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J.Med. Chem. 1994,37, 1889-1893 1889

(R)-Methanandamide: A Chiral Novel Anandamide Possessing Higher Potency andMetabolic StabilityVasiliki Abadji,t Sony uan Lin ,? Gihan Tah a,t Graem e Griffin,* Lesley A. Stevenson,$Roger G. Pertwee,t andAlexandros Makriya nnis*~+ *~School of Pharmacy and Insti tute of Materials Science, University of Connecticut, Storrs, Connecticut 06269, and Departmentof Biomedical Sciences, University of Aberdeen, Aberdeen AB9 lA S, ScotlandReceived February 1 , 1994"

Four chiral congeners of arachidonylethanolamide (anandamide) have been synthesized andevaluated for (a) their abili ty to bind to th e cannabinoid receptor in rat forebrain mem branes an d(b) their pharmacological potency as me asured by t he compo unds' abili ty t o inhibit electrically-evoked contraction s of the m ouse vas deferens. T he lead analog was also teste d for i ts potencyin vivo. Of the analogs tested, (R)-(+)-arachidonyl-l'-hydroxy-2'-propylamide [ ( I t ) -methanan-dam idel exhibited the highest affinity for the cannabinoid receptor wi th aK i of 20f .6nM , 4-foldlower than that of anandamide (Ki= 78 f 2 nM) . Moreover, determ ination of the cannabinoidbinding affinity in the presence and absence of the protease inhibitor phenylmethanesulfonylfluoride (PMSF) revealed th at (R)-me thanan dam ide possesses a remark able stabili ty to ami-nopeptidase hydrolysis. Pharmacological s tudies on mouse isolated vasa deferentia demo nstratedth at all four analogs produce concentration-related inhibition of the twitch response and th e orderof potency is the same as th e rank order of the affinities of these ag onists for cannabinoid bindings i tes . Fur thermore, exper iments wi th mice have dem onstrated tha t (R)-methanandamide alsopossesses cannabimimetric properties in uiuo, as established by the four tests of hypothermia,hypokinesia, r ing imm obility, and antinociception.Introduction

Arachidonylethanolamide (anandam ide;1)was recentlyisolated from porcine brain and identified as a putativeendogenous ligand for the can nabinoid receptor. Thisidentification was based on the a bility of anan dam ide toinhibit both th e specific binding of a tritiated c anna bino idligand to synaptosom al membranes a nd the electrically-evoked twitch response of the mouse vas deferens.lSubseque nt studies demonstrated tha t the pharmacologi-cal activity of anandamide, when administered in viuo,parallels tha t of other cann abinoid receptor ago nis k2Furthermore, anan damide was shown to share the abilityof other c ann abim ime tic agents to inhibit forskolin-stimulated adenylate cyclase both in neuroblastoma celllines tha t naturally express canna binoid receptors and incells transfected with plasmids carrying cannabinoidreceptor DNA.3 More recently, ana ndam ide has also beenshown to exhibit cross-tolerance with (-)-A9-tetrahydro-cannabino l (A9-THC) n the mouse vas deferens4 and tobind, albeit with a lower affinity, to a second type ofcannabinoid receptor expressed in the periphery.5Two considerations have gone into the design of th eanandam ide analogs included in this study. The first ofthese is aimed a t improving the affinity of th e anan dam ideligand for the rec eptor. Th e second is to obtain analogswith a higher degree of metabolic stability. The se tworequirementsare necessitated by th e fact tha t anand amidepossesses only a mo derate affinity fo r the receptor (Ki78 nM) and that it has a short metabolic half-life.Two pairs of stereoisom ers were designed in which ame thyl group was introduced , respectively, in the a- nd

Scheme 10

barachidonic acid

&positions of th e ethanolamine com ponen tof anan dam ide.Th e new chiral analogs were sub sequ ently evaluated fortheir affinities for cannabinoidbinding sites, for resistanceto aminop eptidase hydrolysis, and for their abilities toinhibit electrically-evoked twitches of the mouse vasdeferens. Th e most potent analog was subsequentlysubjected to in uiuo testing.Chemistry

Anandamide 1, and its analogs 2-5 were prepared byreaction of the appropriate amino alcohols with arachidonicacid chloride, which in tur n was obta ined from arachidonicacid by treat me nt w ith oxalyl chloride in the presence ofdimethyl formamide (Scheme 1).** Address correspondence to this author at the Scho ol of Pharmacy,+ School of Pharmacy, University of Conn ecticut.* Department of Biome dical Scienc es, University of Aberdeen.8 Institute of Materials Science , University of Connecticut.a Abstract published in Aduance ACS Abstracts , May 1, 1994.

