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PharmacologyB/ochem/,nry& Behav/m-,VoL 38, pp. 505-512. o Pergamon Pressplc, 1991. Printed in the U.S]_.

3,4-Methylenedioxyamphetamine (MDA) Analogues Exhibit Differential Effects on Synaptosomal Release of 3H- and 3H-5-Hydroxytryptamine

DENNIS J. McKENNA, *l X.-M. GUAN* AND A. T. SHULGINt

*Department of Neurology & Neurological Sciences, Stanford University Medical Center, Stanford, CA 94305 \ p1483 Shulgin Road, Lafayette, CA 94549 x

Received 16 August 1990

McKENNA, D. J., X.-M. GUAN AND A. T. SHULGIN. 3,4-Methylenedioxyamphetamine (MDA) analogues exhibit differential effects on synaptasomal release of ZH-dopamine and SH-$-hydroxytryptamine. PHARMACOL BIOCHEM BEHAV 38(3) 505-512, 1991.--The effect of various analogues of the neurotoxic derivative, MDA (3,4-methylenedioxyamphetamine) on car- rier-mediated, calcium-independent release of ZH-5-HT and 3H-DA from rat brain synaptosomes was investigated. Both enantiomers of the neurotoxic analogues MDA and MDMA (3,4-methylenedioxymethamphetamine) induce synaptosomal release of 3H-5-HT and 3H-DA in vitro. The relea._ of 3H-5-HT induced by MDMA is partially blocked by 10 -6 M . The (+) enantiomers of both MDA and MDMA are mote potent than the ( - ) enantiomers as releasers of both _H-5-HT and 3H-DA. Eleven analogues, dif- feting from MDA with respect to the nature and number of ring and/or side chain substituents, also show some activity in the re- lease exper/rnents, and are more potent as releasers of 3H-5-HT than of 3H-DA. The amphetamine derivatives ( n-)fenfluram/ne, (-+)norfcnfluramine, (±)bIDE, (-*)PCA, and d- are all potent releasers of 3H-5-HT and show varying degrees of activity as 3H-DA releasers. The DOM does not cause significant release of either 3H-monoamine. Possible long-term serotoncrgic neurotoxicity was assessed by quantifying the density of 5-HT uptake sites in rats treated with multiple doses of se- lected analogues using 3H-paroxefine to label 5-I-11 uptake sites. In the neurotoxicity study of the compounds investigated, only .,o ( + )MDA caused a significant loss of 5-HT uptake sites in comparison to saline-treated controls. These results are discussed in terms /,2, f o _of the apparent structure-activity properties affecting 3H-monoaminc release and their possible relevance to neurotoxicity in this se- (_.)t+l _ ties of MDA congeners.

Methylenedioxyamphetamines Dopamine Synaptosomes Neurotoxicity Structure-activity relationships

MDA (3,4-methylenedioxyamphetamiae) and its N-methyl arm- its metabolites (9,44). These agents induce the presynaptic re- logue, MDMA (3,4-methylenedioxymethamphetamine; "Ecsta- lease of DA and serotonin (5-HT) both in vitro (12, 32, 42) and sy") are riag-substimted phenylisopmpylamine derivatives which ia vivo (32, 33, 46). The long-term semtonergic neurotoxicity bear structural and pharmacological similarities to both amphet- can be significantly attenuated either by pretreatment with 5-HT amines and such as (15,38). Both behav- uptake inhibitors such as citalopram (32), prior depletion of en- ioural (7, 18, 19) and pharmacological data indicate that MDMA dogenous monoamines with reserpine, blockade of DA synthesis a_l some of its derivatives may represent a new class of psycho- with -methylparatyrosine (AMT), or prior lesioning of ni- pharmacologic agents whose mechanisms of action are distinct grostriatal dopamine neurons with 6-hydroxydopamine (44). from those of either classical hallucinogens or (15,17). Structure-activity data, although limited, are also consistent MDA, MDMA, and some of their structural congeners arc selec- with a role of DA in mediating the semtonergic neurotoxicity. tively neurotoxic to sewtonergic nerve terminals in rats and pri- For example, the enantiomers of MDA or MDMA are appwxi- mates (3, 14, 20, 24, 28, 31). mately equipotent as releasers of 3H-5-HT from superfused brain Although the exact mechanisms are incompletely understood, slices, but the (+) enantiomers are more potent releasers of 3H- several investigators have proposed that the neurotoxicity induced DA than the ( - ) enanfiomers (12) and are also significantly more by MDMA and its congeners is mediated by dopamine (DA) or potent in producing the long-term 5-HT depletion indicative of

