Indian Journal of Chemistry Vol. 50B, May 2011, pp. 729-733

Note

Synthesis and evaluation of N-(α-benzamido

cinnamoyl) aryl hydrazone derivatives for

anti-inflammatory and antioxidant activities

G Rajitha*, N Saideepa & P Praneetha

Institute of Pharmaceutical Technology, Sri Padmawati Mahila Viswavidyalayam, Tirupati 517 502, India

E-mail: rajitha.galla@yahoo.com

Received 30 November 2009; accepted (revised) 22 March 2011

A series of novel N-(α-benzamido cinnamoyl) aryl hydrazone

derivatives 3a-m have been synthesized and screened for their anti-inflammatory and antioxidant activities. These compounds have been synthesized by refluxing intermediate α-benzamido-cinnamhydrazide with substituted benzaldehydes in the presence

of a few drops of glacial acetic acid. The intermediate α-

benzamido-cinnamhydrazide is prepared by stirring 4-benzyli-dene-2-phenyl oxazole-5-one with hydrazine hydrate in presence of absolute ethanol. The chemical structures of the compounds are

established by IR, 1H NMR and mass spectral data. Of all the compounds screened, compound 3m N'-(5-bromo-4-hydroxy-3-methoxybenzylidene)-2-(benzamido)-3-phenylacrylohydrazide shows anti-inflammatory activity (75%) which is equivalent to that produced by the same dose (100 mg/kg) of phenylbutazone (74%). Compound 3l N'-(4-hydroxy-3-methoxy benzylidene)-2-(benzamido)-3-phenyl acrylohydrazide, shows good anti-inflammatory and antioxidant activity.

Keywords: Acylhydrazone, hydrazone, anti-inflammatory,

antioxidant activity

Hydrazone derivatives are reported to possess

various pharmacological activities such as anti-tumoral

1, anti-mycobacterial

2, anti-microbial

3, anti-

malarial4 and anti-convulsant activity

5. Hydrazone

and acylhydrazone moieties are also the most important pharmacophoric cores of several anti-

inflammatory, antinociceptive and antiplatelet

activities6-8

. Curcumin and dehydrozingerone are

reported to be potent scavengers of oxygen free radicals and also possess good anti-inflammatory

activity9. Both are styryl ketones with similar

substituents on the phenyl ring. In view of these observations it was considered of interest to

synthesize a new class of acylhydrazones in order to

study the possible pharmacophoric contribution of

both the functionalized acylhydrazone units and the styryl carbonyl moiety.

Results and Discussion

4-Benzylidene-2-phenyloxazol-5-one was prepared

by condensing benzaldehyde with benzoyl glycine in presence of acetic anhydride and anhydrous sodium

acetate. The oxazolone was then treated with 99%

hydrazine hydrate in ethanol to give intermediate hydrazide. The intermediate hydrazide upon

nucleophilic addition with substituted benzaldehydes

at 60°C in ethanol yielded the title compounds 3a-m

(Scheme I). The structures of synthesized compounds were confirmed on the basis of

1H NMR, IR and mass

spectral data. All these compounds were screened for

anti-inflammatory and antioxidant activities.

Anti-inflammatory Activity

Anti-inflammatory activity of the title compounds

was carried out by the carrageenan induced edema method of Winter et al.

10 (Table I). 5-Bromo

vanillinyl derivative 3m was found to be equipotent to

standard drug phenylbutazone. Substitution by 4-

hydroxy 3k and vanillinyl 3l derivatives showed moderate activity. Substitution by electron-donating

groups such as 4-methyl 3d and 4-dimethylamino 3f

showed significant activity. Similar results were obtained with paradimethylamino derivative of

acylhydrazone framework11

. Substitution by isopropyl

group 3e resulted in an increase in activity (67%).

Lipid Peroxidation

All the compounds were tested for inhibition of

lipid peroxidation induced by ferric ions in rat brain

homogenate according to the method of Sreejayan and M. N. A. Rao

12 (Table II). Compounds containing

phenolic hydroxyl group were found to be active.

