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Discovery of Fused Triazolo-thiadiazoles as Inhibitors of TNF-alpha: Pharmacophore Hybridization for Treatment of Neuropathic Pain

Introduction Neuropathic pain is a complex, chronic pain state that is usually accompanied by tissue injury. With neuropathic pain, the nerve fibers themselves may be damaged, dysfunctional, or injured. Methods A series of pharmacophoric hybrids of substituted aryl semicarbazides incorporated into a fused triazolo-thiadiazole nucleus were synthesized and evaluated for neuropathic pain activity. After the assessment of neurotoxicity and peripheral analgesic activity, the compounds were evaluated in two peripheral neuropathic pain models, the chronic constriction injury and partial sciatic nerve ligation, to assess their antiallodynic and antihyperalgesic potential. Results Selected compounds exhibiting promising efficacies (4b, 6a, and 7e) revealed median effective dose (ED50) values ranging from 7.62–28.71 mg/kg in four behavioral assays of allodynia and hyperalgesia (spontaneous pain, tactile allodynia, cold allodynia, and mechanical hyperalgesia). Studies carried out to assess the underlying mechanism revealed that compounds suppressed the inflammatory component of the neuropathic pain by inhibiting tumor necrosis factor (TNF)-alpha and preventing oxidative and nitrosative stress. Conclusion Using a hybrid design approach, the present study identified novel chemical compounds that could be a potential lead for the treatment of neuropathic pain.


INTRODUCTION
Neuropathic pain syndromes characterized by neuronal hyperexcitability in damaged areas of the peripheral or central nervous system involve complex pathophysiological processes [1]. The development of inflammatory microenvironment at the site of nerve injury and the release of inflammatory mediators, together with their coupled signaling pathways, contribute to the generation and maintenance of neuropathic pain [2,3]. Following nerve injury, peripheral as well as central sensitization occurs [4,5] with upregulation of tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine, and its receptors in Schwann cells, endothelial cells, and in the dorsal horn of the spinal cord and hippocampus [6][7][8]. TNF-alpha, a key mediator in the inflammation, activates T cells and macrophages, thereby initiating the innate immune response. Consequently, the release of other inflammatory cytokines, sympathomimetic amines, prostaglandins, and nitric oxide (NO) is stimulated, which are involved in sensitization of primary afferent nociceptors [5][6][7][8]. Activated microglia and pro-inflammatory cytokines also induce the generation of free radicals leading to oxidative and nitrosative stresses, which exaggerate pain states [9,10]. 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 [11,12].
In recent years, the condensed bridgehead nitrogen heterocyclic system of triazolothiadiazole, which may be considered as the cyclic analog of thiosemicarbazide and biguanide has received considerable attention. A wide spectrum of biological activities, specifically anticonvulsant [13], analgesic [14], anxiolytic [15], and anti-inflammatory [16] properties has been reported for triazolothiadiazole in the literature. The 1,2,4-triazole core of the above fused system has gained considerable attention regarding its action against neuropathic pain acting through various targets. Many triazole-based P 2 X 7 antagonists [17,18] sodium channel blockers [19], and r-receptor inhibitors [20] have been reported. The literature also reveals many 1,2,4-triazole-based cannabinoid modulators [21,22] possessing antinociceptive efficacies.
The primary focus of our research has been on the design and synthesis of pharmacophoric hybrids of aryl semicarbazones to develop multifunctional leads useful in the treatment of neurological disorders such as epilepsy and neuropathic pain [23][24][25][26]. We have previously reported the cyclization of various aryl semicarbazones to 1,2,4-triazoles, which resulted in improved anticonvulsant activities [27]. Moreover, the literature reveals that acylthiosemicarbazides and their corresponding cyclized 1,3,4-thiadiazole derivatives possess anti-inflammatory [28,29] and analgesic [30] activities.
In view of the above reports, the design and synthesis of pharmacophoric hybrids of aryl semicarbazides into triazolo-thiadiazole templates was accomplished followed by assessment of their antinociceptive potential and underlying mechanism of action. Various reports on the antinociceptive efficacy of 4-aminobutyric acid (GABA) and gabapentin [31] and our research on N-spiro GABA derivatives [32] prompted us to explore the structure-activity relationships of GABA and N-spiro GABA derivatives in the cyclized triazolo-thiadiazole template.  N-(4-Bromophenyl)-6-(4-nitrophenyl)- [1,2,4]triazolo [3,4-

Motor Impairment
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 [33].

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 30 min after the administration of test compounds. 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 [34].

Formalin-Induced Flinching
The test involved intraplantar injection of 25 lL 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) [35]. 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 rats using the chronic constriction injury (CCI) model performed essentially as described by Bennett and Xie [36]. 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 hind limb 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 then closed by suturing the muscle using chromic catgut with a continuous suture pattern. Finally, the skin was closed using silk thread with horizontal-mattress suture pattern.

Induction of Peripheral Mononeuropathy:
Partial Sciatic Nerve Ligation Model As described by Seltzer et al. [37], in anesthetized rats, the left sciatic nerve was exposed at mid-thigh level through a small incision, cleared of adhering muscle tissue, and one-half of the nerve thickness was tightly ligated using 7.0 silk suture. Indomethacin (10 mg/kg, i.p.) was used as a positive control [39]. The percentage protection against inflammation was calculated as: V c -V d /V c 9 100, where V c is the increase in paw volume in the absence of the test compound (control) and V d is the increase of paw volume after administration of the test compound.
For the measurement of TNF-alpha, whole right hind paws were collected at the third hour after carrageenan injection. After rinsing with ice-cold normal saline, they were homogenized at 4°C, and the homogenate was centrifuged at 12,000 rpm for 5 min. The supernatant obtained was assayed using a TNF-alpha enzyme-linked immunosorbant assay (ELISA) kit [40].