U-92, U niversity of Connecticu t, Storrs, CT 06269. Receptor Binding StudiesPrior to assaying the newly synthesized analogs, mem-branes were treated w ith 50 pM phenylmethanesulfonyl

0022-2623/94/1837-1889$04.50/0 0 1994 American Chemical Society

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1890 Journal of Medicinal Chemistry, 1994, Vol.37, No. 12Table 1. Affinities (Ki)f Anandamide and Ita Analogs for theCannabinoidReceptor and Potencies for Inhibition ( E m ) of theTwitch Response in Mouse Vas Deferens

affinity for the potency for inhibitionofcannabinoid receptor,O the twitch response,bKi (nM) ICm (nM)compound PMSF No PMSF No PMSFanandamide (1) 78f 2 >1180 52c2 119f5 >2950 1003 26f5 158f33 474 20f 1.6 28 f3 125 173f 26 268f 101 230

100v)g 80-52c5 6 0 -00u-

4 0 -.-.-rr.- 20sSt

0 -

Notes-

-a Affinity of anandamide and ita analogs fo r th e cannabinoidreceptor was determined using rat brain membranes and 0.8 nM[3H]CP-55940 s the tritiated ligand essentially as previouslydescribed.10 Data wereanalyzedusing nonlinearregression analysis.Ki values were obtained from three different experimenta and areexpressed as th e mean f standard error. *Mean concentrationsproducing a 50% reduction in the amplitude of electrically-evokedcontractions(ICmvalues) have beencalculatedbynonlinearregressionanalysis,using GraphPAD InPlot (GraphPAD Software, San Diego,CA) to plot sigmoidlog concentration-response curves (n =6-8vasadeferentiafor each compound. Percentage nhibitionof electrically-evoked contractions (twitchresponses)inducedby eachdoseof eachcompoundwascalculatedby comparing he mean amplitudeof twitchresponses recorded in th e final minute before the first addition ofthe compoundwith the mean amplitudeof twitch response recorded

14-15 min after each addition. From Pertwee e t al. (ref 4). FromPertwee e t al. (ref 4).D l l T I

u I . , . . . . . I t . . . ....I . . . . , . , . . . . . . . . . . . . . . . . . I . , . . .io-1o 1 0 - ~ 10-8 io- 10-6 10-5 10-4[ L i gand] (M )Figure 1. Equilibrium binding of cannabimimetic anandamideanalogs 2 (A), (O), (A), nd 6 B) to the cannabinoid receptorin rat forebrain membranes. The d ata shown are means fS Efrom three different experiments.

fluoride (PM SF), a common protease inhibitor. Th etreatm ent was based on recent accounts providing evidencefor amidase activity in m embrane preparations th at resultsin the hydrolysis of anandamide but is inhibited byPMSF.718 Although this treatm ent for peptidase inactiva-tion was not mentioned in th e original report describingthe isolation and ch aracterization of anandam ide, we foundit necessary in order to obtain aKi value for anandam idecomparable to th at repo rted previously. Failure to usePMSF results in the hydrolysis of anandamide andconsequently err oneous binding affinities (Table 1). Bymeasuring the affinities of t he ligands for the receptor inthe presence and absence of PMSF, we were, thus, ableto obtain a relative measu re of their m etabolic stab ility.Ra t forebrain membranes were used to assess h e affiiityof th e novel ligands for cannabinoid bind ing sites.9 A well-described filtration assay was employed in w hich specificbinding of th e tritiated ligand CP-55,940 to cann abinoidbinding sites was displaced by increasing con centrationsof th e ligand unde r investigation.gJ0 Fig ure 1depicts the

