Design, synthesis and biological evaluation of some novel benzylidene-2-(4-phenylthiazol-2-yl)...

ORIGINAL RESEARCH

Design, synthesis and biological evaluation of some novelbenzylidene-2-(4-phenylthiazol-2-yl) hydrazines as potentialanti-inflammatory agents

Sanjay Kumar Bharti • Sushil Kumar Singh

Received: 11 April 2013 / Accepted: 9 August 2013 / Published online: 21 August 2013

� Springer Science+Business Media New York 2013

Abstract A series of substituted benzylidene-2-(4-phen-

ylthiazol-2-yl) hydrazines (2a–q) have been synthesized,

characterized and evaluated for their anti-inflammatory

activity by carrageenin-induced hind paw edema (acute

inflammation) and cotton pellet granuloma (chronic

inflammation) methods in rats. In carrageenin-induced hind

paw edema method, compounds 2a, 2b, 2c, 2d, 2h, 2k and

2p at a dose of 20 mg kg-1 body weight, p.o. showed

excellent inhibitions (51.80–86.74 %) in between 1 and

4 h. Similarly, in cotton pellet granuloma method, com-

pounds 2a, 2b, 2c, 2d, 2h, 2k and 2p at a dose of

20 mg kg-1 body weight, p.o. inhibited the granuloma

formation (71.71–90.19 % inhibition) which was compa-

rable to that of standard drug, ibuprofen (90.36 % inhibi-

tion of paw volume at 3 h and 94.02 % inhibition of

granuloma formation). Structure activity relationship

studies showed excellent activity of the compounds con-

taining electron withdrawing group (fluoro, chloro, bromo

or nitro) in phenyl ring at C2 and/or C4 position of thiazole

ring.

Keywords Anti-inflammatory activity �2,4-disubstituted thiazoles � Pharmacophores �Phenacyl bromide � Structure activity relationship (SAR) �Thiosemicarbazone

Introduction

The thiazole ring system in numerous biologically active

molecules has been recognized for various pharmacologi-

cal activities. Thiazole bearing compounds are known to

possess activities viz. antibacterial, antifungal (Kaspady

et al., 2009; Wilson et al., 2001), anti-inflammatory (de

Menezes and Catanzaro-Guimaraes, 1985; Lednicer et al.,

1990), antihypertensive (Patt et al., 1992), anti-HIV (Bell

et al., 1995), antitumor, antifilarial (Gu et al., 1999; Jiang

and Gu, 2000; Kumar et al., 1993a, b), anticonvulsant

(Siddiqui and Ahsan, 2010), anti-ulcer (Muri et al., 2004),

herbicidal, insecticidal, schistosomicidal and anthelmintic

(Metzger, 1984). Bleomycin and tiazofurin (antineoplastic

agents), ritonavir (anti-HIV drug), fanetizole, meloxicam

and fentiazac (anti-inflammatory agents) (Fig. 1), ravuco-

nazole (antifungal agent), nizatidine (antiulcer agent),

imidacloprid (insecticide), penicillin (antibiotic), vitamin

B1 and its coenzyme are some of the examples of thiazole

bearing drugs. Thiazole derivatives are also reported in the

synthesis of sulphathiazole (Borisenko et al., 2006), as

ligand of estrogen receptors (Fink et al., 1999), antagonists

for adenosine receptors (van Muijlwijk-Koezen et al.,

2001), latent pharmacophores for diacylhydrazine of SC-

51089 (a potential PGE2 antagonist) and Src homology 2

(SH2) inhibitors (Buchanan et al., 1999; Hallinan et al.,

2001). Synthesis of thiazole derivatives by various methods

and their pharmacological evaluation have been reported

by many research groups (El Kazzouli et al., 2002; El-

Subbagh and Al-Obaid, 1996; Holla et al., 2003; Kare-

goudar et al., 2008; Kolb et al., 2003; Potewar et al., 2007).

The diverse pharmacological properties of thiazole deriv-

atives have attracted much interest in the development of

pharmacologically active compounds. Recently, we repor-

ted the anti-bacterial and anti-fungal activities of some

S. K. Bharti (&)

Department of Medicinal Chemistry, Institute of Pharmaceutical

Sciences, Guru Ghasidas Vishwavidyalaya (G.G.V.),

Bilaspur 495009, India

e-mail: [email protected]

S. K. Singh

Department of Pharmaceutics, Indian Institute of Technology

(Banaras Hindu University), Varanasi 221005, India

123

Med Chem Res (2014) 23:1004–1015

DOI 10.1007/s00044-013-0708-z

MEDICINALCHEMISTRYRESEARCH

novel Schiff bases containing 2,4-disubstituted thiazole

ring (Bharti et al., 2010). The potential of thiazole bearing

molecules (fanetizole, meloxicam and fentiazac) as anti-

inflammatory agents prompted us to design, synthesize and

evaluate the anti-inflammatory activity of some novel 2,4-

disubstituted thiazole (2a–q). The rationale of designing

target compounds, is based on the structural similarity. The

presence of phenyl/substituted phenyl ring at C4 position

and secondary amine/phenyl group at C2 position of thia-

zole ring (Fig. 2). The effect of electron withdrawing/

donating substituent in the phenyl ring on the anti-inflam-

matory activity has also been described.

Chemistry

The target compounds (2a–q) have been synthesized in

two steps. In the first step, the thiosemicarbazone was

synthesized by condensing equimolar quantities of substi-

tuted aryl aldehyde with thiosemicarbazide. In the second

step, equimolar quantities of thiosemicarbazone obtained

in first step and substituted phenacyl bromide were

refluxed to obtain 2,4-disubstituted thiazole. This reaction

proceeds via the cyclization of thiosemicarbazone to the

corresponding 2,4-disubstituted thiazole. The chemical

structures of the synthesized compounds were established

by spectroscopic (FT-IR, 1H and 13C NMR, Mass) and

elemental analyses. The synthetic route of these com-

pounds has been outlined in Schemes 1 and 2. The dif-

ferent substituents of the compounds have been presented

in Tables 1 and 2.

Pharmacology

Anti-inflammatory activity

All the target compounds were screened for their anti-

inflammatory activity. The activity was evaluated by

measuring the physiological response of animals to the

chemical stimuli. For evaluation of anti-inflammatory

activity, carrageenin-induced hind paw edema (acute

inflammation) in rats and cotton pellet granuloma (chronic

inflammation) methods were performed. The acute toxicity

studies were carried out and the experiments were per-

formed at the dose of 20 mg kg-1 body weight, p.o. The

percentage inhibition by compounds was determined and

compared with that of standard drug ibuprofen.

