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A novel series of 4-Substituted benzylamino-5-Substituted Phenyl-3-amino-1, 2, 4-triazole derivatives were synthesized by reaction of 5-Substituted Phenyl-3,4-diamino-1,2,4-triazole with different aromatic aldehydes. The structures of the compounds were synthesized and characterised by elemental analysis, 1H NMR, Mass and IR Spectra. The title compounds were investigated for anti-inflammatory and analgesic activities using carrageenan induced rat paw edema and acetic induced writhing test respectively. All the test compounds exhibited significant activity, compounds IVg, IVh and IVb showed more potent anti-inflammatory activity when compared with standard (Ibuprofen). All the test compounds were significantly (p<0.05) reduced writhings induced by the acetic acid in mice.
Over the years Non-Steroidal anti-inflammatory (NSAIDS) are among the widely used for the treatment of chronic pain and inflammation in arthritis. The mechanism of action of NSAIDS is the inhibition of prostaglandin synthesis by blocking the metabolism of arachadonic acid by cyclooxygenase enzyme(1). Several agents are known to treat inflammatory disorders; their prolonged use often leads to gastric intolerance, bone marrow depression, water and salt retention(2). There are two types of COX enzyme, namely COX-1 and COX-2. COX-1 is found in most cells as constitutive enzyme and is involved in normal homeostasis. COX-2 is induced in inflammatory cells by an inflammatory stimulus(3). Long term inhibition of COX could result in a shunt to the 5-lipooxygenase (LOX) pathway leading to the formation of leukotrienes, which can induce gastric lesions and ulceration(4). Recent reports evidence that coxibs can lead to adverse cardiovascular effects(5). The carrageenin-induced rat hind paw edema test is a common model for evaluating anti-inflammatory drugs. There a good correlation between efficacy in this model and activity in humans(6).
Therefore, although there are a number of anti-inflammatory and analgesic drugs available in the market, development of novel compounds having anti-inflammatory-analgesic activity with improved profile is still a necessity.
Various 1, 2, 4-triazole derivatives have been reported to possess diverse types of biological properties such as antibacterial(7), antimycobacterium agents(8), antifungal(9), anti-inflammatory(10), anticonvulsant(11), antiviral(12), tyrosine kinase inhibitor(13) , and as a serotonin receptor ligands(14)
Over the years our interest has focused on the synthesis of novel heterocyclic systems which have analgesic and anti-inflammatory activity. We have synthesized various 4-(Substituted benzylamino)-5-Substituted Phenyl-3-amino-1,2,4-triazole Derivatives and examined for their anti-inflammatory and analgesic activity. A considerable number of the prepared compounds have anti-inflammatory-analgesic activity comparable with the reference compounds.
The solvents AR grades were obtained from Sd Fine Chem., Mumbai, and E.Merck, Mumbai. Melting points were determined in open capillaries, using Toshniwal melting point apparatus, expressed in oC and are uncorrected. The IR spectra of the compounds were recorded on thermo Nicolet Nexus 670S series, FT-IR spectrometer using KBr disc.1H NMR were recorded on a Avance-300 MHz instrument using TMS as an internal standard(chemical shifts in Î´,ppm) 13C NMR spectra are recorded at 100 MHZ . The purity of the compounds was checked on silica gel-coated aluminum sheets (Merck, 1.005554, silica gel HF254-361,Type 60, 0.25 mm, Darmstadt, Germany) by thin-layer chromatography
Preparation of Benzaldehyde/p. chlorobenzaldehyde Semicarbazones (I)
A mixture of Semicarbazide hydrochloride (0.02mol) and anhydrous sodium acetate (0.01mol) in 10 ml of water. This was warmed gently till a clear solution was obtained. A solution of benzaldehyde/p-chlorobenzaldehyde (0.01 mol) in 5 ml of rectified spirit was added drop wise to the above mixture while stirring vigorously. It was warmed gently and stirring was continued for 15 minutes. The reaction mixture was cooled, filtered and the product was washed with cold water. Finally the compound was recrystallized and dried.
