Docking Studies and Synthesis of Novel Flavones Screened for Biological Activities like Anticancer and Antioxidant Activity

 

Sushil Dagadu Patil²*, Kardele Vinayak², Balsubraniyan¹ and Shaikh Anwar²

¹NIMS University, Jaipur

²Bhujbal Knowledge City, Department of Pharmaceutical Chemistry, MET Institute of Pharmacy,

Adagaon, Nashik -422003India.

*Corresponding Author E-mail: sushilpharma@rediffmail.com

Flavones are the derivatives of flavonoids, possessing the wider range of biological actions. A novel series of flavonoid Derivatives with the help of substituted benzaldehyde were synthesized and screened for biological activities like anticancer and antioxidant activity. All synthesized derivatives were screened for in-vitro cytotoxicity by Brine shrimp lethality assay in which 1-(2, 4- dihydroxyphenyl)-3-(4-(dimethyl amino) phenyl) prop-2ene-1one.(MCR-001) and 2-(4-(Di-methylamino) phenyl)-7-hydroxy-4-H-chromen-4-one. (MCR-002), 2-hydroxy-2-(4-(methyl (4-nitrostyryl) amino) phenyl)-4-oxochroman-7-yl benzoate. (MCR-004) and 2-(4-dimethyl amino) phenyl)-4-oxo-4H-chromen-7-yl benzoate. (MCR-005) showed highest anticancer activity as compared to 5-fluorouracil due to presence of hydroxy group. Among synthesized compounds, 4-nitro flavonoids compound derivatives were screened for antioxidant activity by using DPPH Scavenging Method. 2-(4-dimethyl amino) phenyl)-2-hydroxy 4-oxochroman-6-yl benzoate (MCR-005) showed highest antioxidant activity as compared to ascorbic acid. This work has revealed the scope and potential of flavonoids for future exploration to synthesize substituted flavonoids and chalcone derivatives by the use of different substituted benzaldehyde.

 

 

INTRODUCTION:

Flavonoid has gained recent interest because of their broad biological and pharmacological activities including anti‐oxidant [1‐4], Anxiolytic[ 5], anti cancer[ 6], analgesic [7‐8], and antimicrobial [9] activities. Flavonoid showed promise of being powerful antioxidants which can protect the human body from free radicals. The function of an antioxidant is to intercept and react with free radicals at a rate faster than the substrate. Since free radicals are able to attack at a variety of target including lipids, fats and proteins, it is believed that they may damage organisms, leading to disease, poisoning and including aging [10]. The newly prepared derivatives were docked into the active site of homology modeled 1GS4, using Vlife Software, MDS.4.3.

 

The chemical structures of the new derivatives were confirmed by elemental and spectral (1H-NMR and Mass) analyses. All compounds were investigated for antioxidant activity and anticancer activity.Docking Score and Hydrogen, pi and hydrophobic bindings shown in Table 1.

 

MATERIALS AND METHODS:

All materials were procured from Sigma Aldrich and Merck specialties Pvt. Ltd. (Mumbai. India). Solvents were dried and distilled being used. Anhydrous sodium sulphate was used to dry the solvents. Thin Layer Chromatography (TLC) analyses were carried out on aluminum plates (Merck) precoated with silica gel 60 F254 (0.2 mm), and spots were visualized with UV light and I2. Liquid intermediates were checked for purity using Gas chromatography (Pack column SE-30, OV-101, and capillary column BP-5). Gravity column chromatography was performed using silica gel (Merck 60). Melting points were taken in open glass capillary using OMEGA melting point apparatus and were uncorrected. Infra red (IR) spectra were recorded on KBr pellets on a Shimadzu 1000 FTIR spectrometer in the range of 4000-200 cm-1, Resolution 2.0 with number of scan - 45. Apodization; Happ - Genzel. Proton (1H) Nuclear Magnetic Resonance (NMR) spectra of compounds were recorded on Bruker Advance II 400 NMR Spectrophotometer using CDCl3 solvent, at SAIF, Punjab University, Chandigarh. Mass spectra of compounds were recorded on API 4000 Q TRAP LC/MS/MS system using electron spray ionization positive ion mass spectrometric technique, at NHRDF, Chitegaon, Nashik. Elemental analyses were performed on a Perkin- Elmer 2400 Analyser and are within ±0.4% of theoretical values. The method followed for the synthesis of flavonoids derivatives has been elaborated here:

 

Figure 1

 

SYNTHESIS:

Synthesis of 1-(2,4- dihydroxyphenl)-3-(4-(dimethyl amino)phenyl) prop-2ene-1one.(MCR-001) A mixture of 2,4- dihydroxyacetophenone (3.04gm, 20mmol) was prepared containing  p- dimethyl amino benzaldehyde (4.47gm, 30mmol), KOH (30gm) in methanol (50ml) and water (25ml). This mixture was stirred at RT for 26-27hr. After stirring mixture was mixed with 32gm acetic acid (30ml) and 300ml water. A clear solution obtained which turned to fresh red and brown solid ppt.The brown

 

solid precipitate was recrystalised from 95% ethanol, giving product (brown colour).

 

Synthesis of 2-(4-(Dimethylamino)phenyl)-7-hydroxy-4-H-chromen-4-one. (MCR-002) Hydrogen peroxide (30%, 20ml) was added dropwise to a DMSO (50ml). A solution of 4’-dimethyl amino 2, 4- dihydroxychalcone (MCR-001) (5gm, 17.6mmol) and KOH (20%, 25ml) at 0°c was prepared by stirring for 4hr at RT. A blackish green solution was formed. After mixing the solution with glacial acetic acid (25ml) and water(300ml), a solid ppt was formed (product). Washing and recrystalisation from 95% ethanol, yellow product observed.

Synthesis of 7-hydroxy-2-(4-methyl (4-nitrostyryl) amino) phenyl)-4-H-chromen-4-one. (MCR-003) Equimolar quantity of MCR-002 compound and p- nitro benzaldehyde was taken in round bottom flask to which 12.5 ml ethanol was added. 0.2 ml of pyridine was added to reaction mixture then refluxed for 3-4 hrs (120-130°C). The mixture was cooled, treated with ethanol and poured into ice water. Precipitate obtained which was filtered out and recrystallized from ethanol.

 

Synthesis of  2-hydroxy-2-(4-(methyl(4-nitrostyryl) amino)phenyl)-4-oxochroman-7-yl benzoate. (MCR-004) 1g (0.003 mol) of (MCR-003) compound was suspended in 20 ml of 5% sodium hydroxide solution in a well corked small conical flask, to which 2g (0.014 mol) of benzoyl chloride was added, with 0.5ml portion of constant shaking under cold condition. Mixture was shaken vigorously for 5-10 min until odour of excess benzoyl chloride disappeared. Reaction mixture was maintained alkaline. The product obtained was filtered, washed with ice cold water and recrystallized with ethanol.

 

Synthesis of 2-(4-dimethyl amino)phenyl)-4-oxo-4H-chromen-7-yl benzoate. (MCR-005)

1g (0.003 mol) of (MCR-002) compound was suspended in 20 ml of 5% sodium hydroxide solution in a well corked small conical flask, to which 2g (0.014 mol) of benzoyl chloride was added, with 0.5 ml portion of constant shaking under cold condition. Mixture was shaken vigorously for 5-10 min until odour of excess benzoyl chloride disappeared. Reaction mixture was maintained alkaline. The product obtained was filtered, washed with ice cold water and recrystallized with ethanol.

 

Synthesis of 2-(4-(4-chlorobenzyl) methyl) amino)phenyl)-2,6-dihydroxychroman-4-one. (MCR-006)Equimolar quantity of  MCR-002 compound and p- chloro benzaldehyde were taken in round bottom flask to which 12.5 ml ethanol was added. 0.2 ml of pyridine was added to reaction mixture then refluxed for 3-4 hrs (120-130°C). The mixture was cooled, treated with ethanol and poured into ice water. Precipitate obtained which was filtered out and recrystallized from ethanol.