HN

N

S

N

SNH C

O

N S

OH

H3C

O O

H3CN

S

Cl

COOH

fanetizole meloxicam fentiazac

Fig. 1 Anti-inflammatory

agents containing thiazole ring:

fanetizole, meloxicam and

fentiazac

HN

N

S

N

SNH C

O

NS

OH

H3CO O

H3C

N

S

Cl

COOH

HCN

R4

R3

R2R1

HNN

S

R5

Fanetizole

Meloxicam

Fentiazac

Common structural motif

2a-q

Fig. 2 Rationale of designing

target compounds (2a–q)

Med Chem Res (2014) 23:1004–1015 1005

123

Results and discussion

Chemistry

The FT-IR spectra of thiosemicarbazones (1a–e) showed

absorption bands in the range of 3140–3375 cm-1 for -NH-

and -NH2, 2950–3070 cm-1 for aromatic C–H and

1563–1635 cm-1 for azomethine group (-CH=N-). The

formation of product is also supported by the absence of

absorption band in the region of 1700–1750 cm-1 (–CHO

group) and the appearance of band at 1563–1635 (for

-CH=N- group). The absorption bands at 3069–3494 cm-1

R1 R2

R3

R4

HCO

H2N HN NH2

S

+

HCNHN

H2NS

EtOH, AcOH

RefluxR1

R2

R3

R4

Thiosemicarbazide Aryl aldehyde Thiosemicarbazones (1a-e)

Scheme 1 Synthetic route

to thiosemicarbazone

HCNHN

H2NS R1

R2

R3

R4

Reflux

MeOH/EtOH+R5

O

Br HCN

R4

R3

R2R1

HNN

S

R5

(1a-e) (2a-q)

Scheme 2 Synthetic route

to 2,4-disubstituted thiazoles

(2a–q)

Table 1 Different substituents of thiosemicarbazones

Comp. no. R1 R2 R3 R4 Molecular formula Mol. wt.

1a H H H H C8H9N3S 179.24

1b F H H H C8H8FN3S 197.23

1c OH H OH H C8H9N3O2S 211.24

1d H H NO2 H C8H8N4O2S 224.24

1e H OCH3 OCH3 OCH3 C11H15N3O3S 269.32

Table 2 Different substituents of 2,4-disubstituted thiazoles

Comp. R1 R2 R3 R4 R5 Molecular formula Mol. wt.

2a H H H H H C16H13N3S 279.36

2b H H H H F C16H12FN3S 297.35

2c H H H H Cl C16H12ClN3S 313.80

2d H H H H Br C16H12BrN3S 358.26

2e H H H H OCH3 C17H15N3OS 309.39

2f H H H H OCF3 C17H12F3N3OS 363.36

2g H H H H CF3 C17H12F3N3S 347.36

2h H H H H NO2 C16H12N4O2S 324.36

2i H H H H Phe C22H17N3S 355.46

2j H H H H CH3 C17H15N3S 293.39

2k F H H H H C16H12FN3S 297.35

2l OH H OH H H C16H13N3O2S 311.36

2m H H NO2 H H C16H12N4O2S 324.36

2n H OMe OMe OMe H C19H19N3O3S 369.44

2o OH H OH H CF3 C17H12F3N3O2S 379.36

2p F H H H Cl C16H11ClFN3S 331.80

2q H OMe OMe OMe Cl C19H18ClN3O3S 403.88

1006 Med Chem Res (2014) 23:1004–1015

123

for -NH-, at 1604–1663 cm-1 for azomethine group were

found in 2,4-disubstituted thiazoles. The broad peaks of

phenolic OH (Ph–OH) at ortho/para position of compound

2l and 2o appeared in the region 3420–3448 cm-1. The 1H

NMR spectra of thiosemicarbazones showed peaks of

aromatic, hydrazide (NH), primary amine (NH2) and imine

(-N=CH-) proton. The 1H NMR spectra of thiazoles (2a–

q) showed sharp singlet at d 7.2–11.5 indicating the pres-

ence of azomethine (-CH=N-) proton and singlet at d7.8–10.5 for hydrazide (NH) proton. The sharp singlet at d3.8–3.9 indicated the presence of –OCH3 group attached to

the phenyl ring (2e, 2g, 2n). The appearance of multiplets

in the range of d 6.9–7.7 was due to aromatic protons.

Moreover, 13C NMR spectra showed the signals in the

range of d 114.4–131.4 ppm and at d 132.8–143.1 ppm due

to aryl carbon and azomethine carbon, respectively. The

peak appearing in the range of d 160–170, 148–150 and

101–106 ppm corresponds to C2, C4 and C5 of the thiazole

ring, respectively. In the mass spectrum, compound 2a

showed peak at m/z 280 (M?1, 100 %), which matches

with its molecular formula C16H13N3S. Similarly, a peak at

m/z 363 (M?, 100 %), m/z 325 (M?1, 100 %), m/z 356

(M?1, 100 %), and m/z 311 (M?, 100 %) was observed for

compound 2f, 2h, 2i and 2l which is in conformity with the

molecular formula C17H12F3N3OS, C16H12N4O2S,

C22H17N3S and C16H13N3O2S, respectively. FT-IR, 1H and13C NMR, Mass spectral data and elemental analysis

results are in agreement with the proposed structures.

Physicochemical, spectral data and results of elemental

analysis of the compounds are given in Sec. 6.

Anti-inflammatory activity

The carrageenin-induced hind paw edema method in rats

was studied to investigate the anti-inflammatory effect of

compounds on acute phase of inflammation. Generally, the

carrageenin-induced inflammatory process in the rats

involves three phases: an initial, second and third phases

caused by the release of histamine and serotonin; brady-

kinin and prostaglandins respectively (Crunkhorn and

Meacock, 1971; Di Rosa, 1972). In the present study the

anti-inflammatory activity of target compounds was

observed at 1, 2, 3, 4, 5, 6, 7, and 8 h after carrageenin

injection. The results of anti-inflammatory activity of target

compounds are presented in Table 3. Some of them

showed moderate to good activity (51.80–86.74 % inhibi-

tion of paw volume). Significant inhibition of edema for-

mation by 56.62, 65.06, 77.10 % (2a); 78.31, 81.92,

85.54 % (2b); 73.49, 78.31, 84.33 % (2c); 69.87, 73.49,

75.90 (2d); 51.80, 62.65, 71.08 % (2h); 54.21, 67.46, 73.49

(2k) and 81.92, 84.33, 86.74 % (2p) (p \ 0.01) after 1, 2

and 3 h was observed. The group which received standard

drug ibuprofen, significantly inhibited the edema formation

by 89.15, 90.36 and 90.36 % after first, second and third

hour respectively. The inhibition of paw edema produced

by compounds at the dose of 20 mg kg-1 (b.w.), p.o. was

found to be comparable to that of ibuprofen (20 mg kg-1

b.w., p.o.). The extended effects up to third hour suggest

that their action mechanism may involve multiple anti-

inflammatory factors and mediators (Martelli, 1977).

The cotton pellet granuloma method has been widely

employed to assess the transudative, exudative and prolif-

erative components of chronic inflammation (Spector,

1969). The dry weight of the implanted cotton pellet corre-

lates well with the amount of granulomatous tissue formed

(Swingle and Shideman, 1972). In this study, some of the

target compounds significantly inhibited the granuloma

formation which is comparable to that of standard drug,

ibuprofen. In particular, compounds 2a, 2b, 2c, 2d, 2h, 2k

and 2p inhibited the granuloma formation by 81.27, 88.44,

87.56, 85.01 %, 71.71, 77.21 and 90.19 % (p \ 0.01),

respectively whereas standard drug inhibited the granuloma

formation by 94.02 % at the dose 20 mg kg-1 b.w., p.o. The

reduction in the dry weights of implanted cotton pellets by

the compounds indicated that it may inhibit the proliferative

phases of inflammation. This may be due to the ability of

compounds in reducing the number of fibroblasts and syn-

thesis of collagen and mucopolysaccharide, which are nat-

ural proliferative agents of granulation tissue formation.

