SYNTHESIS AND EVALUATION OF 41 METHYL CHALCONES FOR THEIR ANALGESIC AND ANTIOXIDANT ACTIVITIES

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SYNTHESIS AND EVALUATION OF 4

1

-METHYL CHALCONES FOR

THEIR ANALGESIC AND ANTIOXIDANT ACTIVITIES

*R. Padma, M. Lakshmi Surekha, Muggu. Muralikrishna and Ch. Ajay

A.M. Reddy Memorial College of Pharmacy, Dept. of Pharmaceutical Chemistry,

Uppalapadu, Andhra Pradesh 522601. India.

Literature review revealed that substituted chalcones and its derivatives

showed different pharmacological activities like antimicrobial,

anticancer, anti-inflammatory, antioxidant activity etc. In the present

study we planned to synthesize substituted 4-methyl chalcones

associated with antioxidant and analgesic activities.

OBJECTIVES

 To synthesize the various new series of various substituted

4-methyl chalcones and to purify the final compounds by appropriate

recrystallization and or chromatographic techniques.

 To characterize all the new compounds by analytical and spectral

analysis.

 To evaluate analgesic activity of the compounds.  Acetic acid induced writhing test in mice.

 To screen the title compounds for in-vitro activities viz., nitric oxide scavenging activity,

reduction of DPPH stable free radical and inhibition of iron induced lipid peroxidation in

rat brain homogenate, hydroxyl radical scavenging activity, superoxide radical

scavenging activity, reducing power assay, ABTS+ radical scavenging activity.

General structure of chalcone

O

 All the chalcones give pink coloration with concentrated H2SO4 (positive Wilson test) and

violet coloration with alcoholic ferric chloride solution.

Volume 8, Issue 13, 966-989. Research Article ISSN 2277– 7105

*Corresponding Author

R. Padma

A.M. Reddy Memorial

College of Pharmacy, Dept.

of Pharmaceutical

Chemistry, Uppalapadu,

Andhra Pradesh 522601.

India.

Article Received on 30 Sept. 2019,

Revised on 20 Oct. 2019, Accepted on 10 Nov. 2019

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 Chalcones on heating with traces of iodine in dimethyl sulfoxide (DMSO) for 2hrs give

the corresponding flavones.

 Chalcones were converted to the corresponding flavonols by their oxidation using

hydrogen peroxide in methanolic sodium hydroxide solution and these flavonols showed

characteristic greenish yellow fluorescence in ethanolic solution as well as with

concentrated sulphuric acid.

General Methods Of Synthesis Of Chalcones

The chalcones are important intermediates in the synthesis of pyrazoles, isoxazoles and

pyrimidines. They can be obtained by the acid or base catalyzed aldol condensation of

2-hydroxy acetophenones with benzaldehydes (Claisen, L. et al., 1881).

Chalcones were prepared by using the following methods

1. Solvent free synthesis

2. Mortar and pestle synthesis

3. Synthesis of chalcones based on Suzuki reaction

4. Solvent free synthesis using silica- sulfuric acid as reagent.

Procedure for mortar and pestle synthesis

The solvent free synthesis of chalcones was carried out by grinding the substituted aromatic

aldehydes and the aetophenone in the presence of solid sodium hydroxide with a mortar and

pestle. In general, the chalcones were obtained in high yield and high purity. The by products

were easily removed by recrystallization. The result indicates a correlation between the

success of the solvent- free synthesis and melting point of the chalcone. (Rajendra K. et al.,

2006).

Procedure for solvent free synthesis using silica- sulfuric acid as reagent

Ketones (2 mmol), p- substituted benzaldehydes (4.2 mol) and silica-sulfuric acid (1.5 g

equal to 4mmol of H+) were mixed thoroughly, placed in a glass tube and capped. The

mixture was heated in an oven at 800C for 2-3.5h. After complete conversion of ketones

monitored by TLC, the mixture was cooled to room temperature. Dichloromethane (20-30

ml) was added and heated for 3-5 minutes. The reagent was removed by filtration. The filtrate

was concentrated and the solid residue was recrystallized from ethanol to afford the pure

products as pale yellow glittering solid. The catalyst was recycled by washing the solid

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for 2h. and can be reusable for another reaction run. They are characterized by comparison of

their physical constants and spectral data reported in literature. (Thirunarayanan G. et al.,

2006).

They can be obtained b y the acid or base catalyzed aldol condensation of 2-hydroxy

acetophenones with benzaldehydes.

For example 1

2-hydroxy acetophenone and benzaldehyde react in the presence of 0.1M NaOH to give the

chalcone.

OH

COCH3

CHO

+

O Acetophenone _Benzaldehyde

Chalcone NaOH

2. Some novel dihydro-α-ionone based chalcones have been synthesized.

O

+

CHO

R1

R2

R3

R4

KF/ Al₂O₃

Microwave

O

R1

R2

R3

R4

3. Reductive trimerization of aromatic aldehydes to 1,2,3 – triaryl – 2 – propen – 1 – ones as

well as rearrangements of aryl ketones to pinacolone analogues were efficiently achieved

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R1

O

R R

1

R1

O R

O

R1

TCS/Zn

R=Me,Ph

TCS/Zn

R=H

 4. Substituted oxathiolone fused chalcones were prepared by condensation of 4-acetyl

-5-methoxy- 2 -oxo- benz (1,3) oxathiole with benzaldehydes under acidic conditions.

