Microwave-mediated synthesis of triazolothiadiazoles as anti-inflammatory, analgesic, and anti-oxidant agents

ORIGINAL RESEARCH

Microwave-mediated synthesis of triazolothiadiazolesas anti-inflammatory, analgesic, and anti-oxidant agents

K. V. Sujith • Balakrishna Kalluraya •

Adithya Adhikari • K. Vijayanarayana

Received: 18 November 2010 / Accepted: 12 January 2011 / Published online: 26 January 2011

� Springer Science+Business Media, LLC 2011

Abstract Efficient approach to the synthesis of a series of

triazolothiadiazole analogs of ibuprofen has been carried out

using microwave energy. Thus a series of 1,2,4-triazolo[3,4-

b]-thiadiazoles 5 were synthesized starting from 4-amino-3-

[1-(4-isobutylphenyl)ethyl]-5-mercapto-1,2,4-triazole 3 and

different aromatic acids using phosphorous oxychloride as

cyclizing agent by microwave and conventional method.

Microwave irradiation reduces both time and reaction

endeavors along with reduced amount of phosphorous

oxychloride. In addition, preliminary results of the biologi-

cal evaluation of these compounds are also reported and they

found to exhibit significant antioxidant, anti-inflammatory,

and analgesic activities with reduced ulcerogenicity.

Keywords Microwave synthesis � Triazolothiadiazoles �Anti-inflammatory � Analgesic � Antioxidant �Ulcerogenic activities

Introduction

The quest for NSAIDs with improved efficacy or safety

profile has gained considerable attention to discover new

analogs. Prompted by the varied biological activities of 1,2,

4-triazoles and 1,3,4-thiadiazoles, and in continuation of

our work on the synthesis of 1,2,4-triazoles derivatives of

ibuprofen (Sujith et al., 2009), a series of 1,2,4-triazolo

[3,4-b]-thiadiazoles were being synthesized having improved

anti-inflammatory and analgesic properties.

Ibuprofen is an important class of drug belongs to

NSAIDs with anti-inflammatory and analgesic activity

(Xiangguo et al., 2006). Long term use of NSAIDs like

ibuprofen has been associated with gastrointestinal com-

plications ranging from stomach irritation to life-threaten-

ing GI ulceration, bleeding, and nephrotoxicity (Allison

et al., 1992; Amir and Kumar, 2007). In this perspective

ibuprofen has been exploited, in fact this molecule has a

carboxylic group as functional group, common to most

NSAIDs which is responsible for its local irritation

(Hawkey et al., 2000). It has been reported that the

derivatization of the carboxyl function of representa-

tive NSAIDs, resulted in an increased anti-inflammatory

activity with reduced ulcerogenic effect (Kalgutkar et al.,

2000; Duflos et al., 2001; Metwally et al., 2007). This

paved the way for the development of variety of hetero-

cyclic compounds, such as triazole and thiadiazole. So we

decided to combine ibuprofen with 1,2,4-triazolo-[3,4-b]-

1,3,4-thiadiazole ring which has been formed as the result

of heterocyclization in order to obtain new derivative with

increased pharmacological activity. In the design of new

compounds, development of hybrid molecules through the

combination of different pharmacophores in one structure

may lead to compounds with increased biological activity.

These observations prompted us to synthesize new triaz-

olothiadiazole scaffold of ibuprofen. The synthesis of the

title compounds was carried out by both conventional

methods as well as by using microwave energy.

Microwave irradiation dominates over usual conven-

tional methods due to its non-hazardous and eco-friendly

nature. The microwave induced organic reaction received

K. V. Sujith � B. Kalluraya (&) � A. Adhikari

Department of Studies in Chemistry,

Mangalore University, Mangalagangothri 574199,

Karnataka, India

e-mail: [email protected]

K. Vijayanarayana

Department of Pharmacy,

Manipal College of Pharmaceutical Sciences,

Manipal 576104, Karnataka, India

123

Med Chem Res (2012) 21:543–551

DOI 10.1007/s00044-011-9569-5

MEDICINALCHEMISTRYRESEARCH

considerable attention due to their simplicity and opera-

tional convenience. The use of microwave irradiation as a

source of heat in synthetic chemistry offers a promising

alternative (Jyothi et al., 2007; Kalluraya et al., 2008).

Microwave assisted heating under controlled conditions is

an invaluable technology because it often dramatically

reduces reactions times, with increased yield (Escalante

and Dıaz-Coutino, 2009). Nowadays, microwave assisted

organic synthesis has become a new era in the field of

synthetic chemistry (Algul et al., 2009).

