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Original article
1 2

GABA derivatives for the treatment of epilepsy and neuropathic pain: A synthetic integration of GABA in 1, 2, 4Triazolo2Hone Nucleus
Q1

3 4 5 6

Yogeeswari Perumal , Sravan Kumar Patel , Ingala Vikram Reddy , Arvind Semwal , Monika Sharma , Matharasala Gangadhar , Matikonda Siddharth Sai , Dharmarajan Sriram
Neuropathic Pain Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, R.R. District-500078, Andhra Pradesh, India

7 8 9 10 11 12 13 14 15 16 17 18 19 20

a r t i c l e

i n f o

a b s t r a c t
Neuropathic pain is characterized by a neuronal hyperexcitability in damaged areas of the peripheral or central nervous system. The neuronal hyperexcitability in neuropathic pain have many common features with the cellular changes in epilepsy. This has led to the use of anticonvulsant drugs for the treatment of neuropathic pain. The present study aims at design and synthesis of two types of -aminobutyric acid (GABA) derivatives incorporated in the 1, 2, 4-triazol-2H-one nucleus and their evaluation for antiepileptic, peripheral analgesic, antiallodynic and antihyperalgesic potential in neuropathic pain models. Most of the synthesized triazole derivatives of GABA produced antiepileptic, antinocciceptive, antihyperalgesic, and antiallodynic actions and hence emerges as a promising lead for new drug development for the treatment of epilepsy and neuropathic pain. 2012 Elsevier Masson SAS. All rights reserved.

Article history: Received 5 March 2012 Accepted 11 March 2012 Available online xxx Keywords: Triazole Antiepileptic Neuropathic pain Analgesic activity Antiallodynic Antihyperalgesic

1. Introduction Neuropathic pain is characterized by spontaneous burning pain accompanied with hyperalgesia and allodynia as a result of a primary lesion, injury or dysfunction in the central or peripheral nervous system [1]. Conventional antiepileptic drugs, such as phenytoin, carbamazepine, topiramate, and lamotrigine have been used to treat neuropathic pain, but recently, some of the newer anticonvulsants, like gabapentin and pregabalin received increased attention as analgesics for treating neuropathic pain [2]. One of the important approaches to control chronic neuropathic pain involves the activation of -aminobutyric acid (GABA) mediated inhibition. Previous reports from our laboratory have shown the effect of various GABA derivatives for the treatment of epilepsy and neuropathic pain [39]. It is well reported that triazole nucleus possess diverse pharmacological activities like anti-microbial [10,11], anti-tubercular [12], anticonvulsant activity [13] and anti-inammatory and analgesic activities [1416]. 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 to exhibit biological activity in four animal models of seizures [17].

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

1, 2, 4-Triazole derivatives have shown to have activity against neuropathic pain through various targets. Carroll et al. have shown few 1, 2, 4- triazole derivatives to have P2X7 antagonistic action and also to inhibit IL-1b release from human THP-1 cells, which were further evaluated for antinociceptive activity in the CCI model of neuropathic pain with a good oral bioavailability [18]. An European patent (EP1921073), described various 1, 2, 4-triazole derivatives as - receptor inhibitors and claimed to have effect against pain, especially neuropathic pain and inammatory pain [19]. An US patent (US20040106614), described 1H-1, 2, 4-triazole-3-carboxamide derivatives having cannabinoid-CB1 receptor agonistic, partial agonistic, inverse agonistic or antagonistic activity and active against neuropathic pain disorders [20]. Another patent (US7572822) described various biaryl substituted triazoles as sodium channel blockers and active against neuropathic pain [21]. In view of the above reports, we synthesized various 1, 2, 4triazole derivatives integrated with GABA aimed at investigating their antiepileptic, peripheral analgesic, anti-allodynamic and antihyperalgesic activities. 2. Materials and methods 2.1. Chemistry Melting points were measured Buchi 530 melting point apparatus (IR) and proton nuclear magnetic were recorded for the compounds in open capillary tubes on a and are uncorrected. Infrared resonance (1 H-NMR) spectra on Jasco IR Report 100 (KBr)

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

47

48

49 50 51 52

Corresponding author. Tel.: +919 705 932 091; fax: +040-66303998. E-mail addresses: pyogee.sriram@gmail.com, pyogee@bits-hyderabad.ac.in (Y. Perumal). 2210-5220/$ see front matter 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biomag.2012.03.001

Please cite this article in press as: Perumal Y, et al. GABA derivatives for the treatment of epilepsy and neuropathic pain: A synthetic integration of GABA in 1, 2, 4Triazolo2Hone Nucleus. Biomed Aging Pathol (2012), doi:10.1016/j.biomag.2012.03.001

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and Brucker Avance (300 MHz) instruments, respectively. Chemical shifts are reported in parts per million (ppm) using tetramethyl silane (TMS) as an internal standard. All exchangeable protons were conrmed by addition of D2 O. 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 silica gel-G (Merck) coated aluminium plates, visualized by iodine vapor and UV light.