1 I10-9 10-7 10-5Concentration (M )Figure 2. Mean concentration-response curves for the anan-damide analogs 2 (A), (O), (A), nd 5 (B) in mouse isolatedvasa deferentia. Each symbol representa the mean value f SEof inhibition of electrically-evoked contractions expressed as apercentage of the amplitude of the twitch response measuredimmediately before the firs t addition of drug to the organ bath(n = 6-8 different vasa deferentia).affi iity of t he novel anandam ide analogs for the cannab-inoid receptor. (R)-(+)-Arachidonyl-l-hydroxy-2-pro-pylamide (4) was shown to exhibit th e highest affinity forcannabinoid b inding sites with aKiof 20 f .6 nM, followedby 3 with a Ki of 26 f 5 nM (Table 1). These ligandspossess, respectively, 4- and 3-fold higher affinities forth e cannabinoid receptor when compared to the parentcompound anandam ide (Ki=78f nM ). However, whencompared with 3and anandamide,4 displays a remarka blemetabolic stab ility toamino peptidase hydrolysis. Analog2 exhibits slightly lower affinity for the cannabinoidreceptor than anandamide, whereas 5 exhibits only amoderate affinity for the receptor with a Ki value 2-foldhigher than t ha t of amandamide.PharmacologicalStudies

Mouse isolated vasa deferentia were used to assess thepharmaco logical activity of each newly synthesized a nan -damide analog. Previous experiments had shown, firstly,th at cannabinoid receptor agonists are highly potent andcan exhibit remarkable stereoselectivity as inhibitors ofelectrically-evoked contractions of this tissue (twitchresponse) and, secondly, h at th e potency of cannabinoidsas inhibitors of th e twitch response c orrelates well withtheir potency as psychotropic agents.J2 As shown inFigure 2, analogs 2-5, all produced concentration-relatedinhibitions of the twitch response. Th e rank ord er ofpotency was the same as the rank order of th e affinitiesof these compounds for can nabinoid binding sites, mea-sured in the presence of PMSF (Table l). Th e findingth at anan damide and ita four analogs can each attenuatethe twitch response when administer ed a t reasonably lowdoses in the absence of a protease inhibitor probablyindicates th at none of these compounds are significantlymetabolized in mouse vasa deferen tia, a t least under theconditions used in th e prese nt investigation.The results from our in vitro experiments suggestedthat, in comparison with anandamide, (R)-methananda-mide (4 ) is more poten t asa cannabim imetic agent aswellas more stable. Additional experiments were carried out

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NotesTable 2. Some in Vivo Effects of (RkMethanandamide and Anandamide in Micea

Jou rna l of Medicinal Chemistry, 1994, Vol. 37, No. 12 1891

(R)-methanandamidedm g vehicle (compound 4)dose (m g/kg iv) 0 1.5625 3.125 6.25number of mice 5 6 6 6body tem perature 0.80 f 0.10 0.27f 0.15 -1.50f 0.20**hypokinesia (counts)' 65.4f 4.7 76.8f 15.5 26.5f 14.1* 10.2f 3.2*tail flick (% )e 3.4f 2.2 6.7i .2 41.5f 10.5* 44.6f 18.4*

