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. 2013 Feb 7;3(1):13–18. doi: 10.5681/apb.2013.003

Analgesic Activity of Some 1,2,4-Triazole Heterocycles Clubbed with Pyrazole, Tetrazole, Isoxazole and Pyrimidine

Shantaram Gajanan Khanage 1,*, Appala Raju 2, Popat Baban Mohite 3, Ramdas Bhanudas Pandhare 4
PMCID: PMC3846043  PMID: 24312806

Abstract

Purpose: In the present study in vivo analgesic activity of some previously synthesized 1,2,4-triazole derivatives containing pyrazole, tetrazole, isoxazole and pyrimidine ring have been evaluated. Methods: Acetic acid induced writhing method and Hot plate method has been described to study analgesic activity of some 1,2,4-triazole derivatives containing pyrazole, tetrazole, isoxazole and pyrimidine as a pharmacological active lead. Results: Thirty six different derivatives containing 1,2,4-triazole ring were subjected to study their in vivo analgesic activity. Chloro, nitro and methoxy, hydroxy and bromo substituted derivatives showed excellent analgesic activity and dimethylamino, furan and phenyl substituted derivatives showed moderate analgesic activity in both of the methods. Compounds IIIa, IIId, IIIf, IIIi, IIIj, IVa, IVb, IVd, IVf, IVh, IVj IV3a and IIj were found to be superior analgesic agents after screening by Acetic acid induced writhing method. Compounds IIIb, IIId, IIIf, IIIh, IIIj, IVa, IVb, IVd, IVf, IVh, IVi, IV3c, IV3e and IIj were showed analgesic potential after screening of Hot plate method. Conclusion: All tested compounds containing 1,2,4-triazole were found to be promising analgesic agents, for this activity pyrazole, tetrazole, isoxazole and pyrimidine leads might be supported.

Keywords: Triazole, Analgesic Activity, Pyrazole, Tetrazole, Isoxazole, Pyrimidine

Introduction

Analgesic and anti-inflammatory drugs are one of the most valuable medicaments that used in many of disease for relief of pain and inflammation. Most analgesic and anti-inflammatory drugs available in the market, still present a wide range of many problems such as efficacy and undesired effects including GIT disorders and other unwanted effects,1 that limit their clinical usefulness and remain to be solved and leaving an open door for new and better compounds.2 This situation highlights the need for advent of safe, novel and effective analgesic and anti-inflammatory compounds.3 1,2,4-triazole received sheer attention of medicinal chemists because of their many therapeutic applications like anticancer,4,5 antimicrobial,6-9 anticonvulsant,10 anti-inflammatory, analgesic,11 antidepressant,12 antitubercular,13 antimalarial14 and hypoglycemic15 activities.

We have reported that 5-methyl-2-[(5-substituted aryl-4H-1,2,4-triazol-3-yl)methyl]-2,4-dihydro-3H-pyrazol-3-one and 5-phenyl-1-[(5-substituted aryl-4H-1,2,4-triazol-3-yl)methyl]-1H-tetrazole had significant anticancer activity specially on renal cancer cell lines (UO-31) as well in vitro antibacterial activity against gram positive bacterial S. aureus NCIM 2079, B. subtillis NCIM 2063 and gram negative bacterial E. coli NCIM 2065, P. aeruginosa NCIM 2863 strains.16 More recently we have reported antimicrobial, antitubercular and anticancer activity of 1-[5-(substituted aryl)-1,2-oxazol-3-yl]-3,5-diphenyl-1H-1,2,4-triazole (4a-g).17 In continuation of our previous work18-20in this article the attempts have been made to explore the analgesic potential of some formerly synthesized 1,2,4-triazoles clubbed with pyrazole, tetrazole, isoxazole and pyrimidine heterocycles.

Materials and Methods

The standard analgesic drugs Ibuprofen and Pentazocine, solvents used for the experimental work were commercially procured from E. Merck India and Qualigens India. Swiss strain albino mice for study were procured from National Toxicology Center, Pune.

Evaluation of analgesic activity

Study protocol was approved by the Institutional Animal Ethics Committee for the purpose of control and supervision of experiments on animals (IAEC, Approval No.1211/ac/08/CPCSEA) before experiment. Swiss strain albino mice of either sex weighing 25–30 g were used for this study. The test compounds were administered intraperitoneally in 10% v/v Tween 80 suspension.