'Requests for reprints should be addressed to Dennis $. McKerma at his present address: Shaman Pharmaceuticals, Inc., 887 Industrial Road, Suite G, SanCarlos, CA 94070.

5O5 506 McKENNA, GUAN AND SHULGIN MD

H o .., o .., ,,,....._ 200 ['.--3 BASAL

(+) MOA (-) MDA (.,,)MDE -LL_ 150 * *

C_ OC_

', "o-_ ( :Ei 50. J

(*) DOM (-) DOn I.,IMOMA

oc_ /---o

o'.__'l_'_'"y.. ° "", withca++ Co++n° 57° c 4.0c I Mu_ UUD^-2 MMOA-a. FIG. 2. Effect of calcium and temperature on the potassium-induced re- lease of 3H-5-HT from rat synaptosomes. *p<0.05 vs. basal; #p<0.05 - (oZ%< oMw,^-2 m,_ Mm_ macognosy, Purdue University, for the generous gift of (-+)noffenfluramine. Other analogues used in the study were syn-

"'o,t4_ a ( rural characterization of several of the analogues used in this study ct, have been described elsewhere (35, 36, 39, 41) or will be pul> FE4fk_ ,-r. MMO^-: lished separately. All other chemicals used were of the highest quality commercially available. FIG. 1. St_ctures of amphetaminederivatives andMDA (3,4-methylene- dioxyamphetamine)analogues investigated in the present study. Synaptosomal Release Preparations mcub No. 3 In order to measure the in vitro synaptosomal release of 3H- stake neurotoxicity (29,33). Moreover, N-ethylation of MDA to give 5-HT and 3H-DA, we used a modification of a method described 5 nfl 1 the analogue MDE (3,4-methylenedioxyethylamphetamine) re- by Bender and co-workers (4). Male Sprague-Dawley rats (200- tion f suits in a compound which is less neurotoxic and less potent as a 225 g; Simousen Labs, Gilroy, CA) were lightly anaesthetized lation releaser of aH-DA in vitro than MDA or MDMA, but is approx-3 with and decapitated. The brains were removed and expre, imately equipotent with these compounds as a releaser of H- cerebral cortex and hippocampus pooled and homogenized in 10 releas 5-HT (12, 25, 29, 43). volumes of ice-cold 0.32 M sucrose with a Wheaton motor-driven trois ( Previous structure/activity studies of MDMA and its conge- Teflon-glass homogenizer (speed 6). The cortical-hippocampal 100 -_ hers have focused primarily on the differential of the tissue was used for the 3H-5-HT release experiments, while pooled suits enantiomers as neurotoxins or as 3H-monoamine releasers, or on mesolimbic and striatal tissue, prepared in an identical manner, or me the influence of the side-chain nitrogen or alpha-carbon substitu- was used for the 3H-DA release experiments. The homogenates Ch ent on these parameters (12,16). In the present study, we investi- were centrifuged at 3000 x g for 10 rain and the supernatant, con- ducte( gated the effects of eleven ring-substituted analogues of MDA mining the crude synaptosomal fraction, was gently decanted and the sy (Fig. 1) on synaptosomal release of 3H-5-HT and 3H-DA in vitro diluted 1:1 with Krebs-Hepes buffer [pH 7.4, composition (mM): systen and evaluated selected derivatives for neurotoxicity in the rat. NaCI, 117; KC1, 4.8; CaCI 2, 2.5; MgCI2, 2.5; Hepes, 25; pargy- , potass The results are compared with the effects of MDA and other am- line, 0.010]. The supematant was recentrifuged at 10,000 x g for additi, phetamine derivatives, some of which are known to exhibit sero- 20 rain, and the final pellet was resuspended in 80 volumes of These tonergic neurotoxicity in animals. Krebs-Hepes buffer. The synaptosomal preparations were main- from mined on ice until used. The synaptosomal preparations (800 gl) preser Mm'ROD were aliquoted into test tubes containing either 3H-5-HT (specif- Re ic activity: 12.5 Ci/mmol, NEN; final concentration, approx. 9 measu and Chemicals nM) or 3H-DA (specific activity: 51.9 Ci/mmol, NEN; final con- preset Drugs used in the in vitro release studies were obtained from centration, approx. 2.3 nM) and incubated at 37°C for 15 min. in bul the National Institute on Abuse [(+ )DOM, (-)DOM, Specific uptake was defined as the total minus blank taken in the were i (-)MDA, (+)MDA,(+)MDMA,(-)MDMA,(+_)MDEI, Sigma presence of 10-4 M fluoxetine in the case of 3H-5-HT, or I0 -4 counte Chemical Co. (St. Louis, MO) [d-methamphetamine, M in the case of aH-DA. After 15 minutes incubation, the po (+-), (+-)para-chloroamphetamine], or Research either buffer or varying concentrations of releasing drugs (0.01- lng ca Biochemicals, Inc. (fluoxetine, bupropion). We are indebted to 10 IxM) or KCI (final concentration: 50 mM) were added and the elate ( Dr. D. E. Nichols, Department of Medicinal Chemistry and Phar- incubation at 37°C continued for an additional 15 min. Following , To MDA ANALOGUES AND SYNAPTOSOMAL [aH]-MONOAMINE RELEASE 507

