Monoamine Oxidase Inhibitory Properties of Some Methoxylated and Alkylthio Amphetamine Derivatives Strucrure-Acfivity RELA TIONSHIPS Ma

ELSEVIER

Monoamine Oxidase Inhibitory Properties of Some Methoxylated and Alkylthio Amphetamine Derivatives STRUcruRE-ACfIVITY RELA TIONSHIPS Ma. Cecilia Scorza,* Cecilia Carrau,* Rodolfo Silveira,* GemId Zapata,Torres,t Bruce K. Casselst and Miguel Reyes,Parada*t *DIVISIÓNBIOLOGíACELULAR,INSTITUTODEINVESTIGACIONESBIOLÓGICASCLEMENTeEsTABlE, CP 11600, MONTEVIOEO,URUGUAY;ANDtDEPARTAMENTODEQUíMICA, FACULTADDECIENCIAS,UNIVERSIDADDECHILE, SANTIAGO,CHILE

ABSTRACT. The monoamine oxidase (MAO) inhibitory propenies of a series of amphetamine derivatives with difTerent substituents at or around rhe para position of the aromaric ring were evaluateJ. i¡, in viero stuJies in which a crude rar brain mirochllndrial suspension was used as rhe source of MAO, several compounds showed a srrong (ICS0 in rhe submicromolar range), selecrive, reversible, time-independenr, and concenrrarion-related inhibition of MAO-A. After i.p. injection, the compounds induced an inerease of serotonin and a decrease of j-hydroxyindoleacetic acid in the raphe nuclei and hippocampus, confinning rhe in virro results. The analysis of structure-activity relationships indicates rhat: molecules with amphetamine-Iike structure and different substitutions nn the aromaric ring are potentially MAO-A inhibitors; substituents at different positions of the aromatic ring moditY the porency but have litde inf1uence "n the selectiviry; substituents at rhe para position sllch as ,lmino, alkoxyl. halogens. or alkylthio produce a significant increase in rhe acrivity; the para-substituent musr be an e1ectron Jonor; hulky ~roups next to rhe para subsriruent Icad ro a Jecrease in the actÍ\'ityi ,ubstiruents loearcd ar posirions more Jistant ,m rhe aromaric ring havc less intluence anJ, even when the subsriruent is '1 halogen (CI, Br), an increase in rhe acrivity "f rhe cllmpound is llbtained. Final!y, rhe MAO-A inhibirory properties of some of rhe compounJs evaluareJ are Jiscussed in relation to: (a) potential antidepressant acri\"Íry, and (b) their reponed hallllcinogenic, neurotoxic, nr anxiolyric effecrs. BIOCHEM rHAR~1ACOL54;lZ:1361-lJ69, 1997. ,© 1997 Elsevier Science Inc,

KEY WORDS. monoamine oxidase; MAO inhibirors; amphetamine Jerivarivesi strueture-aetivity rdation• ships; phenylisopropylamine Jerivarives; antidepressants

MAO§ (EC 1.4.3.4, monoamine:Oz oxidoreductase), monoamines such as catecholamines and 5-HT, exists in which catalyzes the oxidative deamination of a variety of two different forms (MAO-A and -B) that are distinguish• able by their substrate selectivity, inhibitor sensitivity, and amino acid sequence [1-3). Non-selective and irreversible :j: Corresponding author: Dr. Miguel Reyes-Parada. Instituto de Investiga• ciones Biolágicas Clemente Estahle. Divisián Biología Celular, Avenida MAOls have been usetyramine-rich food, § Abhret'ianons: MAO, monoamine oxidase; MAOI, MAO inhibiror; 5-HT. ;erotonin; DMAPEA. 4-dimerhylaminophenethylamine; 5-HIAA. have limited their use (4-6). A better understanding of the 5-hydwxyindoleacetic acid; 50S. sodium ocryl sulfate; DMAPAA. differing activities of the two enzymatic isoforms has led to 4-dimerhylaminophenylaceric acid; PeA. p-chloroamphetamine; FLA• the development of a new generation of antidepressants 527. 2.5-dimerhoxy-4-dimerhylaminoampheramine; MTA, 4-methylthio• based on me selective and reversible inhibition ofMAO·A. aml'heramine; ETA, 4-ethylrhioaml'hetamine; lTA, 4-isopropylthioam• phetamine; Z.4-DMA. 2.4-dimerhoxyamphetamine; ooTFM, 2,5-dime• This class of compounds exhibits antidepressant actions thoxy-4-trifluoromethylamphetamine; 4-EtOA. 4-ethoxyamphetamine; while it appears to interact only weakly with dietary 4-MetOA. 4-merhoxyamphetamine; 3.4-DMA, 3.4-dimethoxyamphet• amine; 2.5-DMA. 2,5-dimethoxyamphetamine; AlEPH-2, 2.5-dime• tyramine (7-9). ., :(, 1 thoxy-4-ethylthioamphetamine; AlEPH-l, 2,5-dimethoxyl-4·methylthio• Several phenylisopropylamines, including amphetamine amphetamine; TMA-2, Z.4.5-trimerhoxyamphetamine; TMA, 3.4,5-tri• itself, have been evaluated as MAOls (10). Generally methoxyamphetamine; ooB. 2,5-dimethoxy-4-bromoamphetamine; 001. Z,5-dirnethoxy-4·ioJoampheramine;ooM, 2,5-dimethoxy-4'methylamphet• speaking, substituents on the aromatic ring of the pheny. amine:roN, 2,5-dimethoxy-4-nitmamphetamine;roA, Z,5-dimethoxy-4-ami• lisopropylamine molecule (in particular at the para posi. noamphetamine; Z-Br-4.5-DMA. 2-bromo-4,5-dimethoxyamphetamine; tion), such as amino groups [11-13), halogens [14], hy• 5-Br-2,4-DMA. 5-bromo-Z.4-dimethoxyampheramine; Z-NOz-4,5-DMA, 2-nitro-4,5-dimerhoxyampheramine; 2-Br-4.5-MDA, 2-bromo-4.5-meth• droxyl (15) and methoxyl groups [16, 17], lead to an ylenedioxyamphetamine; 2-NOz-4.5-MDA, 2-nitro-4,5.merhyleneJioxy• increase in the potency and selectivity towards MAO-A, ampheramine; Z-CI-4,5-MDA, 2-chloro-4.5-merhyleneJioxyampher• with resrect t~ the parent compound. However, except for amine; MDA. 3,4-merhylenedioxyamphetamine; MDMA, 3.4.-merhyl· enedioxymerhampheramine; Rn. raphe nuclei; and He. hippocampus. the studies of Ross and FlorvaU's group [11, 12, 17-20], no Received 26 February 1997; aecepted 19 June 1997. extensive molecular series of MAOls have been studied 1362 M. C. ScOI'%8 el al.

