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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

ARTICLE IN PRESSG Model

BIOMAG44 110

Biomedicine & Aging Pathology xxx (2012) xxxxxx

Available online at

www.sciencedirect.com

Original article

GABA derivatives for the treatment of epilepsy and neuropathic pain: A syntheticintegration of GABA in 1, 2, 4Triazolo2Hone Nucleus2

Yogeeswari Perumal , Sravan Kumar Patel , Ingala Vikram Reddy , Arvind Semwal , Monika Sharma ,Q1Matharasala Gangadhar , Matikonda Siddharth Sai , Dharmarajan Sriram

3

4

Neuropathic Pain Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, R.R. District-500078, Andhra Pradesh, India5

6

a r t i c l e i n f o7

8

Article history:9

Received 5 March 20120

Accepted 11 March 2012

Available online xxx2

3

Keywords:4

Triazole5

Antiepileptic6

Neuropathic pain7

Analgesic activity8

Antiallodynic9

Antihyperalgesic0

a b s t r a c t

Neuropathic pain is characterized by a neuronal hyperexcitability in damaged areas of the peripheral orcentral nervous system. Theneuronal hyperexcitability in neuropathic pain havemany commonfeatures

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 the1, 2, 4-triazol-2H-onenucleus and their evaluation forantiepilep-

tic, peripheral analgesic, antiallodynic and antihyperalgesic potential in neuropathic pain models. Most

of the synthesized triazole derivatives of GABA produced antiepileptic, antinocciceptive, antihyperal-

gesic, 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.

1. Introduction7

Neuropathic pain is characterized by spontaneous burning pain8

accompanied with hyperalgesia and allodynia as a result of a9

primary lesion, injury or dysfunction in the central or periph-0

eral nervous system [1]. Conventional antiepileptic drugs, such as

phenytoin, carbamazepine, topiramate, and lamotrigine have been2

used to treat neuropathic pain, but recently, some of the newer3

anticonvulsants, like gabapentin and pregabalin received increased4

attention as analgesics for treating neuropathic pain [2]. One of the5

important approaches to control chronic neuropathic pain involves6

the activation of-aminobutyric acid (GABA) mediated inhibition.7Previous reports from our laboratory have shown the effectof vari-8

ous GABAderivatives for the treatmentof epilepsy and neuropathic9

pain [39].0

It is well reported that triazole nucleus possess diverse pharma-

cological activities like anti-microbial [10,11], anti-tubercular [12],2

anticonvulsant activity [13] and anti-inflammatory and analgesic3

activities [1416]. In the past we had attempted to synthesize 1,4

2, 4-triazole nucleus to study the effect of cyclization of aryl semi-5

carbazone pharmacophore on anticonvulsant activity and found to6

exhibit biological activity in four animal models of seizures [17].7

Corresponding author. Tel.: +919 705 932 091; fax: +040-66303998.

E-mail addresses: [email protected], [email protected]

(Y. Perumal).

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- triazolederivatives to have P2X7antagonistic action andalso to inhibit IL-1b release from human THP-1 cells, which were

further evaluated for antinociceptive activity in the CCI model of

neuropathic pain witha good oral bioavailability [18]. An European

patent (EP1921073), describedvarious1, 2, 4-triazole derivatives as

- receptor inhibitors and claimed to haveeffect against pain, espe-cially neuropathic pain and inflammatory pain [19]. An US patent

(US20040106614), described 1H-1, 2, 4-triazole-3-carboxamide

derivatives having cannabinoid-CB1 receptoragonistic, partial ago-

nistic, 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, 4-

triazole derivatives integrated with GABA aimed at investigatingtheirantiepileptic, peripheral analgesic, anti-allodynamic and anti-

hyperalgesic activities.

2. Materials and methods

2.1. Chemistry

Melting points were measured in open capillary tubes on a

Buchi 530 melting point apparatus and are uncorrected. Infrared

(IR) and proton nuclear magnetic resonance (1H-NMR) spectra

were recorded for the compounds on Jasco IR Report 100 (KBr)

2210-5220/$ see front matter 2012 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.biomag.2012.03.001
http://dx.doi.org/10.1016/j.biomag.2012.03.001http://dx.doi.org/10.1016/j.biomag.2012.03.001http://www.sciencedirect.com/science/journal/22105220mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.biomag.2012.03.001http://dx.doi.org/10.1016/j.biomag.2012.03.001mailto:[email protected]:[email protected]://www.sciencedirect.com/science/journal/22105220http://dx.doi.org/10.1016/j.biomag.2012.03.001http://dx.doi.org/10.1016/j.biomag.2012.03.001

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BIOMAG44 110

2 Y. Perumal et al. / Biomedicine & Aging Pathology xxx (2012) xxxxxx

and Brucker Avance (300 MHz) instruments, respectively. Chemi-

cal shifts are reported in parts per million (ppm) using tetramethyl

silane (TMS)as an internalstandard. All exchangeableprotonswere

confirmedby addition of D2O. Elementalanalyses (C,H andN) were

undertaken with a Perkin-Elmer model 240C analyzer andall anal-

yses 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.