-3.18f .45**ring immobility ( % ) d 30.5f 4.4 40.2f 4.0 87.4 f 2.8** 93.9f 2.1**

~~anandamide(compound 1)106-1.43 f 0.22**14.7 i .3**64.2i .8**-3.5 i 2.5

a Values have been expressed as means an d Iimita of error as standa rd error. Dunn ett's test has been used to calculate th e significance ofdifferences between the m ean effect of each dru g treatme nt and th e mean effect of the vehicle, Tween 80 (*P 0.05;**P 0.01). * Meandifference between rectal temperatures m easured shortly before drug administration a t ime zero an d rectal temperatures m easured at 10min.c Mean num ber of squares entered (counta) over a 5-min period after placem ent in the ce nter of a walled arena (30 30 cm) marked out in16 squares of equal size (time zero to 5min). Mean percentage time spend immobile on ring between 5and 10min. e Mean difference betweentail flick latency measured at -30min and tail flick latency measured at 12min calculated as percent maximum possible effect. Mice th atdid not respond within 10 s were removed from the apparatus. Mean tail flick latency measured at -30 min was 2.8 f 0.1s (n = 29).to determine whether (R)-methanandamide also possessescannabimimetic properties in vivo. Th is was achieved byestablishing its ability to produce four effects in mice tha ttogether are thought to be predictive of cannabimimeticactivity.l3 These are hypothermia, hypokinesia, ringimmobility, and an tinociception. As found previously foranandamide,14 the peak hypothermic response to (R ) -methan andam ide (3.125or 6.25 mg/kg iv) occurred 10 mi nafter its administration (d ata not shown). Accordingly,drug effects on behavior were monitored over the first 1 0min after adm inistration. Effects on body tem peratu reand nociception were measured imm ediately after comple-tion of these tests. (R)-methanan damideshowed activityin all four tests following its administration at doses of3.125 or 6.25 mg/kg iv (Table 2). The effects of (R)-methanandamide have been compared with those pro-duced by anandam ide (10mg/kg iv) a t the same test times(Tab le 2). Interestingly, this dose of anand am ide showedno detectab le antinociceptive activity, although it did showsignificant activity in th e other tests. Previous experi-ments have shown that, when administered intraperito-neally, anandam ide can produce antinociception in mice,as measured by a paw-licking response in a h ot plate test,as well as hypotherm ia, hypokinesia, an d ring immo bility.2It could well be tha t the discrepancy between o ur resultsand th ose of the previous stu dy reflects some differencein protocol. For example, the dose used or th e time a twhich the test for antinociception was applied in thepresent experiments may not have been optimal foranandamide.Discussion and Conclusion

We have shown th at th e introduction of an (R)-a-m ethylgroup at the methylene carbon adjacent to th e amidenitrogen imparts both higher affinity and metabolicstability to anandam ide. Similarly, th e introduction ofan (S)-P-methyl also imparts improved affinity, butimproves metabolic stability less effectively. The seincreases in affinity are associated with correspondingincreases in pharmacological potenc y as measured by th ecompound's ability to inhibit electrically-evoked contrac-tions of th e mouse vas deferens. Our finding th at (R ) -methana ndam ide can produce hypothermia, hypokinesia,ring immobility, and antinociception in m ice demon stratesthat it also has cannabimimetic properties in vivo.Comparison of the present data (Tables 1 and 2) withresults obtained in previous experim ents4 indicates (R)-methanandamide to be more potent tha n anandamide notonly as an inh ibitor of electrically-evoked contractions of

the mouse vas deferens but also as a hypothermic agent.The relative potencies of these two compounds in theproduction of hypok inesia, ring immob ility, and antinoci-ception in mice remain to be established.A recent publication'5 re ports th e synthesis of the tworacemates corresponding to our four analogs (2-5) andtheir subsequen t evaluation as cannabimimetic agonists.These two racemates were evaluated for their ability tobind to the cannabinoid receptor an d to inhibit forskolin-stimulated adenylate dyclase activity using mammaliancell lines in which the hum an cannabinoid receptor wasexpressed. Th e results obtained from the above studyshowed th at th e two racemic diastereomers exhibit 7- nd2-fold lower binding affinities for the cannanbinoidreceptor an d 4-fold lower potencies in the adenylate cyclaseassay compared to anandam ide. The se results are widelydivergent from those repo rted in this pa per, where on th ebasis of the data for each of the enantiomers, thediastereomeric racemates would be expected to exhibithigher affinities and potencies th an the pa rent ananda-mide. We have no obvious explanation for the observeddifferences between our results and those mentioned inthe above publication. Perh aps the discrepancy could beattributed to he differences between the rat brain receptorpreparation used in our study and the transfected m am-malian cell line preparation in the earlier report.Of the four chiral analogs described in this repo rt, (R ) -methanandamide (4) is clearly the most interesting andsuperior to any an andam ide analog reported to date. Itsfavorable biochemical and pharmacological propertiesinclude higher affinity for th e receptor an d higher agonistcannabimimeticpotency compared to its native prototype.Furth erm ore, this new analog exhibits significant stereo-selectivity an d improved metab olic stability. Such im-proved properties qualify it as a potentially usefulbiochemical probe and pharmacological tool.Experimental Section