O S

O

OCH3 X

S O

O

OCH3

COCH3

+

CHO

X

ACOH H2SO4

ANTIOXIDANT ACTIVITY

 Oxygen plays a vital role in diverse biological phenomena. It is essential for life, but it

can also provoke damaging effects. A free has been defined as any species capable of

independent existence that contains one or more unpaired electrons. Unpaired electron

makes the molecules unstable and highly reactive. Ironically these reactive species are

derived from normal physiological and metabolic processes that are essential to the cell.

The feature of the reaction of the free radicals with non radical is that they usually

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 Normally a balance between oxidative events and antioxidative force maintains the status

quo within living cells. A variety of enzymes help to maintain cells in reduced state

despite the presence of aerobic environment. Thus major cellular reducing agents, such as

ascorbate, glutathione and  - tocopherol are present predominantly in their reduced form

(Slater, 1984; Yagi, 1987). In addition a number of enzymes scavenge and remove these

reactive chemical species. When normal balance is upset, either by loss of reducing

agents or protective enzymes or by increased production of oxidizing species or by both

events simultaneously, the tissue is considered to be under oxidative stress. An

antioxidant is any substance, when present at low concentrations compared to those of

oxidizable substrate significantly delays or prevents oxidation of that substrate. The term

oxidizable substrate includes almost everything found in living cells, including proteins,

lipids, carbohydrates and DNA.

TYPES OF FREE RADICALS

 Free radicals can be defined as chemical species associate with an odd or unpaired

electron (Gupta. V.K. et.al., 1992; Halliwell B, et al., 1990). They are neutral, short lived,

unstable and highly reactive to pair up the odd electron and finally to achieve stable

configuration. Example of free radicals are super oxide (O-2, an oxygen centered radical),

thiyl (RS; a sulphur - centered radical), trichloromethyl (*CCl3, a carbon centered radical)

and nitric oxide (NO) in which the unpaired electron is delocalized between both the

atoms. (Halliwell and Gutteridge, 1990).

Oxygen -Derived Free Radicals (ODFR)

 The free radicals and other reactive oxygen species are continuously produced in the

human body. The term reactive oxygen species is a collective one that includes not only

oxygen-centered radicals such as super oxide radical, singlet oxygen, and hydroxyl

radicals, but also some potentially dangerous non-radicals such as hydrogen peroxide, and

hypochlorous acid (Southern, P.A, et al., 1988).

 There has been a great interest and focus on the role of free radicals, and the reactive

species usually derived from oxygen products in the inflammatory process. Formation of

these highly reactive oxygen species (ROS) is integral to the phagocytic action of

polymorphonuclear leukocytes (PMNs). Stimulation of the cell by chemo attractants such

as LTB4, or products of the complement cascade, results in activation of the

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These include super oxide radical and hydrogen peroxide, and possibly the very reactive

hydroxyl radical (Flohe L, et al., 1985).

 ODFR reactions mediated by activated neutrophils are likely to have a role in vascular

damage, endotoxin shock and burn induced damage. ODFR induced damage in

inflammatory disorder may be mediated through increased neutrophil elastase activity as

well.

 Toxic effects of oxygen derived free radicals (ODFR) include, protein oxidation, lipid

peroxidation (which leads to change in membrane fluidity, membrane permeability and

ultimately cell necrosis), carbohydrate oxidation and nucleicacidoxidation (Halliwell.B.

et.al., 1990).

CHEMISTRY

Materials

Melting points were determined in open capillaries on a Tempo melting point apparatus, and

were uncorrected. UV spectra, were recorded on SYSTRONICS UV/VIS

SPECTROPHOTOMETER, FTIR spectra were obtained using PERKIN-ELMER, Mass

spectra using BXI SYSTEM. Proton NMR spectra were taken is using AVANCE-300 MHz.

Aldehydes were procured from Sigma Aldrich and S.d fine chemicals. All other chemicals

are of AR grades. Purity of the compounds was checked by Thin layer chromatography

(TLC) using precoated plates with Silica gel G, and spots were detected by iodine chamber

and UV chamber.

