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University Microfilms International 300 North Zeeb Road Ann Arbor, Michigan 40106 USA St. John's Road, Tyler's Green High Wycombe, Bucks, England HP10 8HR 77-31,832 BOSSART, Josef F., 1951- CONFORMATIONALLY RESTRAINED ANALOGS: CYCLOBUTYL ANALOGS OF DOPAMINE AND 6-HYDROXYDOPAMINE.

The Ohio State University, Ph.D., 1977 Chemistry, biological

University Microf iims intem ationai, Ann Arbor, Michigan 48ioe | CONFORMATIONALLY RESTRAINED ANALOGS:

CYCLOBUTYL ANALOGS OF DOPAMINE

AND 6-HYDROXYDOPAMINE

Dissertation

Presented in Partial Fulfillment of the Reouirements for

the Dedree Doctor of Philosophy in the Graduate School

of The Ohio State University

By

Josef F* Bossartf B.Sc» Hon,

* * * *

The Ohio State University

1977

Readind Committee: Approved By

Dr, N, Lewis

Dr, D, Miller

Dr, P, Patil

Dr, D, Witiak Adviser Collede of Pharmacy ACKNOWLEDGEMENTS

I would like to thank and acknowledge the support and guidance of mu superviser Dr» Duane D» Miller throughout this study. Not only did he help make mu graduate education 3 rewarding oner but I suspect he made it more fun than it was intended to be.

I would like to thank Prof, Popat N, Patil for his guidance of the biological portions of this research and his manu helpful discussions.

I would like to acknowledge the excellent biological studies of Dr, Robert Ruffolo and Dr, Harold Komiskeu without which this study would not have been complete,

I would like to thank Jack Fowble and Ed Fairchild for their contributions to this research as well as the knowledge and friendship they shared with me.

11 VITA

November 22» 1951 Born - Montrealr Quebec» Canada

1969-1973 B*Sc. Hon. Chemistry Carleton University Ottawa» Canada

1973-1976 Research Assistant ColleSe of Pharmacy The Ohio State University Columbus» Ohio

1976-1977 Teachins Assistant Collede of Pharmacy The Ohio State University Columbus» Ohio

FIELD OF STUDY. Medicinal Chemistry

111 TABLE OF CONTENTS

Fade

ACKNOWLEDGEMENTS ii

VITA i i i

LIST OF FIGURES vi

LIST OF TABLES vi i

INTRODUCTION

Pharmacology of 6-hydroxydopamine 5

Pharmacology of Dopamine 19

OBJECTIVES 37

RESULTS AND DISCUSSION

Synthetic 42

A) Preparation of cis- and trans- 2(2',4'p5'-trihydroxyphenyl)cyclobutyl- amine hydrobromide (^) and ( 42

B ) Preparation of trans- and cis- 2(3'f4'-dihydroxyphenyl)eyelobutylamine (50) and (51)» and trans- and cis- NfN-dimethyl-2(3',4'-dihydroxyphenyl) cyclobutylamine hydrobromide (52) and ( ^ ) 53

C) Rearrangement of 2»4»5-trimethoxypropi- ophenone 61

Biological

A) Pharmacological Assay of ^ and ^ 68

B) Uptake Assay of Analogs ^ and ^ 72

C) Binding Studies of 50» 51» 52 and53 75

IV EXPERIMENTAL 79

Synthetic 81

Biological 102

SUMMARY 106

BIBLIOGRAPHY 108 LIST OF FIGURES

Fiaure Title Fade

1 Conformations of Dopamine 24

2 Rotational Considerations of DopaminerSic ASents 27

3 Newman Projections of Several Riaid Analoas 38

4 Reactions of 2-phenalcyclohutalo;

5 90 MHz NMR Spectrum of 75 58

6 Temperature Study of 53 60

7 Results of Neurotoxicity Studies of 6-hydroxydopaminef ^ and ^ vivo 69 TT 8 Inhibition of H-Norepinephrine Uptake in vivo by ^ and 49 70