Results and discussion

After optimization of the experimental conditions, 4-amino-3-

[1-(4-isobutylphenyl)ethyl]-5-mercapto-1,2,4-triazole 3 was

synthesized by following an easy one-pot protocol (Sche-

me 1). Similarly 6-substituted-3-[1-(4-isobutylphenyl)ethyl]-

1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles 5 were prepared by

the reaction of equimolar mixture of ibuprofen substituted

triazole 3 and aromatic acids 4 employing phosphorus

oxychloride as cyclizing agent under conventional heating

and microwave irradiation method. The main advantage of

microwave-mediated method in this regard is the minimum

use of the hazardous phosphorus oxychloride compared to

conventional method (Scheme 2).

Anti-inflammatory activity was determined by the car-

rageenan-induced paw edema method in Wistar albino rats

using plethysmography (Winter et al., 1962). Diclofenac at

an oral dose of 20 mg/kg served as the standard drug. For

further comparison ibuprofen was additionally used as a

second standard reference. Analgesic activities were eval-

uated on Swiss albino mice by tail-flick method (Di stasi

et al., 1988) in this method heat is used as a source of pain. A

dose level of 10 mg/kg pentazocine and ibuprofen served as

the standard drugs for comparison. The responses produced

in the animal were observed for 30 min intervals and

percentage in reaction time was calculated for analgesic

activity. The in vitro antioxidative potential for the synthe-

sized compounds was studied and the results were com-

pared with a standard antioxidant butylated hydroxytoluene

(BHT). The DPPH (1,1-diphenyl-2-picrylhydrazyl) assay is

used as a preliminary test to investigate the antioxidant

potential of the synthesized compounds. Compound show-

ing better DPPH free radical scavenging property was

investigated for further in vitro antioxidant studies like

hydroxyl radical scavenging, superoxide anion scavenging,

and nitric oxide radical scavenging activity. The DPPH free

radical scavenging activity of the compounds was deter-

mined by using the modified method (Brand-Williams et al.,

1995). The hydroxyl radical scavenging activity was deter-

mined according to the modified methods (Chung et al.,

1997; Nishikimi and Rao, 1972). The superoxide anion

scavenging activity was determined. Sodium nitroprusside

in aqueous solution at physiological pH spontaneously

produce nitric oxide, which reacts with oxygen to produce

nitrite ions, which can be determined by using the Griess

Illosvoy reaction (Garrat, 1964). Acute ulcerogenicity was

determined (Cioli et al., 1979) by evaluating after oral

administration of the test compounds (5c, 5d, and 5g) at an

equimolar dose relative to 200 mg/kg ibuprofen. All ulcer

[0.5 mm were counted and the overall total length was

designated as the ulcer index. Well diffusion method was

employed for the determination of antibacterial and anti-

fungal activity. Staphylococcus aureus (Gram ?ve) and

Eschericia coli (Gram -ve) are the bacteria used for the

antibacterial assay. Similarly Aspergillus was used for

the antifungal assay. But none of the compounds showed any

significant antibacterial or antifungal activity.

All the synthesized compounds 5 were tested for their

anti-inflammatory activity at an equimolar oral dose rela-

tive to 20 mg/kg diclofenac and ibuprofen. The compounds

showed anti-inflammatory activity ranging from 34 to 69%

(Table 1), whereas standard drugs diclofenac and ibupro-

fen showed 75 and 42% inhibition after 3 h, respectively.

Compounds 5a, 5d, 5f, and 5g displayed the highest anti-

inflammatory activity among the set of compounds tested

in this study. Test compounds that exhibited the most

potent anti-inflammatory activity were further evaluated

for their analgesic activity in mice. In general, the tested

compounds showed a better analgesic activity compared to

pentazocine as illustrated in Table 2.

CH3

CH3

CH3N

NN

NH2

SH

CH3

CH3

CH3

COOH

(1)

(3)

+

NH2 NH C

S

NH NH2

(2)

b

a

Scheme 1 Reagents and

conditions: (a) Microwave, 1, 2,

irradiation at 160 W, 4–5 min;

(b) Fusion, 1, 2, reflux with

stirring 4-5 h, 160�C

544 Med Chem Res (2012) 21:543–551

123

From the pharmacological activity results obtained, the

structure–activity relationship can be drawn for test com-

pounds 5a–g. The anti-inflammatory activity data showed

that the compound 5d having furyl group at position 6 of

triazolothiadiazole possesses highest overall activity.

