ARTICLE

2-(4-substituted piperazin-1-yl)-1,8-naphthyridine-3-carboxylic acids:Novel 5-HT3 receptor antagonists with anxiolytic-like activity in rodentbehavioral modelsRadhakrishnan Mahesh, Arghya Kusum Dhar, Ankur Jindal, and Shvetank Bhatt

Abstract: The aim of this study was to investigate the anxiolytic potential of a series of novel carboxylic acid based 1,8naphthyridines as 5-HT3 receptor antagonists. The pA2 values of all the compounds were determined against agonist 2-methyl-5-hydroxytryptamine in longitudinalmusclemyenteric plexus preparations from guinea pig ileum. Compounds with higher pA2

values, particularly those greater than ondansetron, a standard 5-HT3 receptor antagonist, and optimal log P values werescreened in mice by using behavioral tests such as a light–dark (L/D) aversion test, elevated plus maze (EPM) test, and an openfield test (OFT). In the L/D test, compounds 7a, 7b, 7d, 7e, and 7i (2 mg/kg body mass, intraperitoneal) significantly (P < 0.05)increased the latency time to leave the light compartment, total time spent in the light compartment, and the number oftransitions between the light and dark compartments. Compounds 7a, 7d, 7f, 7h, and 7i (2 mg/kg, i.p.) significantly (P < 0.05)increased the time spent in the open arms and the number of entries into the open arms in the EPM test. In addition, compounds7a, 7d, 7e, 7f, and 7h (2 mg/kg, i.p.) significantly (P < 0.05) increased the ambulation scores and the frequency of rearing in the OFT.

Key words: 5-HT3 antagonist, anxiolytic, 1;8-naphthyridine, L/D, EPM, OFT.

Résumé : Le but de cette étude était d'examiner le potentiel anxiolytique d'une série de nouveaux carboxylates de 1,8-naphthyridines en leur qualité d'antagonistes du récepteur 5-HT3. Les valeurs de pA2 de tous les composés ont été déterminéespar rapport a l'agoniste 2-méthyl-5-hydroxytryptamine dans une préparation de plexus myentérique du muscle longitudinal del'iléon de cobaye. Les composés dont les valeurs de pA2 étaient plus élevées, particulièrement ceux dont les valeurs étaientsupérieures a celle de l'ondasétron, un antagoniste standard du récepteur 5-HT3, et dont les valeurs du log P étaient optimales,ont été criblés chez la souris a l'aide de tests comportementaux, notamment le test d'aversion lumière/noirceur (light–darkaversion test, L/D), le labyrinthe en croix surélevé (elevated plus maze, EPM) et le test d'espace ouvert (open field test, OFT). Lors du testL/D, les composés 7a, 7b, 7d, 7e et 7i (2mg/kg demasse corporelle, intrapéritonéale (i.p.) accroissaient significativement (P< 0,05)le temps de latence pour quitter le compartiment éclairé, le temps total passé dans le compartiment éclairé et le nombre detransitions entre les compartiments éclairés et sombres. Les composés 7a, 7d, 7f, 7h et 7i (2 mg/kg, i.p.) accroissaient significa-tivement le temps passés dans les corridors ouverts et le nombre d'entrées dans les corridors ouverts lors du test EPM. De plus,les composés 7a, 7d, 7e, 7f et 7h (2 mg/kg, i.p.) accroissaient significativement (p < 0,05) les scores d'ambulation et le nombre decabrages lors de l'OFT. [Traduit par la Rédaction]

Mots-clés : antagoniste du 5-HT3, anxiolytique, 1;8-naphthyridine, L/D, EPM, OFT.

IntroductionAnxiety, a normal response to stress, is a cardinal symptom of

many psychiatric illnesses closely allied with appropriate fearthat in many cases can become an important pathological, dis-abling, and lasting disorder (Barkow et al. 2004; Airaksinen et al.2005). Anxiety disorders are the most common psychiatric disor-ders in the US, with lifetime prevalence in approximately 30% ofthe population, and affecting 1/8 of the total population world-wide (Kessler et al. 2005). Benzodiazepines (BZDs) (diazepam andrelated drugs) are a class of drug generally prescribed as medica-tion for the treatment of anxiety disorders. However, this class ofdrugs has a wide side effects profile, including sedation and de-pendence, as well as cognitive and psychomotor impairment(Lader and Morton 1991; Whiting 2006). Besides BZDs, selectiveserotonin reuptake inhibitors (SSRIs) (Hidalgo andDavidson 2000;Borsini et al. 2002) and MAO-A inhibitors (De Angelis and Furlan2000; Mai et al. 2003) are effective in treating a wide spectrum ofanxiety disorders. Despite their therapeutic actions, weightchanges, insomnia, drowsiness or sedation, headache, and sexual

dysfunction (Dording et al. 2002; Priest et al. 1992) are the reportedside effects associated with SSRIs and MAO inhibitors. Thisprompted many researchers to explore for new anxiolytics withfewer unwanted effects.