9 Inhibition of H-Norepinephrine Uptake in vitro by 50 and 5jL 73

10 Inhibition of ^H-Dopamine Uptake in vitro by ^ and 51 73

11 Inhibition of ^H-Dopamine Bindina in vitro by 50» 51 » ^ and ^ 77

VI LIST OF TABLES

Table Title Fade

1 Effect of 6-hydroxydopamine on catecholamines and serotonin in the Brains of Rats 3

2 Central Neurotransmitter Depletion by 3a and 3b 5

3 The Activity of 6-hydroxydopamine Analogs: Aromatic Substitution Variants 16

4 The Activity of 6-hydroxydopamine Analogs: Side Chain Variants 17

5 The Activity of Dopaminergic Agents on the Neurons of Helix Aspersa 30

6 Effect of Reaction Conditions on Yield of 61 45

7 Chemical Shifts of the Acetamides 67 and 68 50

8 Stereoselectivity in the Reduction of 72 56

9 Chemical Shifts of the Acetamides 73 and 74 56

10 Spectral Data for A and B 62

11 Possible Structures for A 63

12 Uptake Ki Values for 50 and 72

13 Ki Values for 50r 51 r 52 and 53 76

v n INTRODUCTION

The Pharmacology of 6-Hydroxydopamine

The first appreciation of 6-hydroxydopamine (6-OHDA) (J^)

as a natural product arose with the iji vitro studies of

Senoh and Witkop in 1959 of the enzymatic and nonenzymatic

conversion of dopamine (DA) (2) to norepinephrine Cl,2],

Senoh and Witkop isolated a metabolite of labeled dopamine

which did not show the requisite ratio for conversion

to norepinephrine and which was shown to be 6-OHDA

6-OHDA was synthesized vivo was demonstrated by the

peripheral administration of labeled DA which allowed

isolation of labeled 6-OHDA from the urine of the treated

rats ♦

± 2.

Studies by Porter and Stone» in the early sixties» of the

pharmacological effects of 6-OHDA indicated that unlike

other catecholamines it was capable of causing a long term

depletion of norepinephrine (NE) from the hearts of dogs and mice [3»4]* This NE depletion was later shown to be ouite general for NE stores in a number of tissues and animals though when injected peripherally 6-OHDA caused no depletion centrally [5»6]« This depletion of NE was dose dependent» low doses (1-3 mg/kg i.v.) caused only temporary depletion

1 2

while larder doses <100 md/ka) caused déplacement and lond

term depletion Z71*

The depletion of NE by a variety of false

neurotransmitters had been well established but the lond

lastind depletion caused by 6-OHDA was unioue. This lond

term depletion was postulated by Porter and coworkers to

^rise from the ability of 6-OHDA to destroy or irreversibly

alter NE bindind sites C33* Shortly thereafter it was shown

that 6-OHDA in larde doses caused destruction of the

adrenerdic neuron ultrastucture stained for visualization by

flourescence spectroscopy [7,8,9],

This destruction of neurons by 6-OHDA was shown to be

confined to the nerve terminal and had very little effect,

except in neonatal mice and rats CIO], on the axon and

neuron cell body C8], The specificity of neurotoxicity was

shown to be unioue for catecholamine containind neurons, no

destuction of cholinerdic neurons or other tissue was

observed [8,9]« This same specificity of neurodestuction was

shown to be true for the central nervous system [11], While

6-OHDA did not cause neurodestruction of the central neurons when injected peripherally its central application produced

a similar lond lastind depletion of dopamine and norepinephrine. No depletion of cholinerdic neurons was noted and serotonerdic fibres showed very little effect.