Similarly tested compounds 5a, 5f, and 5g possessing

methyl, 4-bromophenyl, and 2-chlorophenyl, respectively,

showed excellent anti-inflammatory activity. Except com-

pound 5c all the tested compounds exhibited better anti-

inflammatory activity than the parent ibuprofen. The

compounds that showed better anti-inflammatory activity

were further tested for their analgesic activity at an equi-

molar oral dose relative to 10 mg/kg pentazocine. Com-

pounds showed analgesic activity ranging from 30.06 to

75.88%, whereas the standard drug pentazocine showed

77.34% and ibuprofen showed 30.04% inhibition after

1.5 h. It was noted that the compounds showing highest

anti-inflammatory activity also exhibited highest analgesic

activity. Among these compounds, 5e having 2-chloro-5-

nitrophenyl group at position 6 of triazolothiadiazole

showed activity (78.20%, P [ 0.05) more than that of

standard pentazocine, whereas compound 5g (75.88%,

N

N N

NH2

SH

CH3

CH3

CH3

+ R COOH

N

N N

CH3

CH3

CH3

N

S

R (3) (5)

a

R = CH3, C2H5, C3H7, 4-BrC6H5, 2-ClC6H4, 2-Cl-4-NO2C6H3,

O

b(4)

Scheme 2 Reagents and

conditions: (a) 3, 4, POCl3,

Microwave irradiation 160 W,

4–5 min; (b) 3, 4, POCl3, reflux

14–16 h

Table 1 Anti-inflammatory activity of ibuprofen derivatives against carrageenan-induced rat paw edema model in rats

Compounds Change in paw volume in milliliter ± SEMa (% inhibition)

0.5 h 1 h 1.5 h 2 h 2.5 h 3 h

Diclofenac 0.2 ± 2.38 (82)* 0.5 ± 1.01 (65)* 0.6 ± 2.56 (80)** 0.8 ± 4.18 (76) 1 ± 3.9 (73)* 1 ± 0.89 (75)**

Ibuprofen 0.88 ± 4.31 (20) 1.15 ± 2.67 (18)** 2.17 ± 2.69 (25)* 2.11 ± 3.74 (36)* 2.19 ± 4.25 (39) 2.26 ± 2.35 (42)*

5a 0.4 ± 1.13 (64) 0.8 ± 3.02 (43)* 1.1 ± 1.67 (62)* 1.4 ± 2.08 (58) 1.6 ± 3.27 (56) 1.5 ± 4.08 (62)*

5b 1.05 ± 3.22 (5) 1.1 ± 1.14 (22) 1.5 ± 2.14 (49)** 1.8 ± 4.41 (45) 2 ± 1.71 (44) 1.75 ± 2.19 (55)*

5c 1 ± 3.64 (10) 1.2 ± 1.79 (15)* 2.2 ± 1.74 (24) 2.4 ± 1.15 (28) 2.5 ± 4.99 (31)* 2.6 ± 4.19 (34)**

5d 0.8 ± 2.14 (28) 0.6 ± 1.64 (58)** 0.9 ± 3.37 (69) 1.2 ± 0.97 (64)* 1.15 ± 2.31 (68)* 1.3 ± 1.72 (67)*

5e 0.4 ± 0.87 (64) 0.8 ± 1.48 (43)* 1.25 ± 3.05 (57) 1.35 ± 1.54 (59)** 1.5 ± 4.11 (58)** 1.6 ± 3.13 (59)*

5f 2.0 ± 0.94 (–) 1.35 ± 1.71 (4) 1.2 ± 2.84 (59)* 1.1 ± 2.64 (67) 1.2 ± 5.14 (67)** 1.2 ± 4.28 (69)

5g 0.7 ± 3.16 (36)* 1.15 ± 3.26 (18)* 1.3 ± 1.44 (55) 1.35 ± 0.85 (59)* 1.4 ± 2.07 (61) 1.4 ± 1.56 (64)**

a Results are expressed as their mean values (n = 6)

* P \ 0.05, ** P \ 0.01; significant from the control

Table 2 Analgesic activity data of ibuprofen derivatives by tail-flick method

Compounds Before treatment ± SEMa After treatment ± SEMa (% increase in reaction time)

0.5 h 1.0 h 1.5 h

Pentazocine 1.81 ± 0.21 2.18 ± 0.14 (20.44) 2.54 ± 0.28 (40.33) 3.21 ± 0.16 (77.34)*

Ibuprofen 2.13 ± 0.26 2.38 ± 0.29 (11.73) 2.61 ± 0.38 (22.53) 2.77 ± 0.33 (30.04)*

5a 1.50 ± 0.21 1.90 ± 0.42 (26.66) 1.98 ± 0.14 (32.00) 2.14 ± 0.19 (42.66)**

5b 1.53 ± 0.17 1.64 ± 0.11 (7.18) 1.78 ± 0.45 (16.33) 1.99 ± 0.27 (30.06)*

5d 1.62 ± 0.25 1.98 ± 0.19 (22.22) 2.39 ± 0.23 (47.53) 2.65 ± 0.18 (63.58)**

5e 1.56 ± 0.15 1.86 ± 0.38 (19.23) 2.18 ± 0.48 (39.74) 2.78 ± 0.17 (78.20)*

5f 1.73 ± 0.23 2.03 ± 0.37 (17.34) 2.28 ± 0.33 (31.79) 2.45 ± 0.24 (41.61)*

5g 1.41 ± 0.27 1.54 ± 0.12 (9.21) 1.98 ± 0.21 (40.42) 2.48 ± 0.22 (75.88)*

a Results are expressed as their mean values (n = 6)

* P \ 0.05, ** P \ 0.01; significant from the control

Med Chem Res (2012) 21:543–551 545

123

P [ 0.05) and compound 5d (63.58%, P [ 0.01) showed

good analgesic activity. All the tested compounds exhibited

better analgesic activity than the starting compound ibu-

profen. Further comparison, is done by plotting bar dia-

gram for anti-inflammatory (Fig. 1) and analgesic activities

(Fig. 2).