The pathophysiology and precise mechanism of anxiety re-mains unclear. Nevertheless, monoaminergic system (serotonin(5-HT) and nor-epinephrine (NE)) in the brain have been postulatedto play an important role in the pathophysiology of anxiety disor-ders (Zhang et al. 2004). Few preclinical or clinical studies provideevidence to support the hypothesis that anxietymay be associatedwith an alteration or dysfunction of the serotonergic system inthe central nervous system (CNS) (Aghajanian et al. 1990; Revelliet al. 1998; Jindal et al. 2013). Thus, modulation of the serotonergicsystem may be implicated as a promising mechanism for the ac-tion of anxiolytic drugs (Zhang et al. 2004; Jindal et al. 2013). The5-HT3 receptor is a ligand-gated ion channel that mediates therelease of monoamine neurotransmitters (Wolf 2000). The 5-HT3receptor has been identified in the CNS (Pandey et al. 2008). An-tagonists of this receptor, such as ondansetron, granisetron,

Received 3 April 2013. Accepted 29 May 2013.

R. Mahesh, A.K. Dhar, A. Jindal, and S. Bhatt. Department of Pharmacy, FD-III, Birla Institute of Technology & Science, Pilani, Rajasthan 333 031, India.

Corresponding author: Arghya Kusum Dhar (e-mail: arghyakusumdhar@yahoo.in).

848

Can. J. Physiol. Pharmacol. 91: 848–854 (2013) dx.doi.org/10.1139/cjpp-2013-0134 Published at www.nrcresearchpress.com/cjpp on 11 June 2013.

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dolasetron, and tropisetron are commonly used for the treatmentof radiotherapy- or chemotherapy-induced nausea and vomiting(CINV) in patients with cancer (Israili 2001; Walstab et al. 2010).

Numerous studies have indicated that 5-HT3 receptors are ex-pressed in the areas of the brain implicated in anxiety disorders.(Jones 1990; Costall and Naylor 1992; Simpson et al. 1992; Olivieret al. 2000; Walstab et al. 2010). Animal studies have revealed that5-HT3 receptor antagonists exert an anxiolytic effect by blockinglimbic (hippocampus and amygdale) hyperactivity responses (Griebel1995; Millan 2003; Rajkumar and Mahesh 2010). In addition, stud-ies of 5-HT3 knockout mice also supported the involvement of5-HT3 receptors in anxiety, which revealed that 5-HT3 receptorregulates anxiety-related behaviour (Kelley et al. 2003).

Thus, this study was intended to investigate the anxiolytic-likeeffect of a series of carboxylic acid based 1,8 naphthyridines thatwere designed and synthesized as bioisosteric analogs of our ear-lier synthesized nitrile-based 1,8 naphthyridines (Mahesh et al.2004). The compounds were tested in our laboratory for their5-HT3 receptor antagonistic potential in isolated guinea pig il-eum, and the pA2 values were determined against 2-methyl-5-hydroxytryptamine, a 5HT3 receptor agonist (Table 1). In thisstudy, the compounds were selected from the series based ontheir optimal log P and pA2 value, particularly if it was greaterthan the standard 5-HT3 receptor antagonist, ondansetron(Table 1). A behavioral test battery, including the light–dark (L/D)test, elevated plus maze (EPM) test, and the open field exploratorytest (OFT) were used to explore the anxiolytic-like activities of theselected compounds.

