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Synthesis and Cytotoxic Activity of New Chalcones and their Flavonol Derivatives
ISSN: 2161-0444

Medicinal Chemistry
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Synthesis and Cytotoxic Activity of New Chalcones and their Flavonol Derivatives

Rathnakar Reddy Kotha1*, Ravindra G Kulkarni2, Anil Kumar Garige1, Sridhar Goud Nerella3 and Achaiah Garlapati1*
1University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana, India
2SVERI’s College of Pharmacy, Pandhapur, Maharashtra, India
3Molecular Modeling Facility, Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Hyderabad, Telangana, India
*Corresponding Author(s): Rathnakar Reddy Kotha, University College of Pharmaceutical Sciences, Kakatiya University, Warangal-506 009, Telangana, India, Tel: +919949314355, Email: [email protected]
Achaiah Garlapati, University College of Pharmaceutical Sciences, Kakatiya University, Warangal-506 009, Telangana, India, Tel: +919440601057, Email: [email protected]

Received Date: Nov 14, 2017 / Accepted Date: Nov 20, 2017 / Published Date: Nov 25, 2017

Abstract

A series of chalcones and respective flavonols have been synthesized to explore their anti-cancer activities. All the chalcones were synthesized by the reaction between aldehydes and substituted acetophenones in typical base carried Claisen-Schmidt condensation and their corresponding flavonols were synthesized by hydrogen peroxide oxidized Flynn-Algar-Oymada epoxidation and cyclization. The synthesized compounds were characterized by FTIR, 1H NMR, 13C NMR and Mass spectrometry and subjected for cytotoxicity test on MCF-7, HT-29 and HeLa cell lines. Maximum number of compounds demonstrated anti-proliferative activity with IC50 in the range of 18.67-174.3 μM. Compound 3h with a chloro group and 1-phenyl-3(4-methoxy phenyl)-4-pyrazolyl moiety and the flavonol 4a with 3-thienyl group were found to be most potent compounds among all the tested compounds against MCF-7 cell lines with IC50 18.67 and 23.79 μM respectively. The most active compound 3h also showed high docking score of -8.825.

Keywords: Chalcone; Flavonol; Anti-proliferative activity

Introduction

Cancer is a major health problem in developing as well as underdeveloped countries recognized by uncontrolled cell growth [1,2]. Despite, intensive advancement in the treatment of cancer, the present chemotherapy is ineffective because of drug resistance and inability of many drugs to differentiate between normal cells and the cancerous cells [3]. According to the WHO report, cancer is the second leading cause of death globally, and was responsible for 8.8 million deaths in 2015. Approximately 70% of deaths occur in low- and middle-income countries due to cancer [3]. Therefore, development of new molecules with less toxicity, better efficacy and improved selectivity provide an important contribution for the development of safer drugs useful in the chemotherapy of cancer.

Targeted agents have more effective and less toxicity profiles over conventional chemotherapeutic agents. Natural compounds, like chalcones (Figure 1), have been shown to be relatively nontoxic, and certain chalcone moieties can target key molecular events that may lead to carcinogenesis [4]. Chalcones are important bioactive molecules belong to the flavonoid family found in many natural compounds [5]. Chalcones are the precursors of flavonoids and isoflavonoids are an important pharmacophore of various natural products [6]. Chemically, chalcones are 1,3-diaryl-2-propen-1-ones in which two aromatic rings or substituted aromatic rings are joined together by a three carbon α,β unsaturated carbonyl system.

medicinal-chemistry-structure

Figure 1: General structure of chalcones.

Molecular hybridization approach has been gaining special attention from medicinal chemistry, in which two pharmacophores are combined to yield a single molecule with additive biological properties. Literature survey reveals that chalcone-heterocycle hybrids with substituted pyrazole [7], pyridine [8], thiophene [9] moieties exhibited potent anticancer activities in in vitro studies. Chalcone-triazolecoumarin hybrids were reported by Pingaew et al. as anticancer and antimalarial agents [10]. It has been reported that chalcones possess many important biological activities including anti-oxidant [11], anticancer [ 12], anti-bacterial [13], anti-fungal [14], antiparasitic [15], antivascular [16] and anti-inflammatory [17]. Recently Tatiana et al. reported the anticancer activities of chalcone and flavonol derivatives [18]. Due to their anticancer activities, considerable efforts have been dedicated to identify new potential chalcone based drug candidates with in the field of oncology [19]. Keeping in view the biological importance of chalcones with various heterocycles such as pyrazole, pyridyl, thienyl moieties, herein, it is proposed to carry out the synthesis, docking and anticancer evaluation of some chalcone-hybrids with various heterocycles such as pyridyl, furyl, thienyl, substituted pyrazolyl groups and their flavonol derivatives (Figures 1 and 2).

medicinal-chemistry-active-substituted

Figure 2: Biologically active substituted chalcone derivatives from literature reports.

