Structure-Activity Relationships Among the Halogen at Ed Amphetamines (1978)

8/6/2019 Structure-Activity Relationships Among the Halogen at Ed Amphetamines (1978)

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STRUCTURE-ACTIVITY RELATIONSHIPS AMONG THEHALOGENATED AMPHETAMINESRay W. Fuller

The Lilly Research LaboratoriesEli Lilly and CompanyIndianapolis, Indiana 46206

Lowering of brain serotonin by halogenated amphetamines was initiallyobserved by Pletscher et ~ l . , ' - ~ho primarily studied p-chlero-N-methylamphet-amine (p-CMA). A series of halogenated am phetam ines had been prep ared inthe Lilly Research Laboratories by Dr. Jack Mills as poten tial appetite suppres-sant drugs; 4 , s we examined structure-activity relationships among those com-pounds and reported that p-chloroamphetamine (p-CA) was somewhat moreactive than p-CMA in lowering brain serotonin 16 hr after drug injection inra ts 6 . Later studies revealed that p-CMA is extensively converted to p-CA inrats and that most of its in vivo effects may be due to p-CA (see below). Themechanism of action of p C A might initially have been described as obscureand now must be described as complex. O ur studies of p-CA have emphasizedthe use of structural analogs as tools in elucidating and dissociating the multipleactions that p-CA apparently exerts on serotonin neurons.MULTIPLECTIONSF p-CA ON SEROTONINEURONS

The initial observations that the concentrations of both serotonin and 5-hydroxyindoleacetic acid (5-HIAA) in the brain were depressed by p-CMAand p-CA 2 , * suggested that 5-hydroxyindole synthesis was inhibited (agentslike reserpine that deplete serotonin by release increase 5-HIAA as serotonin isexposed to attack by monoamine oxidase) . Brain tryptophan hydroxylase wasnot measured directly because methods were then unavailable, but the haloge-nated amphetamines were shown not to inhibit tryptophan hydroxylation byra t l i v e r2 *? r by Chromobacterium violaceum7 and to have no effect on5-hydroxytryptophan decarboxylation.2 Since the halogenated amphetaminesreleased serotonin from platelets in vitro but prevented its conversion to 5-HIAA,3 the proposal was made that the compounds lowered serotonin in vivoby releasing it from storage granules and lowered 5-HIAA by inhibition ofmono amine oxidase. Th is mechanism left unanswered th e fat e of th e releasedserotonin and was insufficient for explaining the effects produced by the haloge-nated amphetamines, though p-CA probably does release serotonin and inhibitmonoamine oxidase in V ~ V O . ~ - ~ ~anders-Bush et a1.12 made the important ob-servation that p-CA does inhibit tryptophan hydroxylation in vivo in brain, andthis inhibition of 5-hydroxyindole synthesis is probably the major mechanismthat accounts for the lowering of serotonin and 5-HIAA levels. T h e molecularbasis for this effect on tryptophan hydroxylase in vivo is unknown, becausep-CA does not inhibit the enzyme in vitro, except at very high concentrations.p-CA and p-CMA are potent inhibitors of serotonin uptake by brain slices l3and synaptosomes l4 in vitro, and inhibition of serotonin reuptake probably

1470077-8923/78/0305-0147 $01.75/0 @ 1978, The New York Academy of Sciences


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148 Annals New York Academy of Sciencesoccurs in vivo, though such inhibition would not result in depletion of serotoninlevels.Th e actions of p-CA on serotonin neurons are sum marized in TABLE. Theinitial effects probably include inhibition of tryptophan hydroxylation, releaseof serotonin from intraneuro nal storage granules, inhibition of serotonin reup-take by the neuronal mem brane pu mp , and inhibition of serotonin oxidation bymonoamine oxidase. Lowering of serotonin by p-CA may occu r largely throughinhibition of tryptophan hydroxylation, but these actions seem to vary inprominence among various p-CA analogs, as will be discussed. Lowering of5-HIAA may occur via inhibition of tryptophan hydroxylation, inhibition ofmonoam ine oxidase, an d inhibition of serotonin reup take.Not only does p-CA produce rapid depletion of brain serotonin, but theeffects are also of extremely long duration. Sanders-Bush er d . 1 5 first recog-

TABLEACTIONSF p-CA THATMAY NFLUENCE-HYDROWINDOLEONCENTRATION

IN BRAIN ND THATARE POTENTIALLY DISSOCIABLEInfluence on Concentra.tionof

Action Serotonin 5-HIAAInhibition of tryptophan hydroxylation decreaseRelease of serotonin decreaseInhibition of serotonin reuptakeInhibition of monoamine oxidase increaseno changeIrreversible damage toserotonin neuronal function * decrease

decreaseincreasedecreasedecreasedecrease

* All parameters specifically associated with serotonin neurons are decreased:tryptophan hydroxylase, high-affinity uptake of serotonin, serotonin and 5-HIAA con-centrations, and serotonin turnover.

nized that serotonin levels were still depressed for as long as 4 months aftera single injection of p-CA in rats. All param eters of serotonin neural function(tryptophan hydroxylase,l5 serotonin concentration 15, l6 and turnover,I6 5-H1AA,l5# and serotonin uptake l7) are depressed at these long time intervals,which suggests that some serotonin neurons have been destroyed, and Harveyet al. have published histologic evidence for selective neurotoxic effects ofp-CA.l8, 8 The initial effects of p-CA are not permanent, however, and canbe readily reversed for a few hours after p-CA administration *OJQ (TABLE) .Thus, the rapid initial effects and the long-lasting destructive effects of p-CAon serotonin neur ons ca n be readily distinguished.By studying structural analogs of p-CA, we have been able to dissociatethese initial and long-term effects and have further been able to distinguish thefour multiple initial actions listed above. The structures of several of theseanalogs a re shown in FIGURE.