Table 1, C12], Some selectivity is observed between destruction of dopaminerdic and adrenerdic fibres allowind for specificity in central sympathectamy, Table 1

Effect of 6-OHDA on catecholamines and serotonin in the brains of rats (injection of 200 md of 6-OHDA 2 days before sacrifice)«

Controls 6-OHDA injected ud/d ud/d % control

Dopamine 0*85 0*58 68 Norepinephrine 0*54 0*19 35 Serotonin 0*49 0*43 88

The selectivity of the neurotoxicity peripherally and centrally was shown to be due to the hidhly specific uptake of 6-OHDA into adrenerdic and dopaminerdic neurons C131*

That uptake is reouisite for neurotoxicity was demonstrated by the prevention of 6-OHDA induced neurodestruction by pretreatment with uptake blockers such as imipramine and cocaine [14fl5]* This explained why cell bodies and axons» which do not possess uptake mechanisms for 6-OHDA were not subject to destruction*

Whether concentration of 6-OHDA into the neuronal granules is reouisite for its neurotoxicity is not clear*

Pretreatment with reserpine did not prevent neuronal destruction by 6-0HDA» an indication that dranular concentration is not necessary C161* However it has been shown that 6-OHDA is be concentrated to a small extent in the amine containind granules by a reserpine resistant mechanism [171*

As well as havind presynaptic actions 6-OHDA also may effect the postsynaptic receptor* Pretreatment with 6-OHDA caused a decrease in the pressor response of isolated perfused rat mesenteric arteries to NE [18»19], Responses to

calcium chloride and KCl were normal, indicating a specific

rather than deneral depression. Pretreatment with the

alpha-hlocker phentolamine blocked the pressor depression

associated with 6-OHDA administration and led to the

conclusion 6-OHDA reacted irreversibly with the postsynaptic

receptor. In Seneral postsynaptic effects are minimal due to

the nature of the synapse. The postsynaptic receptor is

protected by the bindind of neurotransmitter released by

6-OHDA, Additionally, the 6-OHDA concentration in the

receptor vicinity is low due to the active uptake mechanism

at the pre-synaptic receptor. There appears to be little

destruction of the uptake mechanism by 6-OHDA in the time

scale of neuronal destruction [18,19],

The destruction of an ordan or tissue is a powerful tool

for the study of its physiolodical role. The use of 6-OHDA

as a sympathectant has advantages over those methods usind

surdical procedures or nerve drowth factor antisera [20], By treatment of newborn [21,22] or adult [23] animals it is possible to effect a permanent or temporary peripheral sympathectamy. This property is particularily valuable for the study of the central nervous system. The central administration of 6-OHDA has allowed, by suitable choice of conditions the selective destuction of adrenerdic or dopaminerdic neurons [20], This selective destruction has been used extensively for the mappind of central pathways

[24] as well as for the study of catecholamine influence on behavior and other centrally mediated functions [20], It should be noted that chemical symeathectants related

to 6-OHDA have been developed which allow selective

destruction of the serotonerdic system [25], Both of these

adentsf 5r6-dihydro;

5f7-dihydroxytryptamine (5»7-DHT) (3b) reduce brain

serotonin with only minimal effect on the dopaminerdic and

adrenerdic systems. Table 2,

Table 2 Central Neurotransmitter Depletion by 3a and 3b

5-HT NE DA % control % control % control Brain Brain Brain

5.6-DHT (50ud) 56 84 70 5.7-DHT (50ud) 48 52 91 5.7-DHT (150ud) 17 48 not assayed 5.7-DHT <150ud)+ 21 124 not assayed DMI (25md/kd)