4-Hydroxy derivative 3k showed good activity (52%). Vanillinyl 3l and 5-bromo vanillinyl 3m derivatives

were found to be more active than the standard drug

α-tocopherol. The good activity of these compounds might be due to inductive effect of the methoxy group

ortho to the phenolic group. Substitution by electron withdrawing group, 4-chloro 3b, resulted in slight

decrease in activity whereas substitution by electron

donating groups at the para position such as isopropyl 3e, 4-dimethylamino 3f and 4-methyl 3d resulted in

an increase in activity. 3,4,5-Trimethoxy 3j and 3,4-

INDIAN J. CHEM., SEC B, MAY 2011

730

dimethoxy 3i derivatives showed good activity. 4-

methoxy 3h and 4-acetamido 3g derivatives showed significant activity.

Nitric Oxide Scavenging Activity

The compounds 3a-m were evaluated for anti-oxidant activity against sodium nitroprusside induced

nitric oxide (NO) production, measured by Griess

reagent12

(Table II). Among the compounds in this

series, compounds 3a, 3f and 3g showed significant activity. The vanillinyl derivative 3l showed good

activity (41%).

Reduction of Stable free radical DPPH

All these compounds were screened for stable free radical DPPH scavenging activity by the reported

procedure13

. Table II shows the reactivity of title

compounds with DPPH at 100 µM concentration. Among phenolic compounds vanillinyl derivative 3l

showed highest activity (77%). 5-Bromo vanillinyl derivative 3m showed activity which is comparable to

the activity of α-tocopherol. These results agree with

earlier work indicating that the antioxidant efficiency

of monophenols is strongly enhanced by the introduction of one or two methoxy substitutions in

positions ortho to the phenolic group14

. 4-Dimethyl

CH2COOH

NH2+ COCl C

NHCH2COOH

O

(CH3CO)2O/

CH3COONa

CHO

CH

N O

O

NH2NH2

Ethanol absolute

CH C

NH C

O

C NHNH2

21

CHOR

Glacial acetic acid / ethanol

CH C

NH C

O

C

O

3a-m

NHN CH

R

O

Scheme I

Scheme I

Table I — Anti-inflammatory activity of compounds 3a-m at 100 µM

Compd R Edema

Volume mL (± SD)d

Edema Inhibition

(%)c

3a H 0.43 (0.07)b 36

3b 4-Cl 0.36 (0.01)a 41*

3c 4-F 0.35 (0.02)b 17

3d 4-CH3 0.41 (0.01)b 39*

3e 4-CH(CH3)2 0.22(0.14)b 67*

3f 4-N(CH3)2 0.30(0.02)b 44*

3g 4-NHCOCH3 0.45(0.04)b 33

3h 4-OCH3 0.45(0.04)b 33

3i 3,4-(OCH3)2 0.43(0.07)a 36

3j 3,4,5-(OCH3)3 0.35(0.02)b 17

3k 4-OH 0.27(0.08)a 55*

3l 4-OH, 3-OCH3 0.27(0.09)a 55*

3m 5-Br, 4-OH, 3-OCH3

0.15(0.05)a 75*

Phenylbutazone 0.16(0.05)a 74*

a. Control edema volume = 0.61 (0.06). b. Control edema volume = 0.68 (0.05). c. At 100 mg/kg (p.o) percent edema inhibition calculated by comparing edema volume with that of respective vehicle

treated control animals. d. Edema volume was measured 3 hr after carrageenan injection and expressed as mean ± standard deviation. * Statistically significant (P<0.05 Mann – Whitney)

NOTES

731

amino derivative 3f (35%) showed significant activity. Other compounds were found to be inactive.

Experimental Section

All the melting points reported in this series were determined in open capillaries using Thermonik

Precision Melting Point cum Boiling Point Apparatus

Model C-PMB-2 and are uncorrected. Homogenity of

the compounds was checked by using precoated TLC plates (E. Merck Kieselgel 60 F254). The IR spectra

were recorded using KBr Pellets on a Perkin-Elmer

1760 spectrophotometer (cm-1

). 1H NMR spectra were

recorded on GE Omega 400 MHz spectrometer or

Bruker Avance 300 MHz spectrometer, using

tetramethylsilane (TMS) as an internal standard. Mass

spectra were recorded on a Jeol-JMS-D-300 spectrometer. All solvents were procured from

Aldrich and Sigma and used without further

purification.

Synthesis of αααα-benzamido-cinnamhydrazide, 2

4-Benzylidene-2-phenyloxazol-5-one 1 (0.03 mol)

was stirred with a solution of hydrazine hydrate (0.06

mol) in ethanol (25 mL) for 30 min. The deep yellow

colour of the oxazolone immediately changed to a light yellow coloured solid, which was filtered,

washed and purified by recrystallization from

methanol.