Estimation of Total Nitrite/Nitrate
On day 9 post-CCI, after 2 h of administration of test compounds, the total nitrate/nitrite in the brain and sciatic nerve was estimated according to the reported procedure [9]. The method involved reduction of nitrate to nitrite followed by calorimetric estimation using Griess's reagent. The concentration of nitrite in the supernatant was calculated using a standard curve and expressed as a percentage of the control.

RESULTS
With the aim of synthesizing pharmacophoric hybrids of chemical entities with proven antinociceptive properties, the synthesis of various substituted phenyl semicarbazides (1a-1e), was accomplished from substituted anilines via urea formation as per our previously reported procedure [26]. Upon accomplished as reported in our earlier work [32]. The structures of the synthesized compounds were characterized by both spectral and elemental analysis, and the data were within ±0.4% of the theoretical values.
Following assessment of neurotoxicity of the synthesized compounds by rotarod (data not shown), the non-neurotoxic compounds were further evaluated in acetic acid-induced writhing and formalin-induced flinching models ( Table 1). 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: first phase (0-5 min) occurs due to direct stimulation of nociceptors (C-fibers), whereas the second phase (10-30 min) results following the development of a localized inflammatory response along with activation of N-methyl-Daspartate (NMDA) and non-NMDA receptors and nitric oxide production [42,43]. All tested compounds, except 6e, suppressed the acetic acid-induced writhing response significantly (P\0.01) in comparison with the control ( Table 1) In the CCI model (Fig. 2 were effective only up to 0.5 h, whereas compounds 4e, 5a, 7e, and 8c were effective until 1 h. Compound 6c exhibited irregular activity pattern.
Overall, it appears that in the CCI model of neuropathic pain compounds that showed promising results include 4b, 6a, 7e, and 8e, which were effective in four tests; 4a and 4e, effective in three tests; and 7b, 7c, 8b, and 8c, effective in two tests.
In the PSNL model (Fig. 3), the paw withdrawal durations due to spontaneous ongoing pain were significantly reduced by compounds 4b, 6a, and 7e throughout the experiment, similar to gabapentin. The  The carrageenan-induced paw edema model was used to investigate the probable role of the selective compounds (4b, 6a, and 7e) in the inhibition of inflammatory mediators. A significant reduction in edema was observed for compounds 4b, 6a, and 7e at all the timepoints (Table 3). TNF-a levels quantified in the carrageeenan injected paw were also     (Table 4).

DISCUSSION
The results obtained in the nociceptive assays provide an insight into the structure-activity relationships of the triazolo-thiadiazoles. Functionalization of the aryl ring of the semicarbazide fragment forming triazolo-thiadiazoles with dimethyl substitutions resulted in variable antinociceptive efficacy.
Compounds having a 2,4-dimethyl substituted aryl semicarbazide fragment (6c, 7c, and 8c) reversed one or more nociceptive parameters in both CCI and PSNL animals. Introduction of a 2,5-dimethyl substituted aryl ring proved to be detrimental for the antinociceptive efficacy in both CCI and PSNL animals. Only one compound, 6d, was found to be effective against cold allodynia in PSNL animals. The introduction of electron-releasing 2,6-dimethyl substitutions (4e, 7e, and 8e) resulted in significant attenuation of one or more nociceptive parameters in neuropathic animals. Introduction of an electron-withdrawing halogen (bromo) para to the aryl ring resulted in significant activity against one or more nociceptive assays. Compounds 4a, 5a, 6a, and 7a were effective in CCI animals, whereas compounds 4a, 6a, 7a, and 8a were found to be effective in one or more nociceptive parameters in PSNL animals. Compounds with para chloro-substituted aryl rings (4b, 6b, 7b, and 8b) resulted in significant attenuation of one or more nociceptive parameters in neuropathic animals. Accounting for the nociceptive efficacies observed for triazolo-thiadiazoles in the neuropathic pain models, it can be generalized that unlike 2-propylpentanyl substitution at the R 2 position (5a-5e), 2-aminobutanyl substitution (4a, 4b, and 4e) at the R 2 position was beneficial for nociceptive efficacy. Aryl substitution with a 4-nitobenzyl group (6a-6d) and heteroaryl substitution with an isonicotinyl group (7a, 7b, 7c, and 7e) resulted in significant attenuation of nociceptive parameters. N-spiro GABA substitution (8a, 8b, 8c, and 8e) also proved to be additive for antinociceptive efficacy.
In conclusion, utilizing a pharmacophoric hybrid approach, a series of structurally novel triazolo-thiadiazoles derivatives were synthesized and evaluated for acute antinociceptive, antiallodynic, and antihyperalgesic potential. In the carrageenan-induced paw edema model, in which there is a pronounced local TNF-alpha response, compounds 4b, 6a, and 7e significantly inhibited paw swelling as well as localized TNF-alpha levels in the paw, thereby suppressing the inflammatory component of neuropathic pain. The neuroprotection exhibited by compounds 4b, 6a, and 7e is also associated with their free-radical scavenging abilities and subsequent attenuation of oxidative and nitrosative stresses. ACKNOWLEDGMENTS P. Yogeeswari is the guarantor for this article, and takes responsibility for the integrity of the work as a whole.
Conflict of interest. The authors declare that they have no conflicts of interest and received no funding for this study.
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