Methods

General procedure for the synthesis of (E)-3-(41-Methyl phenyl)-1-(substituted phenyl) prop-2-ene-1ones(compounds, 1-12)

Synthesis of (E)-3-(41-methyl phenyl)-1-(phenyl) prop-2-ene-1-one

Equimolar concentrations of 4-methyl acetophenone (412mg, 0.025mol) and unsubstituted

benzaldehydes (350mg, 0.025 mol) were dissolved in 20ml of methanol. Methanolic Sodium

hydroxide solution (0.03 mol) was added slowly and the mixture was stirred for two hours or

till the completion of the reaction(progress of the reaction checked by TLC). The mixture was

acidified with 0.1 N HCl. Then it was filtered, washed with water, dried and recrystallised

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SCHEME

CHO

+

O H3C

R Room temperature _NaOH

PHYSICOCHEMICAL DATA OF COMPOUNDS (1-12)

Compound 1 : (R=H)

Chemical name : (E)-3-phenyl-1-p- tolyl prop-2-en-1-one

Molecular formula : C17H14O2

TLC : Rf (3:1 benzene: ethyl acetate):0.70

UV (methanol) : 249 nm

Compound 2 : (R=4-Cl)

Chemical name : (E)-3-(4-Chlorophenyl)-1-p- tolyl prop-2-en-1-one

Molecular formula : C16H13OCl

TLC : Rf (3:1 benzene: ethylacetate): 0.86

1

H NMR (CDCl3) : δ 2.4 – 2.5 (d, 3H, CH3), δ 7.27-7.31(d, Hα)J=17.5,

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FTIR (KBr)cm-1 : 3056. 7(Ar-H); 1670.9(C = O), 1561.1 (C = C)

Compound 3 : (R=4-CH3)

Chemical name : (E)-3-(4-methylphenyl)-1-p – tolyl prop-2-en-1-one

Molecular formula : C17H16O

TLC : Rf (3:1 benzene: ethyl acetate): 0.91

1

H NMR (CDCl3) : δ 2.36(s, 3H, CH3), δ 2.43(s, 3H, CH3)

: δ 7.18-7.22(d, Hα) J=17.2, δ 7.35- 7.85(d, Hβ), J=17.2.

: δ 7.0-7.50(d, 10H, Ar-H)

FTIR (KBr) cm-1 : 3061.0 (Ar-H); 1660.3(C = O), 1593.2 (C = C)

Compound 4 : (R= 4- N(CH3)2

Chemical name : (E)-3-(4-dimethylaminophenyl)-1-p-tolylprop-2-en-1-one

Molecular formula : C18H19ON

TLC : Rf (3:1 benzene: ethylacetate) : 0.71

1

H NMR (CDCl3) : δ 2.30 –2.45 (d, 3H, CH3), δ 2.95-3.05(d, 6H,(CH3)2)

: δ 6.88- 6.94 (d, Hα) J=17, δ 7.427.46(d,Hβ), J=17

: 6.56-7.50(m,10H,Ar-H)

FTIR (KBr) cm-1 : 3036.2 (Ar-H); 1655.9(C = O), 1592.6 (C = C)

Compound 5 : (R = 4- OCH3)

Chemical name : (E)-3-(4-methoxyphenyl)-1-p-tolyl prop-2-en-1-one

TLC : Rf (3:1 benzene: ethyl acetate) : 0.81

1

H NMR (CDCl3) : δ 2.34 – 2.35(d, 3H, CH3), δ 3.80-3.90( d, 3H, OCH3)

: δ 6.89-6.94(d, Hα) J=17.3, δ 7.51-7.54(d, Hβ),J=17.3.

: δ 6.85-7.6(d, 10H, Ar-H)

FTIR (KBr)cm-1 : 3041.6 (Ar-H); 1639.7(C = O), 1597.6 (C = C)

Compound 6 : (R=3,4,(OCH3)2)

Chemical name : (E)-3-(3,4-dimethoxyphenyl)-1-p-tolyl prop-2- en-1-one

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1

H NMR (CDCl3) : δ 2.35-2.50 (d, 3H, CH3), δ 3.85-3.95(d, 6H, (OCH3)2)

: δ 6.85-6.91(d, Hα) J=17.2, δ 7.45-7.49(d, Hβ), J=17.2

: δ 6.85- 7.5(m, 9H, Ar-H)

FTIR (KBr)cm-1 : 3047.4 (Ar-H); 1632.1(C = O), 1597.5(C = C)

Compound 7 : (R=3,4,5,(OCH3)3)

Chemical name : (E)-3-(3,4,5-trimethoxyphenyl)-1-p-tolyl prop- 2-en-1-one

1

H NMR (CDCl3) : δ 2.36 (s, 3H, CH3), δ 3.30-3.85(d, 9H,(OCH3)3)

: 7.32- 7.35(d, Hα) J=17, 7.56-7.60(d, Hβ), J=17.

7.1-7.7(m,4H,Ar-H)

FTIR (KBr) : 2957 (Ar-H); 1612.9(C = O), 1568.1 (C = C)

MASS(m/z) : 313.24(M++1)

Compound 8 : (R=2-OH)

Chemical name : (E)-3-(2-hydroxyphenyl)-1-p-tolyl prop-2-en-1-one

Compound 9 : (R=4-OH, 3-OCH3)

Chemical name : (E)-3-(4-hydroxy,3-methoxyphenyl)

-1-p-tolyl prop-2-en-1-one

Compound 10 : (R=4-OH, 3,5 (OCH3)2)

Chemical name : (E)-3-(4-hydroxy,3,5-dimethoxyphenyl)-1-p- tolyl prop-2-en-1-one

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Compound 11 : (R=4-OH, 3-OCH3, 5-Br)

Chemical name : (E)-3-(5-bromo,4-hydroxy-3-methoxy phenyl)-1- p-tolyl prop-2-en-1-

one

Molecular formula : C17H16O3Br

UV (methanol) : 286 nm.