2.2. Pharmacology Lamotrigine and carbamazepine were obtained as gift samples from M/s IPCA Laboratories, India. Swiss albino mice (either sex) with weights ranging from 2025 g were used for the acetic acid induced writhing and formalin test. Wistar rats of either sex (200250 g) were used for both the neuropathic pain models. All 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. Compounds 110 (100 mg/kg, i.p.) were administered in 30% v/v PEG 400. The control group received 30% v/v PEG 400 (10 mL/kg for mice and 2 mL/kg for rats). 2.2.1. Anticonvulsant screening All the test compounds were administered intraperitoneally in a volume of 0.01 mL/g for mice at doses of 100 and 300 mg/kg. Anticonvulsant activity was assessed after 30 min and 4 h of drug administration. Activity in the MES and scPTZ tests was established according to the earlier reported procedures [23,24] and the data are presented in Table 1. Clinically proven antiepileptics such as phenytoin, and ethosuximide also were tested at the same dose levels. 2.2.2. Neurotoxicity screen Rotarod test has been performed to detect the minimal motor decit in mice. Animals were divided into groups (4-8) and trained to stay on an accelerating rotarod that rotates at 10 revolutions per minute [3]. The rod diameter was 3.2 cm. Trained animals (able to stay on the rotarod for at least 2 consecutive trials of 90 s each) were given an i.p. injection of the test compounds at doses of 100 and 300 mg/kg. Neurological decit 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. The dose at which the animal fell off the rod was determined. 2.2.3. Acetic acid induced writhing Mice were divided into groups of six each. Writhing was induced by an intraperitoneal injection of 0.1 mL of 3% v/v acetic acid [25]. Test group mice received acetic acid 30 min after drug-treatment. The number of writhings occurring for a 30 min time period was recorded. For scoring purposes, a writhe was indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. 2.2.4. Formalin test This test involves the injection of a small amount of formalin into subcutaneous tissues, typically the dorsal surface of the rodent paw that gives inches of paw in the early phase (05 min) and the late phase (10-30 min) [26]. In formalin test pain-related behaviors were quantied based on the total time of inching and licking of the paw. Flinching and licking were chosen as measures of pain, because they are more spontaneous than other formalin pain-related behaviors (e.g. favoring and lifting) and consequently, are thought to be more reliable for the quantication of the painrelated behaviors. 2.2.5. Chronic constriction nerve injury (CCI Model) Unilateral mononeuropathy was produced in rats using the CCI model performed essentially as described by Bennett and Xie [27]. The rats were anesthetized with an intraperitoneal dose of pentobarbital sodium (65 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 (ipsilateral) at the mid-thigh level with the right hindlimb serving as the control

115

116 117 118 119 120 121 122 123 124 125 126 127

62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88

2.1.1. General procedure for the preparation of 4 Aryl substituted 1, 2, 4-triazoles (1-5) For the synthesis of 4-aryl substituted 1,2,4-triazoles, GABA was N-protected using phthalic anhydride by taking equimolar quantities of GABA and the fomer in presence of toluene and triethyl amine (as a proton scavenger) [6]. Hydrochloric acid (HCl) was then added, stirred for 30 minutes, ltered and collected the precipitate of N-phthaloyl GABA (m.p 105107 ). Different substituted anilines were reacted with sodium cyanate for 30 min in the presence of water and glacial acetic acid to form substituted ureas, which were converted into semicarbazides by reuxing it with hydrazine hydrate and sodium hydroxide in presence of ethanol for 24 h [22]. The carboxyl group of N-phthaloyl GABA and amino group of semicarbazide were further coupled in the presence of DCC and DMF by stirring the reaction mixture for 24 h. N-protected phthaloyl group was then removed by reuxing the precipitate with hydrazine hydrate in presence of ethanol for 10 h. The reaction mixture was cooled and then conc. HCl was added till precipitate form. To the ltrate pyridine was added till it becomes basic. Further puried by extracting the end product with dichloromethane (DCM) and water mixture in basic condition. Then ethanol and pyridine were distilled off to get deprotected product. The product obtained in was further reuxed in the presence of 4% NaOH to get the cyclised compounds (1-5) The structures were characterized by both spectral and elemental analysis and the data were within 0.4% of the theoretical values.

