, Leu 428	Thr 347
		4OAR	Leu 715, Leu 718, Leu 721, Leu 763, Val 760 , Met 759, Met 756, Leu 887, Tyr 890, Cys 891, Leu 797, Phe 794,Met 801, Phe 778	Thr 894, Gln 725		Phe 778
		4WKQ	Leu 718, Met 793, Leu 792, Leu 788, Phe 856, Met 766, Leu 777, Ala 743, Val 726, Leu 844	Gln 791, Thr 790, Thr 854	Met 793
3.	PR3	3ERT	Phe 404, Met 421, Ile 424, Met 388, Leu 391, Leu 387, Leu 384, Trp 383, Leu 525 Met 528, Cys 530, Ala 350, Leu 346, Met 343, Leu 428	Thr 347
		4OAR	Leu 887, Tyr 890, Cys 891, Phe 794, Leu 797, Leu 715, Leu 718, Leu 721, Leu 763, Val 760, Met 759, Met 756, Trp 755, Phe 778	Thr 894, Gln 725		Phe 778
		4WKQ	Leu 718, Met 793, Leu 792, Leu 844, Leu 788, Phe 856, Met 766, Leu 777, Ile 744, Ala743, Val 726	Thr 790, Thr 894	Met 793
4.	PR4	3ERT	Trp 383, Leu 384, Leu 387, Met 388, Leu 391, Met 343 Leu 346, Leu 349, Ala 350, Phe 404, Met 528, Leu 525	Thr 347	Glu 353, Leu 387
		4OAR	Leu 763, Val 760, Met 759, Met 756, Phe 794, Leu 797, Leu 715, Leu 718, Leu 721, Phe 778, Leu 887, Tyr 890, Cys 891	Thr 894, Gln 725		Phe 778
		4WKQ	Leu 718, Met 793, Leu 792, Leu 844, Leu 788, Phe 856, Met, 766, Leu 777, Ile 744, Ala 743, Val 726	Gln 791, Thr 790, Thr 854	Met 793
5.	PR5	3ERT	Trp 383, Leu 384, Leu 387, Met 388, Leu 391, Met 343 Leu 346, Leu 349, Ala 350, Phe 404, Met 528, Leu 525	Thr 347	Arg 349 Leu 387
		4OAR	Leu 797, Cys 891, Tyr 890, Trp 755, Met 756, Met 759, Val 760, Leu 763, Leu 718, Leu 721, Phe 778	Thr 894 Gln 725	Thr 894	Phe 778
		4WKQ	Leu 718, Pro 794, Met 793, Leu 792, Leu 844, Leu 788, Phe 856, Met 766, Ala 743, Val 726	Thr 790 Thr 854	Met 793
6.	PR6	3ERT	Phe 404, Met 421, Ile 424, Met 388, Leu 391, Leu 387, Leu 384, Trp 383, Leu 525 Met 528, Cys 530, Ala 350, Leu 346, Met 343, Leu 428	Thr 347
		4OAR	Phe 895, Cys 891, Tyr 890, Leu 797, Leu 887, Met 756, Met 801, Met 759, Val 760, Leu 763, Phe 778, Leu 718, Leu 715	Thr 894, Gln 725, Asn 719	Thr 894
		4WKQ	Leu 718, Met 793, Leu 792, Leu 788, Phe 856, Met 766, Ile 744, Ala 743, Val 726, Leu 844	Gln 791, Thr 790, Thr 854	Met 793
7.	PR7	3ERT	Met 343,Leu 346, Leu 525, Ala 350, Leu 353, Leu 536, Leu 539, Trp 383, Leu 384, Leu 387, Met 388, Phe 404, Leu 391, Ile 424, Leu 428, Met 421	Thr 347
		4OAR	Leu 715, Leu 718, Met 801, Met 756, Val 760, Leu 887, Leu 797, Tyr 890, Cys 891, Phe 895	Asn 719 Thr 894	Thr 894
		4WKQ	Met 793, Leu 792, Leu 844, Leu 788, Phe 856, Met 766, Ala 743, Leu 718, Val 728	Thr 790 Thr 854
8.	PR8	3ERT	Trp 383 Leu 383 Leu 387 Met 388 Leu 391, Leu 428 Met 343 Leu 346 Ala 350, Val 533 Cys530, Met 528 Leu 525, Met 421, Ile 424, Phe 424	Thr 347
		4OAR	Leu 726, Leu 721, Leu 718, Phe 778, Leu 763, Met 801,Val 760, Met 759, Leu 887, Met 756, Trp 755	Gln 725 Asn 719
		4WKQ	Pro 794, Met 793, Leu 792, Ala 743, Leu 844, Leu 788, Met 766, Phe 856, Val 726, Leu 718	Thr 790 Thr 854	Met 793
9.	PR9	3ERT	Met 538, Leu 525, Met 421, Phe 404, Ile 424, Met 343, Leu 346, Ala 350, Trp 383, Leu 384, Leu 387	Thr 347, Hid 534
		4OAR	Leu 715, Leu 718, Phe 778, Leu 763, Val 760, Met 759, Met 801, Leu 887, Leu 797, Tyr 890, Cys 891	Thr 894, Gln 725, Asn 719	Thr 894
		4WKQ	Pro 794, Met 793, Leu 792, Ala 743, Leu 844, Leu 788, Met 766, Phe 856, Val 726, Leu 718	Thr 890 Thr 854	Met 793
10.	PR10	3ERT	Ile 424, Met 421, Leu 428, Phe 404, Leu 391, Met 388,  Leu 387, Leu 384, Trp 383, Ala 350, Leu 346, Met 343,  Met 528 , Leu 525	Thr 347
		4OAR	Phe 895, Cys 891, Tyr 890, Leu 797, Leu 887, Met 756, Met 801, Met 759, Val 760, Phe 778, Leu 763, Leu 718, Leu 715	Ser 898, Thr 894, Asn 719
		4WKQ	Leu 718, Leu 844, Met 793, Leu 792, Leu 788, Phe 856, Met 766, Ala 743, Val 726	Gln 791, Thr 790, Thr 854	Met 793
11.	PR11	3ERT	Trp 383, Leu 384, Leu 387, Met 388, Leu 391, Ala 350, Leu 349, Leu 346, Met 343, Leu 525, Met 528	Thr 347
		40AR	Cys 891, Tyr 890, Leu 797, Leu 88, Met 756,Met 759, Val 760, Leu 763, Phe 778, Leu 721, Leu 718,  Leu 715	Thr 894 Gln 725		Phe 778
		4WKQ	Leu 718, Leu 844, Met 793, Leu 792, Leu 788, Phe 856, Met 766, Ala 743, Val 726, Ile 744	Gln 791, Thr 790, Thr 854	Met 793
12.	PR12	3ERT	Leu 539, Leu 536, Ala 350, Leu 346, Met 343, Leu 525, Trp 383, Leu 384,Leu 387, Met 388, Leu 391, Phe 404	Thr 347
		4OAR	Met 756, Met 759, Val 760, Leu 763, Phe 778, Leu 887, Tyr 890, Cys 891, Phe 794, Leu 797, Leu 715, Leu 718, Leu 721	Thr 894 Gln 725		Phe 778
		4WKQ	Leu 718, Leu 844, Met 793, Leu 792, Leu 788, Phe 856, Met 766, Ala 743, Val 726, Ile 744	Gln 791, Thr 854, Thr 790	Met 793