Hence we proposed to synthesize molecules with 2-hydroxy phenyl group and various heterocyclic moieties as part of the chalcone system. Similarly the chalcones were converted to their corresponding flavonols and all the synthesized compounds were evaluated for their anticancer activity. The molecular docking studies were also performed for the synthesized compounds and the results are all presented here.

Materials and Methods

All chemicals and solvents were purchased from commercial sources (Sigma Aldrich, Hymedia and sdfine) used without further purification. All compounds were characterized by spectroscopic data and compared with the data available in the literature. The NMR spectra were recorded in DMSO-d6 or CDCl3. 1H NMR spectra were obtained on a Bruker Advance 3400 (1H: 400 MHz and 13C: 400 MHz) and deuterated DMSO was used as solvent. The chemical shifts were expressed in values parts per million (ppm scale) and the J values were reported in Hertz (Hz). The peak patterns were indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet. The reactions were monitored by Thin Layer Chromatography (TLC) using silica gel 60 F254 plates (Merck). The melting points were determined on a Stuart SMP3 melting point apparatus. Elemental analyses were performed on Elementar vario MICRO CHNS instrument.

Chemicals and cell culture the 3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide (MTT), DMEM (Dulbecco's modified Eagles medium), antibiotic/antimycotic solution, trypsin, EDTA and Phosphate Buffered Saline (PBS) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Fetal Bovine Serum (FBS) was bought from Gibco. 25 cm2 and 75 cm2 flask and 96 well plated purchased from Eppendorf India.). All other reagents were of analytical grade. MCF- 7 (Breast adenocarcinoma cancer cell line) and HT-29 cells (human colorectal cancer cell line), HeLa (Human Cervical Carcinoma Cell line) were purchased from NCCS, Pune.

Experimental

General procedure for the synthesis of substituted hydroxychalcone derivatives (3a-h)

To a solution of KOH (1.12 g, 0.02 mol) in methanol (50 ml) was added substituted hydroxyacetophenones 2a-c (1.36 g,0.01 mol) and substituted hetero aromatic aldehydes 1a-e (2.62 g, 0.01 mol) at 0-5°C [14]. The reaction mixture was stirred overnight at room temperature ( Scheme 1). Then, this reaction mixture was poured over crushed ice and acidified with dil. HCl. The solid formed was filtered and washed with distilled water and dried. The crude product was crystallized with ethanol to afford pure 2-hydroxychalcones.

medicinal-chemistry-synthetic-routes

Scheme 1: Synthetic routes of chalcone and flavonol derivatives.

General procedure for the synthesis of substituted chromone derivatives (4a-g)

To a well-stirred solution of substituted hydroxychalcones 3 (2.0 g, 0.007 mol) in MeOH (20 ml) and aq. KOH (10 ml, 20%), cooled at 5-10°C, was added 30% H2O2 (10 ml) drop wise over 1 hr. The reaction mixture was further stirred for 4-5 hrs and the resulting light yellow reaction mixture was poured on crushed ice and neutralized with dilute hydrochloric acid [14]. The light yellow solid thus obtained was filtered, washed with water and dried. The crude product was crystallized with ethanol to afford pure hydroxychromone derivatives.

Biological assays

Cell culture: The cell lines (MCF-7, HT-29 and HeLa) were maintained in culture with MEM supplemented with 10% Fetal bovine serum (FBS) and the antibiotics penicillin/streptomycin (0.5 mL- 1), in atmosphere of 5% CO2 and 95% air at 37°C. Stock solutions of synthesized chalcones (3a-h) and flavonols (4a-g) were made in DMSO and kept in aliquots at -20°C. For MTT assay, each test compound was weighed separately and dissolved in DMSO, made up the final concentration with media to 1 mg/ml and the cells were treated with series of concentrations from 10 to 100 μm.

MTT assay: Inhibition of cell proliferation by chalcones and flavonols were determined using the methyl thiazolyltetrazolium (MTT) cell viability assay with three independent experiments with six concentrations of compounds in triplicates. MCF- 7, HT-29 and HeLa cells were trypsinized and preformed the tryphan blue assay to know viable cells in cell suspension. Cells were counted by haemocytometer and seeded at density of 5.0 × 103 cells / well in 100 μl media in 96 well plate culture medium and incubated overnight at 37ºC. After incubation, the old media was taken off and added with fresh media 100 μl with different concentrations of test compound in respective 96 well plate. After 48 hrs, the drug solution discarded and the fresh media with MTT solution (0.5 mg/ml) was added to each well and plates were incubated at 37°C for 3 hrs. At the end of incubation time, precipitates are formed as a result of the reduction of the MTT salt to chromophore formazan crystals by the cells with metabolically active mitochondria. The optical density of solubilized crystals in DMSO was measured at 570 nm on a microplate reader. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% values is generated from the doseresponse curves for each cell line [20].