Literature precedents describe the importance of Schiff

bases/hydrazones and 2,4-disubstituted thiazoles as anti-

fungal and anti-inflammatory agents (Bharti et al., 2010; da

Silva et al., 2011; Loncle et al., 2004). Abafungin, rav-

uconazole (antifungal agents), fanetizole, fentiazac, fenc-

lozic acid and meloxicam (anti-inflammatory agents) are

some examples of drugs containing 2,4-disubstitued thia-

zole ring in their structure. 2,4-disubstituted thiazoles

(Holla et al., 2003), imidazolyl thiazoles (Sharma and

Sawhney, 1997), and pyrazolyl thiazoles (Russo et al.,

1993) have also been recognized as anti-inflammatory and

analgesic agents. Karegoudar et al. (2008) synthesized and

evaluated several thiazole derivatives for their antifungal

activity. Bondock et al. (2007) reported some new thiazole

derivatives with good antifungal activity against different

fungal strains. Ravuconazole is reported to inhibit lanos-

terol demethylase, a cytochrome P450 responsible for the

14a-demethylation of lanosterol, thus blocking ergosterol

biosynthesis (the major membrane sterol of fungi) and

leading to its antifungal activity (Odds et al., 2003). Bekhit

et al. (2003) reported the anti-inflammatory activity of 1H-

pyrazolyl derivatives of thiazole. Fentiazac is reported to

inhibit prostaglandin synthesis by inhibiting cyclooxygen-

ase, which converts arachidonic acid to cyclic endoperox-

ides, precursors of prostaglandins (Molina, 1985). The target

compounds are Schiff bases (containing hydrazone/azome-

thine group, -HN–N=CH-) coupled with 2,4-disubstituted

Med Chem Res (2014) 23:1004–1015 1007

123

Ta

ble

3A

nti

-in

flam

mat

ory

acti

vit

yo

fco

mp

ou

nd

s(2

a–

q)

and

ibu

pro

fen

on

carr

agee

nin

-in

du

ced

hin

dp

awed

ema

inra

ts

Com

pound

Mea

nin

crea

sein

paw

volu

me

(ml)

±S

EM

atti

me

T(h

),(%

inhib

itio

n)

12

34

56

78

2a

0.3

6±

0.0

21*

(56.6

2)

0.2

9±

0.1

7*

(65.0

6)

0.1

9±

0.0

12*

(77.1

0)

0.4

3±

0.0

22

(46.9

1)

0.4

6±

0.0

40

(42.5

0)

0.5

0±

0.1

5(3

3.3

3)

0.5

5±

0.0

18

(26.6

6)

0.6

8±

0.0

66

(2.8

5)

2b

0.1

8±

0.0

33*

(78.3

1)

0.1

5±

0.0

24*

(81.9

2)

0.1

2±

0.0

31*

(85.5

4)

0.2

8±

0.0

44*

(65.4

3)

0.3

7±

0.0

35

(53.7

5)

0.4

5±

0.0

15

(40.0

)0.6

0±

0.0

65

(20.0

)0.6

5±

0.0

75

(7.1

4)

2c

0.2

2±

0.0

23*

(73.4

9)

0.1

8±

0.0

16*

(78.3

1)

0.1

3±

0.0

11*

(84.3

3)

0.2

7±

0.0

17*

(66.6

6)

0.2

8±

0.0

10

(62.6

6)

0.3

6±

0.0

25

(52.0

)0.6

1±

0.0

45

(18.6

6)

0.7

0±

0.0

32

(0)

2d

0.2

5±

0.0

17*

(69.8

7)

0.2

2±

0.0

16*

(73.4

9)

0.2

0±

0.0

12*

(75.9

0)

0.3

5±

0.0

26

(56.7

9)

0.4

0±

0.0

37

(50.0

)0.5

2±

0.0

15

(30.6

6)

0.5

4±

0.0

18

(28.0

)0.6

2±

0.0

36

(11.4

2)

2e

0.7

9±

0.0

18

(4.8

1)

0.7

3±

0.0

24

(12.0

4)

0.6

6±

0.0

26

(20.4

8)

0.8

0±

0.0

07

(1.2

3)

0.8

0±

0.0

20

(0)

0.7

5±

0.0

30

(0)

0.7

5±

0.0

40

(0)

0.7

0±

0.0

40

(0)

2f

0.5

2±

0.0

29

(37.3

4)

0.4

6±

0.0

28

(44.5

7)

0.3

9±

0.0

32

(53.0

1)

0.6

0±

0.0

20

(25.9

2)

0.6

2±

0.0

30

(17.3

3)

0.6

8±

0.0

31

(9.3

3)

0.7

0±

0.0

30

(6.6

6)

0.7

0±

0.0

22

(0)

2g

0.8

0±

0.0

37

(3.6

1)

0.7

8±

0.0

20

(6.0

2)

0.7

8±

0.0

21

(6.0

2)

0.8

0±

0.0

26

(1.2

3)

0.8

0±

0.0

35

(0)

0.7

5±

0.0

25

(0)

0.7

5±

0.0

45

(0)

0.7

0±

0.0

75

(0)

2h

0.4

0±

0.0

31

(51.8

0)

0.3

1±

0.0

24*

(62.6

5)

0.2

4±

0.0

06*

(71.0

8)

0.4

5±

0.0

20

(44.4

4)

0.4

8±

0.0

10

(40.0

)0.5

1±

0.0

44

(32.0

)0.6

2±

0.0

20

(17.3

3)

0.6

8±

0.0

45

(2.8

5)

2i

0.7

2±

0.0

23

(13.2

5)

0.6

5±

0.0

20

(21.6

8)

0.5

8±

0.0

30

(30.1

2)

0.7

8±

0.0

23

(3.7

0)

0.7

8±

0.0

12

(2.5

0)

0.7

5±

0.0

15

(0)

0.7

5±

0.0

86

(0)

0.7

0±

0.0

95

(0)

2j

0.7

5±

0.0

34

(9.6

3)

0.7

1±

0.0

16

(14.4

5)

0.7

1±

0.0

17

(14.4

5)

0.8

0±

0.0

31

(1.2

3)

0.8

0±

0.0

35

(0)

0.7

5±

0.0

20

(0)

0.7

5±

0.0

25

(0)

0.7

0±

0.0

35

(0)

2k

0.3

8±

0.0

20*

(54.2

1)

0.2

7±

0.0

17*

(67.4

6)

0.2

2±

0.0

23*

(73.4

9)

0.4

0±

0.0

31

(50.6

1)

0.4

8±

0.0

25

(40.0

)0.5

0±

0.0

18

(33.3

3)

0.5

5±

0.0

28

(26.6

6)

0.6

0±

0.0

35

(14.2

8)

2l

0.7

0±

0.0

32

(15.6

6)