128 129 130 131 132 133 134 135 136

137 138 139 140 141 142 143 144 145 146 147

148 149 150 151 152 153 154 155

89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114

2.1.2. General procedure for the preparation of 5- Aryl substituted 1, 2, 4-triazoles (6-10) For the synthesis of 5-aryl substituted 1, 2, 4-triazoles (6-10), GABA (0.01 moles) was reacted with 0.01 moles (1.25 mL) of phenyl chloroformate in presence of sodium hydroxide solution to form 4-[(phenoxycarbonyl) amino] butanoic acid. The mixture was vigorously stirred for 90 min. The precipitate was then ltered, washed and nally dried in a hot air oven at 52 C. A solution of 0.0005 moles of 4-[(phenoxycarbonyl) amino] butanoic acid in 25 mL of ethanol was treated with 0.0005 moles of hydrazine hydrate and catalytic amounts of 4-dimethylaminopyridine to form hydrochloride salt of 4-[(hydrazinocarbonyl) amino] butanoic acid; (semicarbazide). The mixture was reuxed for 6 h and then concentrated by evaporation. The precipitate was then ltered and kept for air-drying. 0.0025 moles of semicarbazide salt and excess (0.0075 moles) of triethylamine were taken and dissolved in ethanol. To this reaction mixture an equimolar quantity of acid chloride was added and reuxed for 34 h. Then the reaction mixture was concentrated by distilling out ethanol and the product was obtained by keeping in ice-cold condition. The product was ltered and dried. The obtained 4-({[(2-oxo-2-phenylethyl) amino] carbonyl} amino) butanoic acid, was then cyclised to 1, 2, 4-triazole by reuxing in presence of 4% alcohol, for 23 h [17]. The structures were characterized by both spectral and elemental analysis and the data were within 0.4% of the theoretical values.

156 157 158 159 160 161 162 163 164 165 166

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Please cite this article in press as: Perumal Y, et al. GABA derivatives for the treatment of epilepsy and neuropathic pain: A synthetic integration of GABA in 1, 2, 4Triazolo2Hone Nucleus. Biomed Aging Pathol (2012), doi:10.1016/j.biomag.2012.03.001

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Table 1 General structure, antiepileptic activity, neurotoxicity and acute antinocciceptive efcacy of compounds (110)

HN
O

N R2 R1
. R1 R2 Antiepileptic activity and Neurotoxicity Acetic acid (% inhibition) Formalin test (% inhibition) Phase-1 Phase II

Compound

MES 0.5 h 4h

scPTZ 0.5 h 4h

Neurotoxicity 0.5 h 4h

Br

(CH2)3 NH2

100

300

300

300

300

99.0

72.2a

70.1a

Cl
H3C CH3

(CH2 )3 NH2

300

17b

17.5b

45.3a

(CH2)3 NH2

300

300

300

91.5a

47.4a

65a

H3C
4

CH3

(CH2)3 NH2

300

300

94a

15.8b

15.3b

H3C
5

Cl
95.5a 34a 70.8a

(CH2)3 NH2

100

300

300

(CH2 )3 COOH

96a

61.5a

80.1a

(CH2 )3 COOH

300

73a

73.2a

77.5a

8 9

(CH2 )3 COOH (CH2 )3 COOH

O N O
(CH2 )7
NH CH3

78a 87a

75.9a 40.9a

77a 54a

10 Phenytoin Ethosuximide Indomethacin

(CH2 )3 COOH

100

100

300 -

100

100

13b 96a

5.2b 11.3

0b 85.5a

Doses of 100 and 300 mg/kg were administered for anticonvulsant efcacy and neurotoxicity. The gures in the table indicate the minimum dose whereby bioactivity was demonstrated in half or more of the mice (three in each group). The animals were examined at 0.5 and 4 h. The line () indicates an absence of anticonvulsant activity or neurotoxicity at the maximum dose tested. a Signicantly different from vehicle at p < 0.05. b Not signicant at p < 0.05 (one-way ANOVA, followed by post-hoc Bonferroni test). All the compounds were administered intraperitoneally at a dose of 100 mg/kg. Control animals were administered 30% v/v PEG 400.

175 176 177 178 179 180 181 182 183 184 185 186 187

(contralateral). 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 then made with a 4-0 braided silk suture around the sciatic nerve with about 1-mm spacing as reported elsewhere [28]. 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. A sham surgery (n = 4) was performed by exposing the sciatic nerve as described above, but not damaging it. Povidone iodine ointment was applied topically on the wound and gentamicin antibiotic (4 mg/kg) was given intramuscularly for ve days after surgery. The animals were then transferred to their home-cages and left for recovery.

using the transverse processes of L6 as a guide, the left paraspinal muscles were exposed and separated from the spinous processes of L4 to S2 by blunt dissection. The L5 spinal nerve was then exposed at the level of the dorsal root ganglion, and ligated tightly with a 4-0 braided silk suture. Only one tight ligature was made in this model. After conrmation of hemostasis, the wound was then closed by suturing at both muscle and skin levels. A sham surgery (n = 4) was performed by exposing the L5 spinal nerve as described above, but not damaging it. Povidone iodine ointment was applied topically on the wound and gentamicin antibiotic (4 mg/kg) was given intramuscularly for ve days after surgery. The animals were then transferred to their home-cages and left for recovery.