Among the synthesized compounds used in the experi-

ment, compound 5c having propyl group at position 6

of triazolothiadiazole showed strong DPPH scavenging

activity compared to the standard (Table 3). At the con-

centration of 100 lg 5c and standard BHT exhibits 77.89

and 84.79% free radical scavenging activity, respectively.

Where as other compounds failed to scavenge DPPH rad-

ical. The scavenging ability of 5c on DPPH is almost

similar to scavenging activity of standard antioxidant BHT.

So this compound is further tested for in vitro antioxidant

studies like hydroxyl radical scavenging, superoxide anion

scavenging, and nitric oxide radical scavenging activity.

The results of these studies are summarized in Table 4.

Compound 5c exhibited significant hydroxyl radical scav-

enging (62.35%), superoxide anion scavenging (58.57%),

and nitric oxide radical scavenging (52.57%) properties at

100 lg concentration. A bar diagram comparing hydroxyl

radical scavenging, superoxide anion scavenging, nitric

oxide radical, and DPPH radical scavenging activities at

100 lg concentration are shown in Fig. 3.

Compounds which exhibited excellent antioxidant and

anti-inflammatory activities were further tested for their acute

ulcerogenic activity. The tested compounds showed low

ulcerogenic activity ranging from 8.54 ± 0.28 to 14.12 ±

1.90, compared to ibuprofen having high ulcer index

29.37 ± 0.54. The maximum reduction in ulcerogenicity

0

10

20

30

40

50

60

70

80

Diclo Ibu 5a 5b 5c 5d 5e 5f 5g

% in

hib

itio

n

1 Hour2 Hour3 Hour

Fig. 1 Anti-inflammatory activity data of compounds 5

0

10

20

30

40

50

60

70

80

90

Pent Ibu 5a 5b 5d 5e 5f 5g

% in

crea

se in

rea

ctio

n t

ime 0.5 Hour

1 Hour

1.5 Hour

Fig. 2 Analgesic activity data of compounds 5

Table 3 DPPH radical assay of triazolo[3,4-b]-1,3,4-thiadiazoles (5)

Compounds 5a 5b 5c 5d 5e 5f 5g BHT

% Radical scavenged at 100 lg/ml 7.90 5.61 77.89 20.61 4.46 1.71 23.02 84.79

Table 4 Percentage inhibition

of hydroxyl, superoxide anion,

and nitric oxide radical

scavenging activity of 3-[1-(4-

isobutylphenyl)ethy]-6-propyl-

1,2,4-triazolo[3,4-b]-1,3,4-

thiadiazole (5c)

Concentration (lg/ml) 5c (% inhibition)

Hydroxyl radical Superoxide anion Nitric oxide radical

20 26.13 ± 0.23 24.48 ± 0.37 14.27 ± 0.45

40 38.70 ± 0.65 36.36 ± 0.64 26.67 ± 0.54

60 44.23 ± 0.54 42.41 ± 0.36 38.27 ± 0.68

80 56.43 ± 0.34 52.95 ± 0.56 46.69 ± 0.44

100 62.35 ± 0.42 58.57 ± 0.46 52.57 ± 0.86

BHT (100 lg/ml) 68.26 ± 0.46 72.46 ± .48 62.24 ± .0.42

Fig. 3 Comparison of antioxidant properties of 5c to BHT at 100 lg/ml

concentration

546 Med Chem Res (2012) 21:543–551

123

(8.54 ± 0.28) was observed for compound 5c having pro-

pyl group attached to 6th position of triazolothiadiazole.

Other tested ibuprofen triazolothiadiazole derivatives 5d

(12.57 ± 0.47) and 5g (14.12 ± 1.90) also exhibited better

GI safety profile compared to the starting material ibuprofen

(Table 5).

Conclusion

A practical and high-yielding methodology to synthesize

ibuprofen triazole under microwave irradiation has been

developed for the first time. Herein we developed hybrid

molecules through the combination of different pharma-

cophores in one structure which lead to compounds with

increased pharmacological activity. We determined the

microwave-mediated conditions to obtain expected com-

pounds in good yields. Another advantage of microwave

method in the synthesis of triazolothiadiazole is the limited

use of hazardous phosphorous oxychloride compare to the

conventional method. The in-built heterocyclic ring sys-

tem of ibuprofen exhibited increased biological potency.