1

H NMR (CDCl3) : δ 2.25-2.40(d, 3H, CH3), δ 3.629-3.639(d, 3H, CH3)

: 6.82-6.89(d, Hα) 17.6, 7.06-7.1(d, Hβ), J=17.6, 6.45-7.45(m, 6H, Ar-

H)

FTIR (KBr) : 3062.7 (Ar-H); 1656.5(C = O), 1583 (C = C)

MASS(m/z) : 346.04(M++1)

Compound 12 : (R= 4-OH, 3-OCH3 5-I)

Chemical name : (E)-3-(5-iodo,4-hydroxy,3-methoxyphenyl)-1-p-tolyl

prop-2-en-1-one

Molecular formula : C17H16O3I

PHARMACOLOGICAL STUDIES

Analgesic activity

Acetic acid induced writhing test

Mice of either sex approximately 20-25gm are used. An aliquot of 0.25 ml of 0.6% acetic

acid was injected intraperitoneally in each animal. The animal reacts with a characteristic

stretching behavior, i.e. a series of constrictions occur that travel along the abdominal wall,

some times accompanied by turning movements of the body and extension of the hind limbs.

This response is called Writhing.

The mice were divided into 17 groups. Each group contains four animals. One group

consisting of four served as control, while the other groups of four animals received the test

compounds or standard drug. The mice were dosed (100mg/kg) orally with test compounds

one hour before injection of 0.25 ml of 0.6% acetic acid was injected intraperitoneally in each

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IN VITRO ANTIOXIDANT STUDIES

Materials

Sodium nitroprusside, N-napthylethylenediamine dihydrochloride, were obtained from S.d

fine chem., Ltd. 1-1-dipheny1-2-picrylhydrazy1 (DPPH), was obtained from Sigma

Chemicals. All other chemicals were of laboratory grade.

Instruments

Absorbance was measured in SYNSTRONIC UV-VIS-SPECTROPHOTOMETER – 117.

Centrifugation was done using REMI centrifuge meachine.

METHODS

Interaction with stable free radical DPPH (Blios et al., 1958)

Solutions of various test compounds at 100 M concentration were added to 100 M DPPH

in 95% ethanol and the tubes were kept at an ambient temperature for 20 minutes and the

absorbance was measured at 517 nm.

Assay of Nitric Oxide (NO) Scavenging activity (Marcocci, 1994)

Sodium nitroprusside (10M) in phosphate buffer pH 7.4 was incubated with 100 M

concentrations of test compounds dissolved in a suitable solvent (dioxan/methanol) and tubes

were incubated at 25oC for 120 minutes. Control experiment was kept without test compound

but equal amount of solvent, was added in an identical manner. 2 ml of incubation solution

was removed and diluted with 2 ml of Griess reagent. The absorbance of the chromphore

formed during diazotization of nitrite with sulphanilamide and subsequent coupling with

N-napthylethylene diamine was read at 546 nm (Halliwell B. et al., 1987).

Composition of Griess reagent (Sorba et al., 1997)

Sulphanilamide : 1%

N-Napthylethylene diamine : 0.1%

10% orthophosphoric acid : 2%

Distilled water : 100 ml

Inhibition of iron induced Lipid peroxidation

Preparation of rat brain homogenate

Albino wistar rats (180-250g) of either sex were used for the study. Decapitated and removed

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brain tissue with blood. Tissue was weighed and homogenate (10% w/v) was prepared in 0.15

M KCl and centrifuged at 800 g for 10 minutes (Sharma, 1976). The supernatant was used

immediately for the study. (Shivakumar et al., 1992).

Procedure

The incubation mixture contained in a final volume of 1 ml, brain homogenate (400 μl), KCl (150 mM) and ethanol (10 μl) or test compound dissolved in ethanol.

Lipidperoxidation was initiated by adding, fe3+ (100μM) to give the final concentration stated

(Ciutti M, et al., 1991). After incubating for 20 minutes at 370C, reactions were stopped by

adding 2 ml of ice-cold 0.25 M HCl containing 15% trichloroaceticacid, 0.38%

thiobarbituricacid and 0.05% BHT. Following heating at 80oc for 15 minutes samples were

cooled and centrifuged at 800g for 10 minutes. The absorbance of the supernatant was

measured at 532 nm. Percentage inhibition of TBARS (thio barbituric acid reactive

substances) formed by test compounds was calculated by comparing with vehicle only

experiments (Braughler et al., 1993). Iron solutions were prepared fresh in distilled water and

used immediately. Since most buffers trap hydroxyl radical or interfere with iron conversion

(Duncan et al., 1986), the reactions were carried out in unbuffered 0.15M KCl.