193 194 195 196 197 198 199 200 201 202 203 204

188 189 190 191 192

2.2.6. Selective segmental L5 SNL Model A left L5 spinal nerve ligation, as described by Kim and Chung [29], was performed. The rats were anesthetized with an intraperitoneal dose of pentobarbital sodium (65 mg/kg) with additional doses of the anesthetic given as needed. Under aseptic conditions,

2.2.7. Sensory testing after CCI and SNL injury in rats Four nociceptive assays aimed at determining the severity of behavioral neuropathic responses namely allodynia and hyperalgesia were performed. The assays involved measurement of the degree of spontaneous (ongoing) pain and tests of hind limb withdrawal to cold and mechanical stimuli (dynamic mechanical

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allodynia, cold allodynia and mechanical hyperalgesia). A minimum of 10 min separated the testing procedures to reduce the inuence of prior nociceptive testing. The order of testing was as follows:spontaneous pain, dynamic allodynia, cold allodynia and lastly mechanical hyperalgesia. All of the behavioral responses were timed with a stopwatch. 2.2.7.1. Spontaneous pain. Spontaneous pain was assessed for a total time period of 5 min as described previously by Choi et al. [30]. 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. A total number of four rats (n = 4) were assigned to this group. The test consisted of noting the cumulative duration that the rat holds its ipsilateral paw off the oor. The paw lifts associated with locomotion or body repositioning was not counted. Its been suggested that those paw lifts in the absence of any overt external stimuli are associated with spontaneous pain, and are correlative of ongoing pain. 2.2.7.2. Dynamic allodynia. All of the operated rats were assessed for dynamic allodynic response according to the procedure described by Field et al. [31]. 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. A total number of four rats (n = 4) were assigned to this group. A positive dynamic allodynic response consisted of lifting the affected paw for a nite period of time in response to mild stroking on the plantar surface using a cotton-bud. This stimulus is non-noxious to a normal-behaving rat. The latency to paw-withdrawal was then noted down. If no paw withdrawal was shown within 15 s, the test was terminated and animals were assigned this withdrawal time. Hence, 15 s effectively represented no withdrawal. 2.2.7.3. Cold allodynia. The rats demonstrating unilateral mononeuropathy were assessed for acute cold allodynia sensitivity using the acetone drop application technique as described by Caudle et al. [32]. 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 behaviour ceased. A total number of four rats (n = 4) were assigned to this group. Few drops (100200 L) of freshly dispensed acetone were squirted as a ne mist onto the midplantar 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. 2.2.7.4. Mechanical hyperalgesia. Mononeuropathic rats were assessed for mechanical hyperalgesia sensitivity according to the procedure described by Gonzalez et al. [33]. 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. A total number of four rats (n = 4) were assigned to this group. Hindpaw withdrawal duration was measured after a mild pin-prick stimulus to the midplantar surface of the ipsilateral (left) hindpaw. A withdrawal was dened as being abnormally prolonged if it lasted at least 2 s. The mean withdrawal duration was taken from a set of three responses.. 2.2.8. Statistical analysis All data are expressed as means standard error of mean (SEM). The data were analyzed by one-way ANOVA, and bonferronis post hoc test was used for individual comparisons with the control values. Signicance was assigned to a p value of less than 0.05. The