Chemistry. lH NM R spectra were recorded on a BruckerWP-200SY 200 MH z spectrometer using tetramethylsilane asan intern al reference. Elem ental analyses for compounds 2-5are w ithin (&)0.4% of th e calculated values. Specific rotationswere determined with a Perkin-Elmer 241 polarimeter using a1.00-dm cell. T he optically pur e amino alcohols were obta inedfrom Aldrich Chemical Co. (Milwaukee, WI).T he general procedure for th e synthesis of arachidonylamidesis as ollows: A solution of arachidonic acid (200mg,0.66mmol)and d ry dimethyl formamide (0.05 mL, 0.66mmol) in 5 mL ofdry benzene was cooled in an ice-bath and oxalyl chloride (0.12mL,1.32mmol) was adde d dropwise und er nitrogen. Th e reactionmixture was stirred at 25 "C for an additional hour when 5 mL

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1892 Jou rna l of Medicinal Chemistry, 1994, Vol. 37, No. 12of anhydrous T H F was added and th e mixture was cooled in anice-bath. Subsequently, a solution of the ap propriate aminoalcoho l (10-fold excess) in 5 mL of anhydrous TH F was added.After furthe r stirring at oom temperature for 15min, he reactionmixtur e was diluted with chloroform (15mL ), washed successivelywith 10 % HCl and 10 % NaOH solutions, and dried (MgSO,),the solvent removed under vacuum, and t he residue purified bycolumn chromatography on silica gel.Anandamide (1)was synthesized from arachidonic acid (400mg, 1.3 mmol) and ethan olamine in 71 % yi el d colorless oil; Rf0.18 ( 5% MeOH/CHC&);1H NMR (200 MH z, CDCls) 6 (TMS)5.89 (br s, lH ), 5.48-5.27 (m, 8H ), 3.73 (t , J = 4.82 Hz, 2H),3.54-3.39 (m , 2H ), 2.87-2.79 (m, 6H), 2.26-2.00 (m , 6H ), 1.80-1.67 (m, 2H ), 1.40-1.18 (m, 6H ), 0.90 (t, J = 6.57 Hz, 3H).(R)-(-)-Arachidonyl-2'-hydroxy-l'-propylamide 2)wassynthes ized from arachidonic acid (200 mg, 0.66 m mol) and (R )-(-)-l-amino-2-propanol ([(rI2OD -23.5") in 67 % yield colorlessoil; [cY]=D = -9.44' (c = 1,CHCls); Rf 0.3 (5% MeOH/CHCls);1H NMR (200 MH z, CDCb) 6 (TM S) 5.9 (m, l H ), 5.46-5.28 (m,8H ), 3.95-3.86 (m, lH ), 3.50-3.39 (m , lH ), 3.18-3.05 (m, lH ) ,2.86-2.78 (m , 6H ), 2.26-2.01 (m , 6H ), 1.78-1.64 (m , 2H ), 1.43-1.25(m,6H),1.18(d,J=3.17Hz,3H),0.89(t,J=5.87Hz,3H).Anal. (C~ Hsg N0 2) , H, N.(S)-(+)-Arachidonyl-2'-hydroxy-l'-propylamide 3) wassynthesiz ed from arachidon ic acid (200 mg, 0.66 mmol) and (S)-(+)-l-amino-2-propanol [CylMD +23.5') in 63% yie ld colorlessoil; [ a ] % D = +9.44" (c = 1, CHC13); Rf .3 (5% MeOH/CHCb);1H NMR (200 MH z, CDCb) 6 (TM S) 6.42 (m, lH ), 5.46-5.30 (m,8H ), 3.93-3.85 (m, lH ), 3.47-3.36 (m, lH ), 3.16-3.03 (m, lH ),2.83-2.80 (m , 6H ), 2.26-2.01 (m , 6H ), 1.78-1.64 (m , 2H ), 1.39-1.25 (m, 6H), 1.18 (d ,J = .18Hz, 3H),0.89 (t,J = .43Hz, 3H).Anal. (Cd sgN O2 ) C, H, N.