Acute toxicity study

The acute toxicity for the test compounds was determined by the Miller and Tainter method administering the compounds intraperitoneally. LD50 of the test compounds calculated by Miller and Tainter (1944) method,21 initially least tolerated (smallest) dose (100% mortality) and most tolerated (highest) dose (0% mortality) were determined by hit and trial method. After determination of two doses we have selected five doses in between the least tolerated and most tolerated doses were given intraperitoneally to 5 groups of mice, 10 animals in each group. The animals were observed for first 2 hours and then at 6th and 24th hour for any toxic symptoms. After 24 hours, the number of deceased animals was counted in each group and percentage of mortality calculated. From the obtained data determined the LD50of the test compounds by using probit value transformations.

Acetic acid induced writhing method (Abdominal Constriction Test)

The animals were divided into 38 groups of six mice each. The control group of animals was administered with 10% v/v Tween 80 (0.5 ml) suspension. The animals of another group were injected intraperitoneally with standard drug Ibuprofen (10 mg/kg). After 20 min of the administration the test compounds, all the groups of mice were given with the writhing agent 3% v/v aqueous acetic acid in a dose of 2 ml/kg intraperitoneally. The writhing produced in these animals was counted visually for 15 min and the numbers of writhings produced in treated groups were compared with control group. The results of analgesic activity are recorded in Table 1. Analgesic activity in percent was calculated by using following formula.

Table 1. Evaluation of analgesic activity by acetic acid induced writhing method.

Sr. No. Treatment Dose mg/kg Writhing episodes in 15 min (Mean ± S.E.M.) Percent protection
1 Control -- 38.70±0.5547 -
2 Ibuprofen 10 13.19±0.6921** 66
3 IIIa 50 17.28±0.6647** 55
4 IIIb 50 18.24±0.6425** 53
5 IIIc 50 24.89±0.5354** 36
6 IIId 50 15.65±0.7845** 60
7 IIIe 50 26.45±0.8121** 32
8 IIIf 50 16.46±0.5744** 57
9 IIIg 50 27.65±0.5478** 29
10 IIIh 50 18.57±0.4545** 52
11 IIIi 50 16.49±0.6545** 57
12 IIIj 50 16.47±0.2254** 57
13 IVa 50 16.45±0.5456** 57
14 IVb 50 17.49±0.5641** 55
15 IVc 50 21.73±0.5974** 44
16 IVd 50 15.44±0.6133** 60
17 IVe 50 22.45±0.5525** 42
18 IVf 50 15.36±0.6455** 60
19 IVg 50 19.65±0.7322** 49
20 IVh 50 17.22±0.4855** 56
21 IVi 50 19.47±0.6513** 50
22 IVj 50 14.24±0.4745** 63
23 IV3a 100 15.44±0.4454** 60
24 IV3b 100 22.65±0.5546** 42
25 IV3c 100 19.47±0.5941** 50
26 IV3d 100 21.77±0.4423** 44
27 IV3e 100 25.49±0.4473** 34
28 IV3f 100 19.42±0.6651** 50
29 IV3g 100 26.56±0.4329** 31
30 IV3h 100 28.56±0.7325** 26
31 IV3i 100 23.58±0.6423** 39
32 IV3j 100 19.26±0.4523** 50
33 Ih 25 20.54±0.6425** 47
34 Ii 25 22.89±0.5214** 41
35 Ij 25 18.34±0.4527** 53
36 IIh 25 20.57±0.8592** 47
37 IIi 25 22.44±0.6854** 42
38 IIj 25 15.47±0.8957** 60
** P < 0.01 represent significant difference when compared with control groups.
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Protection = 100-[{(No. of writhes in treated mice)/(No. of writhes in untreated mice)}×100]

Hot plate method

The method of Eddy and Leimbach was adopted for the study. The temperature of a metal surface in the hot plate test was set at 55±1.0ºC. The time taken by the animals to lick the fore or hind paw or jump out of the place was taken as the reaction time. Latency to the licking paws or jumping from plate was determined before and after treatment. The latency was recorded at the time of 0 (just before any treatment) and 15, 30 and 60 min after intraperitoneal administration of test compounds. A latency period of 15 sec was defined as complete analgesia as cut off time to prevent damage to mice. The reference compound Pentazocine was administered in a dose of 5 mg/kg. The time course of hot plate latency was expressed as the percentage of the maximum possible effect (%MPE) according to the following formula:

%MPE=[(post drug latency) - (pre-drug latency) /(cut-off time) - (pre-drug latency)]×100

After the treatment of test and reference compounds, the pain thresholds of the animals were observed and presented in Table 2.

Table 2. Evaluation of analgesic activity by Hot plate method.