αααα-Benzamido-cinnamhydrazide, 2: Yield: 84%.

m.p. 165°C; IR (KBr): 3289 (NHNH2), 1644 (C=O),

1631 cm-1

(C=C); 1H NMR (DMSO-d6): δ 4.3 (br s,

2H, NH2), 7.0-8.0 (m, 16H, Ar-H and olefinic), 9.7 (s,

1H, -CONH-N), 9.9 (s, 1H, =C-NHCO).

General method of synthesis of compounds 3a-m

Synthesis of N'-(substituted benzylidene)-2-

(benzamido)-3-phenylacrylo hydrazide, 3a: Equi-

molar amounts of α-benzamido-cinnamhydrazide (0.01 mol) and benzaldehyde (0.01 mol) in absolute

ethanol with a few drops of acetic acid were heated on a water bath at 60°C for 1 hr

15. The mixture was

allowed to cool to RT, filtered, and purified by

recrystallization from methanol. The physical and

analytical data of the synthesized title compounds are given as follows.

2-(Benzamido)-N'-benzylidene-3-phenylacrylohy-

drazide, 3a: Mol. formula C23H19N3O2 Yield: 74%. m.p. 228-31°C; IR (KBr): 3278 (N-H), 3027 (ArC-H),

1647 (C=O), 1601 cm-1

(C=C); 1H NMR (DMSO-d6):

δ 7.3 (s, 1H, PhCH=C), 7.1-8.0 (m, 15H, Ar-H), 8.4 (s, 1H, -N=CH-Ph), 10.1 (s, 1H, =C-NH-CO), 11.7 (s,

1H, -CO-NH-N); MS (EI): m/z 370 (M+±1).

N'-(4-Chlorobenzylidene)-2-(benzamido)-3-phe-

nylacrylohydrazide, 3b: Mol. formula C23H18N3O2Cl Yield: 75%. m.p. 186-89ºC I;R (KBr): 3218 (N-H),

3026 (ArC-H), 1644 (C=O), 1600 cm-1

(C=C); 1H

NMR (DMSO-d6): δ 7.1 (s, 1H, C6H5-CH=), 7.2-8.1 (m, 14H, Ar-H), 8.1 (s, 1H, Ar-CH=N), 9.7(s, 1H,

NHCO), 11.4 (s, 1H, CONHN=).

N'-(4-Flourobenzylidene)-2-(benzamido)-3-phe-

nylacrylohydrazide, 3c: Mol. formula C23H38N3O2F.

Table II — Antioxidant activities of compounds 3a-m at 100 µM

Compd R % inhibition of lipid

peroxidation % scavenging of

nitric oxide % reduction of

DPPH

3a H 32 36 NA

3b 4-Cl 41 28 NA

3c 4-F 60 23 20

3d 4-CH3 70 29 14

3e 4-CH(CH3)2 74 27 30

3f 4-N(CH3)2 75 32 35

3g 4-NHCOCH3 40 33 31

3h 4-OCH3 44 25 28

3i 3,4-(OCH3)2 65 29 12

3j 3,4,5-(OCH3)3 66 28 NA

3k 4-OH 52 27 15

3l 4-OH, 3-OCH3 77 41 77

3m 5-Br, 4-OH, 3-OCH3 73 22 56

α-tocopherol 65 51

INDIAN J. CHEM., SEC B, MAY 2011

732

Yield: 64%. m.p. 225-30ºC; IR (KBr): 3285 (N-H),

2829 (Ar-H), 1635 cm-1

(C=O); 1

H NMR (DMSO-d6):

δ 7.3 (s, 1H, Ph-CH=C), 7.1-8.0 (m, 14H, Ar-H), 8.4 (s, 1H, -N=CH-Ph), 10.1(s, 1H, =C-NH-CO), 11.7 (s,

1H, -CO-NH-N=).

N'-(4-Methylbenzylidene)-2-(benzamido)-3-phe-

nylacrylohydrazide, 3d: Mol. formula C23H38N3O2, Yield: 68%. m.p. 215-17ºC; IR (KBr): 3277 (N-H),

3027 (ArC-H), 1641 (C=O), 1601 cm-1

(C=C); 1H

NMR (DMSO-d6): δ 2.3 (s, 3H, CH3), 7-8 (m, 15H, Ar-H and olefinic), 8.3 (s, 1H, -N=CH-Ph), 10.1 (s,

1H, =C-NH-CO), 11.6 (s, 1H, -CO-NH-N).