Materials and methods

AnimalsSwiss albino mice (25–35 g) were obtained from Hissar Agricul-

tural University (Hissar, Haryana, India) and maintained under

standard conditions: 12.00 h (light) −12.00 h (dark) cycle, temper-ature of 23 ± 2 °C, and relative humidity of 60% ± 10%. The rodentswere housed in standard polycarbonate cages and given free ac-cess to food (standard pellet chow) and filtered water. The animalswere used only once for each experiment and were acclimatizedto the experimental room for 1 h before testing. Experiments onanimals were approved by the Institutional Animal Ethics Com-mittee of the Birla Institute of Technology & Science, Pilani, India(Protocol No. IAEC/RES/14/04).

DrugsDiazepam was obtained from Lancaster chemicals (UK). Ondanse-

tron was obtained from Natco Pharmaceuticals, Hyderabad, India.All of the other chemicals used for synthesis were of analyticalgrade. Diazepam and ondansetron were used as reference stan-dards. All of the NCEs, diazepam, and ondansetron were freshlyprepared before use by dissolving in distilled water. Diazepam(2mg/kg bodymass), ondansetron (2mg/kg), and NCEs (1 or 2mg/kg)were administered intraperitoneally (i.p.) to mice 30 min beforestarting the behavioural observation in each test.

ChemistryThe title compounds 2-(4-substituted piperazin-1-yl)-1,8-

naphthyridine-3-carboxylic acids were designed as bioisostericanalogs of previously synthesized 2-(4-substituted piperazine1-yl)-1,8-naphthyridine-3-carbonitriles (Mahesh et al. 2004). Encouraged bythe promising 5-HT3 antagonism profile of our earlier synthesizednitrile-based 1,8 naphthyridines, it was proposed to design ana-logs. Consequently, the nitrile group of the previously synthe-sized molecules was replaced with its nonclassical bioisosterecarboxylic acid group (Lima and Barreiro 2005) to generate 2-(4-substituted piperazin-1-yl)-1,8-naphthyridine-3-carboxlic acid. Thecompounds were synthesized via the sequence of reactions de-picted in the Supplementary data1 (Fig. S1, Table S1). The startingmaterial, 2-aminonicotinaldehyde (3), was synthesized by heatingnicotinamide and ammonium sulphamate in a neat solvent-freecondition to obtain a pyrido-pyrimidine intermediate, which washydrolyzed by heating with dilute HCl, according to the methodsreported in the literature (Majewicz and Caluwe 1974). Refluxing2-aminonicotinaldehyde with diethyl malonate in ethanol, with afew drops of piperidine as the catalyst, afforded the intermediateethyl 2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxylate (4) (Xiaoet al. 2010). Compound 4was subjected to refluxing in POCl3 witha catalytic amount of DMF to furnish ethyl 2-chloro-1,2-dihydro-1,8-naphthyridine-3-carboxylate (5). Nucleophilic displacementreaction of the chlorine atom of compound 5 was carried outby various substituted piperazines in refluxing acetonitrilewith K2CO3 to obtain the compounds 6a–6o, i.e., substitutionwith phenyl piperazine, resulted in ethyl 2-(4-phenylpiperazin-1-yl)-1,8-naphthyridine-3-carboxylate. Finally, the ester derivatives(6a–6o) were stirred with aqueous NaOH in ethanol. Upon comple-tion of the reaction, the alcohol was evaporated and the reactionmixture was acidified very carefully with a weak acid, such as citricacid, up to pH 4–5 to avoid protonation at the N4 nitrogen of thepiperazine ring, and this furnished the title compounds 7a–7o.

The structures of the synthesized compounds were confirmedby their spectral data.

The spectral data for one representative compound, 2-(4-(4-nitrophenyl) piperazin-1-yl)-1, 8-naphthyridine-3-carboxylic acid(7d), are presented below.

FT-IR (KBr, cm−1): 3290 (broad str of acid –OH), 2931, 2850, 2644,2357, 1719 (broad str of acid C=O), 1625, 1510, 1462, 1350, 1296, and1240. 1H NMR, CDCl3, � (ppm): 8.75–8.73 (dd, 1H, J = 4.8, 2 Hz, C7-Hof naphthyridine ring), 8.12 (s, 1H, C4-H of naphthyridine ring),8.07–8.04 (m, 2H, C6-H of naphthyridine ring), 7.98–7.96 (m, 1H,

1Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/cjpp-2013-0134.

Table 1. The pA2 and log P values of novel 2-(4-substituted piperazin-1-yl)-1,8-naphthyridine-3-carboxylic acids.