statistical software package PRISM (Graphpad Software Inc, San Diego, CA) was used for the analyses. 3. Results and discussion 3.1. Synthesis The synthetic protocols employed for the preparation of 4-aryl substituted triazole (1-5) and 5-aryl substituted triazole (6-10) derivatives are presented in Figs. 1 and 2. For the synthesis of 4aryl GABA substituted 1, 2, 4-triazoles, GABA was rst N-protected using phthalic anhydride [6]. Different substituted anilines were reacted with sodium cyanate in the presence of water and glacial acetic acid to form substituted aryl urea, which were converted into semicarbazides by reuxing it with hydrazine hydrate and sodium hydroxide in presence of ethanol [22]. Amino group of semicarbazide was coupled with carboxyl group of N-phthaloyl GABA in the presence of N, N-dicyclohexyl carbodiimide (DCC) and dimethylformamide (DMF). The product obtained was further reuxed in the presence of 4% sodium hydroxide to get the cyclised compounds (1-5) [17]. For the synthesis of 5-aryl GABA substituted 1,2,4-triazoles (6-10), The starting material, GABA, was treated with phenyl chloroformate in aqueous sodium hydroxide at a range of 05 C to yield 4-(phenoxycarbonyl-l-amino)butanoic acid to yield 70% after crystallization with 95% ethanol. The carbamate, on condensation with hydrazine hydrate in ethanol, gave the 4(hydrazinecarbonyl-l-amino) butanoic acid [3]. Base catalyzed condensation of semicarbazide salt was carried out with equimolar quantity of substituted acid chloride. The product obtained was then cyclised to 1, 2, 4 triazole by reuxing in presence of 4% sodium hydroxide to get the compound 6-10. All the structures were characterized by both spectral and elemental analysis and the data were within 0.4% of the theoretical values. The physical and spectral data of the compounds are as follows. 3.1.1. 3-(3-Aminopropyl)-4-(4-bromophenyl)-1H-1,2,4-triazol5(4H)-one (1) M.P.: 233 C; Yield: 59%; IR (KBr): 3400, 3250,3000, 2860,1740,1650,1600,1580,1475,660 cm-1; 1 H-NMR (DMSOd6 ) (ppm): 8.72 (d, 2H, Ar-H), 7.58 (d, 2H, Ar-H), 7 (s, 1H, NH), 5.11 (s, 2H, NH), 2.55 (m, 2H), 1.69 (m, 2H), 1.53 (m, 2H). Calculated for C11H13BrN4O: C, 44.46; H, 4.41; Br, 26.89; N, 18.85; O, 5.38 found: C, 44.32; H, 4.56; Br, 26.54; N, 18.26; O, 5.16. 3.1.2. 3-(3-Aminopropyl)-4-(4-chlorophenyl)-1H-1,2,4-triazol5(4H)-one (2) M.P.: 230 C; Yield: 78%; IR (KBr): 3380, 3250,3000, 2850,1720,1650,1600,1580,1470,690 cm; 1 H-NMR (DMSO-d6 ) (ppm): 7.43-7.47 (m, 4 Ar-H), 7 (s, 1H, NH), 5.11 (s, 2H, NH), 2.65 (m, 2H), 1.69 (m, 2H), 1.53 (m, 2H). Calculated for C11H13ClN4O: C, 52.58; H, 5.19; Cl, 14.03; N, 22.17; O, 6.33 found: C, 52.67; H, 5.11; Cl, 23.12; N, 22.23; O, 6.30. 3.1.3. 3-(3-Aminopropyl)-4-(2,5-dimethylphenyl)-1H-1,2,4triazol-5(4H)-one (3) M.P.: 211 C; Yield: 54%; IR (KBr): 3360, 3400,3150,3035,1725, 1600, 1550, 1450, 1380, 880,690 cm-1 ; 1 H-NMR (DMSO-d6 ) (ppm): 7.6 (s,1Ar-H), 7-7.4 (m, 4 Ar-H), 6.8 (s, 1H, NH), 5.11 (s, 2H, NH), 2.65 (m, 2H), 2.4 (s,1H),2.1(s,1H).1.69 (m, 2H), 1.53 (m, 2H). Calculated for C13H18N4O: C, 63.39; H, 7.37; N, 22.75; O, 6.50,found: C, 63.28; H, 7.16; N, 22.34; O, 6.64.

270 271

272

273

217 218 219 220 221 222 223 224 225 226 227

274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301

228 229 230 231 232 233 234 235 236 237 238 239 240

241 242 243 244 245 246 247 248 249 250 251 252 253

302 303 304 305 306 307 308 309 310

311 312 313 314 315 316 317 318 319

254 255 256 257 258 259 260 261 262 263 264

320 321 322 323 324 325 326 327 328

265 266 267 268 269

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O O O + H2N OH -H2O Toluene, Triethylamine, HCl O

HO O

O OH

R NH2 NaCNO/H2O R GAA HN NH2 HN O HO O O H N N + O R N O O R H N NH2 DCC O O HN NH NH O O R NaOH/NH2NH2.H2O HN NH2

Hydrazinolysis

NH2NH2.H2O

HN

N NH2 O 4 % NaOH H N H N N H O
Fig. 1. Synthetic protocol for 4-Aryl substituted 1,2,4- triazoles (1-5).

NH2

R R

329 330 331 332 333 334 335 336 337

3.1.4. 3-(3-Aminopropyl)-4-(2,6-dimethylphenyl)-1H-1,2,4triazol-5(4H)-one (4) M.P.: 216 C; Yield: 83%; IR (KBr): 3350, 3400, 3150, 3035,1725, 1600, 1560, 1450, 1375, 870, 760, 690 cm-1 ; 1 H-NMR (DMSOd6 ) (ppm): 7.0-7.3 (m, 3 Ar-H), 7 (s, 1H, NH), 5.11 (s, 2H, NH), 2.65 (m, 2H), 2.12 (s,6H),1.69 (m, 2H), 1.53 (m, 2H). Calculated for C13H18N4O: C, 63.39; H, 7.37; N, 22.75; O, 6.50 found: C, 63.45; H, 7.26; N, 22.53; O, 6.32. 3.1.5. 3-(3-Aminopropyl)-4-(3-chloro-2-methylphenyl)-1H1,2,4-triazol-5(4H)-one (5) M.P.: 189 C; Yield: 64%; IR (KBr): 3360, 3400, 3150,3050,1720, 1600, 1580, 1450, 1370, 880, 700, 690 cm-1 ; 1 H-NMR (DMSO-d6 ) (ppm): 7.6 (m, Ar-H),7.2 (m,Ar-H), 7.4(m,Ar-H), 7 (s, 1H, NH), 5.11 (s, 2H, NH), 2.65 (m, 2H), 2.2 (s,3 H),1.70 (m, 2H), 1.50 (m, 2H).

