In this work, synthetic integration of substituted semicarbazides and various aliphatic, aryl and heteroaryl acids into 1,2,4-triazol-5-ones was accomplished. Following the assessment of neurotoxicity and peripheral analgesic activity, the compounds were evaluated in two peripheral models of neuropathic pain, the chronic constriction injury and partial sciatic nerve ligation to assess their antihyperalgesic and antiallodynic potential. ED50 studies undertaken for selected compounds exhibiting promising efficacies (1c, 3c and 4a) revealed values ranging from 13.21 to 39.85 mg/kg in four behavioral assays of hyperalgesia and allodynia (spontaneous pain, tactile allodynia, cold allodynia, and mechanical hyperalgesia). Mechanistic studies revealed that the compounds suppressed the inflammatory component of the neuropathic pain inhibiting tumor necrosis factor-alpha and preventing oxidative and nitrosative stress.
Tissue damage, inflammation or injury of the nervous system may result in chronic neuropathic pain characterized by spontaneous pain, hyperalgesia and allodynia. Following nerve injury, peripheral as well as central sensitization occurs (1,2) with up-regulation of tumor necrosis factor-alpha (TNF-α) and its receptors in schwann cells, endothelial cells and in dorsal horn of the spinal cord and hippocampus (3–5). TNF-α, a key mediator in the inflammation, stimulates innate immune responses by activating T cells and macrophages that stimulate the release of other inflammatory cytokines, sympathomimetic amines, prostaglandins, and nitric oxide (NO) which are involved in sensitization of primary afferent nociceptors (1–4). Release of pro-inflammatory cytokines following nerve injury also induce generation of free radicals, leading to oxidative and nitrosative stress, which are critically involved in the development and maintenance of neuropathic pain (6–8). Reports suggest that inhibiting TNF synthesis with thalidomide or treatment with anti-TNF neutralizing antibodies at the time of nerve injury blocks the development of hyperalgesia and allodynia in neuropathic animals (9,10).
The triazole nucleus has been well documented to possess wide spectrum of biological activities including antimicrobial (11,12), anticonvulsant (13), anti-inflammatory (11,14), and analgesic (11,14,15) activity. In the past, we had attempted to synthesize 1,2,4-triazole nucleus to study the effect of cyclization of aryl semicarbazone pharmacophore on anticonvulsant activity and found the compounds to exhibit biological activity in four animal models of seizures (16). In recent years, triazole motif has gained considerable attention against neuropathic pain acting through various targets. Many triazole-based P2X7 antagonists (17,18), sodium channel blockers (19) and σ-receptor inhibitors (20) have been reported. Literature also reveals many 1,2,4-triazole-based cannabinoid modulators (21,22) possessing antinociceptive efficacies.
Melting points were measured in open capillary tubes on a Buchi 530 melting point apparatus and are uncorrected. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded for the compounds on Brucker Avance (300 MHz) instrument. Chemical shifts are reported in parts per million (ppm) using tetramethyl silane (TMS) as an internal standard. Mass spectra were measured with a Shimadzu GC-MS-QP5000 spectrophotometer. Elemental analyses (C, H, and N) were undertaken with a Perkin–Elmer model 240C analyzer, and all analyses were consistent with theoretical values (within ±0.4%) unless indicated. The homogeneity of the compounds was monitored by ascending thin layer chromatography (TLC) on silicagel-G (Merck) coated aluminum plates, visualized by iodine vapor and UV light.
General method for the synthesis of substituted N-phenylhydrazine-carboxamides
Substituted acid, 1–4 (1.0 equiv.) was taken in dichloromethane at 0 °C followed by addition of substituted semicarbazide, a–f (1.0 equiv.) (Scheme 1). Under stirring, triethylamine (1.1 equiv.) followed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) (1.1 equiv.) and 1-hydroxybenzotriazole (HOBT) (1.1 equiv.) were added at 0 °C. The reaction mixture was stirred at room temperature overnight. Following completion, the reaction mixture was washed with saturated aqueous NaHCO3 and brine. The organic layer was collected, dried over anhydrous Na2SO4, filtered and evaporated in vacuo to give the product in 78–85% yield.