 

Figure 2. 2D Docking Interaction

a) PR2 with 3ERT; b) PR3 with 4OAR; c) PR2 with 4WKQ

 

ADMET properties:

QikProp was used to determine the ADMET properties. It helps us establish the absorption, distribution, metabolism, and elimination of the compound and provides information related to the onset of action and how the drug crosses the barrier. The ADMET properties help the medicinal chemist make necessary modifications to improve the activity. QikProp determined the different variables such as bioavailability, blood-brain barrier penetration, plasma-protein binding, metabolism, HERG K+ and solvent accessible surface area.

 

Bioavailability prediction:

The parameters that assess oral absorption are the predicted aqueous solubility (logS), the predicted % human oral absorption and agreement to Jorgensen’s famous “Rule of Three (RO3). According to Jorgensen’s RO3, if a compound complies with all or some of the rules (logS > −5.7, Caco- >22 nm/s and # Primary Metabolites< 7), then it is more likely to be orally available. The non-active transport for the gut-blood barrier was assessed from Caco-2 cell permeability, and the studied compounds exhibited a wide range of values.

 

Compound PR6 has shown the most negligible Caco value of 444.253, which infers that it may have the least permeability to the gut-blood barrier compared with other derivatives. All the chalcones, except PR1, have been found to obey Jorgensen’s RO3 with no violations. The predicted aqueous solubility (logS) of the chalcones was within the range of the -6.78 to -3.77 mol dm^–3. The per cent human oral absorption parameter suggests that all the compounds have high human oral absorption.

 

Prediction of blood-brain barrier (BBB) penetration:

QPlogBB assessed the access to the central nervous system. Blood/brain partition coefficients (QPlogBB) of all compounds fall in the range of -2.9 to -0.53, which is recommended limit so that they can penetrate the blood-brain barrier.

