Kavain Analogues As Potential Analgesic Agents
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Pharmacological Reports Copyright © 2012 2012, 64, 14191426 by Institute of Pharmacology ISSN 1734-1140 Polish Academy of Sciences Kavainanaloguesaspotentialanalgesicagents ElaineC.Kormann1,PatriciadeAguiarAmaral2,MicheleDavid3, VeraL.Eifler-Lima4,ValdirCechinelFilho1,FátimadeCamposBuzzi1 1 Chemical-PharmaceuticalInvestigationsCenter(NIQFAR)/CCS,UniversityofItajaíValey(UNIVALI),Itajaí/SC, Brazil 2 LaboratoryofMedicinalPlants(LaPlaM),UniversityoftheExtremeSouthofSantaCatarina(UNESC), Criciúma/SC,Brazil 3 LaboratoryofChemistryTherapeutic(UPRES4090),UniversityofRennes1,Rennes,France 4 LaboratoryofMedicinalOrganicSynthesis(LaSOM),FacultyofPharmacy,FederalUniversityofRioGrande doSul(UFRGS),PortoAlegre/RS,Brazil Correspondence: FátimadeCamposBuzzi,e-mail: [email protected]; VeraL.Eifler-Lima, e-mail: [email protected] Abstract: Background: Kavalactones are pharmacologically active compounds present in preparations of the root trunk of Piper methysticum Forst, known as kava. This work describes the analgesic activity of some synthesized analogues of synthetic kavain, which is the maincomponentofkava. Methods: The essays were initially performed against the writhing test in mice, and the most promising compound was analyzed us- ing other classical models of nociception, including formalin-, capsaicin-, glutamate-induced nociception, the hot plate test, and measurementofmotorperformance. Results: The results indicated that compound 6-(4-fluorophenyl)-4-methoxy-5,6-dihydropyran-2-one (2d) exerts potent and dose- dependent analgesic activity, inhibiting abdominal constrictions caused by acetic acid in mice, and being more active than some ref- erence drugs. It also presented activity in the other models of pain, with the exception of the hot plate test and the measurement of motorperformance. Conclusions: Although compound 2d exerts antinociceptive activity, the mechanism of action remains uncertain, but it does not in- volve the opioid system and does not appear to be associated with non-specific effects such as changes in locomotor activity or motor coordination. Keywords: kavain,lactones,enolethers, d-valerolactones,antinociceptiveactivity Introduction ethylene-a-pyrone skeleton. They are typically 4- methoxy-2-pyrones with a phenyl or styryl substituent at the 6-position [2]. The commercial extracts consist The active constituents for the therapeutic actions of of a mixture of more than 18 different a-pyrones, col- Piper methysticum (kava) extract consist of a group of lectively known as kavapyrones, or kavalactones. The structurally related lipophilic derivatives with an aryl- major constituents are (+)-kavain (1.8%), (+)-methy- Pharmacological Reports, 2012, 64, 14191426 1419 sticin (1.2%), desmethoxyangonin (1.0%), (+)-di- tions. Since some suffering might result from the ex- hydrokavain (0.6%), (+)-dihydromesthysticin (0.5%), periments, the International Association for the Study tetrahydroxyangonin. Minor constituents included of Pain (IASP) Committee for Research and Ethical other kavalactones, chalcones and essential oils [9]. Issues Guidelines [28] were followed, and the experi- Pharmacological studies have revealed that these sub- ments were approved by the Animal Ethics Commit- stances are responsible for the analgesic [1], sedative tee of UNIVALI (protocol no. 609/2007 UNIVALI), [10, 22], anticonvulsive [16] and spasmolytic proper- and all the experiments were conducted according to ties [23] of kava. Four lactones from kava (kavain, di- the principles of the Brazilian College of Animal Ex- hydrokavain, methysticin, and dihydromethysticin) perimentation(COBEA). have been found to possess significant analgesic ef- fects in animal studies, probably by non-opiate path- Chemistry ways [18]. Kavain (1) appears to be the most effective in surface anesthesia, comparable to cocaine in its The solvents were distilled prior to use, following the strength and duration of action [21]. It was selected as standard procedures, and reactions were performed the prototype because of its yield in the plant extract, under nitrogen or argon atmosphere. Silica gel 60 F254 and because it presented analgesic activity in previous plates were used to monitor the synthetic transforma- studies [1]. In this work, we have synthesized kavain tions, with visualization under UV light, 2% KMnO4 (1) and nine analogues, according to a previous report or sulfuric anisaldehyde 2% solutions. Chromato- of an aldolization-lactonization process [3], and graphic purifications were carried out using 70–230 evaluated these compounds in models of pain in mice. mesh silica gel. Melting points were determined on a System Kofler type WME apparatus, and were un- corrected. Infrared (FT-IR) spectra were