II (+)MDMA $ I-)MDMA ,_I'"'(+)MDMA * 1 IsM fluo_ettre, J. I*)MDMA + 1 _.M fkJoxetir_

200 'A

ao I I I I 0.01 0.1 1 1 0 CO_ENmA'rI04 bd4

200 - ; _+)MOUA 200- 'C c. _+)MOA

_180 - . _180 - '_'""(-)MOMA /. // _160- 16o- · l-_uo^ / iV' / _,

\ 140- _- _-12o- - ' ; 12o. ": 100" "100 ' 80 I I I I 80 I I I I 0.01 0.1 1 10 0.01 0.1 I 10 _l'lON _dd] CONCEN_AI')ON _M]

FIG. 3. (A) Synaptosomal mlea_ of 3H-5-HTinduced by MDMA enantiomers and their inhibition by 10 -6 M fluoxetine. (B) Synaptosomal release of 3H-5-HT induced by the enantiomers of MDA and DOM. (C) Synaptosomalrelease of 3H-DAinduced by (+ ) and ( - ) enantiomcrs of MDMA, MDA and DOM.

incubation, the assays were terminated by rapid fdtration through controls were incubated at 37°(2 prior to and after the addition of No. 32 glass fiber fdters (Schleicher & Schuell, Keene, NH) pre- KC1; a duplicate set of tubes was incubated at 37°(2 for 15 min soaked in Krebs-Hepes buffer. The rd[ers were rinsed twice with in the presence of 3H-5-HT to load the synaptosomes, then cooled