General method for the synthesis of substituted 1,2,4-triazol-5-ones (1a–f, 2a–f, 3a–f, 4a–f)
0.05 m of the synthesized hydrazine-carboxamides of step 1 was taken in 4% w/v aqueous sodium hydroxide and heated on water bath for 3–4 h. After the completion of the reaction, the mixture was allowed to cool and then filtered. The filtrate was acidified with 2 m hydrochloric acid. The precipitated solid was filtered, washed thoroughly with water, dried, and recrystallized from ethanol/water.
Swiss albino mice (either sex) with weights ranging from 20 to 25 g were used for the assessment of neurotoxicity, acetic acid-induced writhing and formalin-induced flinching. Wistar rats of either sex (200–250 g) were used for the inflammatory and neuropathic pain models. All the experiments were approved by the Institutional Animal Ethics Committee. Animals were housed six (mice) and four (rats) per cage at constant temperature under a 12 h light/dark cycle (lights on at 7:00 am), with food and water ad libitum.
Minimal motor impairment was measured in mice by the rotarod test. The mice were trained to stay on an accelerating rotarod that rotates at 10 revolutions per minute. The rod diameter was 3.2 cm. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least 1 min in each of the three trials (23).
Acetic acid-induced writhing
Writhing was induced in a group of mice by an intraperitoneal injection of 0.1 mL of 2% v/v acetic acid. Test group mice received acetic acid half an hour after drug-treatment. The number of writhings occurring for a period of 30 min was recorded. For scoring purposes, a writhe was indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. The percentage inhibition of the writhing response was calculated (24).
The test involved intraplantar injection of 25 μL of 1% formalin into the hind paw of mice, which resulted in flinches in the paw in the early phase (0–5 min) and the late phase (10-30 min) (25). Time spent in paw licking and biting was monitored in each 5 min and calculated for both the phases. Test compounds were administered 30 min before the experiment.
Unilateral mononeuropathy – chronic constriction nerve injury model
Unilateral mononeuropathy was produced in the rats using the chronic constriction injury (CCI) model performed essentially as described by Bennett & Xie (26). The rats were anesthetized with an intraperitoneal dose of ketamine (55 mg/kg) and xylazine (5 mg/kg) with additional doses of the anesthetic given as needed. Under aseptic conditions, a 3-cm incision was made on the lateral aspect of the left hindlimb at the mid-thigh level. The left paraspinal muscles were then separated from the spinous processes, and the common left sciatic nerve was exposed just above the trifurcation point. Four loose ligatures were made with a 4-0 braided silk suture around the sciatic nerve with about 1-mm spacing. The wound was closed by suturing the muscle using chromic catgut in a continuous suture pattern. Finally, the skin was closed using silk thread with horizontal-mattress suture pattern. Povidone iodine ointment was applied topically on the wound, and gentamicin antibiotic (4 mg/kg) was given intramuscularly for 5 days after surgery. The animals were then transferred to their home cages and left for recovery.
Induction of peripheral mononeuropathy – partial sciatic nerve ligation model
As described by Seltzer (27), in anaesthetized rats, left sciatic nerve was exposed at mid-thigh level through small incision, cleared of adhering muscle tissue, and one-half of the nerve thickness was tightly ligated using 7.0 silk suture. The wound was closed and dusted with neomycin powder. The animals were then transferred to their home cages and left for recovery.
Sensory testing using nociceptive assays
Compounds (100 mg/kg, i.p.) were administered at t = 0, in 30% v/v PEG 400. The control group of rats received only the vehicle. Gabapentin (100 mg/kg, i.p.) was used as positive control. Paw withdrawal duration (PWD) was observed in spontaneous pain and cold allodynia, and paw withdrawal threshold (PWT) was assessed in tactile allodynia and mechanical hyperalgesia. Paw withdrawal duration is the total time period during which the animal lifts the paw (in the guarded position) after the pain, in this case, during spontaneous pain following surgery and assessment of cold allodynia following acetone spray. Paw withdrawal threshold is the maximum pressure/weight animal can bear after tactile or mechanical stimulus. In the present study, the cutoff value for tactile allodynia was 15 g, whereas for mechanical hyperalgesia, it was 250 g to avoid injury.