Adopting this procedure benzaldehyde semicarbazone (Ia, R=H), m.p. 176-178 oC yield 86% and p-chlorobenzaldehyde semicarbazone (Ib, R = 4-Cl), m.p. 184-186oC, yield 88% were prepared.
Preparation of 2-Amino-5-phenyl/chlorophenyl-1,3,4-oxadiazoles (II)
To the suspension of benzaldehyde semicarbazone (I,0.01 mol) and anhydrous sodium acetate (0.01 mol) in 5 ml of glacial acetic acid. Bromine in acetic acid was added dropwise until light yellow colored compound was obtained. The product was poured into crushed ice, filtered and washed thoroughly with cold water to remove impurities. Adopting this procedure the following compounds were prepared,
2-Amino-5-phenyl-1,3,4-oxadiazole (IIa, R= H), mp. 245-246 oC, yield 64%, Mol.F:C8H7N3O FTIR (KBr) cm-1 3325-3160 (NH2 ), 1613.18 (C=N),1035 (C-O-C); 1H NMR (DMSO-d6): Î´ ppm: 4.2 (2H, NH2), 7.4-7.9 (5H,m, Ar-H),
Preparation of 5-Phenyl/chlorophenyl-3,4-diamino-1,2,4-triazoles (III)
The solution of 2-Amino-5-phenyl-1,3,4-oxadiazole (II,0.01 mol) in 10 ml methanol was taken in a round bottom flask, and hydrazine hydrate (99% , 0.002 mol) was added and refluxed for 3 hours. The reaction mixture was cooled, filtered and the product was washed with cold water. Finally, the compound was recrystallized in methanol and dried. Compound
5-Phenyl-3,4-diamino-1,2,4-triazole (IIIa, R = H), m.p. 232-234 oC, yield 59% 1H NMR (DMSO-d6): Î´ ppm: 8.9 (2H, N-NH2), 4.2 (2H,NH2), 7.38-8.1(5H,m, Ar-H
5-(p-Chlorophenyl)-3,4-diamino-1,2,4-triazole (IIIb, R = 4-Cl), m.p. 235-236 oC, yield 54%.
Preparation of 4-(Substituted benzylamino)-5-phenyl -3-amino-1,2,4-triazoles (IV)
4-(Substituted benzylamino) were prepared following this procedure. Each of the 5-Phenyl-3,4-diamino-1,2,4-triazole (III,0.01 mol) was dissolved in distilled methanol (15 ml) and few drops of glacial acetic acid was added to it and refluxed with various aromatic aldehydes (0.01mol) for 3 hr. The product was cooled and poured into crushed ice.The product was purified by column chromatography by using silica gel as stationary phase and chloroform-methanol as mobile phase.The product obtained in each case was worked out and characterized as their respective 4-(substituted benzylamino)-5-phenyl/chlorophenyl-3-amino-1,2,4-triazoles.
Spectral characterization of the test compounds
IVa. 4-(benzylideneamino)-5-phenyl-4H-1,2,4-triazol-3-amine. FTIR (KBr) cm-1 3298.87 (NH2), 3110.74 (CH=N), 1654.22 (C=N), 1599.81 (C=N).1H NMR (DMSO-d6): Î´ ppm: 4.0 (2H, NH2), 7.53-7.49 (5H,m, Ar-H), 7.44-7.38 (5H, m, Ar-H ),8.09 ( 1H, CH=N); 13C NMR (DMSO-d6) Î´ 125.01,127.50, 128.00, 128.50, 130.10, 130.50, 133.80,146.50, 151.84, 155.82. MS: m/z 264(M+1).