that coulJ contribute to <1 beuer unJerstanJing of rhe rar brain mitochonJrial suspension. The tissue was prepared structural requirements necessary to Jesign new porenr and from whole rat brain (after disc:uding the cerebellum) of selecrive amphetamine-like MAOls. male IIBCE rats weighing 200-240 g as described previ• Methoxylated amphetamines have been rhe object of ously [13J. The mitochondrial MAO activities were deter• many structure-acriviry relarionship studies [see Refs. mined by HPLC with electrochemical derection (HPLC• 21-23 for reviewsJ beca use of rheir grear therapeuric and ED), using selective substrates for the A form (5-HT) and abuse potenrial, and rhe high affinity and selecriviry for B form (DMAPEA) [13, 32J. 5-HT receptors and the 5-HT uprake carrier exhibited by T o determine the inhibition of MAO·A, the incubation some of them [for recenr reviews see Refs. 23 and 24J. In mixture consisted of 25 JLLof a 50 JLM 5-HT solution (final these studies, ir has also been shown rhar the para posirion concentration 2.5 JLMin a final volume of 0.5 mL); 225 JLl appears ro play an essenrial role in rhe porency and of 0.1 M sodium phosphate buffer, pH 7.4; the inhibitor to selectiviry of these Jrugs. be tested at the appropriate concentration (lO nM-lOO On the basis of rhese preceJents, in this work rhe in vitro JLM) in 50 JLL of JistilleJ water¡ and 200 JLL of the selectivity, time dependency, and reversibility of rhe inhi• mitochondrial susrension. For MAO-B, the incubation bition of MAO-A anJ MAO-B inJuced by a series of mixture consisted of 25 JLLof a 100 JLMDMAPEA solution phenylisopropylamines, mainly methoxylated derivatives, (final concentration 5 JLM in a final volume of 0.5 ml); were sruJied. Furrhermore, rhe ex vivo effecrs of some 105 JLLof 0.1 M sodium phosphate buffer, pH 7.4; AldDH compounds were assesseJ by measuring rhe endogenous (0.8 units) and ~.NAD (0.6 JLmol) dissolved in 200 JLLof levels of monoaminergic neurotransmirrers and rheir corre• phosphare buffer; the inhibitor ro be rested at the arpro· sponding MAO metabolites in different rat brain regions. pdate concentration (10 nM-lOO JLM) in 50 JLLof distilled Finally, some structure-acrivity relationships for rhe series water; anJ 120 JLL of rhe mitochondrial suspension. The of drugs srudied were esrablished. reaction was srarted by the aJdirion of the mirochonJrial suspension. The mixtures were incubated for 10 min (MAO-A) and 5 min (MAO·B) at 3¡O in a shaking water MATERIALS AND METHODS bath in open test tubes. The reaction was stopped by adding' Chemicals 200 JLLof 1 M HCIO .•. The mixrure was then centrifuged AII the chemicals useJ were of rhe highesr grade commer• at 15,000 X g for 5 min ar 40, anJ 50 J1Lof supemarant was cially available. T etrahydrofuran (THF) and aceronitrile injected into the HPLC system. Control experiments were (ACN) were Merck HPLC grade. Ampheramine sulfare, performed without inhibitor, and blanks wete run without PCA, monoamines, merabolites, aldehyde dehydrogenase mitochondrial suspension. In all cases, volume adjustments from yeasr (AldDH) and ~-nicorinamide adenine dinucle• were made with 0.1 M phosphate buffer. Each concentra· LHiJe (~-NAD) were from the Sigma Chemical Co. (St. tion of drug was assayed in triplicate. The heights of the Louis, MO, U.5.A.). DMAPAA was from Aldrich (Mil• chromatographic peaks of 5-HT, DMAPEA, and their waukee, WI, U.5.A.). Amiflamine and FLA-527 were respective main MAO metabolites, 5-HIAA and donated by ASTRA AB (Sooertálje, Sweden) and 1• DMAPAA, were used to calculate MAO activity. Product deprenyl was donated by Prof. J. Knoll (Semmelweis Uni• formation was found to be linear with incubation time and versity of Medicine, Hungary). MTA, ET A, ITA, 2,4• enzyme concentration under rhe experimental conditions DMA, and DOTFM were synthesized and donated by Dr. described (data not shown). The IC;o values were de ter· David E. Nichols (Purdue University, U.S.A.). All other mined from plots of inhibirion percentage, calculated in compounds used in this study were synthesized following relation to a sample of the enzyme treated under the same pllhlished procedures [Ref. 25 unless stated otherwiseJ. conditions without inhibitors, versus -Iog [IJ. 3,4-DMA, MDA, 2,5-DMA, DOB, 2-Br-4,5-DMA, 5-Br• 2,4-DMA, 2-Br-4,5-MDA, 2-CI-4,5-MDA, DON, 2-NOz• Chromatographic Conditicnu 4,5-DMA, and 2-NOz-4,5-MDA were available in our laboratory from previous work [26-29J. A CI8 reverse phase column (ODS 250 X 4.6 mm, Other compounds used in this study included 4-EtOA BIOPHASE, U.S.A.), an amperometric detector (BAS [30], 4-MetOA, ALEPH-2, ALEPH-l, TMA-2, TMA, LC-3A), and a two·channel graphic tecorder (BAS) were DOI, DOM, DOA (31), anJ MDMA. All amphetamine used to analyze the reaction mixtures. The mobile phase derivarives were stored and used as salts: hydrochlorides, flow rate was I mL/min, and its composition was 31.5 g hydrobromides, or nitrate (in rhe case of DON). Their citric acid¡ 908 mL bidistilled water; sufficient 12 N NaOH identity was checked by 1H NMR and their purity by to bring the pH value to 3; 200 mg SOS; 42 mL ACN; and elemental analysis. 50 mL THF. Detector sensitivity was 10 nA, and the oxidation potential was fixed at 0.85 V using a glassy carbon working electrodt; versus an Ag/ AgCI reference electrode: . Ef1t)'TlU1tic Assays Time-courses of the MAO·A or MAO·B inhibition by The effects of the phenylisopropylamine derivatives on the drugs wete assessed by preincubating tbe teaction MAO-A or MAO-B activities were studied using a crude mixture with different compounds at an appropriate con· 15) OOTFlvl (J, ",/N~R"Suh5tituteJ CH, O-CHJO-CH1S-CH-(CHCH2-c=CHO-CHz-CH,S-CHrCHJN-(CH)hN-(CH\)zO-CHJHRJHHCH)HO-CHJO-CH)O-CH1S-CHJS-CH,'HHReO-CH1HNOzO-CH1O-CHJCH1O-CH,CFJHHNH,HHO-CH)R7HHH CISrO-CHJO-CH1HR.•Rs AmpheramineslO-CH)CHJO-CHJHH HSrHH HHH HCHz-O-CHzCHrO-CHzCH2~O-CH2HHHHH ,hHHH O-CH1O-CH,CH1O-CHJO-CH¡NOzHHR2CIHSrH ;15MAOH Inhibiwrs 1J63 R,_ O-CHJ Zi)25)24)30)23)22)29)18)11)20)10)1,)13)16)12)14)19)21)6)9)8)4)5)i)31)TMA ALEPH-2TMA-2MDMAMTAFLAITA2Sr-4.5-MDA2Sr-4,;-DMA4-EtOA25-DMAPCA4·MetOAALEPH-I001HA2,4-Dlv1A3.4-DMALX1BOOAOOM5Sr-2,4-DMA2CI-4,5-MDA DON MDA 52i R¡ '--" 28)26)2)3) l-Deprenyl2NOz-4,;-MDA2NOc··l,j-DMA Amphetamine R" 1 R, 1) Amiflamine r r R. Compound TABtE 1. Structural descriptions of the amphetamine derivatives tested