(7a–7o) Basic structure

Compounda Rb pA2c Log Pd

7a C6H5 7.6 3.357b CH2C6H5 6.7 2.897c CH2CH2C6H5 6.5 3.057d p-NO2C6H4 7.2 3.247e m-ClC6H4 7.3 4.017f p-ClC6H4 6.0 4.017g o-ClC6H4 4.6 4.017h o-OCH3C6H4 6.6 3.267i p-OCH3C6H4 7.0 3.267j m-OCH3C6H4 5.0 3.267k p-CH3C6H4 5.9 3.577l o-CH3C6H4 4.4 3.577m CH3 6.3 1.187n CH2CH3 5.4 1.607o CH(CH3)2 5.2 2.06

aA series of compounds synthesized in the laboratory and coded 7a to 7o.bGroup attached to the N4 of piperazine ring of the basic structure.cThe pA2 values are calculated against 2-methyl-5-hydroxytryptamine.dThe log P values are calculated using JMEMolecular Editor (Courtesy of Peter

Ertl, Novartis).

Mahesh et al. 849

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C5-H of naphthyridine ring), 7.18–7.15 (m, 2H, phenyl), 6.84–6.82(m, 2H, phenyl), 4.0–3.95 (m, 4 H, methylene protons adjacent toN1 of piperazine ring), 3.62–3.58 (m, 4H, methylene protons adja-cent to N4 of piperazine ring). MS (ESI –ve mode): m/z (%) = 378.32(M-H+, 100).

Behavioural screening

Light–dark aversion testThe L/D apparatus comprised a box divided into 2 separate com-

partments, occupying 2/3 and 1/3 of the total size, respectively.The larger compartment (light compartment) was illuminated bya 60 W bulb, while the smaller (dark compartment) was entirelyblack and enclosed under a dark cover. The L/D compartmentswere separated by a partition with a tunnel to allow the mousepassage from one compartment to the other (Mi et al. 2005). At thebeginning of the test, the mouse was placed individually at thecenter of the light compartment facing towards the tunnel andwas allowed to explore the entire apparatus for 5 min. The behav-ioral parameters such as latency time to leave the light compart-ment, total time spent in the light compartment, and number oftransitions between the light and dark compartments were ob-served and recorded. A compartment entry was considered validwhen all 4 of the animal's paws were inside that chamber. Theapparatus was thoroughly cleaned with 70% ethanol after eachtrial.

Elevated plus mazeThe EPM test was first evaluated for rats (Pellow et al. 1985) and

later adapted for mice (Lister 1987). In brief, the apparatus con-sisted of a wooden maze with 2 enclosed arms (30 cm × 5 cm ×15 cm) and 2 open arms (30 cm × 5 cm × 0.25 cm) that extendedfrom a central platform (5 cm × 5 cm) to form a “plus sign”. Theplus-maze apparatus was elevated to a height of 25 cm and placedinside a sound-attenuated room. The trial was started by placing amouse on the central platformof themaze facing its head towardsan open arm. The behavioral performances recorded during a5 min test period were as follows: percentage open arm entries,percentage time spent in open arm, and total entries (Klodzinskaet al. 2004). Entry into an arm was considered valid only when all4 paws of the mouse were inside that arm (Biala and Kruk 2008).

The animal's activities were observed and readings were recorded.The apparatus was thoroughly cleaned with 70% ethanol aftereach trial.

Open field testThe apparatus consisted of a wooden box (60 cm × 60 cm ×

30 cm) with the floor divided into 16 squares (15 cm × 15 cm)squares by black parallel and intersecting lines. The apparatuswas illuminated with 60Wbulb suspended 100 cm above it. At thebeginning of the test, the mouse was placed individually at thecenter of the square arena. The ambulation scores (number ofsquares crossed) and frequency of rearing (standing upright onthe hind legs) were recorded for a 5 min period. After each indi-vidual test session the floor was thoroughly cleaned with 70%ethanol.

Statistical analysisResults are the mean ± SEM. The data were analyzed using

one-way analysis of variance (ANOVA) followed by Dunnett's testin Graph pad prism 3. The level of statistical significancewas fixedat P < 0.05.