Calculated for C12H15ClN4O: C, 54.04; H, 5.67; Cl, 13.29; N, 21.01; O, 6.00 found: C, 54.14; H, 5.52; Cl, 13.45; N, 21.01; O, 5.80. 3.1.6. 4-(5-Oxo-3-phenyl-1H-1,2,4-triazol-4(5H)-yl)butanoic acid (6) M.P.: 237 C; Yield: 58%; IR (KBr): 3400, 3100, 2920, 1725, 1700, 1600,1550, 1475, 1450, 880 cm-1 ; 1 H-NMR (DMSO-d6 ) (ppm): 11(s,1H),7.8 3(m,Ar-H), 7.52(m,Ar-H), 7 (s, 1H, NH), 4.08(m,2H),2.30 (m,2H),1.92 (m, 2H), Calculated for C12H13N3O3: C, 58.29; H, 5.30; N, 16.99; O, 19.41 found: C, 58.21; H, 5.28; N, 16.92; O, 19.32. 3.1.7. 4-(3-Benzyl-5-oxo-1H-1,2,4-triazol-4(5H)-yl)butanoic acid (7) M.P.: 197 C; Yield: 61%; IR (KBr): 3380, 3120,2900, 1725, 1700,1660,1600,1550, 1475, 1465, 890 cm-1 ;1 H-NMR (DMSO-d6 ) (ppm): 11(s,1H), 7.2-7.4(m,Ar-H), 7 (s, 1H, NH), 4.1 (m,2H),

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Fig. 2. Synthetic protocol for 5-Aryl substituted 1, 2, 4- triazoles (6-10).

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3.58(s,2H), 2.30 (m,2H),1.92 (m, 2H), Calculated for C13H15N3O3: C, 59.76; H, 5.79; N, 16.08; O, 18.37; found: C, 59.70; H, 5.82; N, 16.22; O, 18.32. 3.1.8. 4-(3-(4-Nitrophenyl)-5-oxo-1H-1,2,4-triazol-4(5H)-yl)butanoic acid (8) M.P.: 212 C; Yield: 56%; IR (KBr): 3300, 3000,2850,1730,1700, 1620,1600,1550,1475,1460,1350 cm-1 ; 1 H-NMR (DMSO-d6 ) (ppm): 11(s,1H), 8.33(m,Ar-H),8.01(m,Ar-H), 7 (s, 1H, NH), 4.1 (m,2H), 2.30 (m,2H),1.92 (m, 2H), Calculated for C12H12N4O5: C, 49.32; H, 4.14; N, 19.17; O, 27.37; found: C, 49.28; H, 4.18; N, 19.22; O, 27.33. 3.1.9. 4-(3-Heptyl-5-oxo-1H-1,2,4-triazol-4(5H)-yl)butanoic acid (9) M.P.: 214 C; Yield: 68%; IR (KBr): 3400, 3000,2950,2920,2850,1730,1720,1465,1450,1375 cm-1 ; 1 H-NMR (DMSO-d6 ) (ppm): 11(s,1H), 7 (s, 1H, NH),4.08(m,2H);2.30(m,2H); 1.9 (m,2H);1.53(m2H), 1.29(m,8H);0.88(m,3H), Calculated for C13H23N3O3: C, 57.97; H, 8.61; N, 15.60; O, 17.82, found: C, 57.92; H, 8.72; N, 15.68; O, 17.68. 3.1.10. 4-(3-(4-Acetamidophenyl)-5-oxo-1H-1,2,4-triazol-4(5H)yl)butanoic acid (10) M.P.: 200 C; Yield: 92%; IR (KBr): 3350,3200,3000, 2850,1740, 1720, 1640,1600,1550,1475 cm-1 ; 1 H-NMR (DMSO-d6 ) (ppm): 11(s,1H), 7.8(m,Ar-H), 7.68(m,Ar-H),7.23 (s1H,NH) 7 (s, 1H, NH), 4.08(m,2H);2.30(m,2H); 2(s,3H);1.9 (m,2H); Calculated C14H16N4O4: C, 55.26; H, 5.30; N, 18.41; O, 21.03; found: C, 55.30; H, 5.34; N, 18.38; O, 21.12.