% Inhibition=100(Control-Treatment)/Treatment

Docking

Dataset preparation: Glide, Version 6.1 (Schrodinger LLC, New York, NY, 2013 Inc. 2012) was used for docking studies. The structure of fifteen synthesized compounds consisting both 2-hydroxychalcones and flavonols were drawn by considering all possible tautomers and were subjected for minimization using OPLS-2005 force field using water as solvent in the GB/SA continuum solvation model in Schrodinger module.

The drawn structures were minimized using Polak-Ribiere Conjugate Gradient (PRCG) method with maximum of 5000 iterations. Van der Waals interaction (8.0), electrostatic (20), and hydrogen bond (4.0) were included in extended contribution. The extensive conformational search was carried out with Mixed torsional/Low-mode sampling method with the use of 100 steps per rotatable bond, maximum number of steps 1000, energy window for saving structures with 5.02 kcal/mol, eliminate the redundant conformers with maximum atom deviation cut-off 0.5 Å and saved 100 structures for each search.

Protein preparation: The 3D coordinates of crystal structure of tubulin was obtained from protein data bank (PDB ID: 1SA0) with resolution of 2.5 Å and co-crystal ligand. Usually deposited crystal structures lack hydrogens, hydrogens were added and also adjusted the bond orders using protein preparation wizard of Schrödinger software graphical user interface Maestro v9.3. Also, refining the structure and minimizing is a major step involved to ensure a stable protein formation. Finally selective protein was minimized to relieve unwanted interactions using OPLS-2005 force field with converge heavy atoms to RMSD 0.3 Ǻ relative to original protein structure.

Docking studies: This step involves actual docking where the given ligands are checked for their binding capability and interactions. The grid and the prepared ligands were placed in respective slots. Further, the process was run as per the requirement. HTVS (High Throughput Virtual Screening), SP (Standard Precision) was chosen. The output was also selected as pose viewer file.

Structure characterization

(E)-1-(2-hydroxyphenyl)-3-(thiophen-2-yl)prop-2-en-1-one (3a): IR νmax (KBr) cm-1: 3308 (OH), 1640 (C=O), 1560 (olefinic C=C). 1H NMR (CDCl3) δ (ppm): 6.96-6.93 (t, J=7.5Hz, 1H), 7.03-7.01 (d, J=8.4Hz, 1H), 7.13-7.10 (t, J=4.2Hz, 1H), 7.42-7.41 (d, J=2.8Hz, 1H), 7.51-7.46 (m, 3H), 7.89-7.87 (d, J=7.6 Hz, 1H), 8.07-8.03 (d, J=15.4 Hz, 1H, olefinic), 12.84 (s, 1H). Elemental analysis: calculated for C13H10O2S; (230); calculated: C, 67.80; H, 4.38; S, 13.92; found: C, 67.68; H, 4.46; S, 13.74. MS: (m/z); 231 [M+1]+.

(E)-1-(5-bromo-2-hydroxyphenyl)-3-(thiophen-2-yl) prop-2-en- 1-one (3b): IR νmax (KBr) cm-1: 3290 (OH), 1610(C=O), 1558 (olefinic C=C). 1H NMR (CDCl3) δ (ppm): 6.94-6.92 (d, J=8.8 Hz,1H), 7.14- 7.12 (dt, J=4.8 Hz, 5.2 Hz, 1H), 7.34-7.31 (d, J=15.2 Hz, 1H, olefinc), 7.44-7.43 (d, J=3.6 Hz, 2H), 7.52-7.50 (d, J=5.2 Hz, 1H), 7.57-7.55 (dd, J=8.8 Hz, 2.4 Hz, 1H), 7.97-7.96 (d, J=2.4 Hz, 1H), 8.09-8.05 (d, J =15.2 Hz, 1H, olefinic), 12.78 (s, 1H); Elemental analysis: calculated for C 13H9BrO2S (309); calculated: C, 50.5; H, 2.93; S, 10.37; found: C, 50.58; H, 2.81; S, 10.17; MS: (m/z); 311 [M+2]+.