N'-(4-Isopropylbenzylidene)-2-(benzamido)-3-phe-

nylacrylohydrazide, 3e: Mol. formula C26H24N2O2, Yield: 74%. m.p. 191-93ºC; IR (KBr): 3218 (N-H),

3026 (ArC-H), 1644 (C=O), 1600 cm-1

(C=C); 1H

NMR (DMSO-d6): δ 1.2 (d, 6H, CH(CH3)2), 2.9 (m, 1H, CH(CH3)2),7.1 (s, H, PhCH=C), 7.3-8.0 (m, 14H,

Ar-H), 8.3 (s, 1H, -N=CH-Ph), 10.1 (s, 1H, =C-

NHCO), 11.6 (s, 1H, CONH-N); MS: m/z 411 (M+).

N'-(4-Dimethylaminobenzylidene)-2-(benzami-

do)-3-phenylacrylohydrazide, 3f: Mol. formula

C25H24N4O2, Yield: 70%. m.p. 204-07ºC; IR (KBr):

3266 (N-H), 3081 (ArC-H), 1652 (C=O), 1597 cm-1

(C=C); 1H NMR (DMSO-d6): δ 2.9 [s, 6H, (CH3)2,N],

6.7 (d, 2H, Ar-H), 7.1 (s, 1H, PhCH=C), 7.3-7.7 (m,

10H, Ar-H), 8.0 (d, 2H, Ar-H), 8.3 (s, 1H, -N=CH-Ph), 10.1 (s, 1H, =C-NHCO), 11.4 (s, 1H, -

CONH-N).

N'-(4-Acetamidobenzylidene)-2-(benzamido)-3-phenylacrylohydrazide, 3g: Mol. formula C25H22N4O3,

Yield: 81%. m.p. 283-85ºC; IR (KBr): 3324 (N-H),

3080 (Ar-H), 1664 (C=O), 1605 cm-1

(C=C).

N'-(4-Methoxybenzylidene)-2-(benzamido)-3-phe-nylacrylohydrazide, 3h: Mol. formula C24H21N3O3,

Yield: 70%. m.p. 137-41ºC; IR (KBr): 3217 (N-H),

3022 (ArC-H), 1644 (C=O), 1603 cm-1

(C=C); 1H

NMR (DMSO-d6): δ 3.8 (s, 3H, OCH3), 7-8 (m, 15H,

Ar-H and olefinic), 8.3 (s, 1H, -N=CH-Ph),

10.1(s,1H,=C-NH-CO), 11.6 (s,1H,-CO-NH-N).

N'-(3,4-Dimethoxybenzylidene)-2-(benzamido)-3-phenylacrylohydrazide, 3i: Mol. formula C25H24N3O4.

Yield: 76%. m.p. 155-56ºC; IR (KBr): 3425 (N-H),

1638 (C=O), 1596 (C=C), 1269 cm-1

(C-O-C); 1

H NMR (CDCl3): δ 3.8 (s, 3H, OCH3), 3.9 (s, 1H,

OCH3), 6.5-8.1 (m, 14H, Ar-H and olefinic), 8.1(s,

1H, Ar-CH=N), 9.6(s, 1H, NHCO); 10.9(s, 1H, CONH-N=).

N'-(3,4,5-Trimethoxybenzylidene)-2-(benzami-

do)-3-phenylacrylohydrazide, 3j: Mol. formula

C26H25N3O5, Yield: 80%. m.p. 220-24ºC; IR (KBr):

3206 (N-H), 3052 (ArC-H), 1643 (C=O), 1627 cm-1

(C=C); 1H NMR (DMSO-d6): δ 3.8 (s, 9H, OCH3), 7-

8 (m, 13H, Ar-H and olefinic), 8.3 (s, 1H, -N=CH-Ph), 10.1 (s, 1H, =C-NH-CO), 11.7 (s, 1H, -CO-NH-

N).