0.6

2±

0.0

23

(25.3

0)

0.5

0±

0.0

37

(39.7

5)

0.7

1±

0.0

13

(12.3

4)

0.7

2±

0.0

42

(10.0

0)

0.7

2±

0.0

45

(4.0

)0.7

3±

0.0

35

(2.6

6)

0.7

0±

0.0

35

(0)

2m

0.7

7±

0.0

17

(7.2

2)

0.6

9±

0.0

18

(16.8

6)

0.5

7±

0.0

25

(31.3

2)

0.7

8±

0.0

08

(1.2

3)

0.7

9±

0.0

25

(1.2

5)

0.7

5±

0.0

38

(0)

0.7

5±

0.0

45

(0)

0.7

0±

0.0

25

(0)

2n

0.6

4±

0.0

40

(22.8

9)

0.5

8±

0.0

10

(30.1

2)

0.5

2±

0.0

14

(37.3

4)

0.6

8±

0.0

40

(16.0

4)

0.6

8±

0.0

10

(15.0

)0.6

9±

0.0

10

(8.0

)0.7

0±

0.0

80

(6.6

6)

0.7

0±

0.0

60

(0)

2o

0.5

0±

0.0

27

(39.7

5)

0.4

1±

0.0

31

(50.6

0)

0.3

4±

0.0

24*

(59.0

3)

0.6

2±

0.0

23

(23.4

5)

0.6

5±

0.0

47

(18.7

5)

0.7

0±

0.0

11

(6.6

6)

0.7

0±

0.0

25

(6.6

6)

0.7

0±

0.0

28

(0)

2p

0.1

5±

0.0

28*

(81.9

2)

0.1

3±

0.0

25*

(84.3

3)

0.1

1±

0.0

35*

(86.7

4)

0.2

3±

0.0

24*

(71.6

0)

0.2

5±

0.0

45

(68.7

5)

0.3

1±

0.0

25

(58.6

6)

0.3

8±

0.0

10

(49.3

3)

0.4

8±

0.0

25

(31.4

2)

2q

0.4

2±

0.0

25

(49.3

9)

0.3

6±

0.0

18*

(56.6

2)

0.2

5±

0.0

15*

(69.8

7)

0.5

2±

0.0

25

(35.8

0)

0.5

3±

0.0

45

(33.7

5)

0.5

6±

0.0

18

(25.3

3)

0.6

4±

0.0

65

(14.6

6)

0.7

0±

0.0

25

(0)

Contr

ol

0.8

3±

0.0

08

(-)

0.8

3±

0.0

08

(-)

0.8

3±

0.0

08

(-)

0.8

1±

0.0

07

(-)

0.8

0±

0.0

10

(-)

0.7

5±

0.0

15

(-)

0.7

5±

0.0

30

(-)

0.7

0±

0.0

25

(-)

Ibupro

fen

0.0

9±

0.0

12*

(89.1

5)

0.0

8±

0.0

11*

(90.3

6)

0.0

8±

0.0

16*

(90.3

6)

0.1

4±

0.0

07*

(82.7

1)

0.2

0±

0.0

22*

(75.0

)0.2

4±

0.0

35*

(68.0

)0.2

5±

0.0

40*

(66.6

6)

0.3

0±

0.0

20*

(57.1

4)

Dose

:co

ntr

ol

(10

ml

kg

-1

b.w

.,p.o

.)an

dst

andar

ddru

gib

upro

fen

(20

mg

kg

-1

b.w

.,p.o

.).

Val

ues

wer

eex

pre

ssed

asm

ean

±S

EM

(n=

5)

*p\

0.0

1is

com

par

edw

ith

the

contr

ol

gro

up

(AN

OV

Afo

llow

edD

unnet

t’s

t-te

st)

1008 Med Chem Res (2014) 23:1004–1015

123

thiazole ring and some of them showing antifungal and

anti-inflammatory activities. The mode of action of target

compounds may also be due to inhibition of ergosterol

biosynthesis and prostaglandin synthesis, respectively due

to their structural resemblance with the above antifungal

and anti-inflammatory agents. Although, further studies are

required to establish their exact mechanism of action.

Structure activity relationship (SAR)

Structure activity relationship (SAR) studies from the

results of the anti-inflammatory activity revealed that

compounds containing electron withdrawing substituent

(fluoro, chloro, bromo or nitro) in phenyl ring at C2 and/or

C4 position of thiazole ring showed excellent anti-

inflammatory activity (Fig. 3). Electron donating group of

phenyl ring at C2 and/or C4 position of thiazole ring did

not show significant anti-inflammatory activity. As the

molecular properties such as membrane permeability and

bioavailability are associated with molecular weight and

partition coefficient of the compounds, the molecular

weight in the range of 279–403 (\500) and calculated

partition coefficient values (clogP) \5 also supporting

good anti-inflammatory activity of the compounds

(Lipinski et al., 1997).

Conclusion

The results of anti-inflammatory activity of compounds 2a,

2b, 2c, 2d, 2h, 2k and 2p by carrageenin induced hind paw

edema (acute inflammation) in rats and cotton pellet

granuloma (chronic inflammation) methods showed

excellent activity when compared with ibuprofen used as

standard. Particularly, compounds 2b, 2c, 2d, 2h, 2k and

2p which are containing electron withdrawing substituent

(fluoro, chloro, bromo or nitro) in phenyl ring at C2 and/or

C4 position of thiazole ring showed excellent anti-inflam-

matory activity.

Experimental protocols

All the chemicals and solvents used for this work were

obtained from S.D. Fine (Mumbai), Merck (Germany), and

Sigma-Aldrich (U.S.A.). The chemicals purchased were

purified by standard methods prior to use. Melting points of

the synthesized compounds were determined in open-glass

capillaries on Stuart-SMP10 melting point apparatus and are

uncorrected. IR absorption spectra were recorded on Shi-

madzu FTIR-8400s using KBr pellets in the range of

4000–400 cm-1, 1H and 13C NMR spectra were recorded on

the JEOL AL300 FTNMR spectrometer operating at

300 MHz and TMS (tetramethylsilane, Me4Si) as an internal

standard. The 1H and 13C NMR chemical shifts were

reported as parts per million (ppm) downfield from TMS.

The splitting patterns are designated as follows; s, singlet; d,

doublet; m, multiplet. Mass spectra were recorded on VG-

AUTOSPEC spectrometer. IR, 1H and 13C NMR and mass

spectra were consistent with the assigned structures. Ele-

mental analyses (C, H, N) were done on a CHN rapid ana-

lyzer. All the new compounds gave C, H and N analysis

within ±0.4 % of the theoretical values. Purity of the com-

pounds was checked by thin layer chromatography (TLC) on

Merck silica gel 60 F254 precoated sheets in chloroform/

methanol mixture and spots were developed using iodine

vapours/ultraviolet light as visualizing agent.

General procedure for the synthesis

of thiosemicarbazones (1a–e)

A mixture of equimolar quantities of substituted aryl

aldehyde (0.01 mol) in ethanol/methanol (20 ml) and thi-

osemicarbazide (0.01 mol) in ethanol (20 ml) was refluxed

on a water bath for 4–6 h in the presence of few drops of

glacial acetic acid. The progress of reaction was monitored

by TLC at appropriate time interval. After completion of

reaction, the solution was cooled, solid thus separated was

washed with ice-cold water and dried. Finally, the product

thus obtained was recrystallized from suitable solvent/

mixture of solvents.