centration, for 30 anJ 60 mino After preincubation, compounds. Groups of male IIBCE rats (N = 6-8) weigh• MAO-A anJ MAO-B activities were measured unJer the ing 200-220 g were injected Lp. with equimolar doses (39.5 same conditions JescribeJ above [13). ~mol of free base) of the drugs. The control group (N = la) The reversibility of the inhibitory process for both forms was injected with saline. The volume injected was 1 mL/kg of MAO was assessed by repeated washing of the mitochon• in each case. Rats were decapitated 1 and 6 hr after injection, drial suspension. MAO-A or MAO-B mixtures Were prein• and the dorsal Rn anJ Hc were dissected out immediately. cubateJ for 10 min with drugs at appropriate conCentra• The tissues were kept at -700 until used (not more than 5 tions. Then the preparations were washeJ three or four days later). The le\'els of monoamines and their metabolites times (centrifugation and resuspension) with 0.1 M sooium were measured by HPLC-ED as previously described [13). phosphate buffer, pH 7.4. Finally, MAO activities were measured again, using HPLC-ED. Control samples, in which the inhibitor solution was replaced by an equal Statistical Analysis volume of water, Were treated in the same way [13). The statistical signitkance was determined using Student's In all cases, the protein content was determined accord• t-test. In al! cases, the significance level was found to be ing to Lowry et al. (33) with BSA as standard. P < 0.05.