IVb. 4-(4-chlorobenzylideneamino)-5-phenyl-4H-1,2,4-triazol-3-amine. FTIR (KBr) cm-1 3296.19 (NH2), 3115.58 (CH=N),1618.43 (C=N), 1H NMR (DMSO-d6): Î´ ppm: 4.2 (2H, NH2), 7.63-7.52 (4H,m, Ar-H), 7.49-7.34(5H, m, Ar-H ), 8.5( 1H, CH=N); 13C NMR (DMSO-d6) Î´ 127.31, 128, 128.50, 134.56, 136.60, 148.15, 151.74, 155.52. MS: m/z 298(M+1).
IVc . 4-(4-methoxybenzylideneamino)-5-phenyl-4H-1,2,4-triazol-3-amine. FTIR (KBr) cm-1 3296.98 (NH2), 3102.45 (CH=N), 1612.43 (C=N). 1H NMR (DMSO-d6): Î´ ppm: 4.0 (2H, NH2), 7.51-7.47 (5H,m, Ar-H), 7.34-7.22 (4H, Ar-H ), 8.0 ( 1H, s, CH=N), 3.7 (3H, OCH3); 13C NMR (DMSO-d6) Î´ 55.30, 113.90, 125.40,128.01, 129.65, 130.30, 151.84, 155.82, 159.90. MS: m/z 294(M+1)
IVd . 4-(4-Hydroxybenzylideneamino)-5-phenyl-4H-1,2,4-triazol-3-amine.FTIR (KBr) cm-1 3297.29 (NH2), 3157 (Ph-OH) 3113.41 (CH=N), 1628 (C=N),.1H NMR (DMSO-d6): Î´ ppm: 6.10 (1H, s, O-H) 3.8 (2H, NH2), 7.45-7.41 (5H,m, Ar-H), 7.39-7.32 (4H, Ar-H ), 8.10 ( 1H, s, CH=N); 13C NMR (DMSO-d6) Î´ 114.70, 128.00, 128.80, 130.40, 135.80, 146.95, 151.35,155.05,157.80. MS: m/z 280(M+1)
IVe. 4-(3,4-dimethoxybenzylideneamino)-5-phenyl-4H-1,2,4-triazol-3-amine:FTIR (KBr) cm-1 3294.38 (NH2Â), 3111.35 (CH=N), 1615.85 (C=N), 1199(C-OCH3).1H NMR (DMSO-d6): Î´ ppm:,7.33-7.45 (5H,m, Ar-H), 7.29-7.24 (3H, d, Ar-H ), 7.08 ( 1H, s, CH=N), 4.0 (2H, NH2), 3.87 (3H, s, OCH3), 3.62 (3H, s, OCH3); 13C NMR (DMSO-d6) Î´ 55.65, 56.32, 107.05, 110.99, 125.40, 128.00, 130.45, 148.03, 149.40, 151.84, 155.50. MS: m/z 324(M+1)
IVf. 4-(benzylideneamino)-5-(4-chlorophenyl)-4H-1,2,4-triazol-3-amine.FTIR (KBr) cm-1 3296.19 (NH2), 3115.58 (CH=N), 1624.46 (C=N), 1588.43 (C=N), 1H NMR (DMSO-d6): Î´ ppm: 3.7 (2H, NH2), 7.63-7.52 (4H,m, Ar-H), 7.49-7.34 (5H, m, Ar-H ), 7.08 ( 1H, s, CH=N). 13C NMR (DMSO-d6) Î´ 125.40, 127.50, 182.60,128.80, 130.10, 130.40, 133.80, 134.50, 146.85, 151.84, 155.72. MS: m/z 297(M+)
IVg. 4-(4-chlorobenzylideneamino)-5-(4-chlorophenyl)-4H-1,2,4-triazol-3-amine.FTIR (KBr) cm-1 3294.38 (NH2), 3111.35 (CH=N), 1635 (C=N), 1598.74(C=N),1H NMR (DMSO-d6): Î´ ppm:3.8 (2H, NH2), 7.53-7.45 (4H,m, Ar-H), 7.29-7.24 (4H, d, Ar-H ), 7.06 ( 1H, s, CH=N). 13C NMR (DMSO-d6) Î´ 125.10, 128.70, 129.34, 129.55, 130.10, 134.24, 134.60, 137.85, 151.58, 155.65. MS: m/z 331(M+)
IVh. 4-(3,4-dimethoxybenzylideneamino)-5-(4-chlorophenyl-4H-1,2,4-triazol-3-amine . .FTIR (KBr) cm-1 3295.23 (NH2), 3115.23 (CH=N), 1614 (C=N), 1H NMR (DMSO-d6): Î´ ppm: 3.8 (2H, NH2), 7.41-7.55 (4H,m, Ar-H),6.5-7.2 (3H, d, Ar-H ),7.11 ( 1H, CH=N), 3.58 (6H, OCH3). 13C NMR (DMSO-d6) Î´ 107.05,110.25, 125.40, 128.70, 129.50, 130.21, 135.75, 146.38, 148.25, 149.35, 151.85, 155.69. MS: m/z 357(M+)
The animals were procured from Mahaveer Enterprises, Hyderabad, India. Male Wister rats (160-210gm) and Albino mice (25-30gm) were used for assessing anti-inflammatory and analgesic activity respectively. The animals were acclimatized for a period of 14 days prior to performing the experiments and maintained under standard husbandry conditions and had free access to food and water ad libitum. The animals were divided into different groups each consist of six animals were fasted overnight prior to the experiments. The protocol of the present study was reviewed and approved by the Institutional Animal Ethical Committee.