Results

Light–dark aversion testThe effects of NCEs, ondansetron, and diazepam on the behav-

iour of mice in the L/D test are presented in Table 2. Treatmentwith compounds 7a–7f, 7h, 7i, and 7m (1 or 2 mg/kg body mass,i.p.), ondansetron (2 mg/kg, i.p.), and diazepam (2 mg/kg, i.p.) sig-nificantly (P < 0.05) increased the time spent by the mice in the litarea as well as the number of transitions, compared with thevehicle control. In addition, diazepam (2 mg/kg, i.p.) treatmentsignificantly (P < 0.05) increased the latency to enter the darkchamber. Treatment with compounds 7a, 7b, 7d, 7e, or 7i (only at2 mg/kg, i.p.) significantly (P < 0.05) increased the latency to enterthe dark chamber. The lower dose of NCEs (1 mg/kg, i.p.) did notproduce any significant change in the latency to leave the litcompartment, when compared with the vehicle control. Theanxiolytic-like effect was more pronounced at the higher dose(2 mg/kg) for all of the tested compounds.

Table 2. Effect of 2-(4-substituted piperazin-1-yl) 1,8-naphthyridine 3-carboxylic acids, ondansetron, and diazepam in a light–darkmouse model.

Latency (s)Time spent in lightchamber (s) Number of transitions

Dose (mg/kg) Dose (mg/kg) Dose (mg/kg)

Compound 2.0 1.0 2.0 1.0 2.0 1.0

7a 48.17±19.45* 40.5±13.84 127.67±12.76* 116.83±7.5* 13.33±1.40* 12.67±0.88*7b 46±7.76* 27.17±9.78 146.33±9.88* 131.75±22.15* 11.20±1.74* 8.83±2.42*7c 6.00±2.07 7.17±2.84 136.67±30.36* 107.17±26.33* 13.5±1.84* 15.6±2.22*7d 36.60±5.15* 26.17±7.80 141.67±13.91* 124.33±10.79* 15.00±1.65* 13.83±1.12*7e 49.20±9.35* 35.20±10.40 131.33±12.14* 129.33±10.44* 16.67±2.48* 15.33±1.37*7f 12.50±3.38 10.00±1.83 147.00±18.64* 90.67±15.03* 17.67±2.21* 13.17±0.83*7g — — — — — —7h 23.33±1.87 19.76±6.84 98.33±10.67* 87.76±16.04* 16.67±2.10* 11±2.19*7i 45.50±8.28* 27.17±9.34 158.67±19.45* 96.33±20.09* 14.00±1.31* 10.00±2.29*7j — — — — — —7k 12.00±2.79 12.50±4.05 87.40±15.12* 71.33±14.61 9.60±1.94* 13.40±2.12*7l — — — — — —7m 10.33±3.91 9.83±1.91 124.75±16.03* 83.50±10.21* 13.17±2.62* 14.60±3.19*7n — — — — — —7o — — — — — —Ondansetron 18±5.21 — 123±5.50* — 11.23±4.00* —Diazepam 73.83±8.90* — 164.83±5.50* — 17.16±9.00* —Control 24.17±3.06 — 63.40±5.29 — 4.67±0.87 —

Note: Values are the mean ± SEM; *, P < 0.05 when compared with the vehicle-treated group (control); n = 6 mice per group.

850 Can. J. Physiol. Pharmacol. Vol. 91, 2013

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Elevated plus mazeThe effects of NCEs, ondansetron, or diazepam on the behavior

ofmice in the EPM test are summarized in Table 3. Treatmentwithcompounds 7a, 7e, 7f, and 7h (1 or 2 mg/kg, i.p.), ondansetron(2 mg/kg, i.p.), or diazepam (2 mg/kg, i.p.) significantly (P < 0.05)increased the time spent in the open arm, when compared withthe vehicle control. Compounds 7d, 7i, and 7m (2mg/kg, i.p., only)significantly (P < 0.05) increased the open-arm entries as wellas the time spent in the open arm. Treatment with diazepam(2mg/kg, i.p.) or ondansetron (2 mg/kg, i.p.) significantly (P < 0.05)increased the open-arm entries and time spent in the open arm,when compared with the vehicle control. Compound 7c onlyshowed a significant (P < 0.05) increase in time spent in the openarms at 2 mg/kg, i.p. The lower dose of the test compounds(1 mg/kg, i.p.) did not induce a significant (P < 0.05) change in theopen-arm entries, when compared with the vehicle control. Treat-ment with compound 7a (1 or 2 mg/kg, i.p.) significantly increasedthe percentage of open-arm entries as well as the percentage of timespent in the open arm, when compared with the vehicle control.