3.2. Neuropharmacology The aim of study was to prepare newer GABA derivatives of 1, 2, 4-triazoles with multiple pharmacological actions effective in the treatment of epilepsy and neuropathic pain. For the anticonvulsant activity, compounds (1-10) were evaluated at dose levels of 100 and 300 mg/kg intraperitoneally in mice by standard anticonvulsant drug development (ADD) program protocols [23,24]. The tests included maximal electroshock seizure test (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure threshold test whose results are presented in Table 1. 4-Aryl substituted 1,2,4-triazoles (1-5) showed activity in both the models indicative of their ability to prevent seizure spread while 5-aryl substituted 1,2,4-triazoles (610) were devoid of any antiepileptic activities. In the MES model, compounds 1 and 5 exhibited longer duration of action until 4 h and showed efcacy at 100 mg/kg at 0.5 h and 300 mg/kg at 4 h, compared to other active compounds, which were active only at 0.5 h at the dose of 300 mg/kg. Compound 5 showed comparable efcacy to phenytoin in MES model, with a better safety prole. In the scPTZ test, four compounds (1, 3, 4 and 5) showed marginal protection, with efcacy at 300 mg/kg at 0.5 h time period only, but comparable to that of ethosuximide. All other synthesized compounds were ineffective in this model. The acute neurological toxicity was determined by the rotorod test [3]. All the compounds except 1 and 3 were devoid of any neurotoxicity. Compound 5 emerged as the most active anticonvulsant in both the models with no neurotoxicity. There was no separation between the anticonvulsant dose and the neurotoxic dose (300 mg/kg) for compound 1 while compound 3 was neurotoxic only at 4 h. Before evaluating the therapeutic potential of the 1, 2, 4-triazole derivatives in animals model of neuropathic pain, compounds were evaluated rst in two acute nocciception models namely acetic acid

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Fig. 3. Effects of compounds in the spontaneous pain assays in CCI and SNL rats. Graphs depict the effects of compounds (100 mg/kg, i.p.) in reversing the spontaneous pain response. The results are shown as mean paw withdrawal duration (PWD SEM) of four rats per group. * denotes p < 0.05, in comparison with the control values (ANOVA followed by post-hoc Bonferroni test).

Fig. 4. Effect of compounds in reversal of the dynamic allodynia in CCI and SNL rats. Graphs depict the effects of compounds (100 mg/kg, i.p.) in reversal of the dynamic allodynia in CCI rats. The results are shown as mean paw withdrawal latency (mean PWL SEM) of four rats per group. * denotes p < 0.05, in comparison with the control values (ANOVA followed by post-hoc Bonferroni test).

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Fig. 5. Graphs show the effects of compounds against cold allodynia in CCI and SNL rats. Graphs depict the effects of compounds (100 mg/kg, i.p) against cold allodynia in CCI rats. The results are shown as either mean paw withdrawal duration (mean PWD SEM) of four rats per group. * denotes p < 0.05, in comparison with the control values (ANOVA followed by post-hoc Bonferroni test).

Fig. 6. Effects of compounds against mechanical hyperalgesia in CCI and SNL rats. Graphs depict the effects of compounds (100 mg/kg, i.p) against mechanical hyperalgesia in CCI rats. The results are shown as mean paw withdrawal duration (mean PWD SEM) of four rats per group. * denotes p < 0.05, in comparison with the control values (ANOVA followed by post-hoc Bonferroni test.

Please cite this article in press as: Perumal Y, et al. GABA derivatives for the treatment of epilepsy and neuropathic pain: A synthetic integration of GABA in 1, 2, 4Triazolo2Hone Nucleus. Biomed Aging Pathol (2012), doi:10.1016/j.biomag.2012.03.001