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Fuller: St ruc ture -Ac t iv i ty Re la t ionships 149

FIGURE. Structures of p-CA and some of its analogs. All of the comp ounds usedwere racemic mixtures of optical isomers.

INFLUENCEF CHLORINEOSITIONN THE RINGIn o u r initial s tudies, we observed n o significant depletion of brain seroto nin

16 hr after administrat ion of ei ther o-chloro- or m-chloroamphetamines , incontras t to the m arked deple tion tha t o ccurred af ter injec t ion of p-CA.' A mor ecareful analysis revealed that these compounds are, l ike amphetamine, rapidlymetabol ized in the ra t .22 The ra t metabol izes amphetamine mainly by para-hydroxylation, and amphetamines that contain a substi tuent in this posit ionpersist in the bra in and other t i ssues much longer than does amphetamineitself .23 Thus, the inabil i ty of the o-chloro and m-chloro compounds to affect

TABLEp-BROMOAMPHETAMINEp-BA)DURATIONF REVERSIBILITYF SEROTONIN DEPLETIONY p-CA AN D

Reversal (% )Hours After Injection ~~of p-CA or p-BA * p-CA p-BA48

2448100873 10

9279200*U ptake inhibitor (fluoxetine hydrochloride) was injected a t this time, and sero-tonin concentration was measured 24 h r later. p-CA data were calculated fromReference 21, and p-BA d ata were from similar unpublished studies.=


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150 Annals New York Academy of Sciencesserotonin in rats, particularly at longer times, could be attributed to their failureto remain in the brain at effective concentrations. Th e metabolic differencesbetween these chlorinated amphetamines in rats can be eliminated hy pretreat-ment with desmethylimipramine z 2 or iprindole 2 4 to block para-hydroxylation.When that is done, m-chloroamphetamine lowers brain serotonin in exactly thesame manner as does p-CA.22s 4 And, in the guinea pig, a species in whichpara-hydroxylation of amphetamines does not occur, m-chloroamphetamine actslike p-CA in lowering brain serotonin.z5 o-Chloroamphetamine, on the otherhand, does not lower but, instead, increases serotonin concentration in des-methylimipramine-treated rats 2 2 and guinea pigs.2e Furthermore, the additionof an o-chloro substituent to p-CA markedly diminishes its ability to lower brainserotonin, although the reduction of 5-HIAA was perhaps even greater withthe dichloro compound.2e Since the dichloro compound is a more potentrnonoamine oxidase inhibitor,ze this finding su ppo rts the idea that inhibition ofmonoamine oxidase does contribute to the decrease of 5-HIAA caused bythese compoun ds.

SUBSTITUENTSTHERTHAN HLORINE N THE RINGp-Bromoamphetamine 2 7 and p-bromomethamphetamine 2R, 29 lower brainserotonin to an extent comparable to the reduction caused by the correspondingp-chloro compo unds. pF luo roa m ph eta m ine decreases serotonin levels, but its

effects do not persist to the same degree as those of p-CA.27s 0 Other pnra-substituted amphetamines have less activity (trifluoromethyl, phenoxy) or noactivity (m ethy l, methoxy ) as serotonin depletors.20Norfenfluramine is an analog of p-CA that bears an m-trifluoromethyl sub-stituent instead of a p-chloro substituent on the ring. Norfenfluramine an dfenfluramine (the N-ethyl compound) lower serotonin in rat brain fo r a verylong time, though they a re slightly less active than p-CA.3*-34 Although Co staand Revuelta 32 have reported that norfenfluramine increases serotonin turnoverinstead of decreasing it, as p-CA does, we find that norfenfluramine causes arapid reduction of tryptophan hydroxylase accompanying the decline in sero-tonin, just as p-CA does (TABLE). Sanders-Bush et al. have reported short-and long-term depletion of serotonin and tryptophan hydroxylase in rat brainafte r injection of fenfluramine.'? In general, the actions of norfenfluram ine an dfenfluramine on brain serotonin neurons may be very similar to those of p-CAand o ther chlorinated amphetamines.