Open field testThe results of the OFT are shown in Table 4. Treatment with

compounds 7a or 7h (1 or 2 mg/kg, i.p. for both), 7d, 7e, or 7f(2 mg/kg, i.p., only), diazepam (2 mg/kg, i.p.), or ondansetron(2 mg/kg, i.p.) significantly (P < 0.05) increased the ambulationscores, when compared with the vehicle control. Moreover, com-pounds 7f and 7h (1 or 2 mg/kg, i.p., for both) and compounds 7a,7d, 7i, and 7m (2mg/kg, i.p., only) significantly (P< 0.05) increasedthe frequency of rearing, compared with the vehicle control.

DiscussionThe present behavioral investigation with various animal

models of anxiety revealed the anxiolytic like effect of novel1,8 naphthyridine derivatives as 5-HT3 receptor antagonists. A3-component model for the interaction of arylpiperazine type li-gands with the 5-HT3 receptor was derived (Cappelli et al. 2002)and it demonstrated that themost essential structural features for

the interaction of arylpiperazines with 5-HT3 receptor were asfollows: (i) the N4 piperazine nitrogen atom, (ii) the heterocylicnitrogen atom, and (iii) the bicyclic heteroaromatic system(Cappelli et al. 2002). All of the compounds (Table 1) were synthe-sized according to a previously described pharmacophore (Rosenet al. 1990; Cappelli et al. 2002; Klodzinska et al. 2004) for the5-HT3 receptor antagonists. The putative anxiolytic activities ofthe synthesized compounds were determined by L/D test, EPMtest, and OFT. The time spent in the lit chamber and in the open

Table 3. Effect of 2-(4-substituted piperazin-1-yl)-1,8-naphthyridine-3-carboxylic acids, ondansetron, and diazepam on the behav-ior of mice in an elevated plus maze test.

No. of entries Time spent (s)

Treatmentgroups

Dose(mg/kg) Open arm Closed arm Open arm Closed arm % OAE % TSOA

7a 2 4.33±0.48* 6.23±0.50 107.13±4.94* 192.87±2.14 41.62±3.89* 35.71±2.71*1 2.83±0.51 5.50±0.96 105.17±6.90* 194.83±4.90 33.97±2.56* 35.05±3.64*

7b 2 3.17±0.68 6.83±0.93 35.83±2.28 264.17±2.28 30.89±2.32 11.94±1.041 2.29±1.08 6.86±1.03 32.14±2.22 267.86±3.95 25.02±3.53 10.71±0.98*

7c 2 2.04±1.12 6.21±1.86 53.15±12.29* 246.85±4.72 24.72±3.66 17.71±6.09*1 1.21±0.67 5.50±1.01 32.13±10.14 267.87±10.11 18.03±7.51 10.71±4.04

7d 2 5.83±1.04* 10.83±0.83 54.5±5.74* 245.5±8.95 34.09±4.13 18.16±1.91*1 2.00±0.57 3.33±0.56 26.33±7.80 273.67±10.11 30.73±7.64 8.78±2.60

7e 2 3.09±1.48 7.16±2.13 51.14±5.52* 248.86±6.95 30.14±5.53 17.04±2.98*1 2.83±2.67 7.10±1.51 44.83±2.04* 255.17±1.11 28.49±7.51 14.94±4.04

7f 2 3.83±0.44* 7.17±1.28 44.33±4.69* 255.67±4.69 36.18±1.78 14.77±1.56*1 2.33±0.67 5.50±2.01 37.83±0.04* 262.17±2.11 29.75±7.51 12.61±2.04

7h 2 5.33±0.99* 7.17±1.07 69.00±13.38* 231.00±8.35 39.84±2.32* 23.00±3.48*1 3.11±0.67 6.50±1.01 54.83±1.14* 245.17±2.51 32.36±7.51 18.27±4.04

7i 2 3.5±0.61* 5.50±1.02 49.17±11.03* 250.83±4.95 30.61±3.45 16.39±3.67*1 3.0±0.50 5.93±1.01 28.00±13.93 272.00±15.11 33.59±4.11 9.33±1.04

7k 2 3.29±1.28 5.16±1.53 41.14±5.42 258.86±8.95 38.93±5.53 13.71±2.98*1 2.43±1.67 5.10±1.21 34.83±2.14 265.17±1.11 32.27±7.51 11.61±2.34