2.1.1. General procedure for the preparation of 4 Aryl substituted

1, 2, 4-triazoles (1-5)

For the synthesisof 4-aryl substituted 1,2,4-triazoles, GABA was

N-protected using phthalic anhydride by taking equimolar quan-

tities of GABA and the fomer in presence of toluene and triethyl

amine (as a proton scavenger) [6]. Hydrochloricacid (HCl) was then

added, stirred for 30 minutes, filtered and collected the precipitate

of N-phthaloyl GABA (m.p 105107). Different substituted ani-

lines 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 refluxing it with hydrazine

hydrate and sodium hydroxide in presence of ethanol for 24h [22].

The carboxyl group of N-phthaloylGABA andaminogroupof semi-carbazide were further coupled in the presence of DCC and DMF by

stirring the reaction mixture for 24h. N-protected phthaloyl group

was then removed by refluxing 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

filtrate pyridine was added till it becomes basic. Further purified

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 refluxed in the presence of 4%NaOH to get the cyclised

compounds (1-5)

The structures were characterized by both spectral and ele-

mental analysis and the data were within 0.4% of the theoretical

values.

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 (0.01moles) wasreactedwith0.01moles(1.25 mL)of phenyl

chloroformate in presence of sodium hydroxide solution to form

4-[(phenoxycarbonyl) amino] butanoic acid. The mixture was vig-

orously stirred for 90 min.The precipitate wasthen filtered,washed

andfinallydriedinahotairovenat52 C.A solutionof 0.0005moles

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 refluxed for 6 h and then concentrated by evap-

oration. The precipitate was then filtered 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 reac-

tion mixture an equimolar quantity of acid chloride was added

and refluxed for 34 h. Then the reaction mixture was concen-

trated by distilling out ethanol and the product was obtained by

keeping in ice-cold condition. The product was filtered and dried.

The obtained 4-({[(2-oxo-2-phenylethyl) amino] carbonyl} amino)

butanoic acid, was then cyclised to 1, 2, 4-triazole by refluxing in

presence of 4% alcohol, for 23 h [17].

The structures were characterized by both spectral and ele-

mental analysis and the data were within 0.4% of the theoretical

values.

2.2. Pharmacology

Lamotrigine and carbamazepine were obtained as gift sam-

ples from M/s IPCA Laboratories, India. Swiss albino mice (either

sex) with weights ranging from 2025g were used for the acetic

acid induced writhing and formalin test. Wistar rats of either sex

(200250g) 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 administeredin 30%v/v PEG 400. Thecontrol

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 0.01mL/g for mice at doses of 100 and 300mg/kg.

Anticonvulsant activity was assessed after 30min and 4 h of drug

administration. Activity in the MES and scPTZtests 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

deficit 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 deficit 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 writhingMice were divided into groups of sixeach. Writhingwasinduced

by an intraperitoneal injection of 0.1mL 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 stretch-

ing 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 forma-

lin into subcutaneous tissues, typically the dorsal surface of the

rodent paw that gives flinches of paw in the early phase (05min)

and the late phase (10-30 min) [26]. In formalin test pain-related

behaviors were quantified based on the total time of flinching andlicking 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 quantification of the pain-

related 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 pento-

barbital sodium (65mg/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
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Y. Perumal et al. / Biomedicine & Aging Pathology xxx (2012) xxxxxx 3

Table 1

General structure, antiepileptic activity, neurotoxicity and acute antinocciceptive efficacy of compounds (110)

NHN

O

R1

R2

.