DMI Desmethylimipramine iCM

6-Hydroxydopamine - Mechanism of Action

The realization that the specificity of 6-OHDA for the

catecholaminerdic systems was dependant on its active

concentration by these neurons has stimulated the study of

the molecular mechanism by which 6-OHDA causes actual neurodestruction [26,27],

It was observed that after administration cWr a neurotoxic dose of 6-OHDA 25% of the administered label was present in the hecH't after 44 days [28] although the f luorescence of

the neurons due to 6-OHDA was maximal one minute after

administration and rapidly decreased to negligible amounts

within an hour [29], Similarly)» the amount of labeled 6-OHDA

found in the supernatant fraction after repeated

centrifugations was neSligable but fully 30-35% of the

administered label was associated with the particulate

fraction [30], These observations led to the proposal that

6-OHDAf a metabolite of 6-OHDA)» or an oxidation product of

6-OHDA bound irreversibly to some component of the

particulate fraction [30],

The search for such an alkylating agent derived from

6-OHDA has focused upon its oxidation products» the Quinones

and adrenochromes, It was initially noted by Senoh and

Witkop that 6-OHDA in basic or neutral solution was rapidly

oxidized to a red solution [31], On the basis of ultaviolet

studies they proposed that 6-OHDA oxidized to the p-ouinone

^ which rapidly cyclized to the indoline via a

1 » 4-addition )» and could further oxidize to the indoline

ouinone Scheme 1 [31],

OH Scheme 1

NH NHg HO HO

HO HO' OH S. Saner and Thoenen proposed that the ouinone ^ or the p-ouinone 4 could he alkulatind cell nucleophiles? Scheme 2

[32], They showed that with decreasing p H the amount of

^H-6-OHDA bound to bovine serum albumin (BSA) decreased and the presence of an antioxidant almost completely inhibited binding? observations consistent with an oxidized product of

6-OHDA being resposible for binding to the BSA* They also demonstrated that acétylation of BSA caused an 81% reduction in binding of >^H-6-GHDA derived products* Dénaturation of the protein reduced binding by only 25%? an indication that inhibition of the ^H-6-ÜHDA binding by acétylation was due to blocking of the reactive nucleophilic portions of the protein rather than a protein dénaturation process [32],

Scheme 2 9"

NHz HO

GH 6H

OH OH

NH. HO

1) 02 2)GH OH OH

NHz HO HO OH OH

Recent work with BSA has indicated that 6-OHDA alone has the ability to crosslink proteins in a concentration dependent fashion [33]* Concentrations of 6-OHDA expected to 8

be found intraneuronal 1« ij2 vivo slave efficient crosslinkind

of BSA while suboptimal concentrations dave very little

crosslinkindf an observation consistent with the observed minimal dose reauirement for neurotoxicity in. vivo. These

investigators also found that the neurotoxin

5,6-dihydroxytryptamine (3a) Save considerable crosslinkind while 5,7-dihydroxytryptamine (3b> ? and dopamine <_^) caused little crosslinkind of BSA under the same conditions [33],

The oxidative fate of 6-OHDA iji vitro was studied by

Blank et air whor employing electrochemical techniouesr showed shortly thereafter that the oxidation pf 6-OHDA at

25°C gave the ouinone ^ C34] but not the auinone as proposed by Senoh and Witkop, Blank et al later showed that at 37°C the auinone 4 cyclized slowly to the indole_7 but not the indoline 5 r Scheme 3 [35],

Scheme 3

HO NH, HO'xx>—-.xo 'OH i

0. slow

- . X O . —

Chemical studies of the fate of oxidation of 6-OHDA have supported the oxidation scheme of Blank, Powell and Heacock isolated 5r6-dihydroxyindole (7.) and 5r6-dihydroxyindoline

(8) as the acetate derivatives following oxidation of 6-OHDA with Dxygenr air or potassium ferricyanide [36], Wehrli et 9

al isolated the same nonacetalated derivatives following

oxidation of 6-OHDA with potassium nitodisulfonate [37],

Swan further showed that the ultraviolet assignments made by

Senoh and Witkop were for the ^-ouinone ^ rather than the

indoline derivative ^ C38], These studies were consistent

with the neslected studies of Harley-Mason in 1953 at which

time he used 6-OHDA for the preparation of

5r6-dihydroxyindole (7> [39], ."Xr? Xo 1 a.