Ex Vivo Studies RESULTS T o evaluate the in ¡.:iva effects of the drugs, monoaminergic neurotransmitter levels were quantified in different rat T able 1 summari:es the structures of the amphetamine CNS regions after the peripheral administration of the derivatives studied. 1364 M. C. Scorza el al.

log100647M 0--0 z ••.•E lf1 ~oQJI-tUtUo 50I conc,25755O A o 749 M o oI.f;E nI< eQJW 856conc.,50 B - e.siie e ii00••..%oa.~o~Eni < 1002575 -o-~-o B ee I O /0 ~ ~ / log10

• ETA o 4-tvletOA <> TMA-2 "Amlflan:'1e lJ I-Deprenyl

FIG. \. Effects of some amphetamine derivatives as MAOIs. Inhibition of MAO.A and ·B is represented by the percentage of inhibition of Jeamination vf 5·HT (Al anJ DMAPEA lB), as measureJ by HPLC·ED. The experimt:nts were performed without en:yme.inhibitor preincubation. \litochondrial suspension was prepared from whole rat brain. Each point is the mean ± SD of three determinations.

In Vitro Studies TABLE 2. le;o Values for MAO inhibition

In lhese ~ludies. lhe effecls ()f lhe compounds lm lhe MAO leso < •.•. aClivilies were evaluated. Figure 1 shows an example ()f the M) curves obtained tor the inhibition of MAO·A (panel A) Compound MAO·A MAO·B and MAO-S (ranel B) l-y some of the compounds. Ami. 1) Amitlamine 2.0 NE tlamine and l-Jeprenyl were included as a positive control 2) I·Deprenyl 50 0.007 3) :\mphelamine 11 NE for the inhibition of ~L-\O-A and MAO-S, respectively. 4) ETA 0.1 29 Several compounds ~h<1weJa strong, selective, anJ concen. 5) MTA 0.2 NE tration-depenJent inhiJ:.ition of MAO-A. 6) ITA 0.4 8.1 T able 2 summari:es the IC;ü values for the inhibition of 7) 4-EtOA 0.2 > 100 8) 4·MetOA borh forms of ~tAO by rhe amphetamine derivatives tested. 0.3 NE 9) PCA 4.0 NE It is noteworthy that most of the compounds (inc\uding 10) 3,4-DMA 20 NE amphetamine itselO \Vere c1early selective as MAO-A 11) 2.5·DMA > 100 NE inhibirors. Additionally, several Jerivatives (4-8, (2) were 12) 2,4·DMA 0.6 NE more potent than amitlamine, the reference MAO-A in• 13) ALEPH·2 3.2 NE 14) ALEPH-l :;,¡ NE hibitor amphetamine Jerivative, \Virh their IC;ü values in 15)ooTF~t NE NE the submicromolar range. Moreover, some Jrugs were 16) FLA 52i NE NE completely devoid of ~tAO inhibitory properties. 17) TMA-2 NE NE 18) TMA T able 3 shows the effects of enzyme-inhibiror preincu• NE NE 19) ooB bation (O, 30, and 60 min) on MAO-A and ·S inhibition 100 NE 20) ooM 24 NE produced by some compounds. AII drugs. except c10rgyline 21) ooN NE NE and l-deprenyl, inhibited enzymatic activity independent1y 22) 001 43 NE of time, since the degree of inhibition was not changed even 23) ooA NE NE 24) 2Br·DMA 9.3 after the longest incul:-ation time. In the case of c\orgyline NE 25) 5Br-DMA 13 NE and l-deprenyl, as exrected for suicide inhibitors, the 26) 2NOz·DMA NE NE inhibition observed after 30- and 60-min preincubation was 27) 2Br-MDA 13 64 significantly greater than that observed with no preincubation. 28) 2NOz-MDA NE 78 29) 2CI-MDA 6.3 The results obtaineJ in the reversibility studies are 38 30) MDMA 30 shown in T able 4. The inhibition of MAO-A after a NE 3l)MDA 9.3 NE ·lO-min preincubation wirh the Jrugs was, in most of the cases, reversed significantly after three washes of the prep' The leso valUd were