 

Prediction of plasma-protein binding:

The binding of the drugs to plasma proteins decreases the amount of drug reaching the blood circulation, affecting drug efficiency. The plasma-protein binding is determined by binding to human serum albumin (log KHSA) (recommended range is −1.5 to 1.5), and from the data, it was found that all the chalcones were in the field of -0.001 to 0.952. So all the compounds are likely to reach the blood circulation freely, thus more available to the target site.

 

Metabolism prediction:

All were within the recommended number (1 to 8 reactions).

 

Prediction of blockage of human ether-a-go-go-related gene potassium (HERG K+) channel:

HERG K⁺ channel blockers are potentially toxic, and the predicted IC₅₀ values often provide reasonable predictions for the cardiac toxicity of drugs in the early stages. All the compounds showed an expected IC₅₀ value above -5 for HERG K+ channels that do not comply with the standard range.

 

Prediction of solvent accessible surface area (SASA):

The measure of the contact area between the solvent and molecule represents SASA (300.0 – 1000.0 Ų), and all the chalcones are within the standard limits. ADMET properties of the synthesised compounds are listed in Table 5.

 

Table 5. ADMET properties of chalcones

S. No.	Chalcones	%Human Oral Absorption	#metab	QPlogS	QPlogHERG	QPP Caco	QPlog BB	QPlog Kp	QPlog KHSA	SASA
1.	PR1	100	1	-6.334	-6.175	3719.508	0.181	-0.608	0.663	564.562
2.	PR2	100	1	-5.39	-6.143	3719.572	0.169	-0.606	0.638	559.52
3.	PR3	100	1	-5.04	-6.094	3719.162	0.115	-0.572	0.556	544.475
4.	PR4	100	2	-4.527	-6.089	965.645	-0.657	-1.643	0.297	551.165
5.	PR5	100	2	-4.685	-6.091	1126.347	-0.587	-1.5	0.365	547.798
6.	PR6	96.36	2	-5.15	-6.16	444.253	-1.027	-2.343	0.411	573.859
7.	PR7	100	2	-4.956	-6.145	3719.72	-0.009	-0.636	0.684	567.587
8.	PR8	100	2	-4.948	-6.12	3717.606	-0.068	-0.539	0.484	572.362
9.	PR9	100	3	-3.772	-6.018	1740.747	-0.328	-1.237	-0.083	528.348
10.	PR10	100	2	-4.051	-5.74	2437.711	-0.06	-1.102	-0.001	518.253
11.	PR11	100	2	-4.726	-5.846	3567.831	0.101	-0.693	0.423	520.909
12.	PR12	100	2	-5.457	-5.818	3571.512	0.262	-0.85	0.555	546.407
13.	Gefitinib	100	5	-5.148	-7.105	1044.674	0.309	-2.683	0.351	759.61
14.	Ulipristal acetate	100	6	-6.785	-5.118	779.371	-0.791	-2.962	0.952	776.869
15.	Hydroxy Tamoxifen	100	4	-5.626	-7.312	642.749	-0.289	-2.458	1.181	739.693

 

 

Table 6. Physicochemical properties of chalcones

S. No.	Chalcones	Molecular Weight	Molecular Volume	LogP	Donor H	Acceptor H	PSA	Rule of Five	Rule of Three
1.	PR1	343.237	947.394	5.103	0	2	26.965	1	1
2.	PR2	298.786	938.485	5.024	0	2	26.965	1	0
3.	PR3	282.332	910.44	4.758	0	2	26.966	0	0
4.	PR4	279.356	923.804	3.588	1.5	3	53.321	0	0
5.	PR5	280.341	917.011	3.795	1	2.75	49.514	0	0
6.	PR6	309.339	967.11	3.763	0	3	71.9	0	0
7.	PR7	278.368	954.328	4.844	0	2	26.965	0	0
8.	PR8	294.367	969.182	4.575	0	2.75	35.261	0	0
9.	PR9	266.317	874.479	3.121	0	4	49.864	0	0
10.	PR10	271.351	856.748	3.485	0	3.5	39.698	0	0
11.	PR11	270.363	864.47	4.41	0	2	27.452	0	0
12.	PR12	304.808	909.882	4.922	0	2	27.436	0	0
13.	Gefitinib	446.908	1335.798	4.314	1	7.7	61.213	0	0
14.	Ulipristal acetate	475.627	1492.76	4.957	0	7	84.942	0	1
15.	Hydroxy tamoxifen	387.521	1344.566	5.751	1	3.5	34.701	1	0

 

Physicochemical properties:

The physicochemical properties determined by QikProp establish the drug-likeness property of the compound. The various physicochemical properties of the twelve chalcone derivatives are listed in Table 6. The lipophilicity QPlogPo/w of the thirty phytoconstituents was within the permissible limit (–2.0 to 6.5), ranging from 3.12 to 5.75. The polar surface area correlating the Van der Waals surface area of polar nitrogen and oxygen atoms were calculated and found that compounds were between 26.96 to 84.94 Å, which is in standard limit 7.0 – 200.0. All the compounds obey Lipinski’s RO5 with no violations, except PR1, PR2 and hydroxytamoxifen, where their log P value ranges above 5. From the above observations, the compounds were considered druglike molecules.