(E)-1-(2-hydroxyphenyl)-3-(4-(4-methoxyphenyl)-1-phenyl-1Hpyrazol- 3-yl)prop-2-en-1-one (3c): IR νmax (KBr) cm-1: 3310 (OH), 1637(C=O), 1564 (olefinic C=C). 1H NMR (CDCl3) δ (ppm): 3.91(s, 3H, OCH3), 6.94 (m, 1H), 7.04-7.02 (d, J=8.4 Hz, 1H), 7.08-7.06 (d, J=8.7 Hz, 2H),7.39-7.41 (d, J=8.4 Hz, 1H), 7.48-7.55 (m, 4H), 7.67- 7.69 (d, J=8.7Hz, 2H), 7.82-7.84 (m, 3H), 8,02-7.88 (d, J=15.3Hz, 1H, olefinic), 8.4 (s, 1H), 12.94 (s, 1H, OH), Elemental analysis: calculated for C25H20N2O3 (396); calculated; C, 75.74; H, 5.08; N, 7.07; found: C, 75.91; H, 5.19; N, 7.01 MS: (m/z); 397 [M+1]+.

(E)-1-(5-bromo-2-hydroxyphenyl)-3-(4-(4-methoxyphenyl)-1- phenyl-1H-pyrazol-3-yl)prop-2-en-1-one (3d): IR νmax (KBr) cm-1: 3294 (OH), 1634 (C=O), 1545 (olefinic C=C). 1H NMR (CDCl3) δ (ppm): 3.89 (s, 3H, OCH3), 6.93-6.91 (d, J=8.8Hz, 1H), 7.08-7.06 (d, J=8.8Hz, 2H), 7.40-7.36 (m, 2H), 7.56-7.49 (m, 3H), 7.65-7.63 (d, J=8.8Hz, 2H), 7.87-7.81(m, 3H), 8.03-7.99(d, J=15.2 Hz, 1H, olefinic), 8.4 (s, 1H), 12.84 (s, 1H). Elemental analysis: calculated for C25H19BrN2O3 (475) calculated: C, 63.17; H, 4.03; N, 5.89; found: C, 63.31; H, 3.94; N, 5.97; MS: (m/z); 477 [M+2]+.

(E)-1-(5-bromo-2-hydroxyphenyl)-3-(1,4-diphenyl-1H-pyrazol- 3-yl)prop-2-en-1-0ne prop-2-en-1-one (3e): IR νmax (KBr) cm-1: 3320 (OH), 1648 (C=O), 1593 (olefinic C=C). 1H NMR (CDCl3) δ (ppm): 6.93-6.91 (d, J=8.8Hz, 1H), 7.40-7.34 (m, 2H), 7.57-7.47 (m, 6H), 7.72–7.70 (m, 2H), 7.85-7.82 (m, 3H), 8.03- 7.99 (d, J=15.2Hz, 1H, olefinic), 8.43 (s, 1H), 12.80 (s, 1H). Elemental analysis: calculated for C24H17BrN2O2 (445); calculated; C, 64.73; H, 3.85; N, 6.29; found: C, 64.41; H, 3.98; N, 6.18; MS: (m/z); 477 [M+2]+.

(E)-1-(5-bromo-2-hydroxyphenyl)-3-(pyridine-3-yl) prop-2-en- 1-one (3f): IR νmax (KBr) cm-1: 3298 (OH), 1636 (C=O), 1560 (olefinic C=C). 1H NMR (CDCl3) δ (ppm): 6.98-6.96 (d, J =8.9Hz, 1H), 7.45-7.42 (m, 1H), 7.61-7.58 (d, J=2.44Hz, 1H), 7.67-7.63 (d, J=15.52Hz. 1H, olefinic), 7.95-7.91 (d, J=15.52Hz, 1H, olefinic), 8.00-8.01 (d, J=2.32Hz, 1H), 8.05-8.03 (d, J=8.07Hz, 1H), 8.70-8.69 (d, J=4.03Hz, 1H), 8.91(s, 1H), 12.58 (S, 1H). 13C NMR (400MHZ, DMSO-d6): δ 192.15, 162.58, 151.50, 150.20, 142.53, 139.34, 134.97, 131.83, 130.24, 123.99, 121.54, 121.00, 120.78, 110. 62 ppm. Elemental analysis: calculated for C 14H10BrNO2 (304); calculated; C, 55.29; H, 3.31; N, 4.61; found: C, 55.41; H, 3.17; N, 4.72; MS: (m/z); 306 [M+2]+.