Compound R1 R2 Antiepileptic activity and Neurotoxicity Acetic acid (%

inhibition)

Formalin test (%

inhibition)

MES scPTZ Neurotoxicity Phase-1 Phase II

0.5 h 4 h 0.5 h 4 h 0.5 h 4 h

1Br

(CH2)3NH2 100 300 300 300 300 99.0 72.2a 70.1a

2Cl

(CH2)3NH2 300 17b 17.5b 45.3a

3

CH3H3C

(CH2)3NH2 300 300 300 91.5a 47.4a 65a

4

H3C

CH3 (CH2)3NH2 300 300 94a 15.8b 15.3b

5

H3C

Cl

(CH2)3NH2 100 300 300 95.5a 34a 70.8a

6 (CH2)3COOH 96a 61.5a 80.1a

7 (CH2)3COOH 300 73a 73.2a 77.5a

8 (CH2)3COOH

N

O

O 0 78a 75.9a 77a

9 (CH2)3COOH (CH2)7 87a 40.9a 54a

10 (CH2)3COOH

NH

CH3

O 13b 5.2b 0b

Phenytoin 100 100 - 100 100 Ethosuximide 300

Indomethacin - 96a 11.3 85.5a

Doses of 100 and 300 mg/kg were administered for anticonvulsant efficacy and neurotoxicity. The figures 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 Significantly different from vehicle at p < 0.05.b Not significantatp < 0.05 (one-wayANOVA, followed by post-hocBonferroni test). All thecompoundswere administered intraperitoneally at a doseof 100mg/kg. Control

animals were administered 30% v/v PEG 400.

(contralateral). The left paraspinal muscles were then separated5

from the spinous processes and the common left sciatic nerve was6

exposed just above the trifurcationpoint. Four loose ligatures were7

then made with a 4-0 braided silk suture around the sciatic nerve8

with about 1-mm spacing as reported elsewhere [28]. The wound9

was then closed by suturing the muscle using chromic catgut with0

a continuous suture pattern. Finally, the skin was closed using silk

thread with horizontal-mattress suture pattern. A sham surgery2

(n = 4) was performed by exposing the sciatic nerve as described3

above, but not damaging it. Povidone iodine ointment was applied4

topically on the wound and gentamicin antibiotic (4 mg/kg) was5

given intramuscularly for five days after surgery. The animals were6

then transferred to their home-cages and left for recovery.7

2.2.6. Selective segmental L5 SNL Model8

A left L5 spinal nerve ligation, as described by Kim and Chung9

[29], was performed. The rats were anesthetized with an intraperi-0

toneal dose of pentobarbital sodium (65mg/kg) with additional

doses of the anesthetic given as needed. Under aseptic conditions,2

using the transverse processes of L6 as a guide, the left paraspinal

muscles were exposed andseparated from thespinous processes of

L4 to S2 by blunt dissection. The L5 spinal nerve was then exposed

at thelevel of thedorsal root ganglion,and ligated tightly with a 4-0

braided silk suture. Only one tight ligature was made in this model.

After confirmation of hemostasis, the wound was then closed bysuturing at both muscle and skin levels. A sham surgery (n = 4)

wasperformed by exposing the L5 spinalnerve as described above,

but not damaging it. Povidone iodine ointment was applied topi-

cally on the wound and gentamicin antibiotic (4 mg/kg) was given

intramuscularly for five days after surgery. The animals were then

transferred to their home-cages and left for recovery.

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 hyperal-

gesia 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 mini-

mum of 10 min separated the testing procedures to reduce the

influence 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 periodof 5 minas describedpreviouslyby 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 floor. The paw lifts associated with locomotion or body repo-

sitioning 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 ini-tial acclimatizationperiod of 10min wasgivento each of therats.A

total number of four rats (n = 4) were assigned to this group. A pos-

itive dynamic allodynic response consisted of lifting the affected

paw for a finite 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 15s, 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 sen-

sitivity using the acetone drop application technique as described

by Caudle et al. [32]. The operated rat was placed inside an obser-vation cage that was kept 5cm 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 (100200L) of freshly dispensed acetone weresquirted as a fine 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

toeach oftherats.A total number offour 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 defined 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

Alldata areexpressed as means standarderror of mean(SEM).

The data were analyzed by one-way ANOVA, and bonferronis post

hoc test was used for individual comparisons with the control val-

ues. Significance was assigned to a p value of less than 0.05. The

statistical software package PRISM (Graphpad Software Inc, San 2

Diego, CA) was used for the analyses. 2

3. Results and discussion 2

3.1. Synthesis 2

The synthetic protocols employed for the preparation of 4-aryl 2

substituted triazole (1-5) and 5-aryl substituted triazole (6-10) 2derivatives are presented in Figs. 1 and 2. For the synthesis of 4- 2