7m 2 3.20±0.08* 7.86±1.03 51.14±5.52* 248.86±6.95 28.93±5.53 17.04±2.18*1 2.13±0.67 7.10±1.51 30.83±12.14 269.17±12.11 23.07±3.51 10.27±2.04

Ondansetron 2 4.83±0.37* 6.67±1.22 41.50±4.35* 258.50±2.32 39.84±3.51 13.83±1.44*Diazepam 2 6.5±1.23* 4.33±0.71 124.5±11.8* 175.5±8.15 60.01±5.53* 41.50±4.38*Control 2.29±0.68 4.4±0.73 26.30±9.94 281.70±9.94 34.23±2.53 8.76±2.16

Note: All values are the mean ± SEM; *, P < 0.05 when compared with the vehicle-treated group; n = 6 mice per group; %OAE, percentage ofopen-arm entries; %TSOA, percent time spent in the open arm in seconds.

Table 4. Effect of 2-(4-substituted piperazin-1-yl)-1,8-naphthyridine-3-carboxylic acids, ondansetron, and diazepam on the behavior of micein an open-field test.

Ambulation score Rearing number

Dose (mg/kg) Dose (mg/kg)

Compound 2.0 1.0 2.0 1.0

7a 331.46±9.81* 319.00±8.30* 17.62±2.89* 14.83±6.007b 249.00±1.48 236.43±3.30 15.17±7.50 08.26±5.537c 304.49±2.92 302.08±8.39 15.32±3.66* 15.17±7.517d 351.50±15.60* 285.00±7.36 21.83±2.45* 18.17±5.517e 328.23±3.50* 253.66±2.35 14.86±6.95 11.17±3.517f 327.00±9.79* 280.16±6.45 19.83±4.04* 15.17±3.11*7g — — — —7h 319.66±7.09* 313.58±5.99* 16.50±2.11* 21.17±7.51*7i 303.67±2.17 291.17±7.80 18.50±2.61* 11.17±7.517j — — — —7k 283.15±2.61 277.60±5.89 12.26±5.53 05.17±2.517l — — — —7m 281.06±4.06 279.30±6.39 18.26±2.43* 12.17±7.917n — — — —7o — — — —Ondansetron 333.17±25.32* — 20.5±3.31* —Diazepam 467.50±15.19* — 40.50±1.02* —Control 276.50±29.23 — 9.83±2.18 —

Note: Values are themean ± SEM; *, P < 0.05 when compared with the vehicle-treated group (control); n = 6 mice per group.

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arm was measured during the L/D test and EPM test respectively,whereas the ambulation score and rearing frequency wererecorded in the OFT. All of these models of anxiety are quitesensitive and relatively specific to anxiolytics, and have beenwidely used to screen NCEs for their anxiolytic potential (Borsiniet al. 1989; Costall et al. 1989, 1993; Kilfoil et al. 1989; Barnes et al.1992; Hogg 1996).

The L/D test is based on the aversive properties of light (Angelisand Furlan 2000; Belzuing et al. 1987) and uses as its index ofanxiety the time spent in the lit area as opposed to the time spentin the dark area. The data from this study is consistent with pre-vious studies (Young and Johnson 1991) showing that the best wayto determine the effect of an anxiolytic agent in mice is the timespent in the lit area, while a decrease in the time spent in the litarea as well as decrease in the number of transitions between litand dark area are characteristic of an anxiogenic response (Laland Emmett-Oglesby 1983; De Angelis 1992). Treatment with com-pounds 7a, 7d, 7e, 7f, diazepam, or ondansetron significantlyincreased the number of transitions as well as the time spent inthe lit area, suggesting anti-anxiety-like effect that is also shownby various anxiolytics and 5-HT3 receptor antagonists (Olivieret al. 2000). It can be observed that the compounds containingelectron withdrawing substituents in the phenyl ring, such as4-nitro (compound 7d), 3-chloro (compound 7e), 4-chloro (com-pound 7f), and an electron releasing substituent such as the4-methoxy (compound 7i) group, showed better activity thanother compounds in the L/D test.