Anti-inflammatory activity using Carrageenan induced rat hind paw edema method
The normal paw volumes of all the rats were measured initially and were divided into seven groups of six animals each and were treated with the vehicle as control (0.5% sodium CMC), standard ibuprofen (50 & 100 mg/kg) and all the test compounds (IVa- IVh) in sodium carboxy methyl cellulose; 0.1 % w/v, 50 & 100 mg/kg b.w.) respectively. Carrageenan (0.1 ml of a 1% suspension in saline) was injected into the sub plantar region of the right hind paw of each rat. The vehicle, Standard and test compounds were administered 30 min prior to the injection of Carrageenan. The swelling (paw volumes) produced after injection of the phlogistic agent was measured in all the rats at 1, 2, 3 and 4 hr after Carrageenan treatment by using plethysmometer (15). A significant reduction in the paw volume compared to vehicle treated control animals was considered an inflammatory response.
% Inhibition= [(VT -V0) control -(VT -V0) treated groups] / (VT -V0) control *100
V0 = paw volume of the rat before administration of Carrageenan
VT = paw volume of the rat after administration of Carrageenan at different time intervals
Acetic-acid induced writhing test in mice: Abdominal construction (writhing movements) induced by intraperitoneal injection of acetic acid was carried out according to the procedures described previously (16). The test samples and reference standard (ibuprofen) was tested at doses of 50 and 100 mg/kg. The test samples and reference drug were administered orally 1 hour before the administration of 0.7% acetic acid in a volume of 10mg/kg i.p. Control animals received an appropriate volume of dosing vehicle. Immediately after injection of the acetic acid, each animal was isolated in an individual cage and the normal of construction was cumulatively counted for a period of 20 minutes. The number of writhings was recorded.
All the results were expressed as Mean Â± Standard deviation (SD). Data was analyzed using one-way ANOVA followed by Dunnett's t-test. P-values < 0.05 were considered as statistically significant.
RESULTS AND DISCUSSION
In the present study, the compounds were synthesized as depicted in the scheme-I, Benzaldehyde semicarbazones I were synthesized by the reactions of Benzaldehyde with semicarbazide HCl. Treatment of compounds I with Br2 in glacial acetic acid undergoes cyclisation to form 2-amino-5-substituted Phenyl-1,3,4-oxadiazole II. The compounds II by reacting with hydrazine hydrate in methanol to form 5-substituted-3,4-diamino-1,2,4-triazole III. Treatment of compounds III with aldehyde derivatives in presence of a few drops of glacial acetic acid as a catalyst to produce Schiff bases IV in good yields.