 

In Vitro Anticancer study by MTT assay:

The results of the cytotoxicity studies are presented in Table 7. Compound PR1, PR2, and PR3 at the highest concentration (200µM) exhibited increased activity, above 80% cytotoxic in nature. On correlating with their docking scores, these compounds have excellently interacted with the three breast cancer targets. Thus the results interpret that the synthesised derivatives might inhibit any of the three targets discussed and exert their anticancer action.

 

 

Table 7. Cytotoxicity studies of the chalcone derivatives

S. No.	Chalcones	% Cytotoxicity
		Concentration  (µM)
		25	50	100	200
1.	PR1	16	30	45	80
2.	PR2	18	38	51	82
3.	PR3	15	30	46	80
4.	PR4	14	28	48	69
5.	PR5	11	25	36	56
6.	PR6	08	22	35	47
7.	PR7	15	33	50	75
8.	PR8	06	14	35	55
9.	PR9	10	24	35	65
10.	PR10	13	25	39	61
11.	PR11	11	27	37	50
12.	PR12	12	25	34	54
13.	Standard	85	88	89	98

DISCUSSION:

The top chalcones were PR2 and PR3, where compound PR2 displayed against 3ERT a docking score of -7.198 kcal/mol and binding energy of -63.55 kcal/mol while comparing with their co-crystal hydroxytamoxifen, which exhibited docking score of -10.152 kcal/mol and binding energy of -83.22 kcal/mol. The amino acids that were responsible for hydrophobic interactions are Phe 404, Met 421, Ile 424, Me, 388, Leu 391, Leu 387, Leu 384, Trp 383, Leu 525 Met 528, Cys 530, Ala 350, Leu 346, Met 343, Leu 428 and amino acid Thr 347 made polar interactions. On the analysis of the binding interactions of chalcones with the target, 4OAR, chalcone PR3 excellently bound with a docking score of -6.683 kcal/mol and binding energy of -68.21 kcal/mol, when comparing with their co-crystal ulipristal acetate with docking score and binding energy as -6.39 and -97.23 kcal/mol respectively. The hydrophobic and polar interactions were performed by the respective amino acids Leu 715, Leu 718, Leu 721, Leu 763, Val 760, Met 759, Met 756, Leu 887, Tyr 890, Cys 891, Leu 797, Phe 794, Met 801, Phe 778 and Thr 894, Gln 725. Pi-pi stacking was observed for the amino acid Phe 778. The best compound that interacted with protein 4WKQ was PR2, which showed a docking score of -7.799 kcal/mol and binding energy as -69.13 kcal/mol. At the same time, their co-crystal (gefitinib) had obtained -8.806 and -95.15 kcal/mol as docking score and binding energy, respectively. Amino acids Leu 718, Met 793, Leu 792, Leu 788, Phe 856, Met 766, Leu 777, Ala 743, Val 726, Leu 844 made hydrophobic interactions and Gln 791, Thr 790, Thr 854, and Met 793 interacted as polar and hydrogen bond contacts with the target 4WKQ (Tables 1-4 and Figures 2-4).

 

CONCLUSION:

Twelve chalcone derivatives were prepared by adopting green synthesis and characterised by spectral analysis. They were computationally analysed for the interactions with three breast cancer targets (3ERT, 4OAR and 4WKQ) and checked for their cytotoxic action by MTT assay. ADMET and physicochemical properties emphasised the drug-likeness and bioavailability of the synthesised chalcones. Among them, the top chalcones were PR2 and PR3, excellently interacting with the targets, per the in vitro studies. PR2 and PR3 have obtained good cytotoxic action against human breast cancer cells. Based on these results, it is concluded that the synthesised chalcones can be utilised as leads as anti-breast cancer agents, which can be verified by in vivo studies as future studies.

 

ACKNOWLEDGEMENTS:

We acknowledge Nitte Deemed to be University, Mangaluru, for the funding to carry out this university sanctioned project. Also, thankful to the authorities of the NGSM Institute of Pharmaceutical Sciences, Mangaluru and NGSMIPS CADD lab for providing requirements for this work. Thanks to CUSAT, Cochin for NMR, Mysore University, Mysuru for Mass and Yenepoya Research Centre for anticancer studies.

 

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Accepted on 08.12.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2215-2222.