T ABLE J. Effects of preincubation on the inhibition of MAO-A and MAO-B by amphetamine derivatives

65.0:!:47.0:!:13.2307M75.03076.977.450.592.0:!:26.482.575.2:!:NONONO40.5o10.1NO60IZ.snoO73.6:!:NONOo80.279.0:!:51.674.4:!: :!:NONONO 3* 3.4Io:!: :!:4.6*:!:4.4:!:0.576.8:!:1:!:2.5 :!::!: :!: 2.2* 3.63393.2:!:5.227.2:!:1.179.2:!: 0.65.41.21.385.1:!:5.24;.5:!: 2.3lA 4.33.6 1.6NO 1NO 2.679.8:!: 1*1.3 81.788.0:!:78.0:!:74.351.427.017.9:!:NOO :!::!:0.5 2.62.72A1.6 5.4 MAO-BPercent of inhibition of MAO-A and MAO-B 2·NO~-4,5-MOAI·Derrenyl4·EtOATMA-ZZ-Br-4.5·0MAClorgylineALEPH·24·MetOAMTA(10-4 (lO-h(10-6(10-7[10-4 (lO-s(l0'~MI MI (lO-sMI(10-4MIMI MI MIMI MAO-A HA (5 X 10-7 MI Compound Preincubation time (min)

CruJe mituchl1nJri;ll "U"rt:'nS1pn~ wt"re rrelncuhltt.·J ;t( J7° ttlr [he times mJicateJ. wnh t:'ach comruunJ al a C"oncentration rhm:, wirhout rreincuhation. JiJ out ,wJuce toml inhibiti,," "1 rh~ en:yme. Percent mh,bitlon ,,1 J~amin"tion oi ;·HT (Z.; ¡.LM) lor MAO-A and DMAPEA (; ¡.LM) ior MAO·B was J~t~rmin~J by HPLC-ED. C'''RYlin~ anJ '~Jerrcnyl were u~cJ as J ("\lSIlI\"e úmrrol. Values are means .= SD llf trirlicue Jeterminations. ND = nll[ Jetermined . ...r.:::l'.25 (tlmrart"J ·""lfh 2·min rreincuhation (StuJent's [-test).

,)hen'e .\ 'ilight rUt 'iignificmt reyersal (yt the en:yme meta\:Yoli:ed hy MAO-A, [he changes obsen'ed in [he inhihition. The inhibirory effect of the irreversible <:ontent l)f the monoamine ::mJ its main MAO metaholite \1.-\0-;\ tnhiht'lr dnrgyline was present after three or fout are probably Jue to inhibition of the emyme acti\·ity. This washes. For ml1st "f the <:ompounds, MAO-A a<:tivity is also supporteJ by the faet that amiflamine rroJ,uceJ showed Jitferent Jegrees úf recovery after washes. basically the same effects. Recovery of )-HT and 5-HIAA levels to near control Ex Vivo Studies values was observed in most cases 6 hr after drug adminis• tration. These ~x vivo findings are in agreement with the T able 5 shows the effects of some amphetamine derivatives reversibility observed in l,irro. Nevertheless, 6 hr after on the 5-HT and 5-HIAA levels in two brain regions, 1 and administration of amiflamine and MT A, increased 5-HT 6 hr after [he ir peripheral administration. In the Rn, all levels were still found, an effect that was e\'en more compounds tested inJuceJ a significant increase in 5-HT pronounced than the effect observed after 1 hr. levels 1 hr after injecrion, whereas a significant Jecrease in In a region rich in serotonergic nerve terminals such as 5-HIAA was obsen:ed in aU <:ases. Since 5-HT is mainly the He, most of the compounds elicited the same changes observed in the Rn. Thus, in He 1 hr after injection, TABLE 4. Reversibility of the inhibition ol MAO-A produced MAO-A inhibition by drugs was evidenced by an increase by some amphetamine derivatives as determined by repeated in 5-HT and/or a decrease in 5-HIAA levels. washing As was seen in the Rn, a reversal of the effects of some compounds was observed after 6 hr. Nevertheless, once Percent of inhibition of MAO-A again amitlamine and MT A produced an increase in 5-HT CompounJ 85.169.142.75.8:!:28.6:!:100washing9.1After :!::!:0.6*:!:8.1t 1.6t3.I:l: :!: 5.3*1.8t97.389.1washingBefore100 :!::!:3.1 l.3 and a concomitant 5-HIAA reduction which persisted 6 hr ETA§ALEPH-2HA2-Br-4,5-DMA4-MetOA4·EtOA Clorgyline[10-4MTAClor~yline§M] after their administration. Like amiflamine, ETA main• taineJ an increased 5-HT level until 6 hr after administra• tion, although 5-HIAA retumed to levels close to the control value at this time.