5 mi Krebs-Hepes buffer and counted in 2 mi Biosafe TM scintilla- on ice to 4°(2 prior to the addition of KCI. tion fluid (Research Products Inc.) in a Packard 1900CA scintil- The effect of MDA and the other analogues on synaptosomal lation counter at 41% efficiency. Effect of releasing agents was release was investigated by loading the synaptosomes with either expressed as a percent of basal release, as follows: % of basal aH-5-HT or 3H-DA for 15 min at 37°(2, then adding varying con- release = (100 - % retained) + 100. By this calculation, con- centrations of releasing drug (0.01-10 IxM) and incubating for a trols (basal release) equal 100%, and assays showing release equal further 15 min. The effect of fluoxetine, a selective 5-HT uptake 100 + % released. Assays were performed in triplicate, and re- blocker, on synaptosomal release induced by MDMA was inves- salts are reported as the means- standard error of the mean, of 3 tigated by first loading the synaptosomes with 3H-5-HT for 15 or more independent experiments, min at 37°(2 as described above; following synaptosomal loading, Characterization of in vitro synaptosomal release. We con- fluoxetine (10 -s M, final concentration) was added and the prep- ducted initial experiments aimed at characterizing the nature of aration incubated for an additional 5 min; then various concentra- the synaptosomal release of tritiated monoamines observed in our 'tions (0.01-10 IxM) of (+)MDMA or ( - )MDMA were added system; specifically, we investigated the effect of calcium and and the incubation was continued for an additional 10 minutes potassium, temperature, and 5-HT uptake blockers on release, in before filtering. Preparations were then filtered and counted as addition to examining the effects of MDA and various analogues, described above. These experiments were conducted using synaptosomes prepared from pooled cortical and hippocampal tissues, incubated in the Neurotoxicity Studies presence of 3H-5-HT. Release of 3H-5-HT from synaptosomes by potassium was Animals and drug administration. Neurotoxicity studies Uti- measuredby incubating synaptosomes for 15 rain at 37°C in the lized male Sprague-Dawley rats (300-325 g; Simonsen Labs, presenceof 3H-5-HT, then adding 1.0 nd 105 mM KCI dissolved Gilroy, CA). Animals were administered repeated doses IP (2 x 5 in buffer, to give a final KCI concentration of 55 mM. Tubes rog/kg/day, for 4 consecutive days) of various MDA analogues, in the were incubated an additional 15 min at 37°(2, then filtered and dissolved in saline. Analogues screened for neurotoxicity were r 10-4 counted as described above. To test for calcium independence of selected on the basis of structural features and their action as aH- ha[ion, the potassium-induced release, the experiment was conducted us- 5-HT or 3H-DA releasers in vitro. Control animals were injected (0.01- ing calcium-free buffer containing 2.5 mM ethyleneglycoltetraac- with saline (1.0 nd/kg). As a positive control, another group of and the etate (EGTA), a calcium chelator, animals received MDA, but a slightly lower dose was used (2 x 3 [lowing To test the effect of temperature on potassium-induced release, mg/kg/day, 4 consecutive days) due to a high rate of mortality ?

508 McKENNA, GUAN AND SHULGIN M

_t

TABLF., I _. lenfluramine EFFECTOFMDAANDMDAANALOGUESONREt.EASEOF3H-S-HTAND --,I,--melhamphetamlne EFI 3H-DAFROMRATBRAINSYNAPTOSOME$* · PGA 20O Drug (1 gM) _H-5-HT _H-DA A & Iv_ Dru 8 0 _1 t Sail (+)MDA 190 ± 4t 183 ± 2tS _u (-+) (__-)PCA 190 ± It 183 ± It _ 160 (+)Methamphetamine 165 4- 4t 180 + 6t _ (-) (+)MDMA 185 ±_ 4t 155 ±_ 9t:[: _ 140 ' (_)(±) (-)MDA 180 ± 4t 133 ± 2tS _