aryl GABA substituted 1, 2, 4-triazoles, GABA was first N-protected 2

using phthalic anhydride [6]. Different substituted anilines were 2

reacted with sodium cyanate in the presence of water and glacial 2

acetic acidto formsubstituted arylurea, which wereconverted into 2

semicarbazides by refluxing it with hydrazine hydrate and sodium 2

hydroxide in presence of ethanol [22]. Amino group of semicar- 2

bazide was coupledwith carboxylgroup of N-phthaloyl GABAin the 2

presence of N, N-dicyclohexyl carbodiimide (DCC) and dimethyl- 2

formamide(DMF).The product obtainedwas further refluxed in the 2

presence of 4% sodium hydroxide to get the cyclised compounds 2

(1-5) [17]. 2

For the synthesis of 5-aryl GABA substituted 1,2,4-triazoles 2

(6-10), The starting material, GABA, was treated with phenyl2

chloroformate in aqueous sodium hydroxide at a range of 0- 2

5 C to yield 4-(phenoxycarbonyl-l-amino)butanoic acid to yield 2

70% after crystallization with 95% ethanol. The carbamate, on 2

condensation with hydrazine hydrate in ethanol, gave the 4- 2

(hydrazinecarbonyl-l-amino) butanoic acid [3]. Base catalyzed 2

condensation of semicarbazide salt was carried out with equimo- 2

lar quantity of substituted acid chloride. The product obtained was 2

then cyclisedto 1,2, 4 triazole byrefluxingin presenceof 4%sodium 2

hydroxide to get the compound 6-10. All the structures were char- 2

acterizedby bothspectral and elementalanalysis andthe datawere 2

within 0.4% of the theoretical values. The physical and spectral 3

data of the compounds are as follows. 3

3.1.1. 3-(3-Aminopropyl)-4-(4-bromophenyl)-1H-1,2,4-triazol-3

5(4H)-one 3

(1) 3

M.P.: 233 C; Yield: 59%; IR (KBr): 3400, 3250,3000, 3

2860,1740,1650,1600,1580,1475,660 cm-1; 1H-NMR (DMSO- 3

d6) (ppm): 8.72 (d, 2H, Ar-H), 7.58 (d, 2H, Ar-H), 7 (s, 1H, NH), 35.11 (s,2H, NH), 2.55 (m,2H), 1.69 (m,2H), 1.53 (m, 2H). Calculated 3

for C11H13BrN4O: C, 44.46; H, 4.41; Br, 26.89; N, 18.85; O, 5.38 3

found: C, 44.32; H, 4.56; Br, 26.54; N, 18.26; O, 5.16. 3

3.1.2. 3-(3-Aminopropyl)-4-(4-chlorophenyl)-1H-1,2,4-triazol- 3

5(4H)-one 3

(2) 3

M.P.: 230 C; Yield: 78%; IR (KBr): 3380, 3250,3000, 3

2850,1720,1650,1600,1580,1470,690 cm;

1

H-NMR (DMSO-d6)3

(ppm): 7.43-7.47 (m, 4 Ar-H), 7 (s, 1H, NH), 5.11 (s, 2H, NH), 2.65 3(m, 2H), 1.69 (m, 2H), 1.53 (m, 2H). Calculated for C11H13ClN4O: 3

C, 52.58; H, 5.19; Cl, 14.03; N, 22.17; O, 6.33 found: C, 52.67; H, 3

5.11; Cl, 23.12; N, 22.23; O, 6.30. 3

3.1.3. 3-(3-Aminopropyl)-4-(2,5-dimethylphenyl)-1H-1,2,4- 3

triazol-5(4H)-one 3

(3) 3

M.P.: 211 C; Yield: 54%; IR (KBr): 3360, 3400,3150,3035,1725, 3

1600, 1550, 1450, 1380, 880,690 cm-1; 1H-NMR (DMSO-d6) 3(ppm): 7.6 (s,1Ar-H), 7-7.4 (m, 4 Ar-H), 6.8 (s, 1H, NH), 5.11 (s, 3

2H, NH), 2.65 (m, 2H), 2.4 (s,1H),2.1(s,1H).1.69 (m, 2H), 1.53 (m, 3

2H). Calculated for C13H18N4O: C, 63.39; H, 7.37; N, 22.75; O, 3

6.50,found: C, 63.28; H, 7.16; N, 22.34; O, 6.64.3

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O

O

O

+

O

OH

H2N -H2O

N

O

OH

O

HO

Toluene,Triethylamine, HCl

NaCNO/H2O

GAAHN

NH2

O

NH2NH2.H2O

HN

HN NH2

O

NH2

R

R

R

N

HO

OO

O

+

DCC

N

O

O

NH

O HN

O

NH

R

HN

HN

NH

O

O

NH2

HN N

NO

NaOH/NH2NH2.H2O

4 % NaOH

Hydrazinolysis

HN

O

HN

NH2

R

R

R

NH2

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

3.1.4. 3-(3-Aminopropyl)-4-(2,6-dimethylphenyl)-1H-1,2,4-9

triazol-5(4H)-one0

(4)