General procedure for the synthesis of 2,4-disubstituted

thiazoles (2a–q)

A mixture of equimolar quantities of thiosemicarbazone

(1a–e) in hot ethanol/methanol and substituted phenacyl

bromide in hot methanol was refluxed on a water bath for

6–8 h. The progress of reaction was monitored by TLC at

appropriate time interval. The excess of solvent was dis-

tilled off and the solid that separated was collected by fil-

tration, suspended in water and filtered to get the desired

product (2a–q). The product was recrystallized from suit-

able solvent/mixture of solvents.

HCN

R4

R3

R2R1

HN

N

S

R5

Electron withdrawing/donating group

Electron withdrawing/donating group

2,4-disubstituted thiazole ring

Schiff base component

Fig. 3 SAR of 2,4-disubstituted thiazoles evaluated for anti-inflam-

matory activity

Med Chem Res (2014) 23:1004–1015 1009

123

Physicochemical, spectral data and results of elemental

analysis of the compounds are given below:

1a. 2-benzylidenehydrazinecarbothioamide

M.P. 154–155 �C; Yield: 82 %; Rf : 0.7 (chloro-

form:methanol, 9:1); solubility: ethanol, methanol, chlo-

roform, DMSO, DMF; IR (KBr, mmax cm-1): 3362 (-NH2),

3236 (-NH), 1606 (HC=N azomethine), 1031 (C=S); 1H

NMR (DMSO-d6, 300 MHz) d (ppm): 7.2–7.5 (m, 5H,

aromatic H), 7.7 (s, 1H, -N=CH), 8.6 (s, 1H, NH); 13C

NMR (DMSO-d6) d (ppm): 121.0–126.5 (Aryl–CH),

132.0–133.6 (Aryl–C), 136.5 (HC=N); % elemental anal-

ysis found (calc.) for C8H9N3S: C, 53.4 (53.6); H, 4.9 (5.0);

N, 23.5 (23.4).

1b. 2-(2-fluorobenzylidene)hydrazinecarbothioamide

M.P. 227 �C; Yield: 75 %; Rf : 0.60 (chloroform:methanol,

9:1); solubility: ethanol, methanol, DMSO, DMF; IR (KBr,

mmax cm-1): 3375 (-NH2), 3298 (-NH), 1552 (HC=N azo-

methine), 1105 (C=S); 1H NMR (DMSO-d6, 300 MHz) d(ppm): 6.9–7.7 (m, 5H, aromatic H), 8.0 (s, 1H, -N=CH),

8.3 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm):

122.1–127.4 (Aryl–CH), 132.0–137.0 (Aryl–C), 137.0

(HC=N); % elemental analysis found (calc.) for

C8H8FN3S: C, 48.8 (48.7); H, 4.0 (4.0); N, 21.4 (21.3).

1c. 2-(2,4-dihydroxybenzylidene)hydrazinecarbothioamide

M.P. 208–209 �C; Yield: 68 %; Rf: 0.70 (chloroform:meth-

anol, 9:1); solubility: ethanol, methanol, DMSO, DMF; IR

(KBr, mmax cm-1): 3541 (-OH, br.), 3220 (-NH2), 3140 (-NH),

1620 (HC=N azomethine), 1087 (C=S); 1H NMR (DMSO-d6,

300 MHz) d (ppm): 7.1–7.4 (m, 3H, aromatic H), 8.2 (s, 1H, -

N=CH), 9.0 (s, 1H, NH), 11.4 (s, 1H, -OH); 13C NMR

(DMSO-d6) d (ppm): 124.5–128.0 (Aryl–CH), 131.0–137.6

(Aryl–C), 144.8 (HC=N); % elemental analysis found (calc.)

for C8H9N3O2S: C, 45.3 (45.4); H, 4.3 (4.2); N, 19.7 (19.8).

1d. 2-(4-nitrobenzylidene)hydrazinecarbothioamide



(KBr, mmax cm-1): 3233 (-NH2), 3164 (-NH), 1570 (HC=N

azomethine), 1075 (C=S); 1H NMR (DMSO-d6, 300 MHz) d(ppm): 7.1–7.4 (m, 4H, aromatic H), 7.6 (s, 1H, -N=CH), 8.3

(s, 1H, NH); 13C NMR (DMSO-d6) d (ppm): 122.5–126.0

(Aryl–CH), 132.0–133.6 (Aryl–C), 136.5 (HC=N); % ele-

mental analysis found (calc.) for C8H8N4O2S: C, 42.7 (42.8);

H, 3.6 (3.6); N, 24.7 (24.9).

1e. 2-(3,4,5-trimethoxybenzylidene)hydrazinecarbothioamide


anol, 9:1); solubility: ethanol, methanol, chloroform, DMSO,

DMF; IR (KBr, mmax cm-1): 3230 (-NH2), 3174 (-NH), 1570

(HC=N azomethine), 1053 (C=S); 1H NMR (DMSO-d6,

300 MHz) d (ppm): 3.9 (OCH3), 7.4 (d, 2H, aromatic H), 7.8

(s, 1H, -N=CH), 8.5 (s, 1H, NH); 13C NMR (DMSO-d6) d(ppm): 56.3 (OCH3), 121.7–126.3 (Aryl–CH), 132.0–133.6

(Aryl–C), 138.4 (HC=N); % elemental analysis found (calc.)

for C11H15N3O3S: C, 49.1 (49.0); H, 5.7 (5.6); N, 15.5 (15.6).

2a. 1-benzylidene-2-(4-phenylthiazol-2-yl)hydrazine

M.P. 180–181 �C; Yield: 80 %; Rf: 0.5 (chloroform:metha-

nol, 9:1); solubility: ethanol, methanol, DMSO, DMF; IR

(KBr, mmax cm-1): 3168 (-NH), 1635 (C=N azomethine),

1575, 1521 (C=N thiazole), 1445, 1383, 1241; 1H NMR

(DMSO-d6, 300 MHz) d (ppm): 7.1–7.6 (m, 10H, phenyl H),

7.8 (s, 1H, thiazole H), 8.1 (s, 1H, -N=CH); 13C NMR

(DMSO-d6) d (ppm): 102.5 (thiazole-C-5), 115.7, 126.2,

129.0 (Ar–CH), 141.1 (HC=N), 148.0 (thiazole-C-4), 170

(thiazole-C-2); MS (m/z, %): 280 (M?1, 100); % elemental

analysis found (calc.) for C16H13N3S: C, 68.70 (68.79); H,

4.67 (4.69); N, 15.02 (15.04).