Compound 5c exhibited significant antioxidant properties.

It was interesting to note that compounds 5a, 5d, 5f, and 5g

were found to have significant anti-inflammatory properties

comparable with the standard drugs. Further these com-

pounds exhibited moderate to good analgesic activity. Most

of the derivatives exhibited better anti-inflammatory and

analgesic activities than the starting compound ibuprofen

with reduced ulcerogenic property.

Experimental

Melting points were determined in open capillary tubes and

are uncorrected. IR spectra were recorded by dispersing the

compounds in KBr pellets on a Shimadzu FT-IR 157

spectrophotometer. 1H NMR spectra were recorded on a

400 MHz Bruker Avance II NMR spectrometer and all the

chemical shift values were reported as d (ppm). 13C NMR

spectra were recorded on a Bruker Avance II 400 NMR

spectrometer. Mass spectra were recorded on a JEOL

SX-102 (FAB) mass spectrometer. Both microwave and

conventional methods are employed for the synthesis of

compounds 3 and 5a–g. Yield obtained from microwave

method is depicted first followed by conventional yield in

the characterization data.

Conventional one-pot method and microwave method

(Scheme 1)

An equimolar mixture of ibuprofen (0.01 mol) and thio-

carbohydrazide (0.01 mol) taken in a 100 ml r.b. flask were

heated on an oil bath till the contents melted. The reaction

mixture was continuously stirred and maintained at a

temperature of 160�C for further half an hour. It was

allowed to cool and treated with dilute sodium bicarbonate

solution in order to remove any unreacted acid left. The

solid was filtered, washed with water, dried, and recrys-

tallized from ethanol to obtain the pure triazole. Yield

62%; m.p. 150–152�C.

An equimolar mixture of ibuprofen (0.01 mol) and

thiocarbohydrazide (0.01 mol) were ground together to get

a uniform mixture. This was zapped inside a 100 ml beaker

and subjected to microwave irradiation on a household

microwave oven operating at 160 W for about 5 min. It

was allowed to cool and treated with dilute sodium bicar-

bonate solution in order to remove any un-reacted acid

left. The solid was filtered, washed with water, dried, and

recrystallized from ethanol to obtain the pure triazole.

Yield 68%; m.p. 150–152�C.

Conventional method and microwave method

(Scheme 2)

A mixture of triazole (3) (0.01 mol), substituted acids (4)

(0.01 mol), and phosphorus oxychloride (20 ml) was

heated for reflux on an oil bath for 14 h. The resulting

reaction mass was poured into crushed ice with stirring.

The solid thus obtained was filtered, washed with dilute

sodium bicarbonate solution, followed by water, dried, and

recrystallized from ethanol.

A mixture of triazole (3) (0.01 mol), substituted acids

(4) (0.01 mol), and phosphorus oxychloride (5 ml) taken in

a 100 ml r.b. flask was irradiated on a microwave oven

at 160 W for 3–5 min. The resulting reaction mass was

poured into crushed ice with stirring. The solid thus

obtained was filtered, washed with dilute sodium bicar-

bonate solution, followed by water, dried, and recrystal-

lized from ethanol.

Table 5 Acute ulcerogenic activity of compounds 5c, 5f, and 5i

Compounds 5c 5d 5g Ibuprofen

No. of Ulcers 5.32 ± 0.14** 5.513 ± 0.17** 7.38 ± 0.48** 15.41 ± 0.54

Ulcer index 8.54 ± 0.28** 12.57 ± 0.47** 14.12 ± 1.90** 29.37 ± 0.54**

The values are expressed in mean ± SEM and the P value ** P \ 0.01 is very significant

Med Chem Res (2012) 21:543–551 547

123

3-[1-(4-Isobutylphenyl)ethyl]-6-methyl-[1,2,4]-

triazolo[3,4-b]-[1,3,4]-thiadiazole (5a)

M.P. 124–26�C, Yield 54 and 48%, IR (KBr) c/cm-1: 2949

(C–H), 1570 (C=N); 1H NMR (CDCl3) d: 0.87 (d, 6H,

J = 6.40 Hz, (CH3)2), 1.77–1.82 (m, 1H, CH), 1.84 (d, 3H,

J = 7.20 Hz, CHCH3), 2.43 (d, 2H, J = 7.20 Hz, CH2–

Ar), 2.63 (s, 3H, CH3), 4.59 (q, 1H, CHCH3), 7.09 (d, 2H,

J = 8.00 Hz, ibu–Ar), 7.30 (d, 2H, J = 8.00 Hz, ibu–Ar);

FABMS (m/z, %): 461 (89), 302 (M? ? 2, 16), 301

(M? ? 1, 100). Anal. Calcd for C16H20N4S: C, 63.96; H,

6.71; N, 18.64; S, 10.67. Found: C, 63.89; H, 6.77; N,

18.59; S, 10.63.