INFLUENCEF SUBSTITUENTSN THE NITROGENAs mentioned previously, the initial observation of serotonin lowering by

halogenated amphetamines was m ade with p-CMA ,'+ which is almost as effec-tive as p-CA In fact, m ore recent studies indicate that p-CMA israpidly and extensively metabolized to p-CA in the rat (TABLE) . We havecompared other N-alkyl derivatives of p-CA and find that they all are lesseffective than p-CA in depleting brain serotonin in rats.', 6c, N-Ethyl, N-iso-propyl, and N,N-dimethyl derivatives lower brain serotonin and are metabolizedby N-dealkylation to p-CA in the rat. Th e exte nt of their conversion to p-CAmay be adequate to account for their lowering of serotonin; that is, they may


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Fuller: St ruc ture -Ac t iv i ty Re la t ionships 151TABLE

RAPID DEPLETIONF BRAIN5-HYDROXYINDOLES AND TRYPTOPHANYDROXYLASEBY m-TRIFLUOROMETHYLAMPHETAMINE (NORFENFLURAMINE) IN RATS*

Brain 5-Hydroxyindoles ( p g / g ) TryptophanMinutes Serotonin 5-HIAA Hydroxylase03060120240

0.552 .02 0.5620.02 24.520.50.4420.02 0.58f0.01 18.9f0.60.31 f0.02 0.52 0.02 19.9 f0.7 t0.2120.005 0.47 O.0 t 16.6k0.20.19 O.O1 t 0.37 kO.01 t 15.320.6* Norfenfluramine hydrochloride (a gift from the A. H. Robins Company) was in-jected intraperitoneally at 15 mg/kg. Brain 5-hydroxyindoles Is and tryptophanhydroxylase were measured fluorometrically. Tryptophan hydroxylase units areexpressed in micrograms of 5-hydroxytryptophan formed per gram of brain per hour.Mean values an d standard erro rs for five rats per group are shown.t p < 0.01,ower than time zero.

al l act via conversion to p-CA. W e have been unable to block this metab olicconvers ion by any drug to de termine whether the N-alkyl compounds arecapable of depleting seroton in o n their own .N-Hydroxyl-p-CA was an analo g of interest because hydrox ylam ines areunstable and chemically reactive. We thought tha t N-hydroxy l -p-CA might bea ( the) neurotoxic metaboli te that has been considered as a possible mediatorof the long-term effects of p-CA. Ho wev er, we fou nd that N-hy drox y-p-C Awas, in fact , rapidly and q uanti tat ively converted to p-C A, so that its effects onbrain sero tonin were indist inguishable fr o m those of P-CA .~'N-Cyclopropylarylalkylamines re irreversible inhibitors of monoamine oxi-d a ~ e , ~ ~ ?n genera l , and N-cyclopropyl-pCA is therefore a unique analog ofp-CA in that the monoamine oxidase-inhibit ing component of i ts activity plays

TABLESEROTONIN EPLETION ND p-CA CONCENTRATIONN RATBRAINAFTERINJECTIONF p-CA OR p-CMA *

Trea tmen t 5-Hydroxytryptamine p-CADrug Interval/(hr) ( p g / g ) (nmol /g)None 0.55k0.02 -p-CA 2 0.32k0.01 t 91f 34 0.28k0.01 t 89+5

8 0.19 20.01 t 8624p-CMA 2 0.37 20.01 t 58k34 0.29f0.01 64248 0.19 20.01 t 7721* Drugs were injected intraperitoneally at 0.1 mmol/kg. Means and standard errorst p < 0.01, ifferent from control group.fo r five rats per group are shown.


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1 5 2 A n n a l s New York A c a d e m y of Sciencesa greater part in its action on serotonin. N-Cyclopropyl-p-CA does not lowerserotonin initially but, instead, produces a delayed and long-lasting depIetion.'O5-Hydroxyindoleacetic acid is lowered immediately, probably due largely toinhibition of monoamine ~ x i d a s e . ' ~ -Cyclopropyl-pCA does cause a rapidreduction of tryptophan hydroxylase in brain, as does p-CA."l Th us, there areinitially two opposing influences on serotonin concentration by this agent :inhibition of serotonin synthesis and inhibition of serotonin degradation, thenet effect being little chan ge in serotonin concentration at first. Th e in tra-neuronal effects of N-cyclopropyl-pCA on serotonin metabolism can be pre-vented by prior administration of an uptake inhibitor to block its entry via themembrane pump.*' Uptake inhibition has no detectable influence on overallinhibition of brain monoarnine oxidase, probably because the monoamine oxi-dase inside serotonin neurons contributes an undetectably small portion to totalbrain activity:" Th us , an up tak e inhibitor removes the inhibition of serotoninsynthesis but not the inhibition of serotonin oxidation, so the result is a rapidincrease in serotonin c~ncent ra t ion.~~lthough serotonin oxidation may nor-mally occur inside serotonin neurons, in the presence of an uptake inhibitor,serotonin presumably is oxidized by monoamine oxidase outside serotoninneurons.