2b. 1-benzylidene-2-(4-(4-fluorophenyl)thiazol-2-

yl)hydrazine



(KBr, mmax cm-1): 1618 (C=N azomethine), 1502 (aromatic

C=C), 833 and 754; 1H NMR (DMSO-d6, 300 MHz) d (ppm):

6.9–7.7 (m, 9H, aromatic H), 7.9 (s, 1H, -N=CH), 8.3 (s, 1H,

NH); 13C NMR (DMSO-d6) d (ppm): 76.5, 114.4 (Aryl–CH),

131.4 (Aryl–C), 132.8 (HC=N), 150.0 (thiazole-C-4), 153.3,

153.6, 159.7, 171.4 (thiazole-C-2); MS (m/z, %): 297 (M?,

100), (M?2, 30); % elemental analysis found (calc.) for

C16H12FN3S: C, 64.55 (64.63); H, 4.05 (4.07); N, 14.17

(14.13).

2c. 1-benzylidene-2-(4-(4-chlorophenyl)thiazol-2-

yl)hydrazine



(KBr, mmax cm-1): 3169 (-NH), 1633 (HC=N- azomethine),

1520 (C=N thiazole), 1126; 1H NMR (DMSO-d6, 300 MHz)

d (ppm): 6.9–7.7 (m, 9H, aromatic H), 8.3 (s, 1H, NH), 11.5 (s,

1H, -N=CH); 13C NMR (DMSO-d6) d (ppm): 102.3 (thiazole-

C-5), 115.7, 126.2, 129.0 (Ar–CH), 140.1 (HC=N), 148.0

(thiazole-C-4), 170 (thiazole-C-2); MS (m/z, %): 314 (M?1,

1010 Med Chem Res (2014) 23:1004–1015

123

100); % elemental analysis found (calc.) for C16H12ClN3S: C,

61.32 (61.24); H, 3.83 (3.85); N, 13.40 (13.39).

2d. 1-benzylidene-2-(4-(4-bromophenyl)thiazol-2-

yl)hydrazine

M.P. 239–240 �C; yield: 67 %; Rf: 0.55 (chloroform:meth-

anol, 9:1); solubility: methanol, DMSO, DMF; IR (KBr, mmax

cm-1): 3394 (-NH), 1563 (C=N azomethine), 1474, 698; 1H

NMR (DMSO-d6) d (ppm): 6.4–7.3 (m, 9H, aromatic H), 7.7

(s, 1H, -N=CH), 8.0 (s, 1H, NH); 13C NMR (DMSO-d6) d(ppm): 38.6, 39.0, 40.3, 126.5, 128.8, 129.7 (Ar–CH), 130.7,

131.8, 132.4 (Ar–C), 134.7 (HC=N), 165.3 (thiazole-C-4),

187.4 (thiazole-C-2); MS (m/z, %): 359 (M?1, 100); % ele-

mental analysis found (calc.) for C16H12BrN3S: C, 53.60

(53.64); H, 3.36 (3.38); N, 11.73 (11.73).

2e. 1-benzylidene-2-(4-(4-methoxyphenyl)thiazol-2-

yl)hydrazine

M.P. 189–190 �C; yield: 72 %; Rf: 0.5 (chloroform:metha-


(KBr, mmax cm-1): 3348, 3271 (-NH), 1621 (HC=N- azome-

thine), 837 and 763; 1H NMR (DMSO-d6, 300 MHz) d(ppm): 3.8 (s, 3H, OCH3), 6.9 (d, 2H, p-anisyl), 7.1–7.6 (m,

5H, phenyl H), 7.7 (s, 1H, thiazole H), 7.8 (s, 1H, NH), 8.0 (d,

2H, p-anisyl), 8.2 (s, 1H, -N=CH); 13C NMR (DMSO-d6) d(ppm): 58.5 (OCH3), 102.5 (thiazole-C-5), 115.7, 126.2,

129.0 (Ar–CH), 141.1 (HC=N), 148.0 (thiazole-C-4), 170

(thiazole-C-2); MS (m/z, %): 310 (M?1, 100); % elemental

analysis found (calc.) for C17H15N3OS: C, 66.00 (66.01); H,

4.86 (4.89); N, 13.57 (13.58).

2f. 1-benzylidene-2-(4-(4-(trifluoromethoxy)phenyl)thiazol-

2-yl)hydrazine




1508 (C=N thiazole), 1255; 1H NMR (DMSO-d6, 300 MHz)

d (ppm): 6.5–7.3 (m, 9H, ArH), 7.8 (s, 1H, thiazole H), 8.0 (s,

1H, NH), 8.4 (s, 1H, -N=CH); 13C NMR (DMSO-d6) d (ppm):

101.3 (thiazole-C-5), 115.0, 129.0 (Ar–CH), 143.1 (HC=N),

148.7 (thiazole-C-4), 151, 155.4, 172.2 (thiazole-C-2); MS

(m/z, %): 363 (M?, 100.0 %), 364 (M?1, 40 %); % elemental

analysis found (calc.) for C17H12F3N3OS: C, 56.23 (56.19);

H, 3.34 (3.33); N, 11.59 (11.56).

2g. 1-benzylidene-2-(4-(4-(trifluoromethyl)phenyl)

thiazol-2-yl)hydrazine

M.P. 194–195 �C; Yield: 66 %; Rf: 0.6 (chloro-

form:methanol, 9:1); solubility: ethanol, methanol, DMSO,

DMF; IR (KBr, mmax cm-1): 3280 (-NH-), 1655 (C=N

azomethine), 1546 (C=N thiazole), 1245; 1H NMR

(DMSO-d6, 300 MHz) d (ppm): 6.8–7.6 (m, 9H, ArH), 7.9

(s, 1H, thiazole H), 8.1 (s, 1H, NH), 8.6 (s, 1H, -N=CH);13C NMR (DMSO-d6) d (ppm): 100.3 (thiazole-C-5),

113.5, 128.6 (Ar–CH), 145.2 (HC=N), 148.0 (thiazole-C-

4), 151.7, 171.1 (thiazole-C-2); MS (m/z, %): 348 (M?1,

100); % elemental analysis found (calc.) for C17H12F3N3S:

C, 58.81 (58.78); H, 3.49 (3.48); N, 12.12 (12.10).

2h. 1-benzylidene-2-(4-(4-nitrophenyl)thiazol-2-

yl)hydrazine



(KBr, mmax cm-1): 3169 (NH), 1572 (C=N azomethine), 1452

(C=N thiazole), 1259; 1H NMR (DMSO-d6, 300 MHz) d(ppm): 7.1–7.3 (m, 9H, ArH), 7.6 (s, 1H, thiazole H), 7.8 (s,


106.0 (thiazole-C-5), 114.6, 123.2, 128.5 (Ar–CH), 141.3

(HC=N), 147.8 (thiazole-C-4), 156.5, 170.1 (thiazole-C-2);

MS (m/z, %): 325 (M?1, 100), 326 (M?2, 15); % elemental

analysis found (calc.) for C16H12N4O2S: C, 59.35 (59.25); H,

3.75 (3.73); N, 17.30 (17.27).