recorded with a Perkin Elmer 1600 spectrometer. Nuclear mag- netic resonance spectra (1H NMR and 13C NMR) were MaterialsandMethods recorded on a Bruker AC-200F spectrometer and per- formed at 300 MHz and 75 MHz, respectively. d Drugs Chemical shifts ( values) are given in parts per mil- lion downfield from tetramethylsilane as the internal The following drugs and reagents were used: acetyl- standard. Mass spectral analyses were performed at salicylic acid (ASA), metamizole (MET), capsaicin the Centre Régional de Mesures Physiques de l’Ouest and glutamate (Sigma Chemical Co., St. Louis, USA); (CRMPO) in Rennes (France). All the compounds formalin, acetic acid (Vetec, Rio de Janeiro, Brazil) (Fig. 1) have already being described by some of the and morphine (Dimorf®), kindly provided by authorsinreference[3]. Cristália (Itapira, São Paulo, Brazil). The compounds studied, as well as the reference drugs, were dissolved in Tween 80 (Merck, AG, Darmstadt, Germany), with the exception of the capsaicin, which was dissolved in ethanol, plus 0.9% of NaCl solution. The final con- centration of Tween and ethanol did not exceed 5% anddidnotcauseanyeffect“perse”. Animals The animals used were Swiss male mice (25–30 g), obtained from the University of Vale do Itajaí (Itajaí, Brazil). They were kept in a temperature-controlled environment (23 ± 2°C) with a 12 h light-dark cycle. Food and water were freely available. The allocation of animals into different groups was randomized, and the experiments were carried out under blind condi- Fig.1. Structureofkavainandthecompoundsevaluated 1420 Pharmacological Reports, 2012, 64, 14191426 Analgesiceffectsofkavainanalogues Elaine C. Kormann et al. PHARMACOLOGY placed in a glass cylinder of 20 cm in diameter, and the time spent licking the injected paw (second phase Abdominalconstrictionresponsecausedby offormalintest)wasdetermined. injectionofaceticacid Capsaicin-inducedpain Abdominal constrictions induced by intraperitoneal (ip) injection of acetic acid (0.6%), consisting of The procedure used was similar to that described pre- a contraction of the abdominal muscle together with viously [25]. Animals were placed individually in a stretching of the hind limbs, were counted according transparent glass cylinders. Following the adaptation to the previously described procedures [10]. The ani- period, 20 µl of capsaicin (1.6 µg/paw) was injected mals were pretreated with 10 mg/kg of all analogues under the skin of the plantar surface of the right hind from kavain (1) or standard drugs via the intraperito- paw, using a microsyringe. The animals were ob- neal route, 30 min before acetic acid injection. The served individually for 5 min following capsaicin in- control animals received a similar volume of vehicle jection. The amount of time spent licking the injected (Tween 80 and 0.9% NaCl). For compounds 1, 2a, 2b, paw was timed with a chronometer and was consid- 2d and 3c the abdominal constriction response was ered as indicative of nociception. The animals were ip analyzed, following pretreatment via the ip route in treated with compound 2d at 1, 3 and 10 mg/kg or sa- three doses (1 to 30 mg/kg) or the standard drugs, but line 1 h before administration of capsaicin. The con- in higher doses (10 to 60 mg/kg) for evaluation of trol animals received a similar volume of 0.9 % NaCl ID50. The enol ether 2d (6-(4-fluorophenyl)-4-methoxy- (10ml/kg, ip). 5,6-dihydropyran-2-one), which was the most active of the compounds tested, was also analyzed orally at Glutamate-inducednociception 100 mg/kg. After the challenge, pairs of mice were placed in separate boxes and the number of abdominal The animals were treated with the compound 2d ip (1, constrictions was cumulatively counted over a period 30 and 60 mg/kg) 30 min before capsaicin injection, of 20 min. Antinociception was expressed as the re- respectively. A volume of 20 µl of glutamate solution duction in the number of abdominal constrictions be- (30 µmol/ paw), made up in phosphate buffered saline tween the control animals and the mice pretreated (PBS), was injected intraplantarly under the surface withcompoundsorstandarddrugs. of the right hind paw as described previously [4]. Af- ter injection with glutamate, the animals were indi- Formalin-inducedpain vidually placed in glass cylinders of 20 cm in diame- ter and observed from 0 to 15 min. The time spent The procedure was similar to that described previ- licking and biting the injected paw was timed with ously [17, 25]. The observation chamber was a glass achronometerandconsideredasindicativeofpain. cylinder of 20 cm in diameter, with a mirror at a 45° angle to allow clear observation of the animal’s paws. Hot-platetest For the formalin-induced pain 20 µl of 2.5% formalin (0.92% formaldehyde) was injected under the surface The hot-plate test