Percentage reversal in spontaneous pain, allodynia, or hyperalgesia was calculated for each animal as defined below (28),
Spontaneous pain was assessed for a total time period of 5 min as described previously by Choi et al. The operated rat was placed inside an observation cage that was kept 5 cm from the ground level. An initial acclimatization period of 10 min was given to each of the rats. The test consisted of noting the cumulative duration for which the rat holds its ipsilateral paw off the floor. Numbers of paw lifts associated with locomotion or body repositioning were not counted (29).
Paw withdrawal in response to mechanical stimuli was measured using Von Frey filaments (UGO Basile, Italy). Each rat was placed on a metallic mesh floor covered with a plastic box. A set of von Frey monofilaments (0.4–15 g), with intensities of mechanical stimulation increasing in graded manner with successively greater diameter filaments, were applied to the plantar surface of the hind paw five times at intervals of 1–2 seconds (30). The weakest force (g) inducing withdrawal of the stimulated paw at least three times was taken as the paw withdrawal threshold with cutoff value at 15 g.
The operated rat was placed inside an observation cage that was kept 5 cm from the ground level and was allowed to acclimatize for 10 min or until exploratory behavior ceased. Few drops (100–200 μL) of freshly dispensed acetone were squirted as a fine mist onto the mid-plantar region of the affected paw. A cold allodynic response was assessed by noting down the duration of paw withdrawal response. For each measurement, the paw was sampled three times and a mean calculated. At least 3 min elapsed between each test (31).
Mechanical paw withdrawal thresholds were assessed with a slightly modified version of the Randall–Selitto method (32) using analgesymeter (UGO Basile). The instrument exerts a force that increases at a constant rate. This force was applied to the hind paw of the rat, which was placed on a small plinth under a cone-shaped pusher with a rounded tip (1.5 mm in diameter) until the animal withdrew its paw. A cut off of 250 g was used to avoid injury. Mechanical paw withdrawal thresholds were calculated as the average of two consecutive measurements.
Determination of the median effective dose (ED50)
To determine ED50 as an estimate of the compounds’ potency, a dose–response curve in various sensory tests was plotted. ED50 was the dose that yielded 50% of the response (33). This value was linearly interpolated between the dose just above and just below the ED50 value.
Carrageenan-induced paw edema and quantification of TNF-α
Paw edema was induced in Wistar rats by intraplantar injection of 50 μL of 2% carrageenan (λ-carrageenan, type IV, Sigma) diluted in saline. The volume of the paw edema (mL) was determined at 0, 60, 120 and 180 min using a water plethysmometer (Ugo Basile) (34). Indomethacin (10 mg/kg, i.p.) was used as positive control. The percentage protection against inflammation was calculated as: Vc − Vd/Vc × 100, where Vc is the increase in paw volume in the absence of the test compound (control) and Vd is the increase of paw volume after injection of the test compound.
For the measurement of TNF-α, the whole right hind paws were collected at the third hour after carrageenan injection. After rinsing with ice-cold normal saline, it was homogenized at 4 °C and the homogenate was centrifuged at 18 300 g for 5 min. The supernatant obtained was assayed using TNF-α ELISA kit (35).
Estimation of total nitrite/nitrate
On ninth day post-CCI, after 2 h of administration of test compounds, the total nitrate/nitrite in brain and sciatic nerve was estimated according to the reported procedure (6). The method involved reduction of nitrate to nitrite followed by calorimetric estimation using Griess reagent. The concentration of nitrite in the supernatant was calculated using standard curve and expressed as percentage of control.
A solution of DPPH was prepared by dissolving 5 mg of DPPH in 2 mL of methanol, and the solution was kept in the dark at 4 °C. Varying concentrations of test compounds (200 μL) were taken in 96-well microplate. Then, 5 μL of methanolic DPPH solution (final concentration 300 μm) was added to each well. After 20 min of incubation, absorbance of the solution was read using an ELISA Reader (EL340 Biokinetic reader; Bio-Tek Instrumentation, Winooski, VT, USA) at a wavelength of 517 nm. A methanolic solution of DPPH served as a control. A dose–response curve was plotted to determine the IC50 values. All tests and analyses were run in triplicate and averaged (36).