M.P.: 216 C; Yield: 83%; IR (KBr): 3350, 3400, 3150, 3035,1725,2

1600, 1560, 1450, 1375, 870, 760, 690 cm-1; 1H-NMR (DMSO-3

d6) (ppm): 7.0-7.3 (m, 3 Ar-H), 7 (s, 1H, NH), 5.11 (s, 2H, NH),42.65 (m, 2H), 2.12 (s,6H),1.69 (m, 2H), 1.53 (m, 2H). Calculated for5

C13H18N4O: C, 63.39; H, 7.37; N, 22.75; O, 6.50 found: C, 63.45; H,6

7.26; N, 22.53; O, 6.32.7

3.1.5. 3-(3-Aminopropyl)-4-(3-chloro-2-methylphenyl)-1H-8

1,2,4-triazol-5(4H)-one9

(5)0

M.P.: 189 C; Yield: 64%; IR (KBr): 3360, 3400, 3150,3050,1720,

1600, 1580, 1450, 1370, 880, 700, 690 cm-1; 1H-NMR (DMSO-d6) 2(ppm): 7.6 (m, Ar-H),7.2 (m,Ar-H), 7.4(m,Ar-H), 7 (s, 1H, NH), 5.113

(s, 2H, NH), 2.65 (m, 2H), 2.2 (s,3 H),1.70 (m, 2H), 1.50 (m, 2H).4

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.: 237C; Yield: 58%; IR (KBr): 3400, 3100, 2920, 1725,

1700, 1600,1550, 1475, 1450, 880 cm-1; 1H-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.: 197C; Yield: 61%; IR (KBr): 3380, 3120,2900, 1725,

1700,1660,1600,1550, 1475, 1465, 890 cm-1;1H-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).

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; 1H-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;1H-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; 1H-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 3

The aim of study was to prepare newer GABA derivatives of 1, 2, 3

4-triazoles with multiple pharmacological actions effective in the 3

treatment of epilepsy and neuropathic pain. For the anticonvul- 3

sant activity, compounds (1-10) were evaluated at dose levels of 3

100 and 300 mg/kg intraperitoneally in mice by standard anticon- 3

vulsant drug development (ADD) program protocols [23,24]. The 3

tests included maximal electroshock seizure test (MES) andsubcu- 3

taneous pentylenetetrazole (scPTZ) seizure threshold test whose 3

results are presented in Table 1. 4-Aryl substituted 1,2,4-triazoles 3

(1-5) showed activityin boththe models indicative of their abilityto 3

prevent seizure spread while 5-aryl substituted 1,2,4-triazoles (6- 4

10) were devoid of any antiepileptic activities. In the MES model, 4

compounds1 and 5 exhibitedlonger durationof actionuntil 4 h and 4

showed efficacy at 100 mg/kg at 0.5 h and 300 mg/kg at 4 h, com- 4

pared to other active compounds, whichwere activeonly at 0.5h at 4

thedose of 300mg/kg.Compound5 showed comparable efficacy to 4

phenytoin in MES model, with a better safety profile. In the scPTZ 4test, four compounds (1, 3, 4 and 5) showed marginal protection, 4

with efficacy at 300mg/kg at 0.5 h time period only, but compara- 4

ble to that of ethosuximide. Allothersynthesizedcompounds were 4

ineffective in this model. 4

The acute neurological toxicity was determined by the rotorod 4

test [3]. All the compounds except 1 and 3 were devoid of any neu- 4

rotoxicity. Compound 5 emerged as the most active anticonvulsant 4

in both the models with no neurotoxicity. There was no separa- 4

tion between the anticonvulsant dose and the neurotoxic dose 4

(300mg/kg) for compound 1 while compound 3 was neurotoxic 4

only at 4 h. 4

Beforeevaluatingthe therapeuticpotential of the1, 2, 4-triazole 4

derivatives in animals model of neuropathic pain, compounds were 4

evaluatedfirst in two acute nocciception models namely acetic acid4

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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 (100mg/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.

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induced writhing and formalin tests in mice. All of the tested com-

pounds (100 mg/kg), except compound 2 and 10, suppressed the2

acetic acid induced writhing response significantly in comparison3

to the control. Compound 1, 3, 4, 5 and 6 showed more than 90%4

inhibition, while indomethacin (5 mg/kg) exhibited 96% percent-5

age inhibition. Compound 1 was the most active in this model with6

99% inhibition.7

In formalin test, time spent in flinchingof pawin the early phase8

(05min) andthe late phase (10-30 min) were measured.Centrally9

acting compounds show inhibition in both the phases, while inhi-0

bition of second phase is characteristic of peripherally acting drugs.