6-Ethyl-3-[1-(4-isobutylphenyl)ethyl]-6-ethyl-[1,2,4]-

triazolo[3,4-b]-[1,3,4]-thiadiazole (5b)

M.P. 80�C, Yield 66 and 58%, IR (KBr) c/cm-1: 2956


J = 6.40 Hz, (CH3)2), 1.12–1.15 (t, 3H, CH3), 1.48–1.51

(q, 2H, CH2), 1.77 (d, 3H, J = 7.20 Hz, CHCH3),

1.77–1.86 (m, 1H, CH), 2.43 (d, 2H, J = 7.20 Hz, CH2–

Ar), 4.51 (q, 1H, CHCH3), 7.13 (d, 2H, J = 8.00 Hz, ibu–

Ar), 7.31 (d, 2H, J = 8.00 Hz, ibu–Ar); FABMS (m/z, %):

315 (M? ? 1, 100), 314 (M?, 57.1), 161 (14.6). Anal.

Calcd for C17H22N4S: C, 64.93; H, 7.05; N, 17.81; S,

10.19. Found: C, 64.87; H, 7.02; N, 17.78; S, 10.21.

3-[1-(4-Isobutylphenyl)ethyl]-6-propyl-[1,2,4]-

triazolo[3,4-b]-[1,3,4]-thiadiazole (5c)



J = 6.40 Hz, (CH3)2), 0.87–0.96 (m, 2H, CH2), 1.57–1.59

(t, 2H, CH2), 1.74 (d, 3H, J = 7.20 Hz, CHCH3),

1.76–1.81 (m, 1H, CH), 2.39 (d, 2H, J = 7.00 Hz, CH2–

Ar), 3.32 (s, 3H, CH3), 4.10 (q, 1H, CHCH3), 7.11 (d, 2H,

J = 8.08 Hz, ibu–Ar), 7.21 (s, 2H, J = 8.08 Hz, ibu–Ar);

FABMS (m/z, %): 329 (M? ? 1, 22.1), 328 (M?, 100), 272

(14.3), 161 (27.6). Anal. Calcd for C18H24N4S: C, 65.81;

H, 7.36; N, 17.05; S, 9.76. Found: C, 65.77; H, 7.33; N,

17.11; S, 9.72.

6-(2-Furyl)-3-[1-(4-isobutylphenyl)ethyl]-[1,2,4]-

triazolo[3,4-b]-[1,3,4]-thiadiazole (5d)

M.P. 118–19�C, Yield 89 and 82%, IR (KBr) c/cm-1: 2968


J = 6.60 Hz, (CH3)2), 1.79–1.85 (m, 1H, CH), 1.89 (d, 3H,

J = 7.32 Hz, CHCH3), 2.43 (d, 2H, J = 7.16 Hz, CH2–

Ar), 4.66 (q, 1H, CHCH3), 6.62 (dd, 1H, Furyl), 7.10 (d,

2H, J = 8.12 Hz, ibu–Ar), 7.13 (d, 1H, Furyl), 7.34 (d, 2H,

J = 8.08 Hz, ibu–Ar), 7.63 (d, 1H, Furyl); FABMS (m/z,

%): 354 (M? ? 2, 34.9), 353 (M? ? 1, 100), 352 (M?,

10.5), 161 (97.7). Anal. Calcd for C19H20N4SO: C, 64.74;

H, 5.71; N, 15.89; S, 9.09. Found: C, 64.68; H, 5.69; N,

15.92; S, 9.07.

6-(2-Chloro-5-nitrophenyl)-3-[1-(4-isobutylphenyl)ethyl]-

[1,2,4]-triazolo[3,4-b]-[1,3,4]-thiadiazole (5e)



J = 6.60 Hz, (CH3)2), 1.79–1.86 (m, 1H, CH), 1.94 (d, 3H,

J = 7.28 Hz, CHCH3), 2.44 (d, 2H, J = 7.16 Hz, CH2–


Ar), 7.36 (d, 2H, J = 8.08 Hz, ibu–Ar), 7.76 (d, 1H,

J = 8.84 Hz, Ar), 8.35 (d, 1H, J = 8.88 Hz, Ar). 8.78 (s,

1H, Ar); FABMS (m/z, %): 443 (M? ? 2, 66.1), 442

(M? ? 1, 100), 441 (M?, 28.1), 406 (52.1), 161 (61.9).

Anal. Calcd for C21H20N5SO2Cl: C, 57.07; H, 4.56; N,

15.84; S, 7.25. Found: C, 56.98; H, 4.54; N, 15.80; S, 7.22.