INFLUENCEF SIDE CHAIN ENGTHThe shorter homolog of p-CA, p-chloro-a-methylbenzylamine,s only aweak inhibitor of serotonin uptake and of monoamine oxidase in vitro anddoes not affect serotonin or 5-H IAA concentration in the brain in viva."? Longerhomologs of p-CA, on the other hand, selectively retain some of the pharmaco-logic actions of p-CA. Th e first longer homolog ( on e additional methylene unitconnecting the phenyl ring an d the amine-bearing ca rb on ) is slightly more activethan p-CA as an inhibitor of rnonoamine oxidase in vi tro and slightly increasesserotonin levels while decreasing 5-HIAA levels in rat brain, presumably dueto weak inhibition of monoamine oxidase in vivo .4* The next longer homolog,with two additional methylene units, is a weaker inhibitor of monoamineoxidase in vitro and does not significantly change serotonin levels in V ~ V O . ~ ~It slightly lowers 5-HIAA levels i n v i v a 4 * Both of these two longer homologsof p-CA retain essentially full activity as a serotonin uptake inhibitor in vitro;this activity probably accounts fully for the 5-HIAA reduction by the longesthomolog and partly for the 5-HIAA lowering by the intermediate homolog.Both of these longer homologs can block the depletion of serotonin by p-CA,demonstrating their ability to inhibit uptake into serotonin neurons i n V ~ V O . ' ~Thus, lengthening the side chain of p-CA abolishes the serotonin-depletingaction but retains other actions of p-CA.

When the @-carbon of p-CA contains two fluorine atoms in place of hy-drogen atoms, the basicity of the amine group is greatly reduced due to thestrong electron-withdrawing property of This unique p-CA analoghas a pK, of 6.8 and so exists at physiologic pH mainly in the neutral forminstead of being virtually completely ionized, as is p C A itself (pK , 9.3). Pre-


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Fuller : Structure-Activi ty Relat ion ships 153sumably, as a consequence of the existence of most molecules of j3,p-difluoro-p-CA in the uncharged form and of nearly all molecules of p-CA as cations,these two substan ces differ strikingly in their biologic dispos ition. T he difluorocompound is localized very heavily in adipose tissue, the tissue in which p-CAitself is presen t in lowest concentration.'$ Because of the localization of &/3-difluoro-p-CA in fat, its concentrations in brain are lower than those of p-CAafter eq uimo lar doses; it must be given at about four times the dose of p-CA toachieve initial concentrations in brain th at are equivalent. Ev en then, th e con-centration of p,&difluoro-p-CA is no t ma intained in the brain, because it ismetabolized much more rapidly and extensively than is p-CA itself in the rat.43Although &p-difluoro-p-CA initially depletes brain serotonin and 5-HIAA tothe same extent as does p-CA itself when brain levels of the two drugs areequivalent, the effects of the difluoro compound on brain 5-hydroxyindoles inthe rat are only transient and disapp ear within 24 hr.43 Prior induction of livermicrosornal enzymes with phenobarbital accelerates the already-rapid metabo-lism of p,/3-difluoro-p-CA and prevents the transient depletion of 5-hydroxy-in d 0 1 e s. ~ ~ his effect of phenobarbital apparently occu rs because the 8,p-difluoro substitution on amphetamines makes the drugs more susceptible todeamination by hepatic enzymes.45

p-CA ANALOGS ITH RIGIDONFORMATIONChlorinated 3-phenylpiperidines are analogs of chloroamphetamines (thoughthey lack the a-methyl group) with rigid conformations in which the nitrogenis part of a second ring structure tha t involves the @ carbon. T h e p-chloro,m-chloro, and m,p-dichloro compounds in this series lower serotonin and 5-HIAA levels in rat brain, but their effect is smaller than that of p-CA and isshort-lasting.46 In other respects, such as tissue distribution and influence ofdesmethylimipramine on brain levels, the chlorinated 3-phenylpiperidines re-semble the ch lo roa m ph e tam ine~ .~~6-Chloro-2-aminotetralin is another analog of p-CA that has a rigid con-formation. This compound also depletes brain serotonin and 5-HIAA, but,

again, its effect is short-lasting and smaller than that of p-CA itself.47 A par-ticularly interesting compound that can be considered as a rigid p-CA analogderived from 6-chloro-2-aminotetralin by the addition of a third ring structureis a compound known as Org 6582 . This compound was reported recently bySugrue et al.lR o be a potent and specific inhibitor of serotonin up take . Wehave compared it to 6-chloro-2-aminotetralin.~QOrg 6582 has lost the abilityof p-CA and 6-chloro-2-aminotetralin to inhibit tryptophan hydroxylase, todeplete serotonin, and to inhibit monoamine oxidase in vivo but retains theability to inhibit serotonin uptake. It th us is a potent a nd specific inh ibitor ofserotonin uptak e with a long duratio n of action.POSSIBLEMECHANISMSF LONG-TERMFFECTSOF p-CA

The long-term depletion of 5-hydroxyindole levels and of tryptophan hy-droxylase and serotonin reuptake capacity suggests that the function of someserotonin neurons has been permanently impaired by p C A , and histologicchanges consistent with neurotoxicity have been observed.l8. Th e mechanism