2i. 1-benzylidene-2-(4-(4-phenylphenyl)thiazol-2-

yl)hydrazine



(KBr, mmax cm-1): 3150 (-NH), 1562 (C=N, azomethine),

1508 (C=N, thiazole), 1257; 1H NMR (DMSO-d6) d (ppm):

7.0–7.6 (m, 14H, Ar–H), 7.7 (s, 1H, thiazole H), 7.8 (s, 1H,

NH), 8.4 (s, 1H, -N=CH); 13C NMR (DMSO-d6) d (ppm):

101.2 (thiazole-C-5), 115.7, 126.2, 129.0 (Ar–CH), 141.1

(HC=N), 148.0 (thiazole-C-4), 167.1 (thiazole-C-2); MS (m/

z, %): 356 (M?1, 100); % elemental analysis found (calc.) for

C22H17N3S: C, 74.44 (74.34); H, 4.84 (4.82); N, 11.85

(11.82).

2j. 1-benzylidene-2-(4-p-tolylthiazol-2-yl)hydrazine




1508 (aromatic C=C), 831 and 748; 1H NMR (DMSO-d6) d(ppm): 6.8–7.6 (m, 9H, Ar–H), 7.7 (s, 1H, thiazole H), 7.8 (s,


101.3 (thiazole-C-5), 115.5, 129.0 (Ar–CH), 143.1 (HC=N),

148.7 (thiazole-C-4), 155.4, 172.2 (thiazole-C-2); MS (m/z,

%): 294 (M?1, 100), 295 (M?2, 30); % elemental analysis

found (calc.) for C17H15N3S: C, 69.64 (69.59); H, 5.17 (5.15);

N, 14.33 (14.32).

Med Chem Res (2014) 23:1004–1015 1011

123

2k. 1-(2-fluorobenzylidene)-2-(4-phenylthiazol-2-

yl)hydrazine




1549 (aromatic C=C); 1H NMR (DMSO-d6) d (ppm):

6.5–7.5 (m, 9H, ArH), 7.9 (s, 1H, -N=CH), 9.6 (s, 1H,

thiazole H), 10.5 (s, 1H, NH); 13C NMR (DMSO-d6) d(ppm): 43.3, 51.2, 109.4 (thiazole-C-5), 116.4, 128.5 (Ar–

CH), 145.1 (HC=N), 148.7 (thiazole-C-4), 155.4, 172.1

(thiazole-C-2); MS (m/z, %): 298 (M?1, 100). % elemental

analysis found (calc.) for C16H12FN3S: C, 64.69 (64.63); H,

4.08 (4.07); N, 14.15 (14.13).

2l. 1-(2,4-dihydroxybenzylidene)-2-(4-phenylthiazol-2-

yl)hydrazine



(KBr, mmax cm-1): 3420 (-OH, br.), 2924 (-NH), 1635

(C=N azomethine), 1566 (aromatic C=C), 1075; 1H NMR

(DMSO-d6, 300 MHz) d (ppm): 7.2–8.0 (m, 8H, ArH), 8.2

(s, 1H, NH), 9.9 (s, 1H, -N=CH), 12.1 (s, 1H, o-OH); 13C

NMR (DMSO-d6) d (ppm): 103.6 (thiazole-C-5), 122–139

(Ar–CH), 143.4 (HC=N), 148.4 (thiazole-C-4), 153 (Ar–C–

OH), 170 (thiazole-C-2); MS (m/z, %): 311 (M?, 100). %

elemental analysis found (calc.) for C16H13N3O2S: C,

61.82 (61.72); H, 4.23 (4.21); N, 13.52 (13.50).

2m. 1-(4-nitrobenzylidene)-2-(4-phenylthiazol-2-

yl)hydrazine


anol, 9:1); solubility: chloroform, DMSO, DMF; IR (KBr,

mmax cm-1): 3120 (-NH), 1622 (C=N, azomethine), 1516

(aromatic C=C), 927, 849, 787; 1H NMR (DMSO-d6) d(ppm): 7.1–7.6 (m, 9H, Ar–H), 7.7 (s, 1H, thiazole H), 7.8

(s, 1H, NH), 8.4 (s, 1H, -N=CH); 13C NMR (DMSO-d6) d(ppm): 105.3 (thiazole-C-5), 115.7, 126.2, 129.0 (Ar–CH),

141.1 (HC=N), 148.0 (thiazole-C-4), 167.1 (thiazole-C-2);

MS (m/z, %): 325 (M?1, 100), 326 (M?2, 35). % ele-

mental analysis found (calc.) for C16H12N4O2S: C, 59.40

(59.25); H, 3.75 (3.73); N, 17.29 (17.27).

2n. 1-(3,4,5-trimethoxybenzylidene)-2-(4-phenylthiazol-2-

yl)hydrazine




1508 (aromatic C=C), 831 and 748; 1H NMR (DMSO-d6,

300 MHz) d (ppm): 6.5–7.3 (m, 7H, ArH), 7.7 (s, 1H,

thiazole H), 7.8 (s, 1H, NH), 8.4 (s, 1H, -N=CH); 13C NMR

(DMSO-d6) d (ppm): 101.3 (thiazole-C-5), 115.5, 129.0

(Ar–CH), 143.1 (HC=N), 148.7 (thiazole-C-4), 151, 155.4,

160.0 (Ar–C-OCH3), 172.2 (thiazole-C-2); MS (m/z, %):

370 (M?1, 100); % elemental analysis found (calc.) for

C19H19N3O3S: C, 61.70 (61.77); H, 5.17 (5.18); N, 11.39

(11.37).

2o. 1-(2,4-dihydroxybenzylidene)-2-(4-

trifluoromethylphenylthiazol-2-yl)hydrazine

M.P. 97–98 �C; yield: 69 %; Rf: 0.5 (chloroform:methanol,

9:1); solubility: ethanol, methanol, DMSO, DMF; IR (KBr,

mmax cm-1): 3448 (-OH, br.), 3271 (-NH), 1602 (C=N

azomethine), 1452 (aromatic C=C), 1250; 1H NMR

(DMSO-d6, 300 MHz) d (ppm): 7.2 (s, 1H, -N=CH),

7.3–7.6 (m, 7H, Ar–H), 8.0 (s, 1H, NH), 9.7 (s, 1H, o-OH);13C NMR (DMSO-d6) d (ppm): 103.6 (thiazole-C-5),

122–139 (Ar–CH), 143.4 (HC=N), 148.4 (thiazole-C-4),

153 (Ar–C–OH), 170 (thiazole-C-2); MS (m/z, %): 380

(M?1, 100); % elemental analysis found (calc.) for

C17H12F3N3O2S: C, 53.93 (53.82); H, 3.20 (3.19); N, 11.18

(11.08).

2p. 1-(2-fluorobenzylidene)-2-(4-(4-chlorophenyl)thiazol-

2-yl)hydrazine


anol, 9:1); solubility: chloroform, methanol, DMSO, DMF;

IR (KBr, mmax cm-1): 3069 (-NH), 1620 (C=N azome-

thine), 1581 (aromatic C=C), 918, 821, 752; 1H NMR

(DMSO-d6, 300 MHz) d (ppm): 7.0–7.2 (m, 4H, o-fluoro-

phenyl), 7.3 (d, 2H, p-chlorophenyl), 7.6 (d, 2H, p-chlo-

rophenyl), 7.7 (s, 1H, thiazole H), 7.8 (s, 1H, NH), 8.5 (s,

1H, -N=CH); 13C NMR (DMSO-d6) d (ppm): 100.3 (thia-

zole-C-5), 116.3, 118, 124.6, 129.3 (Ar–CH), 133.1

(HC=N), 143 (thiazole-C-4), 148, 161.1 (thiazole-C-2); MS

(m/z, %): 332 (M?1, 100), 333 (M?2, 36); % elemental

analysis found (calc.) for C16H11ClFN3S: C, 58.02 (57.92);

H, 3.35 (3.34); N, 12.69 (12.66).