Hydroxyl radical scavenging activity

The scavenging capacity for hydroxyl radical was measured according to the modified

method. The assay was performed by adding 0.1 ml EDTA, 0.01 ml Fecl3, 0.1ml H2O2,

0.36ml Deoxyribose, 1.0ml of test compounds dissolved in methanol (25-125μg/ml), 0.33ml

of phosphate buffer (PH 7.4) and 0.1 ml of ascorbic acid in sequence. The mixture was then

incubated at 370 C for 1 hour. A 1.0 ml of portion of the incubated mixture was mixed with 1

ml of 10% TCA and 1 ml of 0.5%TBA to develop the pink chromogen, which was measured

at 532nm. (Mecrod J.M. et al., 1969).

Reducing power

The reducing power of test compounds was determined by Oyaizu method. Different

concentration of test compounds (25,50, 75,100,125 µM) was mixed with phosphate

buffer(2.5ml, 0.2M, PH 6.6). The mixture was incubated at 500C for 20 min. 2.5ml of 10%

TCA was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The

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solution (0.1%) was added and absorbance was measured at 700 nm. The absorbance of

reaction mixture indicates reducing power (Oyaizu M. et al., 1986).

Superoxide scavenging activity

Superoxide scavenging activity of the compounds was determined by Mcord and Fridorich

method, which depends on the light induced superoxide generation by riboflavin and the

corresponding reduction of nitro blue tetrazolium (NBT). The concentrations assay mixture

contained different test compounds, ethylene diamine tetra acetic acid (6µM), NBT (50µM),

riboflavin (2µM), & phosphate buffer (7.8 PH) to give a total volume of 3ml. The tubes were

uniformly illuminated with an incandescent light (40 volts) for 15 min & measured the

optical density at 560nm. (Elizabeth K. et al., 1996)

ABTS+ radical cation scavenging activity

The ABTS+ radical cation scavenging activity of test compounds, ascorbic acid (as standard)

was determined as mentioned. Briefly, 5.0 ml of 140nm potassium per sulfate over night in

the dark place to yield the ABTS+ radical cation. Prior to use in the assay, the ABTS+ radical

cation diluted with 50% ethanol for an initial absorbance of 0.700 (1:88 ratio) at 734nm. Free

radical scavenging activity was assessed by mixing 1.0 ml diluted ABTS+ radical cation with

10µl of test antioxidant & monitoring the change in absorbance at 0, 0.5, 1min & again at

5min intervals until a steady state was achieved. The antioxidant capacity of tested

compounds was expressed as IC50, the concentration necessary for 50% reduction of ABTS+.

(Re R., et al., 1999).

RESULTS AND DISCUSSION

Chemistry

Due to the great importance of α,β- unsaturated carbonyl moiety in broad range of natural and

synthetically designed products, the development of novel synthetic methods remain

interesting in research area to synthesize chalcones and their derivatives. A vila et al.,

reported that chalcones synthesized by condensation of acetophenone and ring substituted

aldehydes under alkaline conditions, is attractive since it specially generates (E)-isomer,

normally in high yield. These Chalcones are also reported to be a geometrically pure and with

trans configuration (J Hα-Hβ=15.5-16.0) as observed from 1H NMR spectra. (A Vila et al.,

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In the present study, ring substituted chalcones were synthesized by Claisen - Schmidt

condensation using 4-methyl acetophenone and substituted aromatic aldehydes.

The chalcones were synthesized by a base catalyzed claisen- Schmidt reaction between a

substituted benzaldehyde and a substituted acetophenone. Mechanistically, the reaction

involves formation of a carbanion from the acetophenone in the presence of the base NaOH,

followed by nucleophillic attack by the carbanion on the carbonyl carbon of the benzaldehyde

and subsequent loss of water to give the chalcone. Substituted chalcones (1-12) were

prepared by reacting 4-methyl acetophenone with the corresponding substituted aromatic

aldehydes in the presence of methanolic sodium hydroxide at room temperature. Some

substitutions such as 4-methoxy(5), 3,4,5 – trimethoxy[7] and Unsubstituted derivative(1)

took longer reaction time (10-12 hr). Majority of the compounds were obtained in less than

two hours. All the compounds were purified by recrystallization from methanol and ethyl

acetate as solvents.

Other methods for the synthesis of chalcones, are

1. Solvent free synthesis

2. Mortar and pistle synthesis

3. Synthesis of chalcones based on Suzuki reaction.

4. Solvent free synthesis using silica-sulfuric acid as reagent.

Twelve compounds (1-12) were synthesized with the yields generally ranging from 50-72%,

4- methyl, and 4- dimethylamino derivatives were obtained at highest yields (53-72%).

The physical data such as melting points and yields are given in the table 1.

The chalcone derivatives of the present study were characterized by UV,IR, 1H NMR, and

mass spectral analysis. The UV absorption peaks were observed in the region of 250 – 340

nm. The IR spectra of compounds (2,3,4,5,6,7 and 11) displayed bands at 3062.7 – 3022.1

cm-1 due to C-H (Ar-H) stretching, 1670.9 – 1612.9 cm-1 due to C=O stretching, 1602.6 –

1568.1 cm-1 due to C=C stretching.