 

Synthesis of 1-(2,4- dihydroxyphenl)-3-(4-(dimethyl amino)phenyl) prop-2ene-1one.(MCR-001 )  IR: 3059, 2916 (Ar-CH), 2821 (-CH), 1681(Ar-C=C), 1778 (-C=O), 3350 (-OH)

1H-NMR: 7.3-8.5(Doublet (2H) Benzene -CH ), 5.35 (Singlet Ar-OH ), 7.4 (Doublet Ar-CH) 3.09( Singlet (6H) CH₃)_.

 

Synthesis of 2-(4-(Dimethylamino)phenyl)-7-hydroxy-4-H-chromen-4-one(MCR-002)

IR: 2900, 3049, 2819(Ar-CH), 729, 825 Benzene (p), 1678(-C=O), 3317(-OH)

1H-NMR: 6.69-7.18 (Doublet (2H)Ar -CH), 5.40 (Singlet Ar-OH), 3.08 (Singlet (6H) CH₃)_, 7.5-7.6( Doublet –CH)

 

Synthesis of 7-hydroxy-2-(4-methyl (4-nitrostyryl) amino) phenyl)-4-H-chromen-4-one (MCR-003)

IR: 3080 (Ar -C-H), 1446, 1539(NO₂), 1705 (C=O), 3309 (Ar-OH), 1670 (Ar-C=C)

1H-NMR: 6.69-8.40(Doublet Ar-CH), 5.69 (Singlet Ar-OH), 3.08 (Singlet (6H) CH₃), 6.78(Doublet-CH)

 

Synthesis of 2-hydroxy-2-(4-(methyl (4-nitrostyryl) amino) phenyl)-4-oxochroman-7-yl benzoate (MCR-004)

IR:  2825, 2991, 3072 (Ar -C-H), 1166, 1294(C-N), 1496, 1531, 1454 (NO₂), 1712, 1703 (C=O), 1598 (Ar-C=C) 711, 815 (Benzene), 1788(-COOR)

1H-NMR: 6.69-8.1 (Doublet Ar-CH), 5.69 (Singlet Ar-OH), 3.09 (Singlet (6H) CH₃),

 

Synthesis of 2-(4-dimethyl amino) phenyl)-4-oxo-4H-chromen-7-yl benzoate (MCR-005)

IR: 2823, 3030, 3070 (Ar -C-H), 1708 (C=O), 1670 (Ar-C=C), 812-707( Benzene), 1166,1128 (C-N), 1791(-COOR)

1H-NMR: 7.1-8.2 (Doublet(6H)Ar-CH), 5.38 (Singlet Ar-OH), 3.1 (Singlet (6H) CH₃)

 

 

Table no.1

Sr. No.	Hydrogen, pi and hydrophobic bindings	Dock Score
MCR-001		-27.29
MCR-002		-6.12
MCR-003		-8.83
MCR-004		-41.45
MCR-005		-14.00
MCR-006		-43.24
MCR-007		-33.76
Reference		-21.00

 

Table no 2

Sr. no.	Molecular Formula	Mol.Wt. gm/mol	% Yield	M.P.(0C)	Mobile Phase	Rf  value.
1	C17H17NO3	283.32	73%	48o-50oC	nH : EtA  7:5	0.8
2	C17H15NO3	299.32	62%	62º-65ºC	nH : EtA  7:5	0.698
3	C24H18N2O5	420.41	58%	58º-60ºC	nH : EtA            5:5	0.733
4	C31H24N2O7	542.52	55%	80º-82ºC	Ben :meth 7:3	0.615
5	C24H19NO4	393.43	53%	90º-93ºC	Ben : meth 7:3	0.646
6	C23H20ClNO4	409.86	55%	75º-78ºC	nH : EtA           5:5	0.600

EtA: Ethyl acetate, nH: n-Hexane, Ben : Benzene,   meth: methanol.