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154 Annals N e w York Academy of Sciencesby which p-CA exerts these long-lasting effects is not known, but attention hasfocused o n the possibility that a neurotoxic m etabolite is form ed fro m p -CA.50-52Despite this attention, no compelling evidence that a metabolite of p-CA playsany part in its actions on serotonin has emerged. If such a metabolite is in-volved, it most likely is formed inside the serotonin neuron, because p-CA itselfis selectively taken up into serotonin neurons, but there is no a priori rcasonthat a metabolite would be, and because a metabolite so highly reactive that itwould destroy a neuron is unlikely to reach the site of action in brain if it wereformed in a peripheral tissue, such as the liver. Since no evidence in favor ofa toxic metabolite of p-CA has been uncovered, perhaps the idea that none isinvolved deserves more consideration. p-CA itself may cause irreversible dam-age to the serotonin neuron by virtue of being concentrated extensively insidethe neu ron over a prolonged time. Baliga et have shown that amphetaminescan inhibit protein synthesis by interfering with initiation; p-CA was moreactive than amphetamine itself in this regard. Perhaps p-CA attains concentra-tions inside the serotonin neuron high enough to inhibit the formation of keycell proteins and persists at these high concentrations for a long duration as aconsequence of continual reup take by the amine pum p on the serotonin neuro nalmem brane. This mechanism fo r the long-lasting effects of p-CA would becompatible with the observations that an uptake inhibitor can reverse serotonindepletion (and tryptophan hydroxylase lowering) by p-CA initially and that thereversibility gradually declines to zero within 32-48 hr after p-CA injection.This period may be the time fo r wh ich the serotonin neuro n ca n survive withoutthe resynthesis of essential proteins requ ired fo r general cell fun ction . Th e datasummarized in TABLE2 may be instructive in considering mechanisms bywhich serotonin depletion by p-C A and analogs becom es irreversible. O nemight anticipate that p-CA an d p-brom oam phetam ine would be metabolized atdifferent rates , so that their effects would become irreversible at different ratesif the irreversible effects were d ue to a toxic metabolite. Th is possibility wou ldbe particularly applicable to metabolism on the phenyl ring, for instance, theformation of an arene oxide intermediate du e to attack by a mono-oxygenase.osHowever, the rates at which the effects of these two compounds become irre-versible were virtually identical. Th is finding would b e compatible with a the orythat interference with some essential cellular process for a critical period isinvolved; that period would be independent of the inhibitory agent. Th e abilityof some analogs of p-CA (e.g., 6-chloro-2-aminotetralin,p-fluoroamphetamine,/3-/3-difluoro-p-CA) to lower serotonin initially but not for a long duration mayreflect a relatively po orer affinity fo r the up take pu mp , so that these analogs arenot maintained inside serotonin neurons at adequate concentrations for suffi-ciently long periods to pro duc e irreversible damage.

S U M M A R YpChloroamphetamine and various analogs influence brain serotonin neuronsthrough multiple actions. Co mp ariso n of these compounds has permitted thedistinction between short-term and long-term depletion of serotonin and amonginhibition of tryptophan hydroxylation, release of serotonin, inhibition ofserotonin reuptake, and inhibition of monoamine oxidase as mechanisms in-volved in the actions of these agents on serotonin neurons.


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Fuller: Structure-Activity Relationships 155ACKNOWLEDGMENTS

I am g ra te fu l to Drs . J ack Mi ll s an d Bryan B . Mol loy and the i r as soc iatesfor the synthes is of the p-CA ana logs ; to Drs . Dav id T. W o n g , C. J o h n P a r l i ,an d E. M a r t i n G i l for col laborat ive ass is tance in var ious phases of our studies;an d to H ar o l d D. S n o d d y , K en n e t h W . P e r r y , J o h n C. Baker , and SusanH em r i ck for thei r exper imental work.

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Sci. 2: 828-833.2. PLETSCHER, ., G. BAR THO LINI,. BRUDERER , . P . B URKA RD K.F. GEY.1964. Chlorinated arylalkylamines affecting the cerebral metabolism of 5-hydroxytryptamine. J. Pharmacol. Exp . Th er. 145: 344-350.3. PLETSCHER,., M. D A PRADA , . BARTHO LINI ,. P. BURKARD& H. BRUDERER.1965. Two types of monoamine liberation by chlorinated aralkylamines. LifeSci. 4: 2301-2308.4. OW EN , . E., JR . 1963. Psychopharmacological studies of some 1-(chloropheny1)-2-aminopropanes. 1. Effects on appetite-controlled behavior. J. P ha rm . Sci. 52:5 . OW EN , . E., JR. 1963. Psychopharmacological studies of some 1-(chloropheny1)-