1

HNMR spectra were taken for compounds (2,3,4,5,6,7, and 11) which also supported the

structures assigned. These compounds displayed a doublet at δ 7.37- 7.12 (d, Hβ) a doublet at

δ 7.05-6.82 (d, Hα) due to hydrogens of the α,β- unsaturated carbonyl group and multiplets in

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2.36- 2.43due to methyl substitution on the ring. Compound 4 displayed doublet at 2.95-3.05

due to dimethyl amino substitution. Compound 5 displayed doublet at 3.80-3.90due to

methoxy substitution. The structure of the compounds was also assigned by mass spectral

analysis which showed(M++1) peaks of the compounds. The mass spectra of the compound 7

showed a characteristic molecular ion peaks (M++1) at 313.24 and the compound 11 showed

a characteristic molecular ion peaks (M++1) at 347.05.

PHARMACOLOGICAL STUDIES

Analgesic activity (Acetic acid induced writhing test)

Naturally occurring and synthetic chalcones have shown promising biological activiy and

safety profile and have shown potential for use as lead compounds for the discovery of

analgesic and anti-oxidant agents(Ni, L et al., 2004). A number of chalcones and their

derivatives have also been found to inhibit the synthesis of nitric oxide (NO) and

prostaglandins (PG), which are products of nitric oxide synthase (NOS) and cyclooxygenase

(COX) pathways, respectively (Ahmad, S et al., 2006).

Correa. R et al reported that some synthetic chalcones, or those derivatives derived from the

abundant natural product 2-hydroxy-4,6-dimethoxy acetophenone, exhibit pronounced

antinociceptive effect in the mice writhing test( correa, R et al., 2001).

Anti-nociceptive activity was expressed as the reduction in number of abdominal

constrictions between the control animals and the mice treated with the test compounds.

Compous-buzzi et all reported the, synthesis of a series of Acetamidochalcones and evaluated

for anti-nociceptive activity using mice writhing test. All compounds showed good activity

when compared to standard drugs, acetylsalicylic acid and acetaminophen.

Lucky O. et al reported the synthesis of chalcones and evaluated for anti-inflammatory

activity. All compounds showed good to moderate activity when compared to standard drugs.

The electron withdrawing groups, 4-chloro substitution displayed relatively low

anti-inflammatory activity. Present studies, the 4-chloro derivative showed good analgesic

activity.

The unsubstituted aromatic ring containing chalcones were proved have less analgesic

activity. The substitution of aromatic ring at 4th position by methyl group exhibited potent

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induced writhing in mice, at 100mg/kg body weight and data is given in table 2.The

4-dimethyl amino (4), and 3,4,5-trimethoxy(7) derivatives showed highest inhibition (73-71%)

on acetic acid induced writhings in mice. The unsubstituted derivative showed 52%

inhibition.4-methyl derivative (3) showed the potent activity (70%) when compared to

standard drug aspirin (69%) thus indicating the importance of electron releasing substituent’s.

The electron withdrawing groups such as 4-chloro derivative (2) showed good activity (69%).

and 5-bromo vanillinyl derivative (11) also showed good activity (69%). The other phenolic

derivatives like 2-hydroxy (8), 4-hydroxy-3-methoxy (9), 4-hydroxy-3,5-dimethoxy

derivatives (10) showed good activity (54-64%). The other derivatives showed moderate

activity.

IN VITRO ANDIOXIDANT STUDIES

Reduction of DPPH by test compounds

Interest in the involvement of reactive oxygen species (ROS) in various disorders has been

increasing. In particular, they were thought to play an important role in the development of

inflammatory disorders. A radical scavenging antioxidant reacts rapidly with

1,1-diphenyl-2-picrylhydrazyl stable free radical(DPPH). Hence the ability of the test compounds to

scavenge the stable radical has been determined (Halliwell et al., 1990).

Antioxidant reacts with DPPH and convert it to 1,1- diphenyl-2-picrylhydrazine. The change

in the absorbance produced in the reaction measures the antioxidant properties.

Reactive oxygen species are thought to play an important role in the development of

inflammatory diseases (Rajkumar et al., 1995). The nitrogen centered stable free radical 1,1-

diphenyl-2-picrylhydrazyl(DPPH) has often been used to characterized phenolic antioxidants.

It is reversibly reduced, and due to its unpaired electrons, densely coloured. This property

makes it suitable for spectrophotometric studies. A scavenging antioxidant reacts with DPPH

stable free radical and convert it to 1,1- diphenyl-2-picrylhydrazine. The change in

absorbance produced in this reaction has been used to measure antioxidant properties.