Percentage scavenging was calculated according to the following equation.
Results and Discussion
The reaction sequence employed for the synthesis of titled compound is presented in Scheme 1. The synthesis of the starting material, N4-(substituted phenyl) semicarbazides (a–f) was accomplished from substituted anilines via urea formation as per our previously reported procedures (16). Coupling of the substituted N4-(substituted phenyl) semicarbazides with various aliphatic, aryl and heteroaryl acids with the aid of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT) resulted in the synthesis of N1-(substituted)-N4-(substituted phenyl) hydrazine-carboxamides. Cyclodehydration of the same in alkaline medium resulted in N1-(substituted)-N4-(substituted phenyl)-1H-1,2,4-triazol-5(4H)-ones (1a–f, 2a–f, 3a–f, 4a–f).
Pharmacology and structure–activity relationships
Following the assessment of neurotoxicity of the synthesized compounds by rotarod (data not shown), all the non-neurotoxic compounds were further evaluated for their acute antinociceptive efficacy in acetic acid-induced writhing and formalin-induced flinching model. The acetic acid-induced writhing model is a chemical pain test used to evaluate acute antinociceptive function, whereas the formalin model is a tonic inflammatory pain model used to distinguish peripherally and centrally acting compounds. It is characterized by two phases—the first phase (0–5 min) occurs due to direct stimulation of nociceptors (C-fibers), whereas the second phase (10–30 min) results following development of a localized inflammatory response along with activation of NMDA (N-methyl-d-aspartate) and non-NMDA receptors and nitric oxide production (37–39). All the tested compounds except 1a, 1f, and 2e suppressed the acetic acid-induced writhing response significantly (p < 0.01) in comparison with control (Table 1). The standard drug indomethacin exhibited the highest percentage inhibition (96.12%). Compounds 1c, 1d, 2d, 3c, 3d, and 4a were the most active compounds with percentage inhibition 90% or above. In formalin-induced flinching model, 18 of 25 compounds showed significant suppression in both the phases except compounds 1e, 1f, 2c, 3b, and 3f being effective only in the second phase. Indomethacin significantly reversed second phase of the formalin assay (67.14%). Compound 2e was inactive in both the assays.
Table 1. Acute antinociceptive efficacy of the 1,2,4-triazol-5-ones
Acetic acid-induced writhinga
Vehicle-treated animals received 30% v/v PEG 400 in water.
*Each value represents the mean ± SEM of six mice significantly different from the control at p < 0.05 (one-way anova, followed by post hoc Dunnet test) at a dose of 100 mg/kg.
bIndomethacin was taken as positive control at 5 mg/kg.
The 1,2,4-triazol-5-ones were further evaluated in two well-established peripheral neuropathic pain models – the CCI model and the partial sciatic nerve ligation (PSNL) model. Percentage reversal in spontaneous pain, tactile allodynia, cold allodynia, and mechanical hyperalgesia was assessed on ninth day post-surgery.
In the CCI model (Figure 1), compounds 1c, 3c, 3d, 4c, and 4d completely reversed the spontaneous pain response throughout the time period of testing (0.5–2.0 h) similar to gabapentin. Compound 4e exhibited activity up to 1 h. The onset of action of compounds 2c and 4a was at 1 h. Other compounds were ineffective in this test. Four compounds (3a, 3c, 4a, and 4b) were active in attenuating the tactile allodynia throughout the 2 h experiment. Compound 1a was active only up to 1 h of the experiment. All other compounds were ineffective in this test. In the cold allodynia produced in CCI rats, significant reversal of paw withdrawal durations was observed at all time-points by the administration of compounds 2c, 3f, 4a, and 4f. Gabapentin was also found to be effective at all the time-points. The onset of action for compounds 1c and 3c was at 1 h. Compounds 4d and 4e were effective only up to 1 h of the experiment.