Sevencompounds (1, 3, 5, 6, 7, 8 and 9) showeda significant inhibi-2

tionin boththe phases, except compound2, which wassignificantly3

active only in the secondphase. Indomethacin (5mg/kg), which is a4

peripherally acting drug also showed a significant inhibition (85%)5

in phase 2. The most active compounds were 8 (for phase 1, with6

75%inhibition)and 6 (forphase 2, with80% inhibition).Compounds7

4 and 10 were inactive in both the phases.8

Two peripheral neuropathic pain models, the chronic constric-9

tion injury (CCI) and L5 spinal nerve ligation (SNL) models in rats0

were used to assess the antihyperalgesic and antiallodynic poten-

tial of the synthesized compounds. In the CCI model, the left sciatic2

nerve proximal to the trifurcation point was constricted with four3

loose ligatures using 3-0 braided silk thread, while in the SNL4

model, a tight ligation was tied around the L5 spinal nerve using5

3-0 braided silk thread. On the 9th day post surgery, four sensory6

assays aimedat determining the severity of behavioral neuropathic7

responses, namely allodynia and hyperalgesia, were performed at8

30, 60, 90, 120, and 150 min postdrug administration. The assays9

involvedmeasurementof thedegree of spontaneous (ongoing) pain0

and tested for hind limb withdrawal (PWD) to cold and mechan-

ical stimuli (dynamic mechanical allodynia, cold allodynia, and2

mechanical hyperalgesia). All of the compounds were tested at a3

single dose of 100 mg/kg.4

In the CCI model, compounds 3, 4 and 6 completely reversed5

the spontaneous pain response throughout the time period of 30-6

150 min. These compounds had onset of action at 30min, and7

showed more efficacies when compared to carbamazepine (Fig. 3).8

Compounds 8 and 9 were active only from 60 to 120min. Com-9

pound 8 was active at 60 and 120 min, while 7 was active only at0

60 min. Carbamazepine reversed the spontaneous pain response

only till 60 min significantly and lamotrigine was devoid of any2

activity. Compound 1, 2, 5 and 10 were totally inactive.3

Administration of six compounds (3, 4, 6, 7, 9 and 10) completely4

reversed the dynamic allodynic response in CCI rats throughout5

the entire 150 min as the standard drug carbamazepine (Fig. 4).6

Compound 1 had efficacy between 30-60 min. Compounds 2, 5 and7

8 were found to be totally inactive. Lamotrigine was active only at8

30 min.9

The PWDs in response to cold stimuli in CCI rats were sig-0

nificantly reduced by the administration (100mg/kg, i.p.) of

compounds 3, 4, 6, 7, 9 and 10, throughout the entire 150 min2(Fig. 5). Compounds 3, 6 and 9 had onset of action at 60 min; while3

4, 7 and 10 were efficacious from 30 min. Carbamazepine was effi-4

cacious onlytill 60 min. All other compounds including lamotrigine5

were found to be ineffective in this test.6

Hyperalgesia evoked by a mechanical pin-prick stimulus in CCI7

rats was effectively attenuated at all time-points of study by com-8

pounds 6 and 10 (Fig. 6). Compounds 3, 7 and 9 were active from9

60 to 150min. Compound 1 was effective between 30-60 min. All0

other compounds (2, 4, 5, and 8) including lamotrigine and carba-

mazepine were found to be completely inactive in this assay.2

Spontaneous pain in the SNL model was completely reversed3

by compounds 3, 4 and 6 at the dose tested over 150 min period,4

and showed more efficacy than carbamazepine (Fig. 3). Compound5

1 was active between 90150 min, while compound 7 was active6

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 inefficient 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 significantly and lamotrigine was active

only at 90min.

Four compounds (4, 6, 7 and 10) were active in attenuating

the dynamic allodynic response in SNL rats throughout the test-

ing period of 150 min as the standard drug carbamazepine (Fig. 4).

Compound 3 was active between 60-150min, while compounds 2

and 9 wereactivebetween30-60min. None of thecompounds were

as effective as standard drug carbamazepine. Compounds 1, 5 and

8 were totally inactive. Lamotrigine was effective only at 90 min.

Coldallodynia producedin SNL ratswas significantlyreducedby

the administrationof compounds 4, 6 and 10, throughout the entire

150min (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 stimu-

lus was significantly attenuated till 150 min by four compounds 3,

4, 6 and 10 (Fig. 6). Compound 2 was effective only at 30min, while

compounds1, 8 and 9 wereeffective onlyat 60 min.Carbamazepinewas found to be effective from 60-150 min, while lamotrigine was

effective only at 60min. Compounds 2, 5 and 7 were found to be

ineffective.