(E)-1-(5-bromo-2-hydroxyphenyl)-3-(furan-2-yl) prop-2-en-1- one (3g): IR νmax (KBr) cm-1: 3300 (OH), 1635 (C=O), 1516 (olefinic C=C). 1H NMR (CDCl3) δ (ppm): 6.57-6. 55 (t, J=1.58Hz, 1H), 6.83- 6.81 (d, J=3.30Hz, 1H), 6.94-6. 91(d, J=8.92Hz, 1H), 7.47-7.43 (d, J=15.03Hz, 1H, olefinic), 7.59-7.55 (m, 2H), 7.71-7.68 (d, J=15.03Hz, 1H, olefinic), 8.01-8.00 (d, J=2.20Hz, 1H), 12.82 (s, 1H). 13C NMR (400MHZ, DMSO-d6): δ 192.14, 162.46, 151.36, 145.82, 138.85, 131.97, 131.83, 121.31, 120.57, 117.87, 116.92, 113.07, 110.45 ppm. Elemental analysis: calculated for C13H9BrO3 (293); calculated; C, 53.27; H, 3.09; found: C, 53.07; H, 2.98; MS: (m/z); 295 [M+2]+.

(E)-1-(5-chloro-2-hydroxyphenyl)-3-(4-(4-methoxyphenyl)-1- phenyl-1H-pyrazol-3-yl)prop-2-en-1-one (3h): IR νmax (KBr) cm-1: 3300 (OH), 1640 (C=O), 1516 (olefinic C=C). 1H NMR (CDCl3) δ (ppm): 3.90 (s, 3H, OCH3), 7.10-6.98 (m, 3H), 7.43-7.38 (m, 2H), 7.55- 7.51 (m, 2H), 7.68-7.65 (m, 1H), 7.75 (m, 1H), 7.86-7.80 (m, 4H), 8.05- 8.01(d, J=16Hz, 1H, olefinic), 8.54 (s, 1H), 12.83 (s, 1H). Elemental analysis: calculated for C25H19ClN2O3 (430); calculated; C, 56.34; H, 3.27; found: C, 56.07; H, 3.17. MS: (m/z); 432 [M+2]+.

3-hydroxy-2-(thiophen-2-yl)-4H-chromen-2-one (4a): IR νmax (KBr) cm-1: 3310 (OH), 1610 (C=O). 1H NMR (CDCl3) δ (ppm): 6.90 (s, 1H), 7.25-7.23 (t, J=5.4Hz, 1H), 7.43-7.39 (t, J=8Hz, 1H), 7.59-7.56 (d, J=8.4HZ, 1H), 7.63-7.61 (dd, J=0.8Hz, 4.8Hz, 1H), 7.72-7.67 (dt, J=1.6HZ, 2.8HZ, 1H), 8.03-8.02 (dd, J=0.8Hz, 3.6Hz, 1H), 8.25-8.23 (dd, J=1.6Hz, 8.0Hz, 1H). Elemental analysis: calculated for C13H8O3S (244); calculated; C, 63.92; H, 3.30; S, 13.13; found: C, 64.01; H, 3.41; S, 13.01. MS: (m/z); 245 [M+1]+.

6-bromo-3-hydroxy-2-(thiophen-2-yl)-4H-chromen -2-one (4b): IR νmax (KBr) cm-1: 3370 (OH), 1612 (C=O). 1H NMR (CDCl3) δ (ppm): 7.24- 7.22 (t, J=3.54Hz, 1H), 7.36 (s, 1H), 7.48-7.46 (d, J=8.80Hz, 1H), 7.63-7.62 (d, J=4.64Hz, 1H), 7.76-7.74 (d, J=8.68Hz, 1H), 8.02- 8.01 (d, J=2.56Hz 1H), 8.34 (s, 1H). Elemental analysis: calculated for C13H7BrO3S (323); calculated; Elemental analysis: calculated for C 13H9BrO2 (323); calculated; C, 48.32; H, 2.18; S, 9.92. found: C, 48.22; H, 2.08; S, 10.01 MS: (m/z); 325 [M+2]+.

3-hydroxy-2-(4-4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-yl)- 4H-chromen-4-one (4c): IR νmax (KBr) cm-1:3298 (OH), 1613 (C=O). 1H NMR (CDCl3) δ (ppm): 3.88 (s, 3H, OCH3), 7.04-6.97 (m, 3H), 7.41- 7.37 (m, 2H), 7.53-7.47 (m, 4H), 7.87-7.77 (m, 4H), 8.52 (s, 1H), 10.04 (s, 1H) Elemental analysis: calculated for C25H18N2O4 (410); calculated; C, 73.16; H, 4.42; N, 6.83; found: C, 75.52; H, 5.18; N, 7.01 MS: (m/z); 411 [M+1]+.