DISCUSSION

In this paper, we report the MAO inhibitory properties of a series of amphetamine derivativ~s with substituents such CruJe mih'chundn,! "t1:"rt:'tbltln wa", preincuhateJ for ID min wirh inhibitor, .lnJ as halogens. alkylthio and methoxyl groups at or around the th~n the rrerar;)fipn wa ..•'.\ilsheJ rhree times hy ccntrifu~atiun ómJ resusren~ion. para position. Our results show that some of these modifi• \tAl);A .lcri"iry I)f ,he rrepu:lrlon .mJ nf rhe control exrerimenrs \Vas measureJ hy HPLC-Ell lJ,m~ 1-HT .1S ,decri,·~ 'lJbstrar~_ E"ch vallJ~ ¡, rhe m~an = SD "f cations on the amphetamine structure can lead to an triplicates. increase in- the potency of the derivatives as MAO·A 't~ Si~niticmrly Jilferent irom "I",tore washin~" \"allJ~s (as J~r~rtnineJ by inhibitors, as compared with the parent compound. Al• SruJ~nt', '-tesr): • P < 2.21. t P < 0.0;, .mJ t l' <: 0.001. ~ Exrerim~nr, wirh ETA .mJ c1

e • TABLE 5. Effects of some Jerivatives on 5-HT and 5-HIAA endogenous levels in the raphe nuclei anJ hippocampus

-f0068.048.269.563.134.250.720.158.638.0130.0105.8103.J1OI.280.1645.698.537.0240.41157.0165.0127.3¡'tI131.0116.8131.393.9189.5180.5176.5128.0::187.0I hr90.112392.174.3 ::-:::-:::13.i*322 hr6hr ::I-:::17.0t -:::IO.3t 7.5*8.1*4.7*15.0*99.2:: 17.5*34.0::14.5*14.6*16.5*10.7* :: ::-:::27.8*hr 22.6*tl4.ot8.4*t27.0*1O.ft127.9-:::11.4*II.3*t ::365*19.1t 6.8*12.6*7.723.3*t31.0*133.0* S.ot6.6t35.5*27.0*22*31.3*t 2.3.4*t~3.I*t14.0*t18.7*36.7t Ii8.0*t432.6::260.3 5-HIAA:: 30 810:: 3655·HT :: 61.570.2 Saline4-ErOA-f-:-'lerOA4·MetOAMTAHAMTA-f-EtOASaline Hirrocampus Amitlamine Raphe nuclei CompounJ

:\ntJn,lls,~'l·n..· mjl:etcJ I.r. u'nh ,In l'quimniar Ju..;e llt the (IIrnr"unJs \ ~Q'; J.Lm~llj'kg). Each \";llu\: rerr~~nrs ,he mean ~ SD (N = 6-10 in each grour), exrresseJ a~ J rercent dt' ,he Lllnrr(ll ,!.!rnur rTt';lfl·J wirh .•,.Iioc. TIlt: ,-a1tu:s llf rhe (1-ntrnl ~rt'''r ,lre t:"xrr~,,<,J in ng/}!. n( tissue.

- "':'!!!nlficmr J.,feH"nU' (1) ....- : .•....•..;. ~rll\lt"nr·'" '-te"") ¡,f rTt.';lh:J ~n'tJr fflun the .."linc ~r\IUr· '-"'" t ::-=:,t:nltk:lt1t Jdft"renCl" (1''--' ...'...."i. ~ruJen(·'" r-ft:..•t) t\t th~ A·hr ~r(\lIr frllm ,he lphr gn\up.