Structural analysis of these two classes of compounds reveals

that in the anticonvulsant efficacy when the aryl function is

changed from C4 (1-5) to C5 (6-10) position or the GABA function-

ality was changed from C5 to C4 position, bioactivity was found to

be lost. In the acute nociceptive screening also the 4-aryl deriva-

tives were found to be more efficient than the 5-aryl derivatives.

In contrast to these observations, in the neuropathic pain models

(CCI & SNL), the5-aryl derivatives were more potentthanthe 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, antinoc-

ciceptive, antihyperalgesic and antiallodynic activities of 1, 2,

4-triazole derivatives of GABA. 4-Aryl substituted triazole deriva-

tives have a better antiepileptic profile compared to that of 5-aryl

substituted triazole derivatives. While the 5-aryl substituted 1, 2,

4-triazoles weremore efficacious thanthe 4-aryl counterparts with

regard to the antihyperalgesic and antiallodynic properties. Over-

all 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 conflict of Q

interest.

Acknowledgements

The authors wish to thank the Department of Biotechnology

(DBT), New Delhi (India) for funding the project.

References

[1] Merskey H, Bogduk N. Classification of chronic pain. International associa-tion for the study of pain task force on taxonomy. Seattle: IASP Press; 1994,

p.20914.

8/2/2019 BIOMAG_44

11/11

Please cite this article in press as: Perumal Y et al GABA derivatives for the treatment of epilepsy and neuropathic pain: A synthetic


BIOMAG44 110


[2] Jensen TS, Baron R. Translation of symptoms and signs into mechanisms inneuropathic pain. Pain 2003;102:18.

[3] YogeeswariP, RagavendranJV, Sriram DJV,NageswariY, KavyaR, Sreevatsan N,et al. Discovery of 4-Aminobutyric acid derivatives possessing anticonvulsantand antinociceptiveactivities: a hybrid pharmacophore approach. J Med Chem2007;50:245967.

[4] Yogeeswari P, Sriram D, Sahitya P, Ragavendran JV, Ranganadh V.Synthesis and anticonvulsant activity of 4-(2-(2,6-dimethylphenylamino)-2-oxoethylamino)-N-(substituted)butanamides: a pharmacophoric hybridapproach. Bioorg Med Chem Lett 2007;17:37125.

[5] Ragavendran JV, Sriram D, Kotapati S, Stables J, Yogeeswari P. Newer GABA

derivatives for the treatment of epilepsy including febrile seizures: a bioisos-teric approach. Eur J Med Chem 2008;43:26505.

[6] Ragavendran JV, Sriram D, Patel SK, Reddy IV, Bharathwajan N, Stables J, et al.Design and synthesis of anticonvulsants from a combined phthalimide-GABA-anilide and hydrazone pharmacophore. Eur J Med Chem 2007;42:14651.

[7] Yogeeswari P, Menon N, Semwal A, Arjun M, Sriram D. Discovery of moleculesforthe treatmentof neuropathicpain:synthesis,antiallodynicand antihyperal-gesic activities of 5-(4-nitrophenyl)furoic-2-acid hydrazones. Eur J Med Chem2011;46:296470.

[8] Semwal A, Yogeeswari P, Ragavendran JV, Sriram D, Stables J. Discovery of isa-tinimino derivatives as new leads for neuropathic pain treatment: an isomericmodification approach. Int J Drug Des Discov 2010;1:16987.

[9] Yogeeswari P, Ragavendran JV, Sriram D, Kavya R, Vanitha K, Neelakantan H.Newer N-phthaloyl GABA derivatives with antiallodynic and antihyperalgesicactivities in both sciatic nerve and spinal nerve ligation models of neuropathicpain. Pharmacol 2008;81:2131.

[10] Bohm R, Karow C. Biologically active triazoles. Pharmazie 1981;26:2437.[11] Gumrukcuoglu U, Serdar M, Sevim A, Demirbas N. Synthesis and antimicrobial

activitiesof somenew 1,2,4-triazole derivatives. Turk J Chem2007;31:33548.

[12] Mir I, Siddiqui MT, Camire A. Antituberculosis agents. I. Alpha-(5-(2-Furyl)-1,2,4-triazol-3-ylthio) acethydrazide and related compounds. Tetrahedron1970;26:52358.

[13] Modzelewska-Banachiewicz B, Banachiewicz J, Chodkowska A, Wjtowicz J,Mazur L. Synthesis and biological activity of new derivatives of 3-(3,4-diaryl-1,2,4-triazole-5-yl)propenoic acid. Eur JMed Chem 2004;39:8737.