Treatment Average Reaction Time in seconds before treatment (Mean ± S.E.M.) Reaction time in seconds after treatment (Mean ± S.E.M.) %MPE
15 min 30 min 60min
Control 4.75±0.1547 4.75±0.1583 4.75±0.2563 4.75±0.4941 -
Pentazocine 4.70±0.4012** 7.70±0.5211** 9.67±0.4302** 11.81±0.3254** 69.02
IIIa 4.56±0.5221** 7.23±0.6326** 9.23±0.5745** 10.76±0.4369** 59.38
IIIb 4.63±0.4785** 7.49±0.2234** 9.87±0.3865** 11.15±0.5356** 62.87
IIIc 4.67±0.4523** 6.52±0.6542** 9.34±0.5413** 9.53±0.4356** 47.04
IIId 4.63±0.6374** 7.46±0.7585** 9.52±0.2658** 10.47±0.7541** 56.31
IIIe 4.69±0.4474** 6.45±0.5428** 8.36±0.6756** 9.27 ±0.5854** 44.42
IIIf 4.65±0.4469** 7.42±0.3769** 9.49±0.5369** 10.71±0.5775** 58.55
IIIg 4.56±0.5854** 6.70±0.4459** 8.49±0.7474** 9.27±0.6525** 45.11
IIIh 4.43±0.3544** 7.50±0.7585** 9.57±0.6456** 10.52 ±0.6785** 57.61
IIIi 4.71±0.4675** 6.39±0.3646** 9.03±0.5236** 9.46±0.5359** 46.16
IIIj 4.68±0.7548** 7.56±0.5636** 9.52±0.6552** 11.37±0.6426** 64.82
IVa 4.72±0.5957** 7.68±0.4523** 9.56±0.3356** 11.09±0.5517** 61.96
IVb 4.67±0.4358** 7.45±0.6984** 9.16±0.3548** 10.59±0.5478** 57.30
IVc 4.55±0.4785** 6.59±0.4578** 9.44±0.5349** 9.69±0.8689** 49.18
IVd 4.58±0.5878** 7.66±0.6689** 9.50±0.4245** 11.56±0.6741** 66.98
IVe 4.39±0.4589** 6.89±0.7481** 8.59±0.6478** 9.75 ±0.4589** 50.51
IVf 4.66±0.5958** 7.53±0.3549** 9.80±0.3358** 11.61±0.6358** 67.21
IVg 4.45±0.5869** 7.72±0.5269** 9.58±0.5895** 10.46±0.5692** 56.96
IVh 4.41±0.5321** 7.80±0.4781** 9.57±0.5696** 10.82 ±0.3324** 60.52
IVi 4.64±0.2966** 7.56±0.3826** 9.45±0.7851** 10.89±0.5369** 60.32
IVj 4.52±0.2853** 7.70±0.5239** 9.43±0.4665** 10.44±0.6745 ** 56.48
IV3a 4.64±0.4789** 7.34±0.3545** 9.12±0.3456** 10.12±0.2985** 52.89
IV3b 4.56±0.5884** 7.11±0.5789** 8.36±0.6398** 9.47±0.6489** 47.03
IV3c 4.67±0.3956** 7.49±0.5212** 9.27±0.3369** 10.76±0.6245** 58.95
IV3d 4.73±0.4525** 7.13±0.2845** 9.10±0.3235** 9.46±0.5469** 46.05
IV3e 4.59±0.5823** 6.93±0.5861** 9.14±0.6478** 10.74 ±0.4369** 59.07
IV3f 4.54±0.4969** 7.23±0.5326** 9.50±0.3756** 10.32±0.6542** 55.25
IV3g 4.49±0.5478** 7.02±0.4689** 8.48±0.5225** 8.93±0.5692** 42.24
IV3h 4.61±0.4526** 6.80±0.4369** 8.55±0.5236** 9.51 ±0.5236** 47.16
IV3i 4.67±0.6325** 6.56±0.3989** 8.47±0.5364** 9.37±0.6346** 45.49
IV3j 4.71±0.5963** 7.31±0.2845** 9.46±0.3623** 10.20±0.4126 ** 53.35
Ih 4.46±0.6522** 7.25±0.4545** 8.55±0.2625** 9.88±0.4121** 51.18
Ii 4.29±0.3532** 7.45±0.4666** 9.12±0.3644** 9.83±0.5481** 51.72
Ij 4.34±0.5418** 7.75±0.6641** 9.85±0.4236** 10.24±0.3678** 55.34
IIh 4.54±0.6645** 6.96±0.4425** 8.86±0.3356** 9.87±0.4356** 50.95
Iii 4.59±0.5414** 6.45±0.3784** 8.22±0.3541** 9.45±0.4678** 46.68
IIj 4.74±0.4775** 7.35±0.4125** 9.45±0.5925** 10.96±0.6486** 60.62
Dose: 25 mg/kg for compounds Ih-Ij, 50mg/kg for IIIa-j and IVa-j, 100mg/kg for IV3a-j and 5mg/Kg for Pentazocine. ** p<0.01 represent the significant difference when compared control group.
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Statistical analysis

Data were presented as arithmetic mean±SEM. Statistical analysis was performed by one way variance (ANOVA) followed by Dunnett’s test. ‘‘p’’ value of less than 0.05 was considered as statistically significant.