6-(4-Bromophenyl)-3-[1-(4-isobutylphenyl)ethyl]-[1,2,4]-

triazolo[3,4-b]-[1,3,4]-thiadiazole (5f)



J = 6.64 Hz, (CH3)2), 1.76–1.80 (m, 1H, CH), 2.05 (d, 3H,

J = 7.24 Hz, CHCH3), 2.51 (d, 2H, J = 7.16 Hz, CH2–


Ar), 7.30 (d, 2H, J = 8.00 Hz, ibu–Ar), 7.83 (d, 1H,

J = 8.92 Hz, Ar), 7.86 (d, 1H, J = 8.88 Hz, Ar); 13C

NMR (CDCl3) d: 20.11, 22.61, 30.02, 36.29, 44.62, 127.52,

128.66, 129.35, 129.64, 133.13, 139.21, 140.29, 150.61,

153.27, 165.52; FABMS (m/z, %): 442 (M? ? 2, 82.9),

440 (M?, 100), 161 (37.3). Anal. Calcd for C21H21N4SBr:

C, 57.14; H, 4.79; N, 12.69; S, 7.26. Found: C, 57.08; H,

4.81; N, 12.65; S, 7.23.

6-(2-Chlorophenyl)-3-[1-(4-isobutylphenyl)ethyl]-[1,2,4]-

triazolo[3,4-b]-[1,3,4]-thiadiazole (5g)



J = 6.60 Hz, (CH3)2), 1.79–1.86 (m, 1H, CH), 1.93 (d, 3H,

J = 7.28 Hz, CHCH3), 2.44 (d, 2H, J = 7.20 Hz, CH2–


Ar), 7.36 (d, 2H, J = 8.08 Hz, ibu–Ar), 7.40–7.44 (t, 1H,

Ar), 7.47–7.51 (t, 1H, Ar), 7.56 (d, 1H, J = 8.04 Hz, Ar),

7.88 (d, 1H, J = 7.82 Hz, Ar); FABMS (m/z, %): 398

(M? ? 2, 60), 397 (M? ? 1, 100), 161 (43). Anal. Calcd

for C21H21N4SCl: C, 63.54; H, 5.33; N, 14.11; S, 8.07.

Found: C, 63.48; H, 5.31; N, 14.08; S, 8.05.

548 Med Chem Res (2012) 21:543–551

123

Anti-inflammatory activity

Anti-inflammatory activity was determined by the carra-

geenan-induced paw edema method in Wistar albino rats

by using plethysmography following the method of Winter

et al. Diclofenac at an oral dose of 20 mg/kg served as

the standard drug for comparison. The test compounds

(20 mg/kg) were administered 30 min prior to adminis-

tration of carrageenan (0.1 ml of 1% w/v) in the plantar

region of the paw. The paw volumes were measured at 1, 2,

and 3 h after carrageenan administration. The percentage

of inhibition was calculated by using the following equa-

tions and the results are presented in Table 1. All the

pharmacological data are expressed as mean ± SEM.

% Inhibition ¼ 1� Vt

Vc

� �� 100

where Vt average edema volume of test compounds; Vc

average edema volume of control.

Analgesic activity

Analgesic activities were evaluated on Swiss albino mice

by tail-flick method. In this method heat is used as a source

of pain. A dose level of 10 mg/kg pentazocine and ibu-

profen served as the standard drugs for comparison. The

responses produced in the animal were observed for 30 min

intervals and percentage in reaction time was calculated for

analgesic activity, from this percentage increase in reaction

time (as index of analgesia) at each time interval is cal-

culated using the following equation and the results are

presented in Table 2.

% Increase in reaction time ¼ RTAT

RTBT� 1

� �� 100

where RTAT reaction time after treatment; RTBT reaction

time before treatment.

Antioxidant activity

The in vitro antioxidative potential for some of synthesized

compounds were studied and the results were compared

with a standard antioxidant butylated hydroxytoluene

(BHT). The DPPH (1,1-diphenyl-2-picrylhydrazyl) assay is

used as a preliminary test to investigate the antioxidant

potential of the synthesized compounds (Table 4). Com-

pound showing better DPPH free radical scavenging

property was investigated for further in vitro antioxidant

studies like hydroxyl radical scavenging, superoxide anion

scavenging activity and nitric oxide radical scavenging

activity. The DPPH free radical scavenging activity of the

compounds was determined by using the modified method

of Brand-Williams et al. The hydroxyl radical scavenging

activity was determined according to the modified method

of Chung et al. Using the method of Nishikimi et al. the

superoxide anion scavenging activity was determined.

Sodium nitroprusside in aqueous solution at physiological

pH spontaneously produce nitric oxide, which reacts with

oxygen to produce nitrite ions, which can be determined by

using the Griess Illosvoy reaction.