Mechanical hyperalgesia was significantly attenuated at all the time-points by 3c similar to gabapentin. Compounds 3a and 4c were active up to 0.5 h, whereas compounds 1c and 4a were active up to 1 h. Overall, it appears that, in the CCI model of neuropathic pain, compounds that showed promising results include 3c and 4a effective in four tests, 1c in three tests and 2c, 4c, 4d, and 4e active in two tests.
In the PSNL model (Figure 2), the paw withdrawal durations due to spontaneous ongoing pain were significantly reduced by compound 3c throughout the experiment similar to gabapentin. The compounds 2c and 4d exhibited activity only up to 0.5 h, whereas the compounds 3d, 3f, 4c, and 4e were active up to 1 h of the experiment. Compound 1c that significantly reversed the pain response in the CCI rats was inactive in the PSNL model. The tactile allodynia produced by PSNL was effectively reversed by compounds 1c, 3a, and 3c at all the time-points like gabapentin. Compounds 3d, 4a, and 4c were active up to 1 h of the experiment. Cold allodynia produced by the PSNL model was completely reversed by the compound 3f. Compound 4d was active only up to 0.5 h of the experiment. The compounds 1c, 2c, and 3c had onset of action at 1 h. Compounds 4a, 4e, and 4f active in the CCI model were found to be ineffective in attenuating cold allodynia in the PSNL model. Compounds 1c, 2c, and 4a significantly reversed mechanical hyperalgesia at all the time-points like gabapentin. Compound 3a was active only up to 0.5 h of the experiment, whereas compounds 3c and 4c were active in first 1 h of the experiment. Overall, it appears, in the PSNL model, compounds that exhibited promising results included 3c effective in four tests, 1c, 2c, and 4c effective in three tests and 2f, 3a, 3d, 3f, 4a, and 4d effective in two tests.
The results obtained in the nociceptive assays provide an insight into the structure–activity relationships of the 1,2,4-triazol-5-ones. Functionalization of the aryl ring of semicarbazide fragment with dimethyl substitutions proved to be advantageous for antinociceptive efficacy. Compounds having 2,4-dimethyl substituted aryl semicarbazide fragment (3a and 4a) significantly reversed the nociceptive assays. Introduction of 2,5-dimethyl substituted aryl ring proved to be detrimental for the antinociceptive efficacy (1b, 2b, and 3b) in both CCI and PSNL animals. Only 4b was found to be active against tactile allodynia in CCI model. Introduction of electron releasing 2,6-dimethyl substitution (1c, 2c, 3c, and 4d) resulted in significant attenuation of one or more nociceptive parameters in neuropathic animals. Introduction of electron withdrawing halogen (chloro in 4e, bromo in 3d and 4d) para to the aryl ring resulted in significant activity against spontaneous pain and cold allodynia. Trifluoromethyl substitution meta to the aryl ring was detrimental for activity except in case of 3f and 4f which were effective in spontaneous pain and cold allodynia.
In general, among 2-propyl pentyl substituted triazolones (1a–1f), two compounds (1a and 1c) significantly alleviated one or more nociceptive responses in CCI and PSNL rats. 4-phenoxyphenyl substitution in aryl triazolones (2a–2f), proved to be detrimental for the activity except compound 2c which was found to be effective in one or more nociceptive assays in neuropathic animals. Heteroaryl triazolones having 5-nitro-2-furyl and 2-pyrazinyl substitution exhibited pronounced activity against one or more nociceptive parameters in the neuropathic animals. Among 3a–3f compounds, four compounds exhibited significant reversal of nociceptive assays in neuropathic animals. All the compounds having 2-nitro-5-furyl substitution (4a–4f) were found to be effective in one or more nociceptive testings.
Compounds exhibiting more than 90% reversal in one or more of the nociceptive assays (1c, 3c, and 4a) were taken further for ED50 studies. In the CCI model, compound 3c reversed spontaneous pain and tactile allodynia with an ED50 value of 13.21 and 39.85 mg/kg at 2 and 0.5 h, respectively. Compound 4a reversed cold allodynia with an ED50 value of 14.94 mg/kg at 0.5 h. Compound 1c reversed mechanical hyperalgesia with an ED50 value of 29.68 mg/kg at 2 h. In the PSNL model, compound 3c reversed spontaneous pain and mechanical hyperalgesia with an ED50 value of 24.18 and 16.56 mg/kg, respectively, both at 1 h. In tactile and cold allodynia, 4a came out to be the most active compound with an ED50 value of 18.42 and 31.98 mg/kg at 0.5 and 2 h, respectively.