6-bromo-3-hydroxy-2-(4-4-methoxyphenyl)-1-phenyl-1Hpyrazol- 3-yl)-4H-chromen-4-one (4d): IR νmax (KBr) cm-1:3300 (OH), 1610 (C=O). 1H NMR (CDCl3) δ (ppm): 3.89 (s, 3H, OCH3), 6.90-6.88 (m, 1H), 7.03-7.00 (m, 2H), 7.41-7.39 (m, 1H), 7.55-7.50 (m, 2H), 7.67-7.63 (m, 1H), 7.89-7.79 (m, 2H), 8.38-8.37(d, J=2.4Hz, 1H), 8.58 (s, 1H), 8.85 (s, 1H),10.07 (s, 1H). Elemental analysis: calculated for C25H17BrN2O4 (489); calculated; C, 61.36; H, 3.50; N, 5.72 found: C, 61.15; H, 3.62; N, 5.84. MS: (m/z); 491 [M+2]+.

6-bromo-2-(1,4-diphenyl-1H-pyrazol-3-yl)-3-hydroxy-4Hchromen- 4-one (4e): IR νmax (KBr) cm-1: 3402 (OH), 1609 (C=O). 1H NMR (CDCl3) δ (ppm): 6.77-6.75 (m, 1H), 7.56-7-7.38 (m, 4H), 7.70-7.61 (m, 3H), 7.92-7.80 (m, 4H), 8.36-8.37(m, 1H), 8.60 (s, 1H), 8.87 (s, 1H). Elemental analysis: calculated for C24H15BrN2O3 (460); calculated; C, 62.76; H, 3.29; N, 6.10 found: C, 62.84; H, 3.18; N, 6.19. MS: (m/z); 462 [M+2]+.

6-bromo-3-hydroxy-2-(pyridine-3-yl)-4H-chromen-4-one (4f): IR νmax (KBr) cm-1: 3352 (OH), 1609 (C=O). 1H NMR (CDCl3) δ (ppm): 7.54-7.40 (m, 2H), 7.81-7.79 (d, J=8.55 Hz, 1H), 8.37 (s, 1H), 8.68 –8.58(m, 3H), 9.45 (s, 1H). 13C NMR (400 MHZ, DMSO-d6): 172.20, 154.22, 150.74, 148.56, 142.94, 139.29, 137.05, 134.99, 128.06, 127.14, 123.44, 122.01, 120.27, 118.22 ppm. Elemental analysis: calculated for C 14H8BrNO3 (318); calculated; C, 52.86; H, 2.53; N, 4.40 found C, 52.95; H, 2.39; N, 4.48 MS: (m/z); 320 [M+2]+.

6-bromo-2-(furan-2-yl)-3-hydroxy-4H-chromen-4-one (4g): IR νmax (KBr) cm-1: 3284 (OH), 1613 (C=O). 1H NMR (CDCl3) δ (ppm): 6.80–6.79 (t, J=1.4Hz, 1H), 7.31-7.30 (d, J=3.2Hz, 1H), 7.70-7.68 (d, J=8Hz, 1H), 7.92-7.89 (m, 1H), 8.04 (S, 1H), 8.167-8.162 (d, J=2Hz, 1H), 10.197 (s, 1H, OH, D2O Exchangeable). Elemental analysis: calculated for C13H7BrO4 (307); calculated; C, 50.84; H, 2.30 found C, 50.72; H, 2.41 MS: (m/z); 309 [M+2]+.

Results and Discussion

Chemistry

All the chalcones 3a-h were synthesized by base catalyzed aldol condensation type reaction by using appropriate heterocyclic aldehydes 1a-e and 2-hydroxy acetophenones 2a-c. 3M sodium hydroxide was employed for the removal of proton from 2-hydroxy acetophenones and all the reactions were carried at 0-5°C and stirred at room temperature. Upon acidification of the reaction mixture, desired chalcones were obtained, which were purified by recrystalization to afford chalcones (3a-h) in 50-80% yield. All the 2-hydroxy chalcones except 3h in Flynn- Algar-Oymada epoxidation and cyclization using alkaline hydrogen peroxide resulted in cyclized flavonols 4a-g which were purified by recrystallization to get the desired flavonols in 54-90% yield. All the details of characterization have been included in Tables 1 and 2.

equation
Comp. R1 Ar Yields (%) MP (°C) Rf
3a -H equation 65 85-87 0.71
3b -Br equation 78 77-79 0.66
3c -H equation 70 170-172 0.78
3d -Br equation 84 180-182 0.74
3e -Br equation 65 184-186 0.68
3f -Br equation 77 132-134 0.64
3g -Br equation 50 75-77 0.55
3h -Cl equation 61 144-146 0.74

Table 1: Physical data of synthesized chalcone derivatives.

equation
Comp. R1 Ar Yields (%) MP (°C) Rf
4a -H equation 68 199-201 0.73
4b -Br equation 76 210-212 0.81
4c -H equation 70 161-163 0.66
4d -Br equation 90 180-182 0.69
4e -Br equation 60 170-172 0.61
4f -Br equation 67 200-202 0.76
4g 1 Br equation 54 205-207 0.72

Table 2: Physical data of synthesized flavonol derivatives.