It is not clear which species, the p-ouinone 4 or an

oxidized product of the indole _7 midht be responsible for

the neurotoxicity of 6-OHDA, That the indole is involved in

the neurotoxicity seems reasonable on the basis of the observed neurotoxicity of the 5,6- and

5,7-dihydroxytryptamines (3a) and (3b) for serotonerdic neurons [25,40], However, 5,6-dihydroxyindole is formed only very slowly at 37°C vitro [35], inconsistent with the observed rapid bindind of a 6-OHDA metabolite to cell particulate fractions.

With an understandind of the fate of 6-OHDA under physiolodical conditions, Adams and coworkers investidated the fate of 6-OHDA injected centrally, Usind brain implanted electrodes [41,42] they observed that 6-OHDA was rapidly oxidized to the p^ouinone [43], After the rapid initial formation of auinone there existed a steady state balance of 10

6-OHDA to p-RUinone in a 60/40 ratio. Sinii la rl^ii f injection of pure ^-auinone stave rise to a rapid reduction to 6-OHDA and a similar 6-OHDA/auinone eouilibrium ratio of 60/40

[43]. The concentration of both components were reduced rapidly with a half life of approximately 25 min» maintaining however the ubiouitous 60/40 ratio. Under the same conditions dopamine had a half life of approximately

150 min. They found some evidence for the formation of an indole product» but this could not be completely substantiated [43].

With the appreciation of the alkylating potential of the auinone 4 derived from 6-OHDA there have been a number of studies attempting to isolate and characterize such alkylated proteins. Using adrenochrome as a model substance

Powell and Heacock have isolated an N-acetyIcysteine conjugate from ^ vitro alkylation of adrenochrome C44»45].

Adams' group studied the same problem and found that dopamine could be alkylated» as the auinone by a number of nucleophilic species [46]. They noted that the o-auinone reacted readily with free sulfhydryl but not amino groups and this alkylation process occured more auickly than the cyclization process or the reduction by ascorbic acid»

Scheme 4. The ascorbate reduction process was» however» faster than cyclization to the aminochrome and prevented its formation. The possibility that 6-OHDA could be formed in vitro and i£i vivo from dopamine o_-auinone was examined.

Although a hydroxyl function could be added to the auinone this process was slow and was completely inhibited by the 11

presence of ascorbic acid in concentrations found

intraneuronally♦ This led to the conclusion that jjT. vivo

formation of 6-OHDA was very unlikelyr an interesting

observation in lidht of several proposals correlating

endogenous synthesis of 6-OHDA with schizophrenia [47-49]*

Scheme 4

NHg'SseorbIc acid

RNHg / \ R S H slow \ fost

h O''^**S^^SR

This work was extended to 6-OHDA and it was found that

6-OHDAf in an oxidized form, reacted irreversibly at p H? with glutathione (GSH)* Alkylation was shown to occur rapidly ^ vitro at p H7 to form the 2-GSH derivative

C50f51]. Quite recently this in vitro GSH adduct has been purified and shown to be 2* Adams' group has also demonstrated, with high pressure chromatography and electrochemical detection, that the same product arises in vivo after 6-OHDA administration and accounts for approximatly 0*2% of the administered dose [50]* This conjugate was shown to underdo rapid oxidation and cyclization to an indole derivative* 12

HO^-CHg-NH^-ÇH-NHÇ-CHjCHjj-CH-CO^ O ÇH2 O NHg

a

A second theory of the neurotoxicity of 6-OHDA based on its facile oxidation has been presented by Heikkila and and

Cohen [52,53], In the process of 6-OHDA oxidation the complementary reduced species has been shown to be the reduction product of oxyden» hydroaen peroxide, Hydroden peroxide itself is a toxic species, and its presence could be responsible for the observed neurodestruction, while the alkylatind ability of the auinone would be an artifactual process. Scheme 5,

Scheme 5

SOD Removal of O j

HO + 02 HO

Studies of catecholamine uptake vitro, which is inhibited by 6-OHDA induced neurodestruction, showed that adents capable of preventind hydroden peroxide formation or capable of its scavendind prevented uptake inhibition. Thus 13

catalase, a hydroden peroxide scavendind enzyme? prevented

the catecholamine uptake inhibition caused by 6-OHDA?

dialuric acid or hydroden peroxide [52], The addition of

ascorbic acid was found to increase the inhibition of

dopamine uptake jj] vitro caused by 6-OHDA but had no effect

on uptake by itself [53], The explanation presented for this

observation was that ascorbic acid caused a continuous

reductive redeneration of 6-OHDA from the auinone 4 thus

allowind it to adain be oxidized and denerate more peroxide.