EPM is considered a well established model of unconditionedanxiety for detecting anxiolytic/anxiogenic-like activity by inves-tigating aspects of physiological and pharmacological behavior.In the EPM test, increased number of entries and time spent in theopen arms of the maze are the most reliable indicators of de-creased anxiety or for indicating the anxiolytic-like activity of acompound, while anxiogenic substances have the opposite effect(Fernandes and File 1996; Dubinsky et al. 2002; Sienkiewicz-Jaroszet al. 2003). In our study, treatment with these 1,8 naphthyridinederivatives produced anxiolytic-like effects in the EPM test, asevidenced by a significant increase in both open-arm entries andtime spent in open arms, compared with the vehicle control.These results are consistent with previous studies that suggest5-HT3 receptor antagonists increased the number of entries andtime spent in the open arms, and exhibited anxiolytic activity inthe EPM test (Blackburn et al. 1993; Artaiz et al. 1995; Hewlett et al.2001; Zhang et al. 2001). In addition, diazepam was used as a stan-dard reference, and it also showed an anxiolytic effect in theEPM test. It can be noted that analogs containing an electron-withdrawing group such as 4-nitro (compound 7d), 3-chloro (com-pound 7e), and 4-chloro (compound 7f), as well as an electronreleasing group such as 2-methoxy (compound 7h), 4-methoxy(compound 7i), and 4-methyl (compound 7k) groups in the phenylring, showed lower activity compared with the unsubstitutedphenyl ring counterpart (compound 7a) in the EPM test. The openfield test is widely used for the screening of anxiolytic/anxiogenicdrugs. Normal aversion of a rodent to the brightly lit open areaproduces anxiety and fear, which is characterized by alteration inthe behavioral parameters of the animal in the open field. Previ-ous reports have suggested that anxiolytic compounds have atendency to reduce the fearful behaviors of rodents in the OFT(Mechan et al. 2002). Treatment with compounds 7a, 7d, or 7hincreased the ambulation score and rearing frequency in the OFT,indicating the anxiolytic effect of these compounds. Analogs thatcontain electron-withdrawing substituents in the phenyl ring,such as 4-nitro (compound 7d), 3-chloro (compound 7e), 4-chloro(compound 7f), and electron releasing substituents such as2-methoxy (compound 7h), induced better anxiolytic-like activitythan other compounds, in the OFT. In general, compounds withconsiderably higher pA2 values, such as 7a (pA2:7.6), 7d (pA2:7.2),7e (pA2:7.3), 7h (pA2:6.6), and 7i (pA2:7.0) exhibited significant

anxiolytic-like activity in the L/D test, EPM test, and OFT. Thepreliminary attributes of the anxiolytic-like activities of thesenovel 1,8-naphthyridine derivatives could be (i) log P values of 2.01to 3.35, which are optimal for blood–brain barrier permeability(Ter Laak et al. 1994), (ii) 5-HT3 receptor antagonism (pA2 value)greater than ondansetron (6.6), which is a standard 5-HT3 receptorantagonist.

In conclusion, the results from this study demonstrate the an-xiolytic activity of 1,8 naphthyridine carboxylic acid derivatives in3 validated animal models of anxiety. The precise mechanismbehind the anti-anxiety like effect of these 1,8 naphthyridine de-rivatives remains to be fully explored. However, an increase in thelevel of serotonin through blockade of the 5-HT3 receptor (Schafer1999) could possibly contribute to the anxiolytic-like effect ofthese compounds (Greenshaw 1992). The 5-HT3 receptor locatedwithin a neuro-anatomical region might be indirectly regulatingthe 5-HT transmission (Kilpatrick et al. 1989). The blockade of5-HT3 receptors facilitates 5-HT neurotransmission (Rajkumar andMahesh 2010). 5-HT3 receptor antagonists block the postsynaptic5-HT3 receptors that mediate a fast excitatory potential in thelimbic brain regions (Sugita et al. 1992). Although the cascade ofcellular events that follow this fast transmission blockade re-mains unclear, an overall anti-anxiety-like behaviour is plausible(Rajkumar andMahesh 2010). Therefore, our findings reiterate thedevelopment of 5-HT3 receptor antagonists as a possible alterna-tive approach for the treatment of anxiety disorders. Hopefully,future studies with molecular techniques will help in better un-derstanding of the possible mechanism underlying the anxiolyticlike effect of these 1,8 naphthyridine carboxylic acid derivatives atthe cellular level, which may lead to agents that are useful forcontrolling anxiety disorders.

AcknowledgementsThis study was financially supported by grants from the Indian

Council of Medical Research (ICMR), New Delhi, India.

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