(±(±)N)Fenorfeflurnflamurainemine 180186 ± IItt 113120 ± 3t5t _ 120 *J (±)EDMA 175 ± It 120 ± 7t (-)MDMA 170 ± 8t 115 ± ItS 100 sim5 of 3 (±)MM(-)DOMDA 110072 ± 42t 111135 ±- 6 80 I I . . I [ [ (Fig (-+)MEDA 145 -+ 23? 113 ± 5t 0.01 0.1 I 10 1 the ((±+))MMDDOMA-3B 100185 ± 04t 112112 -+ 62t _CENTRATI(_0_M) I rele; (±)MDE 170 ± It 110 + 2 deri (±)MMDA-2 103 ± 3 106 ± 6 chlo (±)HMDMA 117 ± 1? 105 ± 5 beer (±)G-5 100 ± 2 100 ± 16 _fenfluramlne ofs_ (±)4-T-MMDA-2 115 ± 4t 92 ± 4t ---IB--methamphetamine All: (±)DMMDA-2 130 _-2 7t 90 ± 6 I PCA pote 200 - A _ d-mi *Values are percent of basal 3H-monoaminereleased at a drug concen- B to tt tration of I _taM,± standarderror of the mean (see the Method section), cies Results are given in rank orderof potency for 3H-DArelease (column 3]. 18 0 and tp

Effects of Experimental Conditions on In Vitro$ynaptosomal synaptosomal release of 3H-5-HT are shown in Fig. 3A and B; Effec Release the effects of these enantiomers on synaptosomal release of aH- Bindi In our experimental system, the synaptosomal release induced DA are shown in Fig. 3C. The ( + ) enantiomers of MDA and by 55 mM KCI was Ca ++-independent (Fig. 2). The potassium- MDMA are more potent releasers of both tritiated monoamines 'E induced release was temperature-dependent; the percent of con- than the corresponding (-) enantiomers (Fig. 3). The enanfi- satun trois induced by 55 mM KCI at 37°C was 145%, while at 4°C, omers of MDMA and MDA are approximately equipotent releas- with the amount of release in the presence of potassium was equiva- ers of aH-5-HT at a concentration of 1 IxM (Fig. 3A and B, Table 3B, c lent to basal levels. The basal levels at the lower temperature 1) but the (+) enantiomers of both compounds are significantly analy were significantly less than basal levels at 37°C (Fig. 2). These more potent releasers of aH-DA than the corresponding (-) affini results show that basal release, as well as potassium-stimulated enantiomers (Fig. 3C, Table 1). These results are consistent with meas; release, is temperature-dependent, previously reported findings (12, 28, 32). The release of aH-5- ences HT by both enantiomers of MDMA was significantly reduced in line c Effects of MDA and MDA Analogues on In Vitro Synaptosomal the presence of 10-6 M fiuoxetine (Fig. 3A). These results pro- mg/kj vided evidence that the release being measured was at least par- spect Release dally carrier-mediated. Neither enantiomer of DOM induced a with r The effects of the enantiomers of MDMA, MDA and DOM on significant release of aH-DA at any concentration tested (Fig. 3C); (Tabl MDA ANALOGUES AND SYNAPTOSOMAL [aH]-MONOAMINE RELEASE 509

TABLE 2 _U_A EFFECOTFMDA ANDMDAA.NAL_UESONsH-PAROXETINBEINDING ,,-I--MMOA3

200 'A ,-'MI-'--eDM-.uuMDoA2saa

Drug Kd (nM) B_,_ (pmol/g tissue) 18 0 - ®-E[_ ,_ -®-HUOUA Saline 0.23 4-4. o0.0.3 o,, 38,o4-2,.1., 160 £./' 140 ../ ,,=/ __[ (+_)MMDA 0.26 _4- 0.03 40 -_+3.4 _ -_-,o.s j _ (+_)DMMDA 0.24+0.05 33+2.6 _ ._J, _ 'el (+_)EDMA 0.23 _ 0.04 32 · 4.1 _ 120 ·p