(R)-(+)-Arachidonyl-l'-hydroxy-2'-propylamide 4) wassynthesiz ed from arach idonic acid (700 mg, 2.3 mmol) an d (R)-(-)-2-amino-l-propanol ([(Ul2OD -18') in 69% yield colorlessliquid; [(r]=D =+10.9' (c= 1,CHCls);Rf0.3 5% MeOH/CHCb);1H NMR (200 MH z, CDCls)6 (TMS) 5.57 (m, lH ) , 5.47-5.30 (m,8H ), 4.14-4.02 (m , lH ) , 3.71-3.48 (m, 2H) , 2.84-2.81 (m , 6H ),2.24-2.01 (m , 6H ), 1.77-1.65 (m , 2H ), 1.39-1.26 (m , 6H ), 1.17 (d,J = 3.46 Hz , 3H ), 0.89 (t ,J = 6.12 Hz, 3H ). Anal. (CzsHssNOz)C, H, .(S)-(-)-Arachidonyl-l'-hydroxy-2'-propylamide (5) wassynthesiz ed from arac hidonic acid (200 mg, 0.66 mmol) and (SI-(+)-2-amino-l-propanol ([aI2OD +18") in 65% yield colorlessliquid; [ a ] = ~ -10.9' (c = 1,CHCls);Rf0.3 5% MeOH/CHC&);1H NMR (200 MH z, CDCls)6 (TM S) 5.61 (m, lH ) , 5.47-5.30 (m ,8H ), 4.14-4.02 (m , lH ), 3.71-3.49 (m , 2H ), 2.84-2.78 (m , 6H ),2.41-2.04 (m , 6H ), 1.79-1.69 (m , 2H ), 1.39-1.29 (m , 6H ), 1.18 (d ,J = 3.40 Hz, 3H ), 0.89 (t, J = 6.02 Hz, 3H ). Anal. (CzsHssNOz)C, H, N.Binding Assay. Rat forebrain membranes were preparedaccording to the procedure of Devane et al.B Th e binding of th enovel probes to the cannabinoid receptor was assessed aspreviouslydescribed?JO except that the membranes were trea tedwith PMSF. Mem branes, previously frozen a t -80 "C, werethawed on ice. T o he stirred suspension was added five volumesof 25 mM Tris-HC1buffer, p H 7 .4,5 mM MgC12, 1mM EDTAcontaining50pM PMS F (made fresh daily in 2-propanol). After30 min, the membranes were pelleted, the supernatant wasdiscarde d, and t he pellet was resuspende d in five volumes of thePMSF containing buffer. At the end of the second 30-minincubation the m embranes were pelleted and washed three tim eswith 25 mM T ris-HC1buffer, pH 7.4,5 mM M gC12,l mM ED TAto remove the excess, unreacted PM SF. They were subsequentlyused in the binding assay as ollows: ca. 50pg of PM SF- treate dmembranes was incubated in 25 mM Tris-HC1buffer, pH 7.4, 5mM M gC12,l mM ED TA containing0.1% bovine serum albuminalong with 0.8 nM [3HlCP-55,940 and varying concentrations ofthe cannabim imetic analogs to a final volume of 0.2 mL. Assayswere incubated in Regisil-treated culture tubes for 1h a t 30 "Cand terminated by the addition of 0.25 mL of 25 mM Tris-HC1buffer, pH 7.4,5 mM M gC12,l mM EDT A containing 5% bovineserum albumin. Th e assays were immediately filtered on GF /Cfilters using aBran dell M -24 cell harves ter. Following four washeswith 25 mM T ris buffer, pH 7.4,5 mM MgC12,l mM E DTA , thefilters were collected, shaken for 1h with 2 mL of 0.1 5% sodium