The net effect would be to increase the hydroden peroxide

concentration while minimizind the amount of the potential

alkylatind auinone.

More recently emphasis has shifted to a study of the

importance of the superoxide and hydroxyl radicals in

mediatind 6-OHDA autoxidation and its neurotoxic properties

[54-56], Catalysis of superoxide radical dismutation to

hydroden peroxide and oxyden by superoxide dimutase (SOD)

was shown to inhibit the autoxidation of 6-OHDA? but not

6-aminodopamine (6-ADA)? and prevent neurodestruction by the

former? but not the latter. The presence of the hydroxyl

radical scavender? l-phenyl-3-(2-thiazolyl)-2-thiourea

(PTTU) prevented the autoxidation of both 6-OHDA and 6-ADA

and the neurotoxic properties of both compounds. It has been

reported that PTTU by a different mechanism prevents protein

alkylation? allowind no conclusions to be drawn on the

importance of the hydroxyl radical [57],

Various droups have studied the involvement of the

_p-auinone? the indole? hydroden peroxide? the superoxide 14

radical and the hydroxyl radical in the mediation of the effects of 6-OHDA and 6-ADA, Although no common mechanism has been exclusivly shown to be involved it does appear that oxidation of 6-OHDA is in one way or another is reouisite for neurotoxic activity. It may be that it is the cumulative effect of hydroSen peroxide and alkylation which is responsible for the neurotoxic properties of of 6-OHDA and

6-ADA, Certainly the best approach to discriminating between the ouinone and peroxide mechanisms would be by the study of a compound which could underdo only one of these processes

[57], An interestind compound that should have supplied such information, 2,5-dimethyl-6-hydroxydopamine (10), which should not be alkylated was found to not be taken u p in into the neuron. It was also found that this compound reacted irreversibly with proteins and was an irreversible inhibitor of catecholamine-O-methyl transferase (COMT) [58], 6-Hydroxydop3mine Analods

A large number of analogs of 6-OHDA have been prepared to

study the factors influencing its neurotoxic properties*

Listed in Table 3 are those analogs of 6-OHDA whch differ

from 6-OHDA in their aromatic substitution* It is apparent that neuronal uptake of an agent is reouisite for neurotoxicity? thus a number of agents that might be expected to cause neurodestruction are not found to do so because of poor uptake properties. It should be noted that all compounds which were found to be neurotoxic caused release as well as short term depletion of catecholamines while the corollary was not true? not all those agents which caused release were neurotoxic*

A general structure activity relationship may be derived for neurotoxicity* A phenethylamine structure which contains at least three hydroxyl groups in an orientation such that a

^-ouinone may form appears to be requisite for sympathectant properties* At least one of the hydroxyl groups may be replaced by an amino group without a loss in activity? as in compounds 22? 2^? and 24* An exception to the above is compound ^ which although it possesses the correct orientation of hydroxyl groups is not taken up into the neuron *

Compound 19? a potential metabolite of 6-hydroxydopamine and the enzyme catechol-O-methyl transferase has unusual properties. It has no long term depleting effects on 16

Table 3 - The Activity of 6-hydroxydopamine Analods* Aromatic Substitution Variants

NH;