DPPH radical scavenging assay

The DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical

scavenging activity of all the triazolothiadiazoles (5a–g)

was determined. Freshly prepared 2 ml of 0.1 mM DPPH

solution in methanol was added to 1 ml of methanolic

solution of synthesized compounds (100 lg/ml), taken in

different test tubes and the volume was adjusted using

methanol. It was mixed and vortexed thoroughly and left in

dark for 30 min. The absorbance of stable DPPH was mea-

sured at 517 nm. The DPPH control was prepared using the

same procedure by replacing the sample by methanol. Rad-

ical scavenging activity was expressed as the inhibition

percentage and was calculated using the equation of DPPH

radical scavenging activity.

% Radical scavenged ¼ Ac � Atð ÞAc

� 100

where Ac absorbance of the control; At absorbance of the

test compounds.

Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity was determined

according to the modified method of Chung et al. The Fenton

reaction mixture containing 200 ll of 10 mM FeSO4�7H2O,

200 ll of 10 mM EDTA, and 200 ll of 10 mM 2-deoxyri-

bose was mixed with 1.2 ml of 0.1 M phosphate buffer (pH

7.4) containing 100 lg/ml concentration of 5c. Freshly

prepared 200 ll of 10 mM H2O2 was added to the mixture

and incubated for 4 h at 37�C. Later, 1 ml of 2.8% TCA and

1 ml of 1% TBA were added and placed in boiling water

bath for 10 min. The mixture was brought to room temper-

ature and centrifuged at 2000 rpm for 5 min and absorbance

was measured at 532 nm. The percentage of hydroxyl rad-

ical scavenging activity was calculated by employing the

following formula and compared with the standard BHT.

% Hydroxyl radical scavenging

¼ Absorbance of sample

Absorbance of blank� 100

Superoxide anion scavenging activity

Using the method of Nishikimi et al. the superoxide anion

scavenging activity was determined, wherein a mixture of

Med Chem Res (2012) 21:543–551 549

123

1 ml of NBT (156 lM NBT in 100 mM phosphate buffer,

pH 7.4), 1 ml NADH (468 lM in 100 mM phosphate

buffer, pH 7.4), and 100 lg/ml concentration of 5c was

prepared. To this mixture 100 ll of PMS solution (60 lM

PMS in 100 mM phosphate buffer, pH 7.4) was added to

start the reaction. The reaction mixture was incubated at

25�C for 5 min, and the absorbance was measured at

560 nm against blank. Decreased absorbance of the reac-

tion mixture indicated increased superoxide anion scav-

enging activity. BHT was used as standard.

Nitric oxide radical scavenging activity

Sodium nitroprusside in aqueous solution at physiological

pH spontaneously produce nitric oxide, which reacts with

oxygen to produce nitrite ions, which can be determined by

using the Griess Illosvoy reaction. Griess Illosvoy reagent

was slightly modified using naphthylethylenediamine dihy-

drochloride (0.1% w/v) instead of 1-naphthylamine (5%).

The reaction mixture (3 ml) containing 2 ml of 10 mM

sodium nitroprusside, 0.5 ml of phosphate buffer saline (pH

7.4, 0.01 M), and 0.5 ml of 5c was incubated for 150 min at

25�C. The reaction mixture was mixed with 1 ml of sulph-

anilic acid reagent (0.33% in 20% glacial acetic acid) and

allowed to stand for 5 min for the completion of diazotiza-

tion reaction. The resultant mixture was then added with

1 ml of naphthylethylenediamine dihydrochloride (0.1%)

and allowed to stand for 30 min in diffused light. The

absorbance of the pink colored chromophore was measured

at 540 nm against the corresponding blank solution. Scav-

enging capacity of 100 lg/ml concentration of 5c was

compared with standard drug BHT.

Ulcerogenic activity

Acute ulcerogenicity was determined according to Cioli

et al. Animals were divided into different groups consisting

of six animals in each group. Compounds showing good

anti-inflammatory were selected and tested for ulcerogenic

activity. Ulcerogenic activity was evaluated after oral

administration of the test compounds (5c, 5d, and 5g) at an

equimolar dose relative to 200 mg/kg ibuprofen. Control

group received only 0.5% CMC solution. Food, but not

water, was removed 24 h before administration of the test

compounds. After the administration, the rats were fed with

normal diet for 17 h and then killed. The stomach was

removed and opened along the greater curvature, washed

with distilled water and cleaned gently by dipping in nor-

mal saline. The mucosal damage was examined by means

of a magnifying lens. All ulcer[0.5 mm were counted and

the overall total length was designated as the ulcer index

(Table 5).

Acknowledgments The authors are thankful to UGC for the

financial assistance and to the Head, SAIF, CDRI, Lucknow for

providing mass and 1H NMR spectral data.

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Microwave-mediated synthesis of triazolothiadiazoles as anti-inflammatory, analgesic, and anti-oxidant agents

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