The IR spectra of hydroxychalcones showed carbonyl absorption in the range of 1600-1650 cm-1 and an olefinic C=C in the range 1516- 1593 cm-1. Flavanol showed OH absorption in the range of 3284-3402 cm -1 and C=O in the range 1609-1613 cm-1 respectively. In the 1H NMR spectra of hydroxylchalcones, OH peak appeared as singlet in the range of δ 12.58-12.80, olefinic protons H-a and H-b appeared as doublets or multiplets in the range of δ 7.31-7.34 and δ 8.05-8.09 respectively. Transstereochemistry of propenone moiety of chalcones was confirmed by coupling constant of vinyl hydrogen (15-16 Hz) indicating formation of trans-chalcones. In flavonols OH peak appeared as a singlet in the range of δ 7-10. The parent ion peak appeared on the positive mode in the mass spectrum confirms the chalcones and flavonols.

Cytotoxicity activity

The cytotoxicity studies of eight chalcone and seven flavonol derivatives were evaluated on three human cancer cell lines namely MCF-7, HT-29, HeLa using MTT assay in vitro [20]. Semi log plot of concentration of compounds versus % inhibition of cell lines corroborated the determination of IC50 values of compounds using Microsoft Excel Figure 3. The cytotoxicity of all the tested compounds were compared against Cisplatin as standard compound which showed cytotoxic activity with IC50 value of 4.05 μM against MCF-7 and HeLa cell lines and 6.05 μM against HT-29 Cell lines. The results of anticancer activity of all tested compounds are summarized in Table 3 and 4.

medicinal-chemistry-Dose-response

Figure 3: Dose response relationship of 3h in the number of MCF-7 viable cells. The IC50 concentration of tested compounds is also depicted.

equation
Comp. R1 Ar MCF-7 HT-29 HeLa Docking score
 
   
 
 
 
3a -H equation 33.29 52.63 112.36 -6.768
3b -Br equation 44.15 42.02 114.02 -6.913
3c -H equation 150.10 100.23 - -6.478
3d -Br equation 85.51 156.22 77.52 -7.874
3e -Br equation 23.48 78.13 158.61 -8.595
3f -Br equation 28.17 89.08 146.20 -6.328
3g -Br equation 156.22 104.04 173.67 -6.363
3h -Cl equation 18.67 99.38 - -8.825
Cisplatin     4.05 6.05 4.05  

Table 3: Anticancer activity of synthesized chalcone derivatives (IC50 in μM).

equation
Comp. R1 Ar MCF-7 HT-29 HeLa Docking score
4a -H equation 23.79 77.54 148.82 -6.388
4b -Br equation 32.08 89.13 - -6.140
4c -H equation 40.01 92.11 118.54 -5.953
4d -Br equation 55.31 109.34 174.73 -5.623
4e -Br equation 28.08 62.88 - -6.369
4f -Br equation 37.17 80.08 - -5.877
4g -Br equation 95.51 110.11 - -5.953
Cisplatin     4.05 6.05 4.05  

Table 4: Anticancer activity of synthesized flavonol derivatives (IC50 in μM).

Among all tested compounds, cyclized flavanols have demonstrated enhanced activity over their corresponding 2-hydroxy chalcones against MCF-7 and HT-29 cell lines which indicates the restricted binding site which aides in accommodating flat structures. This is evidenced by the activity of profiles of flavonols, compounds 4a, 4b, 4c, 4d and 4g were found to be more active than corresponding 2-hydroxy chalcones (3a, 3b, 3c, 3d and 3g). Only two compounds 3e and 3f were more active than corresponding flavonols against MCF-7 and HT-29 cell lines. Maximum 2-hydroxy chalcones were active in cytotoxicity trials against HeLa cell lines except 3c.