2-aminopropanes. 11. Effects on avoidance and discrimination behavior. J.Pharm . Sci. 52: 684-688.6 . FULL ER, . W., C. W. HINES& J. MILLS. 1964. Lowering of rat brain serotoninlevels by arylalkylamines. Fe d. Proc. 23: 146.7. FULL ER, . W., C. W. HINES& J. M ILLS. 1965. Lowering of brain serotoninlevel by chloroamphetamines. Biochem. Pharmacol. 14:483-488.8. SANDERS-BUSH,. & F. SULSER.1970. p-Chloroamphetamine: in vivo investiga-tions o n the mechanism of action of the selective depletion of cereb ral sero-tonin. J. Pharmacol. Exp. Ther. 175: 419-426.9. FULLER,R. W 1966. Serotonin oxidation by rat brain monoam ine oxidase:inhibition by 4-chloroam phetam ine. Life Sci. 5: 2247-2252.10. GALLAGER,. W. & E. SANDERS-BUSH.973. In vivo measurement of the releaseof 5-hydroxytryptamine (SH T) fro m the hippocampus of the rat: effect of104-1284, pargyline and p-chloroamphetamine (P C A ). Fed . Proc. 32: 303.11 . TRULSON,M. E. & B. L. JACOBS.1976. Behavioral evidence for the rapid re-lease of CN S serotonin by PCA and fenfluramine. Eu r. J. Pharm acol. 3 612. SANDERS-BUSH,., J. A. BUSHING F. SULSER.1972. p-chloroamphetamine-inhibition of cerebral ,tryptophan hydroxylase. Biochem. Pharm aco l. 21:1501-1 5 10.13. CAR LSSO N, . 1970. Structural specificity fo r inhibition of ["CI-5-hydroxy-tryptamine uptake by cerebral slices. J . Pharm . Pharmcol. 22: 729-732.14. WONG,D. T . , J .3 . HORNC& R.W. FULLER.973. Kinetics of serotonin accu-mulation into synaptosomes of rat brain -eff ec ts of amphetamine a nd chloro-amphetam ines. Biochem. Ph arm aco l. 22: 3 11-322.IS. SANDERS-BUSH,., J. A. BUSHING F. SULSER.1972. Long-term effects of p-chloroamphetamine on tryptophan hydroxylase activity and on the levels of5-hydro xytryptam ine an d 5-hydroxyindole acetic acid in brain. Eu r. f .Pharmacol. 20: 385-388.16. FULLER,R. W. & H. D. SNODDY.1974. Long -term effects of 4-chloroam phet-

679-683.

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156 Annals New York Academy of Sciences

17.18.19.20.21.22 .23.24 .25 .26 .27 .28.29 .30.3 1.32.33.34.35 .

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am ine on brain 5-hydroxyindole metabolism in rats. Neuropharmacology 13:SANDERS-BUSH,., J. A. BUSHING F. SULS ER. 1975. Long -term effects of p-chloroamphetamine an d related drugs on central serotonergic mechanisms. J.Pharmacol. Exp. Ther. 192: 33-41.HARVEY,. A., S.E. MCMASTER L. M. YUNGER.1975. p-Chloroamphetamine:selective neurotox ic action in brain. Science 187: 841-843.HARVEY,. A. & S. E. MCMASTER.1976. Neurotoxic action of para-chloro-amphetamine in the rat as revealed by Nissl and silver stains. Psychopharmacol.FULLER,. W., K. W. PERRY& B. B. MOLLOY.1975. Effect of 3-(p-trifluoro-methylphenoxy) -N-methyl-3-phenylpropylaminen ,the depletion of brain sero-tonin by 4-chloroamphetam ine. J. Pharmacol. Exp. The r. 193: 796-803.FULLER,. W., K. W. PERRY& B. B. MOLLOY.1975. Reversible and irreversiblephases of serotonin depletion by 4-chloroamphetamine. Eu r. J. Pharmacol.FULLER,R. W., R. J. SCHAFFER,B. W. ROUSH& B. B. MOL LOY . 1972. D ru gdisposition as a factor in the lowering of brain serotonin by chloroamphet-amines in the rat. Biochem. Pharmacol. 21: 1413-1417.FULLER,. W. & C. W. HINES. 1967. Tissue levels of chloroamphetamines inrats and mice. J. Pha rm. Sci. 56: 302-303.FULLER, . W. & J. C. BAKER.1974. Long-lasting reduction of brain 5-hydroxy-tryptamine levels by 3-chloroamphetamine and 4-chloroamphetamine in iprin-dole-treated rats. J. Pha rm. Pharmacol. 2 6 12-914.FULLER,. W. 1972. Species difference in the lowering of brain 5-hydroxy-tryptamine by meta-chloroamphetamine. J. Pha rm. Pharmacol. 24: 88 .FULLER, . W., H . D. SNODDY, . W. ROUSH& B. B. MOLLOY.1973. Furtherstructure-activity studies on the lowering of brain 5-hydroxyindoles by 4-chloroamphetamine. Neuropharmacology 12: 33-42.FULLER, . W., J. C. BAKER, . W. PERRY& B. B. MOLLOY.1975. Comparisonof 4-chloro-, 4-bromo- and 4-fluoroam phetam ine in rats: drugs levels in brainand effects o n brain serotonin metabolism. Neuropharmaco logy 14: 739-746.LEONARD,. E . & S.A. SHALLICE. 971. Some neurochemical effects of amphet-amine, methylamphetamine and p-bromomethylamphetamine in the rat. Brit.J. Pharmacol. 41: 193-212.M A G Y A R ,. & J. KNOLL. 1975. Para-substituted amphetamines and brain sero-tonin. Pol. J. Pharmacol. Pharm . 27(Suppl.) : 139-143.HARVEY,. A., S. E. MCMASTER R. W. FULLER.977. Comparison betweenthe neurotoxic and serotonin depleting effects of various halogenated deriva-tives of am phetam ine in the rat. J. Pharm acol. Exp . Ther. In press.COSTA,E., A. GROPPETTI& A. REVU ELTA . 971. Action of fe dw ar ni n e onmo noam ine stores of ra t tissues. Brit. J. Pharm acol. 41: 57-64.COSTA, . & A. REWELTA . 1972. Norfenfluramine and serotonin turnover ratein the rat brain. Biochem. Pharmacol. 21:2385-2393.CLINESCHMIDT,. V., J. A. TOTARO, . C. MCGUFFIN& A. B. PFLUEGER. 976.Fenfluram ine: long-term reduction in brain serotonin (5-hydroxytryptamine) .Eur. J. Ph arrnacol. 35: 21 1-214.HARVEY,. A. & S. E. MCMASTER . 975. F enfluramine: evidence for a neu ro-toxic action on midb rain and a long-term depletion of serotonin. Psycho-pharmacol. Com mun. 1: 217-228.MILLER,F. P., R. H. Cox, JR., W. R. SNODGRASSR. P. MAICKEL. 970. Com-parative effects of p-chloro-N-methylamphetamine n rat brain norepinephrine,serotonin and 5-hydroxyindole-3-acetic cid. Biochem. Pharm acol. 19: 435-442.G ~ L , . M. & K. PATTERSON.973. Rapid nonisotopic assay of tryptophan-5-hydroxylase activity in tissues. Ana l. Biochem. 52: 625-628.