Notesdodecyl sulfate, and counted in a B eckman liquid scintillationcounter to determine the bound ligand. Nonspecific bindingwas assessed from tubes containing 250 nM desacetyllevonant-radol. Data were colledted from three different experimentsperformed with duplicate determinations. ICm values werecalculatedby nonlin ear regreasionanalysisusing the comm erciallyavailable program GraphPad InPlot (GraphPad Software, SanDiego, CA) and conve rted toKi values using the assumptions ofCheng and Prusoff.16Pharmacological tudies n Vitro. Vasa deferentia wereobtained from albino MF1 mice and mounted in 4-mL organ

baths at an initial tension of 0.5 g using the me thod describedby Pertwee etal.' Th e baths contained Mg2+-freeKrebs solutionkept at 37 'C an d bubbled with 95% 0 2 and 5% COS. Isometriccontrac tions were elicited by electrical field stim ulatio n througha platinum electrode attached to th e upper end of each bath andastainless steel electrode attached to the lower end. Th e stimuliwere generated by a Grass S48 stimulator and then amplified(Med-Lab channel attenuator) and finally divided to yieldseparate outputs to four organ baths (M ed-Lab Stimusplitter).Contractions were registered on a computer (A pple MacintoshLC) using a data recording and analysis system (Ma cLab) th atwas linked via preamplifiers (Macbridge) to Dynamometer UF1transducers (Pioden Controls Ltd.). Tissues were stimulatedwith 0.5-8 trai ns of thre e pulses of 110% ma xima l voltage (trainfrequency, 0.1 Hz; pulse duration, 0.5 ms). Each tissue wassubjected to several periods of stimulation. Th e first of thesebegan after the tissue had equilibrated bu t before drug admin-istration and continued for 11min. Drug addition (10 pL) wasmade immediatelyafter this f i t timulation period. Su beq uen tstimulation periods lasted 5 min a t the end of which the bathcon tents were washed out by overflow and a high er dose of drugadded. Ten m inutes was allowed toelapse between drug ad ditionsand onset of stimulation. Drug concentrations producing a 50%reduction in the a mplitude of electrically-evokedcontractions(ICW values) were calculated by nonlinear regression analysisusing GraphPad InPlot (GraphP ad Software, San Diego, CA).Anandamide analogs were mixed with two parta of Tween 80 byweight and dispersed in 0.9 % w/v aque ous NaCl solution (saline)asdescribed previously for AB-THC.ll They were stored at -20"C and protected from light at all times.Pharmacological tudies n Vivo. These were carried outwith male MF1mice weighing 23-29 g. Dr ugs were mad e up as

described above and were injected intravenously at time zero.'The degree of hypokinesia was measured immediately afterinjection by placing a mouse in th e center of a walled arena (30X 30 cm) marked ou t in 16 squares of equal size and then notingthe number of squares entered between time zero and 5 min.Ring imm obility was monitored by placing a mouse on a 5.5-cmhorizontal ring mounted on a 16-cm stalk a t 5 min and notingthe proportion (% ) of the total ime spent on the ring (5 min)th at th e anima l rema ined immobile.17 Body temper ature wasrecorded using a therm istor probe (YSI 402) which was inserted3 cm into the rectum. Except when held in the hand for themeasurement of body temperature, the mice were kept unre-strained, each in a separate cage. Rectal temperature wasmeasured shortly before time zero and again a t 10 min. Changesin body temperature were calculated by subtracting th e secondof these values from the first. Antinociception was measured bymeans of a tail flick test, in which time taken (latency) for alightly restraine d m ouse to flick ita tailaway from a radian t heatstimulus was noted. Th e method is based on the test describedby D'Amour an d Smith.'s Mice were subjecte d to the tail flicktest a t -30 min (control latency) and a t 12 min (test atency). Th emaximum possible ail flick latency was 10s asmice th at did notrespond within this time were removed from the apparatus toprevent tissue damage. Antinociceptionwas alculatedaspercentmaximum possible effect by expressing the ratio (test atency -control latency)/(l@ scontrol latency) as a percentage.'# Eachmouse underwent all four tests. Ambient temperatur e was keptbetween 20 and 22 "C. Values have been expressed as meansand limits of error as stand ard errors. Dunnett's test has beenused tocalculate he significanceof differen cesbetween the m eaneffect of each drug trea tmen t an d the mean effect of th e vehicle,Tween 80.

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Notes Journal of Medicinal C hemi stry, 1994, Vol. 37 , No. 12 1893Acknowledgment. This work wa s supported by grantsDA-3801, DA-152, and DA-7215 (to A.M.) from th eNational Institute on Drug Abuse and by Wellcome Trus tGr ant 034924 (to R.P.). We would like to thank Dr. L. S.Melvin of Pfizer, Inc. fo r providing us with desacetyllevo-nantradol.