Lone chloro derivative compound 3h is the most potent against MCF-7 cell line with IC50 value of 18.67 μM (Figure 4) while 3b is most potent against HT-29 cell line (IC50 42.02 μM). Both 3a and 4a are more potent than their corresponding bromo derivatives 3b and 4b. However this claim is untrue for 3d (bromo derivative) which is more potent than 3c against MCF-7 cell line. Three 2-hydroxy chalcone derivatives 3e, 3f and 3h are found to be highly potent against MCF-7 with IC50 less than 30 μM and 100 μM against HT-29cell line. Among the bromo derivatives, five membered ring at Ar showed varied activity, 2-thienyl moiety as in 3b found to be moderately active and when it is replaced by pyrazole scaffold, nearly as in 3d, the activity was reduced by half and improved as in the case of 3e. Only one compound was synthesized with 2-furyl moiety (3g), which is found to be least active among all the compounds tested against MCF-7 cell line. None of the compounds were highly potent against HeLa cell line, all the compounds either were moderately active or poorly active and only compound 3d showed cytotoxicity activity with IC50 77.52 μM.

medicinal-chemistry-study-incubation

Figure 4: Effect of compound 3h on cell morphology during study incubation with MCF-7 cells.

A similar profile of activity could also be noticed with flavonol derivatives 4a-g. Unsubstituted compound 4a with 2-thienyl ring was found to be highly potent against both MCF-7 and HT-29 with IC50 23.79 and 77.54 μM respectively. Substitution at R1 by bromo lead to 4b, which exhibited a little less potency. Replacement of 2-thienyl group by another five membered ring pyrazole improved the activity as in 4e, but resulted in decrease in activity with 4-methoxy substitution on phenyl ring as in 4d against MCF-7 cell line and the same activity profile could be seen against HT-29 cell line for 4e as that of 4a. 2-Furyl derivative 4g, was least active against all the three cell lines and only 3-pyridyl derivative 4f was nearly equal potent like 4b (Figures 5 and 6).

medicinal-chemistry-binding-orientation

Figure 5: The binding orientation of the Ligands to tubulin protein is shown. a) Surface view of the active compound 3h with tubulin receptor for the highest docking score. b) Surface view of the active compound 4b with tubulin receptor.

medicinal-chemistry-interaction-diagrams

Figure 6: 2D ligand interaction diagrams of compound 3h (a) and 4b (b) with tubulin.

Molecular docking

The tubulin is involved in many cellular functions and its dynamic activity is controlled by many of compounds and proteins, including colchicine and stathmin family proteins, which provoked us to study the targeting of this protein by synthesized molecules. Ravelli et al. [21] reported that the structure, at 3.5 Å resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3 [22]. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Hence targeting this receptor with the novel compounds for enhancing the desired activities is one of the possible options. The Molecular docking of synthetic compounds revealed the binding interactions with receptor (PDB: 1AS0). Among all, the synthesized compounds, 3h [(E)-1-(5- chloro-2-hydroxyphenyl)-3-(4-(4-methoxyphenyl)-1-phenyl-1Hpyrazol- 3-yl)prop-2-en-1-one] and 4b, [3-hydroxy-2-(thiophen-2-yl)- 4H-chromen-2-ones] have shown high docking scores of -8.825 and -6.388 than other compounds (Figures 5 and 6; Tables 3 and 4).

Conclusion

The present study revealed the synthesis of chalcones from different aldehydes and corresponding hydrogen peroxide cyclized flavanols. All the purified compounds were screened for their anti-cancer activity against MCF-7, HT-29 and HeLa cell lines. Compounds 3h and 3b in chalcone series exhibited potent cytotoxic activity against MCF- 7 and HT-29 cell lines with IC50 of 18.67 and 42.02 μM respectively. Among the flavonols, compounds 4a and 4e demonstrated potent cytotoxicity with IC50 activity of 23.79 and 62.88 μM. Further synthesis and cytotoxicity studies of more such derivatives and other supportive studies like appoptosis studies probably infer probable mechanism of cyctotoxic activities of chalcones and flavanols. The docking result of the most active compound in chalcone series 3h is in accordance with its potency.

Conflict of Interest

Author RRK is thankful to AICTE for the award of fellowship under Quality Improvement Program.

Acknowledgements

The authors acknowledge Dr A. Mallika, Scientist, Molecular Modeling Group and Mr. N. Sridhar Goud, Research Scholar, NIPER, Hyderabad, for their help in molecular docking studies.

References

Citation: Kotha RR, Kulkarni RG, Garige AK, Nerella SG, Garlapati A (2017) Synthesis and Cytotoxic Activity of New Chalcones and their Flavonol Derivatives. Med Chem (Los Angeles) 7:353-360. DOI: 10.4172/2161-0444.1000480

Copyright: © 2017 Kotha RR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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