85-90.

Bull. 12: 62-64.

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FULLER, . W., K. W. PERRY, . C. BAKER, . J . PARLI,N. LEE, W. A. DAY&B. B. MOLLOY. 974. Comparison of the oxime and the hydroxylamine deriva-tives of 4-chloroamphetamine as depletors of brain 5-hydroxyindoles. Biochem.Pharmacol.23:3267-3272.MILLS, ., R. KATTAU,. H. SLATER R. W. FULLER.968. N-Substituted cyclo-propylamines as monoamine oxidase inhibitors. Structure-activity relationships.Dopa potentiation in mice and in vitro inhibition of kynuramine oxidation. J .Med. Chem. 11: 95-97.FULLER, . W. 1968. Kinetic studies and effects in vivo of a new monoamheoxidase inhibitor, N-[2-(o-chlorophenoxy)-ethyl]-cyclopropylamine. iochem.Pharmacol. 17: 2097-2 106.FULLER,R. W. & B. B. MOLLOY. 1973. Effects of N-cyclopropyl-4-chloro-amphetamne on brain serotonin metabolism in rats. Res. Commun. Chem.Pathol. Pharmacol. 6: 407-418.FULLER,R. W. & K. W. PERRY. 1977. Further studies on the effects of N-cyclopropyl-p-chloroamphetamine in rat brain. Neuropharmacology 1 6 495-497.FULLER, . W., K. W. PERRY,D. T. WONG& B. B. MOLLOY.1974. Effects ofsome homologues of 4-chloroamphetamine on brain serotonin metabolism.Neuropharmacology 13: 609-614.FULLER, . W., H. D. SNODDY B. B. MOLLOY.1973. Effect of p,p-difluoro-substitution on the disposition and pharmacological effects of 4-chloroamphet-amine in rats. J. Pharmacol. Exp. Ther. 184: 278-284.FULLER, . W., J. C. BAKER, . W. PERRY& B. B. MOLLOY.1974. Effect ofphenobarbital on the metabolism and serotonin-depleting action of 4-chloro-p,,%difluoroamphetamine in rats. Arch. Int. Pharmacodyn. 208: 274-278.

FULLER, . W. & B. B. MOLLOY.1976. The effect of aliphatic fluorine on aminedrugs. I n Biochemistry Involving Carbon-Fluorine Bonds. R. Filler, Ed. :77-98. American Chemical Society. Washington, D.C.FULLER, . W., B. B. MOLLOY,. C. BAKER, . W. PERRY& W. A. DAY. 1975.Biochemical pharmacology of some chlorinated 3-phenyl-piperidines in rats.Arch. Int. Pharmacodyn. 214: 263-270.FULLER, . W., K. W. PERRY,. C. BAKER B. B. MOLLOY.1974. 6-Chloro-2-aminotetralin, a rigid conformational analog of 4-chloroamphetamine: phar-macologic properties of it and related compounds in rats. Arch. Int. Pharma-codyn. 212: 141-153.SUCRUE, . F., I. GOODLET S. E. MIREYLEES.976. On the selective inhibitionof serotonin uptake in vivo by Org 6582. Eur. J. Pharmacol. 40: 121-130.FULLER, . W., D. T. WONC,H. D. SNODDY F. P. BYMASTER. 977. Com-parison of the effects of 6-chloro-2-aminotetralin and of Org 6582, a relatedchloroamphetamine analog, on brain serotonin metabolism in rats. Biochem.Pharmacol. 2 6 1333-1337.G ~ L , . M. 1976. The effects of intraventricularly administered para-chloro-amphetamine and its analogs. Psychopharmacol. Bull. 12: 52-54.SHERMAN,., E. M. G ~ L ,. W. FULLER B. B. MOLLOY. 1975. Effects ofintraventricular p-chloroamphetamine and its analogues on cerebral 5-HT.Neuropharmacology 14: 733-737.SANDERS-BUSH,. 1976. Long-term effects of para-chloroamphetamine oncentral serotonergic mechanisms. Psychopharmacol. Bull. 12: 57-59.53. BALIGA, . S., J. ZAHRINGER,. TRACHTENBERG,. A. MOSKOWITZ H. N.MUNRO.1976. Mechanism of d-amphetamine inhibition of protein synthesis.Biochim. Biophys. Acta 442: 239-250.54. PARLI,C. J . & B. SCHMIDT.1975. Metabolism of 4-chloroamphetamine to 3-chloro-4-hydroxyamphetamine n rat: evidence for an in vivo N M shift ofchlorine. Res. Commun. Chem. Pathol. Pharmacol. 10: 601-604.