The odd electron in the DPPH free radical showed a strong absorption maximum at 517 nm

and purple in colour. when the odd electron of DPPH radical becomes paired with a hydrogen

from a free radical scavenging antioxidant to from the reduced DPPH. The decolourisation is

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The compounds were (1-12) screened for reduction of DPPH at 25, 50, 75,100 and 125µM

concentration. The data is given in table 3. At 125 µM concentration some of the compounds

like 3,4,5- trimethoxy, 2- OH, 4- dimethyl amino, and 4- OH, 3- OCH3 showed highest

activity(84 - 92%) 4-methoxy(5), 4-OH-3,5-dimethoxy showed moderate activity (64-65%).

It is interesting to note that, the 3,4,5- trimethoxy(7), 4-hydroxy – 3- methoxy vanillinyl(9)

and 4-dimethylamino derivatives showed significant activities at lower concentrations and

showed IC50 values of 60µM, 60 µM, and 75 µM respectively as compared to standard.

Scavenging of Nitric oxide free radical

Nitric oxide (NO) is an important modulator of physiological and pathological function in the

cardiovascular, neuronal and immune systems and it is involved as an intracellular signal and

defensive cytotoxin in the nervous, muscular, cardiovascular and immune systems.

Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric

oxide (Marcocci et al., 1994). This nitric oxide reacts with oxygen to produce nitrite ions

which can be estimated using griess reagent (Gutteridge et al., 1987). Scavengers of nitric

oxide (NO), compete with oxygen leading to reduced production of nitric oxide.

All the compounds were tested for their ability to scavenge nitric oxide at 25, 50, 75, 100,

125 µM concentrations. The data is given in table 5.

Many compounds, such as 5- iodo vanillinyl and bromo vanillinyl, 4- dimethylamino(4),

derivaties showed (82 - 86%) potent inhibitory activity on nitric oxide scavenging at 125 μM

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Among the tested compounds 4-methyl(3), 2-hydroxy(8), 3,4-dimethoxy(6), 4-

hydroxy-3,5-dimethoxy (10) derivatives (78-71%) showed good activity and the other compounds the un

substituted (1), 3,4,5 – trimethoxy (7), 4-Hydroxy-3-methoxy(9), 4-chloro(2), showed

moderate activity.(43-65%). It is interesting to note that substitution by phenolic hydroxyl

groups and other electron releasing substituents on the 41-methyl chalcones resulted in

pronounced inhibition of nitric oxide. When both he rings of chalcone are substituted by 4-

methyl groups, the compound exhibited lowest IC50 value.

IC50 values of the title compounds were calculated and 4-dimethylamino(4), and 5-bromo

vanillinyl and 5-iodo vanillinyl (11,12) derivatives were found to be more active with 65 µM,

77 µM, and 85 µM respectively. 4-methyl(3), and 4-methoxy(5) derivatives showed IC50

values of 85 µM and 95 µM.

Effect of compounds (1-12) on iron induced lipid peroxidation in rat brain homogenate

Ferric stimulates lipid peroxidation through various mechanisms, as the generation of

hydroxyl radical (Richmond et al., 1979), decomposition of lipid peroxides (Chase et al.,

1987), forming perferryl (or) ferryl species (Koppel et al., 1984) and free radical reaction

mechanisms. The role of lipid peroxidation in various diseases such as cancer, inflammatory

diseases etc was clearly established (Rajkumar et al., 1993). Compounds that inhibit lipid

peroxidation by interfering with the chain reaction of peroxidation and by scavenging

reactive free radical mediated tissue damage could be of great therapeutic importance.

All compounds were tested for their inhibitory effect on ferric induce lipid peroxidation at 25

µM, 50 µM, 75 µM, 100 µM, 125 µM concentration and data is given in table 6.

Compounds like 4- methoxy (5), and 4- dimethyl amino (4) showed IC50 values at 90 µM,

and 110 µM, respectively. Some of the compounds 4-hydroxy-3,5- dimethoxy (10) derivative

showed similar activity (110 µM) as compared to standard.

Scavenging of hydroxyl radical

Hydroxyl radical were generated by using the ferric ions (Fe3+)/ ascorbic acid reaction

system. The detection of hydroxyl radicals was carried out by measuring the amount of

formaldehyde generated from the oxidation of dimethyl sulfoxide. the reaction mixture

contained 0.1 mM EDTA, 167 µM Fe3+, 0,33mM DMSO in phosphate buffer of 50mM

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added finally to initiate the reaction. Trichloro acetic acid (17%, w/v) was added to terminate

the reaction. The contents were observed spectrophotomatically at 412nm for the detection of

formaldehyde. Ascorbic acid was used as reference compound for comparative study.

The compounds were tested for their ability of hydroxyl radical scavenging activity at

25,50,75,100 and 125µM concentration. Data is given in table 7.

The phenolic derivative, 4-hydroxy- 3,5-dimethoxy analog (10) showed highest

activity(91%), at 125μM concentration among all substituted derivatives. 4-methoxy(5), 4-

dimethylamino(4) and 4-hydroxy-3-methoxy(9) showed good activity(78-72%).

3,4-dimethoxy and 3,4,5-trimethoxy(6,7) also showed good activity.(69-69%) which suggest that

the importance of substitution methoxy group.

The substitution with electron donating groups such as 4-methyl (3), and showed good

activity (65%).