N'-(4-Hydroxybenzylidene)-2-(benzamido)-3-phe-

nylacrylohydrazide, 3k: Mol. formula C23H19N3O3. Yield: 80%. m.p. 272-74ºC; IR (KBr): 3284 (N-H),

2990 (ArC-H), 1646 (C=O), 1631 cm-1

(C=C); 1

H

NMR (DMSO-d6): δ 5.9 (s, 1H, Ar-OH), 6.8 (d, 2H, Ar-H), 7.0 (s, 1H, C6H5-CH=), 7.1-8.0 (m, 10H, Ar-

H), 7.6 (d, 2H, Ar-H), 8.5 (s, H, Ar-CH=N), 9.6

(s,1H, NHCO), 10.9 (s, 1H, CONH-N).

N'-(4-Hydroxy-3-methoxybenzylidene)-2-(benzami-do)-3-phenylacrylohydrazide, 3l: Mol. formula

C24H21N3O4. Yield: 61%. m.p. 242-45ºC; IR (KBr):

3292 (N-H), 3005 (ArC-H), 1644 (C=O), 1606 cm-1

(C=C); 1H NMR (DMSO-d6): δ 3.8 (s, 3H, OCH3),

6.8-8.0 (m, 14H, ArH and olefinic), 8.3 (s, 1H, -

N=CHPh), 9.5 (s, 1H, OH), 10.1 (s, 1H, =CNHCO), 11.7 (s, 1H, -CONH-N).

N'-(5-Bromo-4-hydroxy-3-methoxybenzylidene)-2-

(benzamido)-3-phenylacrylohydrazide, 3m: Mol.

formula C24H21N3O4Br, Yield: 72%. m.p. 249-51ºC; IR (KBr): 3267(N-H), 1639 (C=O), 1593 cm

-1 (C=C);

1H NMR (DMSO-d6 + CDCl3): δ 3.9 (s, 3H, OCH3),

6.8 (s, 1H, C6H5-CH=), 7.0-7.6 (m, 12H,Ar-H), 9.4 (s, 1H, NHCO), 11.6 (s, 1H, -CONH-N).

Acknowledgement

The authors are thankful to IICT, Hyderabad for providing

1H NMR spectral analysis of the

compounds and Department of Chemistry, SVU,

Tirupathi for providing IR spectral analysis of the

compounds.

References

1 Abadi A H, Eissa A A H & Hassan G S, Chem Pharm Bul, 51, 2005, 838.

2 Dharmarajan S, Perumal Y & Kasinathan M, Bioorg Med

Chem, 15, 2005, 4502. 3 Savini L, Chiasserini L, Travagli V, Pellerano C, Novellino E,

Cosentino S & Pisano M B, Eur J Med Chem, 39, 2004, 113. 4 Walcourt A, Loyevsky M, Lovejoy D B, Gordeuk V R &

Richardson D R, Int J Biochem Cell Biol, 36, 2004, 401. 5 Ragavendran J, Sriram D, Patel S, Reddy I, Bharathwajan N,

Stables J & Yogeeswari P, Eur J Med Chem, 42, 2007, 146. 6 Silva G A, Luciana M M, Costa, Fernanda C F, Brito, Ana L

P, Miranda, Eliezer J, Barreiro, Carlos A M & Fraga, Bioorg

Med Chem, 12, 2004, 3149.

7 Almasirad M, Bakhtiari D, Shafiee A, Abdollahi M, Zamani M J, Khorasani R & Esmaily, J Pharm Pharmaceut Sci, 8(3), 2005, 419.

NOTES

733

8 Anna C, Cunha M, Figueiredo L M, Tributino L P, Miranda C, Castro B, Carlos A M, Fraga Cecilia B V,

dSouza F, Ferreira J & Barreiro, Bio Org Med Chem, 11, 2003, 2051.

9 Sreejayan & Rao M N A, J Pharm Pharmcol, 46, 1994, 1013.

10 Winter C A, Risley F A & Nus G W, Proc Soc Exp Biol Med, 111, 1962, 544.

11 Lima P C, Lima L M, Silva K C M, Leda P H O, Miranda A L

P, Fraga C A M & Barreior E J, Eur J Med Chem, 35, 2004, 113.

12 Sreejayan & Rao M N A, J Pharm Pharmacology, 49, 1997, 105.

13 Blios M S, Nature, 181, 1958, 1199. 14 Natella F, Nadini M, Di Felice M & Scaccini C, J Agric Food

Chem, 47, 1999, 1453. 15 Nalepa K, Zednikova G, Marek J & Travnicek Z, Monatsh

Chem, 130, 1999, 471.