Most of the test compounds exhibited significant reversal in acetic acid-induced writhing model supporting their role as peripherally acting analgesics. The significant suppression of flinching in both the phases of formalin assay (Table 1) suggested the mediation of anti-inflammatory pathways. The probable role of the selective compounds (1c, 3c, and 4a) in inhibition of inflammatory mediators was investigated using carrageenan-induced paw edema model where edema induced by carrageenan results from the local action of multiple inflammatory mediators, starting with the stimulation of TNF-α and keratinocyte chemokines (KC), subsequently causing the release of IL-1beta, and in turn, stimulating the release of prostanoids and sympathomimetic amines (40). A significant reduction in edema was observed for compounds 3c and 4a at all the time-points (Table 2). TNF-α levels quantified in the carrageenan-injected paw were also found to be inhibited by compound 3c and 4a. Following nerve injury, subsequent generation of free radicals leads to oxidative and nitrosative stress that exaggerates pain states. The putative role of nitric oxide (NO) in the pathophysiology of chronic nerve ligation as evident by significant increase in nitrite and nitrate levels in both brain and sciatic nerve led us to estimate the levels of nitrite, metabolite of NO in brain and sciatic nerve of CCI rats. There was no significant reduction of nitrite in the brain of CCI rats as compared to vehicle-treated animals with compounds 1c, 3c, and 4a. However, a significant reduction was observed in the sciatic nerve of the CCI animals as compared to vehicle-treated group with 3c and 4a, indicating inhibition of local NO. The free radical scavenging efficacies of the compounds (1c, 3c, and 4a) in the DPPH assay indicated reduction in oxidative stress (Table 3).
Table 2. Percent protection in carrageenan-induced paw edema and inhibition of TNF-α in the carrageenan-injected paw
% Protection in carrageenan-induced paw edema
% inhibition of TNF-αb
TNF-α, tumor necrosis factor-alpha.
*Significance at p < 0.05 compared to vehicle (one-way anova followed by Dunnet Test, n = 4).
aIndomethacin was tested at the dose of 10 mg/kg i.p.
bPercent inhibition of TNF-α in paw of carrageenan-stimulated mice as compared to vehicle-treated controls. Compounds were tested at the respective minimal ED50 dose given i.p.
21.1 ± 1.4
66.6 ± 2.7*
69.9 ± 1.7*
Table 3. DPPH scavenging activity and effect of compounds on nitrosative stress in CCI rats
DPPH scavenging activitya
% Inhibition of nitrosative stress (nitrite)b
CCI, chronic constriction injury.
*Significance at p < 0.05, compared to vehicle control (one-way anova followed by Dunnet test, n = 4). Compounds were tested at the respective minimal ED50 dose given i.p.
aDPPH radical scavenging activity of test compounds. Values are represented as % scavenging calculated from the average of triplicate experiments.
bPercent inhibition of nitric oxide in brain and sciatic nerve as compared to vehicle-treated control.
12.3 ± 1.4
24.6 ± 1.6
6.2 ± 1.1
61.6 ± 1.2*
7.1 ± 0.2
62.9 ± 2.1*
Conclusion and Future Directions
This study reports the synthesis, acute antinociceptive, antihyperalgesic, and antiallodynic activities of some novel 1,2,4-triazol-5-one derivatives. Efficacy of the compounds in both acute and chronic models of pain and the proposed contribution of inflammatory reactions in the generation and maintenance of neuropathic pain after nerve injury led us to explore the possible role of 1,2,4-triazol-5-one derivatives in the suppression of inflammatory component of neuropathic pain. Significant reduction in carrageenan-induced paw edema by 3c and 4a supported their anti-inflammatory profile. Both the compounds with aryl substitution significantly inhibited TNF-α levels in carrageenan-injected paw homogenates. Compound 1c with aliphatic substitution exhibited decline in antinociceptive efficacy.