References(1)Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson,L. A.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.;Mechoulam,R. Isolation and Structure of a Brain Constituent Th atBinds to the Cannabinoid Receptor. Science 1992,258,1946-1949.(2) Fride, E.; Mechoulam, R. Pharamcological Activity of the Can-nabinoid Receptor Agonist,Anandamide,a Brain Constituent. Eur .J. Pharmacol. 1993,231,313-314.(3) Vogel, 2.;Barg, J.; Levy, R.; Saya, D.; Heldman , E.; Mechoulam ,R. Anandamide, a B rain Endogenous Compound, Interacts Specif-ically with Cannabinoid Receptors and Inhibits Adenylate Cyclase.J. Neurochem. 1993,61,352-355.(4) Pertwee, R. G.; Stevenson,L . A.; Griffin, G. Cross-tolerancebetweenDelta-9-tetrahydrocannabinol and the Cannabimimetic agentsCP-55,940, WIN 55,212-2 an d Anandamide. Br. J . Pharmacol.(5) Munro, S.;Thom as, K. L.; Abu-Sh aar, M. Molecular Char aderiz a-tion of a Peripheral Receptor for Cannabinoids. Na tur e 1993,365,61-65.(6) Corey,E. J.; Cashman,J.R.; Kantner, S. S.;Wright,S.W. RationallyDesigned, Poten t Competitive Inhibitors of Leukotriene Biosyn-thesis. J. Am. Chem. SOC.984,106, 1503-1504.(7) Deutsch, D. G.; Chin, S.A. Enzymatic Synthesis and Degradation

of Anandamide, a Cannabinoid Receptor Agonist. Biochem.Pharmacol. 1993,46, 791-796.(8) Childers, S. R. Personal Communication. 1993 InternationalCannabis Research Society, Ontario, Canada.(9) Dev ane, W. A.; Dysarz, F. A., 111; Joh nso n, R. M.; M elvin, L. S.;Howlett, A. C. Determination and Characterization of a Cannab-inoid Receptor in Rat Brain. Mol. Pharmacol. 1988,34,605-613.

1993,110, 1483-1490.

Charalambous,A.; Guo,Y.;Houston , D. B.; H owlett, A. C.; Comp ton,D. R.; Martin, B. R.; Makr iyannis, A. 5-azido-A*-THC: A NovelPhotoaffinity Label for the Cannabinoid Receptor. J .Med. Chem.1992,35,3076-3079.Pertwee, R. G.; Stevenson, L. A.; Elrick, D. B.; Mechoulam, R.;Corbett, A. D. Inhibitory Effects of Certain Enantiomeric Can-nabinoids in the M ouse Vas Deferens and the M yenteric PlexusPreparation of Guinea-pig Small Intestine. Br. J .Pharmo col. 1992,105,980-984.Pertwee, R. G. Th e Evidence for the Existence of CannabinoidReceptors. Gen. Pharmacol. 1993,24,811-824.Martin, B. R.; Compton, D. R.; Thomas, B. F.; Prescott, W. R.;Little, P. J.; Razdan, R. K.; Johnson, M. R.; Melvin, L. S.;Mechoulam,R.; Ward, S.J.Beharvio ral, biochemical andmo lecularmo deli eva luat ion s of cannabinoid analogs. Pharmocol. Biochem.Behav. 1991,40,471-478.Pertwee, R. G.; Stevenson, L. A. Effect of pretreatm ent with delta-9-tetrahydrocannabinol on t he ability of certain cannabimimeticagents to induce hypothermia in mice. In: Temperature Regula-tion. Recent Physiologicaland PharmocologicalAdvances; Milton,A. S., Ed.; Birkh iuser: Basel, 1994; pp 177-182.Felder, C. C.; Briley, E. M.; Axelrod, J.; Simpso n, J. T.; Mackie,K.; Devane, W. A. Anand amide, a n endogenous cannabimim eticeicosanoid, binds to the cloned human cannabinoid receptor andstimulates receptor-mediated signal transduction. Proc. Nat l .Acad. Sci. U.S.A. 1993,90, 7656-7660.Cheng, Y. C.; Prusoff, W. H. Relationship betw een the inhibitionconstant (KJ and the concentration of inhibitor which cause8 50per cent inhibition ( E m ) of an enzymatic reaction. Biochem.Pharmacol. 1973,22,3099-3102.Pertwee, R. G. Th e ring test: A quantitative m ethod for assessingthe catalep tic effect of cannabis in mice. Br. J.Pharmacol. 1972,DA mou r, F. E.; Smith , D. L. A method for determin ing loss of painsensation. J . Pharmacol. Exp. Ther. 1941, 72,74-79.Compton, D. R.; Johnson, M. R.; Melvin, L. S.; Martin, B. R.Pharm acological profile of a series of bicyclic cannabinoid analogs:Classificationas cannabimimetic agents. J .Pharmacol. Exp. Ther.

46,753-763.

1992,260, 201-209.