The substitution by electron withdrawing groups such as 4-chloro(2) derivatives exhibited

moderate activity (59%). 5-bromo vanillinyl and 5-iodo vanillinyl derivatives also showed

moderate activity.

It is interesting to note that, among these compounds, the 4-Hydroxy-3,5-dimethoxy(10),

vanillinyl (3), 4-dimethylamino (4), 4-methoxy (5) derivatives showed significant activities at

lower concentrations and showed IC50 values of 60µM, 80µM, 95µM and 100µM

respectively.

Assay for Reducing power

Hydroxyl radicals were generated by using the ferric ions (Fe3+)/ ascorbic acid reaction

system. The detection of hydroxyl radicals was carried out by measuring the amount of

formaldehyde generated from the oxidation of dimethyl sulfoxide. the reaction mixture

contained 0.1 mM EDTA, 167 µM Fe3+, 0,33mM DMSO in phosphate buffer of 50mM

(pH7.4) and 0.1ml test compounds (1mM) solution. Ascorbic acid (150µl. 10mm in phosphate

buffer) was added finally to initiate the reaction. Trichloro acetic acid (17%, w/v) was added

to terminate the reaction. The contents were observed spectrophotomatically at 412nm for the

detection of formaldehyde. Ascorbic acid was used as reference compound for comparative

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All the compounds were tested for their ability of Reducing power activity at 25.50,75,100,

and125µM concentration. Data is given in table 8.

Among these compounds 5-bromo vannillinyl analog (11) showed highest activity(85%). The

phenolic compounds like 2-hydroxy(8), and 5-iodo vanillinyl (12) derivatives showed good

activity.(73%-72%). and 4-hydroxy-3,5-dimethoxy(10) derivatives showed significant

activity (70%).

The 4-dimethylamino(4), and 4-methyl(3), derivatives showed significant activity

(70%,70%), other substituted derivatives, such as 4-methoxy (5), 3,4-dimethoxy(6), 3,4,5-

trimethoxy(7) also showed good inhibitory activities ( 62%, 65%,56%) and the unsubstituted

derivative(1) showed moderate activity(48%) at 125µM concentration.

IC50 values of the title compounds were determined and 5-bromo vanillinyl(11), 4-OH,

3,5-dimethoxy (10), 4-methyl (3) and 4- dimethylamino(4) derivatives showed IC50 values of 75

µM, 80 µM, 85µM and 90 µM respectively, while the standard drug, Ascorbic acid exhibited

Scavenging of Superoxide radical

Super oxide scavenging activity of the test compounds was determined by Mcord &

Fridovich method, which depends on light induced superoxide generation by riboflavin and

the corresponding nitro blue tetrazolium (NBT). It was hypothesized that drugs might

scavenge free radicals by donating the allylic hydrogen of the unsaturated lactone ring. It was

found that the stoichiometry of the reaction between test compounds and superoxide radical

generated from KO2 in DMSO was 2 to 1.

The compounds were tested for their ability of superoxide radical scavenging activity at 25,

50,75, 100 and 125µM concentration. Data is given in table 9.

The 4-hydroxy-3,5-dimethoxy derivative(10) showed appreciable activity(82%). The

substitution by electron with drawing group such as 4-chloro(2) exhibited good activity.

(63%).

The phenolic compound, 2-hydroxy (8), showed significant activity (62%) at 125μM. The

activity of this derivative might be due to the presence of phenolic group which has ability to

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The substitution by an electron donating groups such as 4-methyl(3), 4-methoxy(5) and 4-

dimethyl amino derivatives showed moderate activities (45%, 54%, and 59%).

The 5-bromo(14) and 5-iodo(15) vanillinyl derivatives showed moderate activity(53% and

48%). 4- hydroxy-3,5-dimethoxy(10), 2-hydroxy(8), 3,4,5-trimethoxy(7) and 4- dimethyl

amino (4) showed IC50 values of 80µM, 85µM, 85µM, and 105 µM respectively. Where as

the standard, α- tocopherol showed IC50 value of 95 µM.

CONCLUSION

Twelve analogs of 4-methyl chalcones were synthesized and characterized by IR, 1HNMR,

and Mass spectral analysis. Substituted aromatic aldehydes were condensed with 4-methyl

acetophenone to yield the title products. All the synthesized compounds were evaluated for

their analgesic activity by using acetic-acid induced writhing method. Among the compounds

evaluated 4-dimethyl amino (73%), 3,4,trimethoxy benzaldehyde (71%) and

5-bromovanillinyl derivatives (69%) showed highest activity. All the compounds were tested

for the antioxidant activities like reduction of DPPH stable free radical, nitric oxide

scavenging activity, inhibition of lipid peroxidation, hydroxyl radical, superoxide ABTS+

scavenging activity and reducing power method. 4-dimethyl amino, 2-hydroxy, 4-hydroxy-3,

5- dimethoxy, 5-bromovanillinyl and 5-iodo vanillinyl derivatives showed good antioxidant

activity in all in vitro models.

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