forms nf MAO, they presented a dear ~e1ecti\"ity t(lr extent in the cases of MT A vs ALEPH-I anJ ETA vs MAO-A. These resulrs suggest that MAO-B may have ALEPH-2. more ,;rringent structural reljuirements, presumahly ar the The introJuction of a halngen atom at posltlon ·2 active site, ro accept srructures Jiffering from those nf its increaseJ rhe porency as an MAO-A inhibiror (2Br-4,5• physinlogical substr:1res. In our case, rhis precluJeJ an DMA vs 3,4-DMA; 2CI-4,5-MDA vs MDA). This last analysis nf the stmctural reljuiremenrs necessary for a finJing is in ;lgreement wirh rhe results obtained by Aorvall compound to exhibit ~L'\O-B inhibirory properties. et al. for the amitlamine series (ll, 12, 18). Regarding the potency ot the Jerivatives as tvIAO-A On the llther hanJ, some electron donor character of the inhibirors, the most potent compounds (ET A, 4-EtOA, substituents at the para position seems to be necessary to MT A, 4-MetOA, and ITA) were obrained when only rhe inhibit the en:yme, because strongly electron accepting para posirion ,)f the aromaric ring was substitureJ. The grollps like rrifllloromerhyl or nitro completely abolished addition of bulky substiruents aJjacent to this position the activity of the compounds (ALEPH-l vs DON, DOM decreased potency. Thus, rhe presence of a second me• vs DOTFM). Nevertheless, the electronic properties of the rhoxyl group at the meta position proJuced a compound para substituents seemeJ to have an optimum that was 60-fold less potent than the monosubstituted Jerivative reached in the sulfur-containing compounds (MTA vs (see 4-MetOA vs 3,4-DMA). Moreover, two substituents 4-MetOA, ET A vs 4-EtOA, ALEPH-2 vs TMA-2). occupying both meta positions completely abolisheJ rhe RegarJing the si:e of the para substituent, our results activity of the compound (4-MetOA vs nvtA). Substitu• show that ethoxy- or ethylthio-substituted compounJs were ents locali:ed at more distant positions from the para more effective than methoxyl- or methylthio-substituted carbon atom had less intluence nn the activity of MAO-A Jerivatives (4-EtOA vs 4-MetOA, ETA vs MTA, inhibirors (4-MetOA vs 2,4-DMA), whereas compounds ALEPH-2 vs ALEPH-l). This indicates that the enzyme with groups at positions other than para presented less may better accommodate compounJs with larger substitu• activity than the parent molecule (amphetamine vs 2,5• ents at the para position. However, when the alkyl chain DMA). lnterestingly, the presence of tW() methoxyl groups containeJ more than two carbon atoms, a decrease in at positions 2 and 5 induced a greater Jecrease in the potency was seen (ET A vs IT A). activity than might be expected from the analysis of the No changes were observed in MAO-A and -B inhibition influence of each substituent separately. For insrance, after different preincubation times (O, 30, and 60 min), 3,4-DMA was approximately 60-fold less potent than 4• inJicating that the blockade of both forms of MAO was not MetOA, whereas 2,4-DMA haJ a potency similar to that of time dependent and suggesting that the compounJs were the monosubstituted derivative. However, when three me• reversible MAOIs. Reversibility studies confirmed this as• thoxyl groups were placed at position Z, 4, and 5 in the sumption. showing a significant recovery of MAO-A activ• same molecule, rhe inhibitory activity disappeared com• ity after repeated washing of the preparation. These results plerely (TMA-2). Similar rrends were nbserveJ in rhe case indicate that the in vitro Mf\O inhibition by these amphet• of PCA vs IX}l or DOB (pe.-\. has approximately the same amine derivatives is reversible and essentially different from potency as an MAO-A inhibitor as the para brominated that produced by suicide inhibitors like clorgyline or l• and indinateJ Jerivatives (14, 34, 35)), anJ ro a lesser Jeprenyl. In aJdition, these Jata also show that the inhi- Su"stituted Amrhetamines as MAO Inhi"itors 1361 bition was not due to substrate eompetltlon, sinee the subtypes (38 -40). Our results show that aIthough some of inhibitors were nat metabolized by the enzyme, but rather these compounds have strong MAOI properties, this activ• resulted from a real blockaJe of enzyme aetivity. ity should not playa dominant role in the proouction of The ex t [1'0 stuJies essentially eonfirmed the in vi[TO psyehedelic effects. since it does not eorrelate with the ohservations. Some of the most potent MAO-A inhibitors hallucinogenic poteney reported in humans (as is the ease as evaluateJ in 1 itTO inJuced significant and reversible for their 5-HTz:\J2C affinity) [25, 38, 41J. Nevertheless, increases and Jecreases of 5-HT and 5-HIAA levels, interactions with the enzyme should be considered when respectively. These effects were observed in both seroton• subjeetive Jifferences in the reported effects of this class of ergic eell hxlies and nerve terminal enriehed regions, compounds in humans are analyzed. prohably ret1ecting the MAO-A inhibition. Regarding the On the other hand, compounJs like MDA, MDMA, and duration oi the tóffeets, most of the eompounds were PCA are recogni:eJ serotonergic neurotoxins [23. 