2q. 1-(3,4,5-trimethoxybenzylidene)-2-(4-(4-

chlorophenyl)thiazol-2-yl)hydrazine




1508 (aromatic C=C), 831, 748; 1H NMR (DMSO-d6,

300 MHz) d (ppm): 6.6–7.3 (m, 6H, ArH), 7.7 (s, 1H,

thiazole H), 7.8 (s, 1H, NH), 8.4 (s, 1H, -N=CH); 13C NMR

(DMSO-d6) d (ppm): 101.3 (thiazole-C-5), 115.5, 129.0

(Ar–CH), 143.1 (HC=N), 148.7 (thiazole-C-4), 151, 155.4,

160.0 (Ar–C-OCH3), 172.2 (thiazole-C-2); MS (m/z, %):

1012 Med Chem Res (2014) 23:1004–1015

123

404 (M?1, 100). % elemental analysis found (calc.) for

C19H18ClN3O3S: C, 56.64 (56.50); H, 4.50 (4.49); N, 10.43

(10.40).

Experimental procedure for anti-inflammatory activity

Animals

Male albino rats (Charles foster strain) weighing 140–180 g

were randomly housed in groups of five in polypropylene

cages at an ambient temperature with a 12 h light: 12 h dark

cycle. The animals were allowed free access to laboratory diet

(M/s Hindustan Lever Ltd., Mumbai, India) and water

ad libitum. The animals were fasted overnight before the

experiment. Experiments were performed in accordance with

the current guidelines for the care of laboratory animals and

the ethical guidelines for the investigation of experimental

pain in conscious animals (Zimmerman, 1983).

Acute toxicity studies

The acute toxicity studies for all the test compounds were

carried out in albino rats (weighing 140–180 g) which were

fasted overnight. The dosage was varied from 50 to

200 mg kg-1 b.w. The animals were observed for 24 h for

any signs of acute toxicity such as increased or decreased

motor activity, tremors, convulsion, sedation, lacrimation

etc. No mortality of the animals was observed even after

24 h. Hence, the LD50 cut off value of the test compounds

was fixed as 200 mg kg-1 b.w. and 1/10th of cut off value

(i.e. 20 mg kg-1 b.w.) was taken as maximum screening

dose for the evaluation of anti-inflammatory activity.

Carrageenin induced hind paw edema in rats

(acute inflammation) method

The anti-inflammatory activity of compounds on carra-

geenin-induced rat paw oedema was determined according

to the method described by Winter et al. (1962). The

experimental animals were divided into nineteen groups,

each containing five animals. First group received sterile

normal saline (control) and the second group received

standard drug ibuprofen (20 mg kg-1 b.w., p.o.). The 3rd–

17th groups were administered the test compounds (at a

dose of 20 mg kg-1 b.w. suspended in 10 ml kg-1 of 2 %

gum acacia) orally. After 30 min of administration of test

compounds, 0.1 ml of 1 % (w/v) carrageenin was injected

subcutaneously in the subplantar region of the left hind

paw. The right paw served as a reference to non inflammed

paw for comparison. The initial paw volume was measured

within 30 s of the carrageenin injection by plethysmome-

ter. The relative increase in paw volume was measured in

control, standard and test compounds at 1, 2, 3, 4, 5, 6, 7

and 8 h after the carrageenin injection. The difference

between initial and final readings was taken as the volume

of oedema and the percentage inhibition by the compounds

was calculated using the formula (Kouadio et al., 2000):

% Inhibition ¼ 1� dt

dc

� �� 100

where dt is the difference in paw volume in the compound-

treated group and dc the difference in paw volume in the

control group.

Cotton pellet granuloma (chronic inflammation)

method

The method of Mossa et al. (1995) was used for this study

which involves surgical insertion of sterilized cotton pellet

(30 mg in weight) subcutaneously into the groin of rats

using ether as an anaesthetic agent. Nineteen groups of five

rats in each group, were included in the study. After

shaving off fur, the animals were anaesthetized and

administered the same doses of compounds, vehicle and

ibuprofen as in the carrageenin-induced rat paw edema test.

Table 4 Anti-inflammatory activity of compounds (2a–q) and ibu-

profen on cotton pellet induced granuloma in rats

Compound Mean increase in weight

of pellets (mg) ± SEM

% inhibition

2a 23.5 ± 1.8* 81.27

2b 14.5 ± 1.5* 88.44

2c 15.6 ± 0.62* 87.56

2d 18.8 ± 0.94* 85.01

2e 125.5 ± 1.65 0

2f 121.3 ± 2.22 3.34

2g 122.7 ± 2.12 2.23

2h 35.6 ± 2.4 71.71

2i 125.2 ± 3.56 0

2j 123.5 ± 1.24 1.59

2k 28.6 ± 1.5* 77.21

2l 120.0 ± 3.24 4.38

2m 120.0 ± 2.82 4.38

2n 118.5 ± 1.11 5.57

2o 115.0 ± 4.0 8.36

2p 12.3 ± 1.6* 90.19

2q 111.5 ± 2.45 11.15

Control 125.5 ± 3.0 –

Ibuprofen 7.5 ± 1.2* 94.02

Dose: control (10 ml kg-1 b.w., p.o.) and standard drug ibuprofen

(20 mg kg-1 b.w., p.o.); values were expressed as mean ± SEM

(n = 5)

* p \ 0.01 is compared with the control group (ANOVA followed

Dunnett’s t-test)

Med Chem Res (2014) 23:1004–1015 1013

123

The compounds (2a–q), vehicle and ibuprofen were

administered to respective groups of the animals for seven

consecutive days. All the animals were sacrificed on the

eighth day with an over dose of ether. The pellet and the

surrounding granuloma were dissected out carefully, made

free from extraneous tissues and dried overnight in an oven

at 60 �C to a constant weight. The weight of the granuloma

tissue was obtained by determining the difference between

the initial (30 mg) and the final weight of the cotton pellet

with its attached granulomatous tissue. The mean weight of

the granuloma tissue formed in each group and the per-

centage inhibition were determined. The results have been

presented in Table 4.

Statistical analysis

The mean ± standard error of the mean (SEM) was deter-

mined for each parameter. The data was subjected to one-

way analysis of variance (ANOVA) followed by Dunnett’s

t test. The results were considered significant if p \ 0.01.

Acknowledgments The authors are grateful to the Head, Depart-

ment of Chemistry, Faculty of Science, Banaras Hindu University

(BHU), Varanasi, India for 1H and 13C NMR spectrometry, Indian

Institute of Chemical Technology (IICT), Hyderabad for mass spec-

trometry. S.K. Bharti is grateful to University Grants Commission

(UGC), New Delhi for the award of senior research fellowship.

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