 

 

PHARMACOLOGICAL EVALUATION:

Brine shrimp lethality bioassay was performed in laboratory. Complexes has solubility problem so it should be dissolved in DMSO for the preparation of drug solution. Following results were obtained by which LC₅₀ was calculated. These results were compared with standard drugs i.e. 5-FU. The positive control was done with DMSO.

·         30 active shrimps (larvae) were added into each test tube

·         The surviving (larvae) shrimps were counted after 24 hours and lethality concentration LC50 was assessed [11] result shown in table 3.

 

Table no.3-Brine shrimp lethality bioassay

Compound	LC50 µg/ml
MCR-001	965.65
MCR-002	852.02
MCR-003	681.00
MCR-004	990.26
MCR-005	1022.00
5-FU	690.37

 

Determination of Antioxidant activity:

DPPH radical scavenging activity:

The DPPH assay method is based on the reduction of DPPH, a stable free radical. The free radical DPPH with an odd electron gives a maximum absorption at 517 nm (purple colour). When Antioxidants react with DPPH, which is a stable free radical becomes paired off in the presence of a hydrogen donor (e.g., a free radical-scavenging antioxidant) and is reduced to the DPPHH and as consequence the absorbance’s decreased from the DPPH. Radical to the DPPH-H form, results in decolorization (yellow colour) with respect to the number of electrons captured. More the depolarization more is the reducing ability. This test has been the most accepted model for evaluating the free radical scavenging activity of any new drug. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form (Diphenyl picryl hydrazine; non radical) with the loss of this violet colour (although there would be expected to be a residual pale yellow colour from the picryl group still present).[12]result shown in Table 4.

 

Table no. 4

Compound	IC50in μg/ml
MCR-001	7.8
MCR-002	6.97
MCR-003	5.9
MCR-004	8.12
MCR-005	6.02
Ascorbic acid	5.91

 

Compound: 2-(4-dimethyl amino)phenyl)-4-oxo-4H-chromen-7-yl benzoate. (MCR-005)

 

IR spectra:

 

(MCR-005)

 

Fig. IR spectra of compound MCR-005

 

TABLE-IR Interpretation of compound MCR-005

 

 ¹H NMR spectra of compound MCR-005

Fig. ¹H NMR spectra of compound MCR-005

 

Resolved ¹H NMR spectra of compound MCR-005

 

Fig. Resolved¹H NMR spectra of compoundMCR-005

 

TABLE-¹H NMR interpretation of compound MCR-005

Sr.No.	δ (ppm)	Splitting	Assignment
1	7.1-8.2	Doublet(6H)	-CH(aromatic)
2	5.38	Singlet	-OH
3	3.1	Singlet	CH3.

 

DISCUSSION:

(MCR-001) (MCR-004)and (MCR-007) these compounds shows good docking score as compared to the standard 5-florouracil i.e. compounds shows less dock score than standard. All these compound shows good interaction like hydrogen, pi and hydrophobic binding. Entire compound shows good anticancer activity. Novel flavonoid derivatives were synthesized and screened for biological activities like anticancer and antioxidant activity. All synthesized derivatives were screened for in-vitro cytotoxicity by Brine shrimp lethality assay in which (MCR-001),(MCR-002),

 

(MCR-004) and (MCR-005) showed highest anticancer  activity as compared to 5-fluorouracil due to presence of hydroxy group. Among synthesized compounds, 4-nitro flavonoids compound derivatives were screened for antioxidant activity by using DPPH Scavenging Method. 2-(4-dimethyl amino) phenyl)-2-hydroxy 4-oxochroman-6-yl benzoate (MCR-005) showed highest antioxidant activity as compared to ascorbic acid.

 

This work has revealed the scope and potential of flavonoids for future exploration to synthesize substituted flavonoids and chalcone derivatives by the use of different substituted benzaldehyde.

 

ACKNOWLEDGEMENTS:

The authors are very thankful to the management of MET’s Institute of Pharmacy for providing infrastructural facilities to carry out the research work. Also we are very thankful to SAIF, Punjab University for providing analytical instrumentation Facility.

 

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Received on 18.04.2015         Modified on 30.04.2015

Accepted on 11.05.2015         © AJRC All right reserved