42-44J, "hnrt-actin!!. elicirmg llOly minm nr no changes in brain whereas ~1TA, which was reported to have effects similar to inJollevels ,.;hr aiter their aJministration. The long-Iasting those ll( MDMA in a drug Jiscrimination paradigm and ef(ects of amitbmme (Table 5) have heen relateJ to its sume commonaliries in the mechanism of action, Le. neuron-selec:ive Jction (201. This CllmpounJ anJ/m its serotonin release properties [23,24,45), did not elicit 5-HT N -demethyhteJ metabolite ean he transported into the neurotoxicity. It may be possible that the differenees "erotonergic neurnns via the 5-HT uptake earrier and is, between MTA and MDMA are relateJ to their Jifferent therefore, m.'re ;1Ctivewithin these neurans compareJ with ,lctivities as MAO-A inhibitors. In this case, the enzyme orher neunn, ;lnJ c:ells. Snme other analogues. e.g. ~1TA inhibition proJuceJ by MT A and the eoncomitanr increase which also ;-roJuct'J changes in 5-HT anJ 5-HIAA 6 hr nf the neurotramminer in the surrounJings of the nerve aiter its inie~:i(1I1.:TIayhave this neuro[1-sclecti\"ity, .IS wd\. terminal might rre\-ent the initial rapiJ Jecrease of 5-HT, The ~IAl'[ rrorerties of rhe compounJs e\'aluateJ here which ,eems to "e a neeessary conJition to produce the ;lfe basicalh" in agreement with previouslv reporteJ Jata. long-term neuroroxicity induceJ by compounJs ,;uch ,as Thlls. the 11'e' values JescribeJ here (m amrhetamine /10), MDA or MDMA /46). This woulJ not be true in the case amitlamine. FL\ 527 [11/, PCA (141. ;mJ 4-MetOA (16J of PCA, \vhich has considerable MAO-A inhibitory activ• are similar t,) tho-e pllhlisheJ by Jifferenr authms using ity. but there is some eviJence indicating that PCA and Jiverse meth,xlolo!!ies. :\JJitionally, the strllcture-activity MDMA "erotonergic neurotoxieity is initiated by different relationship \lhaineJ showed similar tenJencies compared mechanisms (46-48). with previous rer"rtS in terms of seIectivity (12. 191, the As rerorted, ~ITA is a potent and selective non• importance "f the substitllent at the para position of the neurotoxie serotonin releasing anJ uptake bIocking agent aromatic rin~ ()i the amrhetamine molecule [11-14. 16. (23. 4jJ. Our results indicate that this compound is aIso a 19I,thenature'lfthesubstituent[ll, 14, 16. 17.20.36. 37J, potent anJ seIecti\-e MAO-A inhibitor. Therefore, MT A and the type ()( inreraction with the en:yme (10. 12, 16-18, combines in the same molecuIe the two characteristic 20. 361. features of the most reeently developed antidepressants, In summa~', our results show that: molecules with an making this Jerivative an interesting candidate for further amphetamine-like sttueture and different substitutions on evaluatian in this sense. * the aromatic ring are potentially MAO-A inhibitors; sub• FinaIly. it has been been reported reeently that another stituents at Jifferenr positions of the aroma tic ring modify compounJ, ALEPH-2, exhibits an anxioIytic-like profile in rhe potenC\" but hJ.ve linle influence on the seIectivity; rodents. as evaluareJ in Jifferent behavioral moJeIs, anJ 'iubstituents .It the 4 (para) position such as amino, alkoxyl. possesse~ an unusual pharmaeologicaI profile (49, j()). Its halngens. nr llkvlthio produce a significanr increase in the MA.o-A inhibitory properties JemonstrateJ in this work activity; the ¡'Jara-substituenr must be an electron Jonor; shouId be regarded in the framework af the analysis of its bulky group next w the para substituent leaJ to a decrease putative anxiolytie action, eonsidering the acknowleJged in the acti\"lty; suhtituents lacated at more Jistant posi• superposition that exists between the activities of anxiolyt• tions on the aroma[Íc ring have less influence anJ, even ics and antidepressants (51]. when the sut-stituent is a halogen (CI, Br). an increase in the activity 1'1- rhe LompounJ is obtained; for the evaIuated We woulJ like lO tMnk the Neurochemis!ry Division staff (IlBCE) lor series. the iJeal Iir11philic character and si:e of the para• thcir invalUllble help and technicalsupport. This work was supported in substituent \\ere found for the alkylthio Jerivatives; and the pare Iry CONICYT (Cruguay) Grants 94/038 and 96/2005 (Fondo MAO-A inhll-ition induced by the derivatives stuJied was Ckmente Estable), and LIdn American :\etwOrk on Natural Bioactive reversible in all ca~es. Compounás (LANBlO) , PEDEClBA (Uruguay), and FOND• SeveraI aJJitional considerations should be made about EC'r:T (Chile) Granr 89/915_ the compounds that were studied. Some nf the drugs evaluated. such as 4-MetOA, DOB, DOI, DOM, ALEPH-l, * Marona-l.:wicka D ;m.1 Nichols DE, Th.: .:ffect of selective sewtonin ar ALEPH-2. have l-een described as having hallucinogenic releasinR agenrs in the chronic milJ stress m,,JeI uf Jepression in rars. In: properties in humans (25). Classically, these effects have Z6rh Annual Meeting, S,.'Ciety for Neutoscience Ansrrdcts, Vol. 22, Pan 1, been associateJ with the activation of 5-HT lA/ze receptor Washington, OC, 16-1\ Novernher 1996, r. \81. 1368 M. C. Scona et al.

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