Results and Discussion

Acetic acid induced writhing method

Abdominal construction responses induced by acetic acid is a sensitive procedure to establish efficacy of peripherally acting analgesics, it may causes increase in the level of PGE2 and PGF2a by intraperitoneally administration of acetic acid. The analgesic activity was expressed as percentage of protection. All tested compounds exhibited activity in a dose range of 25-100 mg/kg. Writhing episodes and percent protection of tested compounds for analgesic activity are summarized in Table 1. Compound IIIa, IIId, IIIf, IIIi, IIIj, IVa, IVb, IVd, IVf, IVh, IVj IV3a and IIj (Figure 1 and 2) were found to be superior analgesic agents with 55, 60, 57, 57, 57, 57, 55, 60, 60, 56, 63, 60 and 60% analgesic activity respectively as compared to other tested compounds. Chloro, nitro, methoxy, hydroxy and bromo substituted derivatives exhibited excellent analgesic activity where as dimethylamino, furan and phenyl substituted derivatives showed moderate analgesic activity. Substituted phenyl ring present on isoxazole, pyrimidine and 1,2,4-triazole nucleus might have attributed crucial role to show analgesic activity. Compounds with substitution of chloro, methoxy, bromo on 4th position of phenyl ring present on isoxazole, pyrimidine and triazole nucleus showed maximum analgesic activity. Compounds IIId, IVd, IVf, IVj, IV3a and IIj were exhibited comparative analgesic property (up to 60% protection) with standard drug Ibuprofen (66%) as illustrated in Table 1. because of 4-methoxy, 4-chloro, 2-chloro, 2,4-dimethoxy groups present on phenyl ring of pharmacologically active isoxazole, pyrimidine, and triazole chalcone heterocycles.

Figure 1.

Figure 1.

Structures of compounds of scheme III, IV and IV3.

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Figure 2.

Figure 2.

Structures of compounds of scheme I and II.

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Hot plate method

All compounds tested by Eddy’s hot plate method exhibited activity in a dose range of 25-100 mg/kg. The analgesic activity measured by central analgesia. Pentazocine 5 mg/kg significantly increased the hot plate latency producing a highest %MPE at 69.02. Compounds IIIb, IIIj, IVa, IVd, IVf, IVh, IVi, and IIj significantly increased the hot plate latency when compared to the control group. The highest antinociception induced by compounds IIIj, IVd and IVf at dose of 50 mg/kg were observed with 64.82, 66.98 and 67.21 %MPE respectively. The analgesic activity of compounds IIIb, IIId, IIIf, IIIh, IIIj, IVa, IVb, IVd, IVf, IVh, IVi, IV3c, IV3e and IIj were comparable to Pentazocine after 15, 30 and 60 min. 2-chloro, 3-nitro, 4-chloro, 4-methoxy, 4-bromo, 4-hydroxy and 2,4-dimethoxy substituted analogs exhibited dynamic analgesic activity. 4-dimethyl amino and 2-furyl substituted compound of tested series of 1-(3,5-diphenyl-1H-1,2,4-triazol-1-yl)-3-(substituted aryl) prop-2-en-1-one (Chalcones) were also acquired higher hot plate latency with 10.76 and 10.74 sec respectively after 60 min. Compounds from series of 1-[5-(substituted aryl)-1,2-oxazol-3-yl]- 3,5-diphenyl-1H-1,2,4-triazoles and 6-(substituted aryl)-4-(3,5-diphenyl-1H-1,2,4-triazol-1-yl)-1,6-dihydropyrimidine-2-thiol obtained excellent analgesic potential.

Conclusion

The results of the present investigation reveals that the increase in analgesic activity is attributed to the presence of 2-chloro, 3-nitro, 4-chloro, 4-methoxy, 4-bromo, 4-hydroxy and 2,4-dimethoxy groups on phenyl ring of isoxazole, pyrimidine, pyrazole, tetrazole and triazole. All tested heterocycles posses central and peripheral analgesic property. Perceptibly the comparative evaluation of active compounds will required in the further studies, the data reported in this article may be helpful guide for the medicinal chemist who is working in this area.

Acknowledgments

Authors are highly thankful to National Toxicology Center, Pune, for providing animals to caring out analgesic activity and acute toxicity study. We are also grateful of Principal M.E.S. College Pharmacy, Sonai and Prashant Patil Gadakh, Secreatary, Mula Education Society for providing excellent research facilities for this work.

Conflict of interest

All the authors report no conflicts of interest.

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