All five of the neurotoxic derivatives showed a similar degree of '-_l,'II'--MMOAa42 0A potency as releasers of 3H-5-HT (Fig. 4A, Table 1) with ,; MMOA..a8 d-methamphetamine showing the weakest activity. With respect 160 ..-.-o_o_ e to the induction of aH-DA release, however, differential poten- B -·-rr.,a_ andd-methamphetamine were approximately equipotent releasers 14 0 - · -i-i1_1_ cieswere observed (Fig. 4B ). ( ± )para-Chloroamphetamine (PCA) 150 -. -_VO. A ,, ,,;_ { of aH-DA, while (±)fenfluramine and (±)MDE were approxi- . I11'4-T-MMOA2 ' / _1[ muteweakerlyequipotactivityentcomparedto each othtoer,metbhutampbothhetaminderivative eands showPCA.ed muchHow- _130120 - N-a-S i///, _/// s release, relative to basal controls, at concentrations of I p.M and _ 110 -_ -- _ .... 10 _Vl. (±)Norfenfluramine was more potent than ( ±)fenflur- < amine at inducing 3H-DA release at I and 10 p.M, but the differ- S 100 -- -- _, :, -" '" enceever' wthasesesignificantderivativesonlstilly at inducI0 pe.M.d significant levels °f 3H-DA ;If 90 _ _Sam_ Of the nine additional MDA analogues tested, nearly all were 80 ] [ [ more active as releasers of 3H-5-HT than of aH-DA (Fig. 5A and B; Table 1). The most active of the 3H-5-HT releasing drugs were 0.1 1 10 (-+)MMDA-3B, (±)MMDA, and (±)EDMA; the others all co_ncm[_Mi showed significantly less potency at a concentration of I IxM (Table 1). All of the analogues tested were much less potent as FIG. 5. (A) Synaptosomal release of 3H-5-HTinduced by slxucturalaha- inducersof aH-DA release, but the three compounds named above logues of MDA and MDMA. (B) Synaptosomal release of 3H-DA in- showedthe most activity as aH-DA releasers as well, even though duced by structural analoguesof MDA and MDMA. All compounds listed this activity was markedly reduced in comparison to their effect are racemic. on 3H-5-HT release (Fig. 5, Table 1).

Effectof Selected Analogues on Density of _H-Paroxetine ( ± )DMMDA-2 had somewhat reduced B_, values compared to BindingSites controls, but this difference did not reach statistical significance.

The Kd and Bmax values obtained from Scatchard analyses of DISCUSSION sat_"afion-binding assays with 3H-paroxetine in animals treated with (_+)MDA or various analogues [( 4- )EDMA, ( ± )MMDA- The major finding of the present study is that those compounds 3B, or (±)DMMDA-2] were compared with saline controls and which other investigators have shown are the most potent seroto- analyzed by one-way ANOVA. The Kd provides a measure of the neqgic neurotoxins in vivo show the greatest potency as releasers affinity of the labelled for its receptor, while the Bm_ of H-DA rather than of H-5-HT. This can be seen in Table 1, measures the density of the uptake sites. No significant differ- where the compounds investigated are ranked according to their ences in Ka values were found between treated animals and sa- potency as in vitro aH-DA releasers. For example, the S(+ ) linecontrols. The Bm,,, value of rats treated with ( _ )MDA (2 x 3 enantiomers of MDA and MDMA are both more potent neurotox- nag/kg,4 consecutive days) was significantly reduced with re- ins than the R(-) enantiomers (12) and this relative potency is spect to saline controls, but the B,n_ values of animals treated reflected by their relative ranking as aH-DA releasers. Similarly, with the analogues were not significantly different than controls MDE displays a relatively low potency for inducing aH-DA re- (Table 2). Animals treated with the analogues (±)EDMA and lease, and requires an approximately 4-fold higher dose to induce J ,3