[14] Hassan HYA, El-Shorbagi NA, El-Koussi AO, Abdel-Zaher O. Synthesis of tria-zoles for biological activity. Bull Pharm Sci 1994;17:2739.

[15] Tozkoparan BG, Akaty G, Yesilada E. Synthesis of some 1,2,4-triazolo[3,2-b]-1,3-thiazine-7-ones with potential analgesic and anti-inflammatory activities.Farmaco 2002;57:14552.

[16] Mohamed BG, Abdelalim M, Abdelalim MA, Hussein MA. Synthesis of 1-acyl-2-alkylthio-1,2,4-triazolobenzimidazoles with antifungal, anti-inflammatoryand analgesic effects. Acta Pharm 2006;56:3148.

[17] Shalini M, Yogeeswari P, SriramD, Stables JP.Cyclizationof thesemicarbazone 5template of aryl semicarbazones: synthesis and anticonvulsant activity of 4,5- 5diphenyl-2H-1,2,4-triazol-3(4H)-one. Biomed Pharmacother2009;63:18793. 5

[18] Carroll WA, Douglas MK, Arturo PM, Alan SF, Ying W, Diana L, et al. Novel and 5potent 3-(2,3-dichlorophenyl)-4-(benzyl)-4H-1,2,4-triazole P2X7 antagonists. 5Bioorg Med Chem Lett 2007;17:40448. 5

[19] Jagerovic CM, Goya L, Mara P, Zueras D, Cuberes A. 1,2,4-Triazole derivatives 5as sigma receptor inhibitors. European Patent 2008 1921073. 5

[20] Lange JHM, Kruse CG, Mccreary AC, Stuivenberg HHV. 1H-1,2,4-Triazole-3- 5carboxamide derivatives having cannabinoid CB1 receptor agonistic, partial 5agonistic, inverse agonistic or antagonist activity. US Patent 2004 0106614. 5

[21] Chakravarty PK, Zuegner III, Parsons WH, Brenda P, Bishan Z, Min KP. Biaryl 5substituted triazoles as sodium channel blockers. US Patent 2009; 7572822. 6

[22] Pandeya SN, Yogeeswari P, Stables J, Synthesis. anticonvulsant activ- 6ity of 4-bromophenyl substituted aryl semicarbazones. Eur J Med Chem 62000;35:87986. 6

[23] Porter RJ, Cereghino JJ, Gladding GD, Hessie BJ, Kupferberg HJ, Scoville 6B, et al. Antiepileptic drug development program. Cleveland Clin Quart 61984;51:293305. 6

[24] Kupferberg HJ. Antiepileptic drug development program: a cooperative effort 6of government and industry. Epilepsia 1989;30:S648. 6

[25] Siegmund E, Cadmus RA. A method for evaluating both non-narcotic and nar- 6cotic analgesics. Proc Soc Exp Biol 1957;95:72931. 6

[26] Hunskaar S, Hole K. Theformalin test in mice: dissociationbetween inflamma- 6tory and non-inflammatory pain. Pain 1987;30:10314. 6

[27] BennettGJ, XieYK. A peripheralmononeuropathyin ratthat producesdisorders 6of pain sensation like those seen in man. Pain 1988;33:87107. 6

[28] Obara I, Mika J, Schafe MKH, Przewlocka B. Antagonists of the kappa-opioid 6receptor enhance allodynia in rats and mice after sciatic nerve ligation. Br J 6Pharmacol 2003;140:53845. 6

[29] Kim S, Chung JM. An experimental model for peripheral neuropathy produced 6by segmental spinal nerve ligation in the rat. Pain 1992;50:35563. 6

[30] Choi Y, Yoon YW, Na HS, Kim SH, Chung JM. Behavioral signs of ongoing pain 6and cold allodynia in a rat model of neuropathic pain. Pain 1994;59:36976. 6

[31] Field MJ, McCleary S, Hughes J, Singh L. Gabapentin and pregabalin, but 6not morphine and amitriptyline, block both static and dynamic components 6of mechanical allodynia induced by streptozocin in the rat. Pain 1999;80: 63918. 6

[32] Caudle RM, Mannes AJ, Benoliel R, Eliav E, Iadarola MJ. Intrathecally adminis- 6tered choleratoxin blocksallodyniaand hyperalgesia inpersistentpain models. 6

J Pain 2001;2:11827. 6[33] GonzalezMI, Field MJ,Hughes J, Singh L. Evaluation of selectiveNK(1)receptor 6

antagonist CI-1021 in animal models of inflammatory and neuropathic pain. J 6Pharmacol Exp Ther 2000;294:44450. 6

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