In general, the IR spectra of the compounds II reveal absorption bands at around 3225.23, 1614,1035 cm-1 resulting from the NH2, C=N & C-O-C functions respectively due to asymmetric & symmetric vibrations of the primary NH2 group. In the IR spectra of compounds IV, shows C=N stretching vibrations at around 1625 cm-1 and NH2 shows two peaks absorption at around 3295 & 3115 cm-1.The characteristic NH2 protons of compound II were detected around Î´ 4.2 ppm. The two -NH2 triazole were detected around Î´ 4 & 8 ppm. Absence of NH2 peak around Î´ 7 to 8 ppm in compounds IV indicates the formation of Schiff bases with aldehyde derivatives.
Anti-inflammatory activity: The anti-inflammatory activity of the all the synthesized compounds were studied at doses of 50 & 100 mg/kg (b.w) using carrageenan-induced paw edema model in rats and the results were presented in table 1. From the data, it appears that all test compounds significantly (p< 0.01) reduced carrageenan-induced paw edema in a dose dependent manner.
Based on these results, all the synthesized 1, 2, 4 triazole derivatives exhibited a significant anti-inflammatory activity against carrageenan induced paw edema. Among the tested compounds, IVg, IVb, IVf and IVh were considered to have potent anti-inflammatory activity and was comparable with standard anti inflammatory drug, ibuprofen. It was found that, IVg revealed slightly enhanced anti-inflammatory properities than those of the corresponding derivatives with the percentage inhibition of 58.9%. The results indicated that the introduction of electron-withdrawing groups resulted in increased activity. Prescence of Cl on the aryl ring results in increased activity
Carrageenan-induced rat paw oedema is a suitable test for evaluating anti-inflammatory drugs which has frequently been used to assess the anti-oedematous effect of natural products (17). Development of oedema in the paw of the rat after injection of Carrageenan is a biphasic event. The initial phase observed during the first hour is attributed to the release of histamine and serotonin. The second phase of oedema is due to the release of prostaglandins, protease and liposome(18,19). Ibuprofen, a COX-inhibitor at the dose of 50 mg/kg, significantly reduced the paw oedema. This indicates action against release of histamine, serotonin and kinins in early phase, while later phases are suspected to be arachidonate metabolites producing an oedema dependent on mobilization of neutrophils(18). The reduction in paw volume after administration of all the 4-(substituted benzylamino)-5-phenyl/chlorophenyl-3-amino-1, 2, 4-triazole indicated the anti-inflammatory activity of the test compounds which is might be due to the inhibition of either release or synthesis of prostaglandins, histamine and serotonin.
Acetic-acid induced writhing test in mice
The analgesic activity of the synthesized compounds was studied at doses 50 & 100 mg/kg (b.w) using acetic acid induced writhing test in mice and the results were showed in table 3. The abdominal constriction response induced by acetic acid is a sensitive procedure to establish peripherally acting analgesics. This response is thought to involve local peritoneal receptors. The number of writhing movements during a 20 min observation in the control group was 58.4 Â±1.75. All the test compounds were significantly (p<0.05) reduced writhings induced by the acetic acid in mice. Among all the test compounds, IVg, IVh, and IVb more significantly (p<0.01) reduced pain induced by acetic acid writhing responses. The number of writhing episode in treated mice decreased significantly and was compared to ibuprofen. The analgesic activity of IVg and IVh were more active in decreasing the number of writhing and showing the highest activity
In conclusion, all the new 1, 2, 4 triazole derivatives showed promising anti-inflammatory activity when compared to the standard drug. It has been felt necessary, from the results of the present preliminary investigations that there is a need for further advanced studies, at least on the few of the test compounds which are found to be superior. The possibility of these compounds for selective COX-2 inhibitor activity will be investigated in our future studies.
The first author is thankful to AICTE for providing GATE fellowship. The authors are also thankful to IICT, Hyderabad for providing spectral data.