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induced writhing and formalin tests in mice. All of the tested compounds (100 mg/kg), except compound 2 and 10, suppressed the acetic acid induced writhing response signicantly in comparison to the control. Compound 1, 3, 4, 5 and 6 showed more than 90% inhibition, while indomethacin (5 mg/kg) exhibited 96% percentage inhibition. Compound 1 was the most active in this model with 99% inhibition. In formalin test, time spent in inching of paw in the early phase (05 min) and the late phase (10-30 min) were measured. Centrally acting compounds show inhibition in both the phases, while inhibition of second phase is characteristic of peripherally acting drugs. Seven compounds (1, 3, 5, 6, 7, 8 and 9) showed a signicant inhibition in both the phases, except compound 2, which was signicantly active only in the second phase. Indomethacin (5 mg/kg), which is a peripherally acting drug also showed a signicant inhibition (85%) in phase 2. The most active compounds were 8 (for phase 1, with 75% inhibition) and 6 (for phase 2, with 80% inhibition). Compounds 4 and 10 were inactive in both the phases. Two peripheral neuropathic pain models, the chronic constriction injury (CCI) and L5 spinal nerve ligation (SNL) models in rats were used to assess the antihyperalgesic and antiallodynic potential of the synthesized compounds. In the CCI model, the left sciatic nerve proximal to the trifurcation point was constricted with four loose ligatures using 3-0 braided silk thread, while in the SNL model, a tight ligation was tied around the L5 spinal nerve using 3-0 braided silk thread. On the 9th day post surgery, four sensory assays aimed at determining the severity of behavioral neuropathic responses, namely allodynia and hyperalgesia, were performed at 30, 60, 90, 120, and 150 min postdrug administration. The assays involved measurement of the degree of spontaneous (ongoing) pain and tested for hind limb withdrawal (PWD) to cold and mechanical stimuli (dynamic mechanical allodynia, cold allodynia, and mechanical hyperalgesia). All of the compounds were tested at a single dose of 100 mg/kg. In the CCI model, compounds 3, 4 and 6 completely reversed the spontaneous pain response throughout the time period of 30150 min. These compounds had onset of action at 30 min, and showed more efcacies when compared to carbamazepine (Fig. 3). Compounds 8 and 9 were active only from 60 to 120 min. Compound 8 was active at 60 and 120 min, while 7 was active only at 60 min. Carbamazepine reversed the spontaneous pain response only till 60 min signicantly and lamotrigine was devoid of any activity. Compound 1, 2, 5 and 10 were totally inactive. Administration of six compounds (3, 4, 6, 7, 9 and 10) completely reversed the dynamic allodynic response in CCI rats throughout the entire 150 min as the standard drug carbamazepine (Fig. 4). Compound 1 had efcacy between 30-60 min. Compounds 2, 5 and 8 were found to be totally inactive. Lamotrigine was active only at 30 min. The PWDs in response to cold stimuli in CCI rats were signicantly reduced by the administration (100 mg/kg, i.p.) of compounds 3, 4, 6, 7, 9 and 10, throughout the entire 150 min (Fig. 5). Compounds 3, 6 and 9 had onset of action at 60 min; while 4, 7 and 10 were efcacious from 30 min. Carbamazepine was efcacious only till 60 min. All other compounds including lamotrigine were found to be ineffective in this test. Hyperalgesia evoked by a mechanical pin-prick stimulus in CCI rats was effectively attenuated at all time-points of study by compounds 6 and 10 (Fig. 6). Compounds 3, 7 and 9 were active from 60 to 150 min. Compound 1 was effective between 30-60 min. All other compounds (2, 4, 5, and 8) including lamotrigine and carbamazepine were found to be completely inactive in this assay. Spontaneous pain in the SNL model was completely reversed by compounds 3, 4 and 6 at the dose tested over 150 min period, and showed more efcacy than carbamazepine (Fig. 3). Compound 1 was active between 90150 min, while compound 7 was active

between 30 to 90 min. Two compounds 2 and 9 were effective only at 30 min and 60 min respectively. Compounds 5, 8 and 10 were totally inefcient in reducing spontaneous pain. Compound 3 emerged as the most effective compound in reducing spontaneous pain. In this test, carbamazepine reversed the spontaneous pain response only till 90 min signicantly and lamotrigine was active only at 90 min. Four compounds (4, 6, 7 and 10) were active in attenuating the dynamic allodynic response in SNL rats throughout the testing period of 150 min as the standard drug carbamazepine (Fig. 4). Compound 3 was active between 60-150 min, while compounds 2 and 9 were active between 30-60 min. None of the compounds were as effective as standard drug carbamazepine. Compounds 1, 5 and 8 were totally inactive. Lamotrigine was effective only at 90 min. Cold allodynia produced in SNL rats was signicantly reduced by the administration of compounds 4, 6 and 10, throughout the entire 150 min (Fig. 5). Compounds 1 and 3 were active from 60-90 min; while compound 9 was active only at 60 min. Carbamazepine was effective only till 90 min of testing, while compounds 2, 5, 7 and 8 were totally inactive like lamotrigine. Mechanical hyperalgesia evoked by pin-prick in SNL rat stimulus was signicantly attenuated till 150 min by four compounds 3, 4, 6 and 10 (Fig. 6). Compound 2 was effective only at 30 min, while compounds 1, 8 and 9 were effective only at 60 min. Carbamazepine was found to be effective from 60-150 min, while lamotrigine was effective only at 60 min. Compounds 2, 5 and 7 were found to be ineffective. Structural analysis of these two classes of compounds reveals that in the anticonvulsant efcacy when the aryl function is changed from C4 (1-5) to C5 (6-10) position or the GABA functionality was changed from C5 to C4 position, bioactivity was found to be lost. In the acute nociceptive screening also the 4-aryl derivatives were found to be more efcient than the 5-aryl derivatives. In contrast to these observations, in the neuropathic pain models (CCI & SNL), the 5-aryl derivatives were more potent than the 4-aryl derivatives with regard to the duration of action and protection in various behavioural assays of neuropathic pain. 4. Conclusion The present study reports the synthesis, antiepileptic, antinocciceptive, antihyperalgesic and antiallodynic activities of 1, 2, 4-triazole derivatives of GABA. 4-Aryl substituted triazole derivatives have a better antiepileptic prole compared to that of 5-aryl substituted triazole derivatives. While the 5-aryl substituted 1, 2, 4-triazoles were more efcacious than the 4-aryl counterparts with regard to the antihyperalgesic and antiallodynic properties. Overall results show that the GABA integrated tiazole deivatives have a potential to be a promising lead for new drug development for the treatment of epilepsy and neuropathic pain. Disclosure of interest The authors have not supplied their declaration of conict of Q2 interest. Acknowledgements The authors wish to thank the Department of Biotechnology (DBT), New Delhi (India) for funding the project. References
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