510 McKENNA, GUAN AND SHULGIN ME

? an equivalent long-term reduction in 5-HT, comparedto MDMA dimethoxy-3,4--phenylisopropylamin(3e)5,36, 39), (25). However, the compounds (± )fenfluramine and its N-de- Structurally, thesecompoundsare intermediatesbetweentheDOM- ethyl congener, (-)norfenfluramine, provide striking exceptions like compounds and the MDA-like compounds, in that they in- , 1. to this apparent correlation. Our study shows thatfenfluramine is corporate the methylenedioxy ring, together with one or two less potent than ( + )MDMA as a 3H-DAreleaser, but according additional methoxy ring substituents(cf. Fig. 1). The human psy- 2. to one study (45) fenfluramine is approximately 3 times more chopharmacologyof these compounds is also apparently interme- toxic than MDMA. In an effort to explain this discrepancy, we diate between that of the "entactogenlc" MDMA-like drugs, and t alsotested ( _ )norfenfluramine, the primarymetaboliteof fenflur- the "hallucinogenic" DOM-like drugs (40). There is little or no amine (6). Although our results indicate that (± )norfenfluramine information available on their behavioral effects in the drug dis- 3. is more potent than (-*-)fenfluramineat inducing aH-DA release crimination assay. The present study provides the furstinforma- , from synaptosomes, this difference is significantonly at the high- tion on the possible neurotoxicity and synaptosomal releasing est concentration used (Fig. 4B, Table 1). The fact that fenflur- ability of compounds in this intermediateclass. While the results amine and its metabolite, norfenfluramine, areneurotoxicyet lack from the in vivo neurotoxicity studies presentedhere must be re- potency as aH-DA releasers is evidence that the serotonergicneu- garded as preliminary, they indicate that the presence of one or s 4. rotoxicity of these amphetamine derivatives may involve mecha- two methoxy substituents on the ring, in additionto the methyl- nisms unrelated to (or in addition to) endogenousDA release. An enedioxy substituent, abolishesthe serotonergicneurotoxicityand alternativepossibility that has yetto receiveextensive experimen- dramatically attenuates the capacity of these agents to release 5. tal investigation is that the endogenous releaseof aH-DA from synaptosomes (Table 2, Fig. 5B); these derivatives (NE), rather than DA, is the primary mechanism mediating the still retain considerable potency as releasers of 3H-5-HT, how- neurotoxicity of these agents, ff NE or its metabolites mediates ever (Table 1, Fig. 5A). If the synaptosomalreleaseof aH-DA the neurotoxicity of these drugs, this would also explain the ap- were a reliable index of serotonergicneurotoxicity, then the ana- ! parent discrepancy between the anatomical distribution of the se- logues in this intermediate class would be expectedto lack neu- 6. rotonergic lesions (cortex, hippocampus) and the relatively low rotoxicity;however, someof theseagents displaysimilarpotencies ' 7. abundance of DA-containing neurons in these regions, to(±)fenfiuramine, (_ )noffenfluramine,and(± )MDEas 3H-DA A secondary finding of this study is that relatively narrow releasers and these compounds are known to displayserotonergic structural determinants can result in large differential effects neurotoxicity. Although none of the analogues tested showed ev- on the ability of MDA and its congeners to release 3H-5-HT idence of neurotoxicity at the comparatively low doses used in 8. and 3H-DA from synaptosomes. Centrally active phenyl- thispreliminary study, it is possible that at higher doses they may isopropylaminescan be separatedinto two classes, differing with exhibit activity as serotonergic neurotoxins. respect to their probable mechanisms of action and behavioral The results found with some of the analogueswhich bear an 9. andpsychological effects. The hallucinogenDOM, along with its even closer structural relationship to MDMA also deserve com- halogenatedcongeners DOB (2,5-dimethoxy-4-bmmo-phenyliso- merit. The additionof a single methylene to the ring alkylidene- propylamine) and DOI (2,5-dimethoxy-4-iodo.phenylisopropyl- dioxy substituent to give the analogue (±)EDMA (3,4- I0. amine)represent a class of hallucinogenicphenylisopropylamines ethylenedioxymethamphetamine,Fig. 1) results in a compound which exhibit high affinity for 5-HT2receptors (10,13) and act at which lacksneurotoxicityand shows markedly attenuated3H-DA these postsynaptic sites primarily as agonistsor partial releasing ability, but is comparable to (-)MDMA as a releaser 11. (22,27). Compounds in this class also generalizeto LSD or other of 3H-5-HT. Interestingly, this compound lacks detectable psy- hallucinogens in the drug discrimination test (1) and exhibit a chotropic effects in man at doses up to 200 mg orally. Addition human psychopharmacology typical of LSD-like hallucinogens of a ring-methoxygroup to this compoundto give the compound 12. (37). Although investigationsare limited, compounds in thisclass MEDA (Fig, 1) attenuatesits potency asa 3H-5-HTreleaser, but are apparentlynot serotonergicneurotoxins. One study used ira- does not further diminish its already low potency as a aH-DA re- munocytochemistry to compare the neurotoxicityof PCA (para- lesser. Extension of the side-chain of MDMA by one carbon to 13. chloroamphetamine) and MDA with the phenylisopropylamine give the homologue HMDMA (41), or replacementof one meth- hallucinogen, DOM (2,5-dimethoxy-4-methyl-phenylisopropyl- ylenedioxyoxygen withsulfurtogive thecompound4-T-MMDA-2 , amine) and found no evidence for serotonergicneurodegeneration (Fig. 1) essentially abolishes the aH-5-HT and aH-DA releasing in animals treated with the latter compound (5). capability, as well as the central psychotropic effect in man 14. The second class is typified by MDMA and its congeners; (Shulgin and Jacob, unpublished). The possible neurotoxicityof these compounds typically displaya low affinityfor 5-HTzrecep- these analoguesis not addressed in the present study. 15. tors (2,23), induce the presynaptic releaseof 5-HT and DA from The results reported here indicate that synaptosomalrelease of nerveterminals, and frequentlydo display serotonergicneurotox- aH-DA shows some relationshipto serotonergic neurotoxicityin icity, at least in some animals under some dose regimens. Drugs vivoof MDA and its congeners; however, some derivatives, no- in this class exhibit similarities to both hallucinogensand stimu- tably fenfluramine and noffenfiuramine, are conspicuous excel>- 16. !antsin the drag discriminationassay (7, 18, 19)and their human tions to this apparent relationship, indicating that additional psychopharmacology has been characterized as "entactogenic' mechanisms must play a role in mediating the semtonergic neu- rather than hallucinogenic (15,17). Structurally, compounds in rotoxicity of these agents. the fa'st (DOM-like) class are 2,5-dimethoxy-4-substituted phe- 17. nylisopropylamines [the nature and polarity of the 4-substituent can vary greatly without loss of central activity;cf. (37)], while ACre4OW__S compounds in the second (MDMA-Iike) class typically have a We thankMs. MaryKellerfor assistancein the preparationof the 18. methylenedioxyring substitution, manuscript.Twoof us (D.J.M.. X-M.G.)thankDr. S. J. Peroutkafor Some of the analogues investigated in the present study be- constructivediscussionandfor financialsupportin theformof postdoc- long to yet a third class of centrally active phenylisopropylamine torsi fellowships. Thisworkwas supported in part by the Stanley Foun- 19. derivatives, typified by the compounds MMDA (3,4-methylene- clarionand NationalInstitutesof HealthgrantsNS 12151-14and NS dioxy-5-methoxy-phenylisopropylamine) and DMMDA (2,5- 23560-03. 20. MDA ANALOGUF._ AND SYNAPTOSOMAL [3H]-MONOAMINE RELEASE 511

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· 512 McKENNA, GUAN AND SHULGIN Pnan

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