55. FULLER, . W., H. D. SNODDY J . C. BAKER. 977. Unpublished results.


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158 Annals New York Academy of Sciences

DISCUSSIONDR. A. S. H O RN : Ha ve you studied the various isomers of these com -pounds? If so, do they have differential actions?D R. FULLER:ll of the compounds that we have studied were administeredas mixtures of the D- and th e L-isomers. It would be interesting to know i f theyhave differential effects; however, we havent given this a high priority, becauseseveral years ago we found that the two isomers of p-chloroamphetamineappeared to be identical with respect to their effects on brain serotonergicneurons.D R. E. SANDER S-BUSH: ave yo u looked at o the r possible antagonist effectsof fluoxetine on p-chloroamphetamine or its derivatives? I ask this question

because there is some debate about the mechanisms underlying the initial de-crease in tryptophan hydroxylase activity seen after p-chloroamphetamine injec-tions. C an this effect be reversed if animals are pretreated with fluoxetine?DR. FULLER:n fact, all parameters that are depressed after p-chloro-amphetamine treatment can be reversed by the administration of fluoxetine.For example, brain serotonin levels and tryptophan hydroxylase activity arenormal within 4 h r after fluoxetine treatment. If the reversal in trypto ph anhydroxylase activity is dependent on the synthesis of new enzyme protein, thatimplies that there is a more rapid rate of synthesis than one would anticipateon the basis of values for tryptophan hydroxylase enzyme turnover describedpreviously in the literature. However, there is also no evidence that th e reversalof p-chloroamphetamine-induced nhibition of tryptophan hydroxylase is relatedto differential inhibition by p-chloroamphetamine o r its metabolites. In s ho rt,then, I really dont know what mechanisms might account for the ability offluoxetine to rapid ly reverse th e effects of p-chloroamphetamine.Some indirect evidence indicates to us that p-chloroamphetamine might actdirectly on serotonin-containing neurons, rath er tha n via a neurotoxic metaboliteof the compo und. Fo r example, in on e study, we reasoned th at if the metabolitewas formed via a change in the ring structu re of p-chloroamphetamine, a com -pound like p-bromoamphetamine might be converted to a neurotoxic metaboliteat a rate different fr om tha t of p-chloroamphetamine. Since the ra te of develop-ment of irreversibility of p-chloroamphetamine an d p-bromoam phetamine w ereidentical, we now think that p-chloroamphetamine exerts its effects directly onthe neuron.D R. E. M. GAL: We have some evidence that pchloroamphetamine pro-duces a strong inhibition of tryptophan hydroxylase activity either in v ivo orin v i m , if you use the natural cofactor fo r the enz ym e in the assay. Hav e Y O Uo r Dr. Sanders-Bush tried this cof acto r?The fact that p-chloroamphetamine-induced inhibition oftryptophan hydroxylase activity cannot be reversed by dialyzing tryptophanhydroxylase obtained from brain homogenates would argue against the possi-bility tha t p-chloroamphetamines effects only oc cur in th e presence of a naturalcofactor but not a n unnatural one. Hence, it seems unlikely that there is anydirect inhibition of tryptophan hydroxylase by either p-chloroamphetamine ora m etabolite that would be present in o ur preparation.

DR. ANDERS-BUSH:We have also found that p-chloroamphetamine de-creases tryptophan hydroxylase activity in v i vo whether we use Dr. Fullersassay or the procedure developed by Dr. GBI.

D R. FULLER:


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Fuller : Structure-Activi ty Relat ion ships 159DR. GAL: There is some evidence that the more active form of the trypto-phan hydroxylase enzyme molecule must be phosphorylated. Is there any possi-bility that this process might be interfered with shortly after p-chloroamphet-

amine administration?DR.FULLER: think that something like your hypothesis might be possible.DR.J . H. GROWDON:mmediately after injection of p-chloroamphetaminein normal animals, one sees a rather complex set of changes in motor behaviorthat can be reversed up to 30-45 min later by injecting these animals withfluoxetine.

Structure-Activity Relationships Among the Halogen at Ed Amphetamines (1978)

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