Compound Destruct*® Deplet.b Uptake^ Ref*

(11) R1=R2=R3=0H - + ++ 59,60,61 (12) R1=R2=R4=0H + ++ ++ 59,60,61 (13) R1=R2=R5=0H - + + 60,61 (1) R1=R3=R4=0H + ++ ++ 4,59,60,61 (14) R1=R3=R5=0H N + - 60,61 (15) R2=R3=R4=0H - ++ ++ 59,60 (16) R1=0CH3 R3=R4=0H N + N 59 (17) R2=0CH3 R1=R4=0H N + N 59 (18) R3=0CH3 R2=R4=0H N ++ ++ 60,61 (19) R4=0CH3 R1=R3=0H * + - 60,61,62 (20) R1=N02 R3=R4=0H - ++ N 59 (21) R3=N02 R1=R4=0H N + N 59 (22) R1=NH2 R3=R4=0H + ++ ? 4,58,59 (23) R3=NH2 R1=R4=0H + ++ N 59 (24) R4=NH2 R1=R3=0H + ++ N 59 (25) R1=0H R2=0CH3 R4=NH2 N - N 59 (26) R2=NH2 R1=R5=0H N - N 59 (27) R1=R2=R3=R4=0H + ++ + 60,61 (28) R1=R3=R4=0H R2=CH3 N NN 58 (29) R1=R3=R4=0H R5=CH3 N NN 58 (10) R1=R3=R4=0H R2=R5=CH3 NN 57

a) Ability to cause Iona term depletion of norepinephrine ^ 1 vivo, ie, destruction <+>-causes Iona term depletion, (-)-no Iona term depletion noted, (N)-not reported, (*)-reported to deplete dopamine but not norepinephrine b) Ability to cause short term depletion of norepinephrine <++)-aood depletion, (f)-poor depletion, (-)-no depletion, (N)-not reported c) Ability to inhibit norepinephrine uptake (++)-aood inhibition, (+)-poor inhibition, (-)-no inhibition, (N)-not reported 17

Table 4 - The Activity of 6-hydroxydopamine Analogs! Side Chain Variants

HO OH

Compound® Destruct* Deplet, Uptake Ref»

(30) R1=R2=CH3 N - 61 (31) R3=CH3 + ++ ++ 59,60,61 (32) R4=R5=CH3 N N N 63 (33) R4=0H - - - 60,64 (34) R3=C02H + N N 65,66

a) See Table 3 for ledend

adrenerdic neurons but is hidhly effective in depletind dopaminerdic neurons C62II» This is rather remarkable as this compound should not be able to form a p-ouinone. The only compound in Table 3 that midht have allowed some comment on the molecular mechanism of 6-hydroxydopamine, compound jhO was found not to be taken u p into the neuron [57],

A number of analods which deviate from 6-OHDA in their side chain substitution have been studied. Table 4» The

N,N-dimethyl analod 30 was found to be inactive because of poor uptake properties as was the 6-norepinephrine analod

33, The lack of neurotoxicity of 33 is not as remarkable as 18

its lack of affinity for the uptake mechanism. It has been

proposed that this lack of affinity may be due to an

unfavourable conformation imposed by intramolecular hydroSen

bondinS C603, The alpha-methyl analod 3^ was found to be as

potent as 6-OHDA indicating that the additional methyl droup has no effect on uptake or neurotoxicity.

The 6-hyroxydopa analod 34 has a number of interestind properties. Unlike 6-OHDA it was able to cross the blood-brain barrier and effect a temporary central sympathectamy, It was shown that 3^ had no depletind effects itself but was converted to 6-OHDA, Studies with the optical isomers has revealed that the (-)-isomer was far more potent in depletind central and peripheral neurons than the

< + )-isomer C65»66II,

Studies with 6-aminodopamine analods have revealed that they possess a similar structure activity relationship [67], 19

Dopamine as a Central Neurotransmitter

The initial perception of dopamine (^) soley as an

intermediate in the biosynthesis of norepinephrine (NE) and epinephrine in the central and peripheral nervous systems has gradually been expanded [68r69], The discovery in 1957 that dopamine constituted u p to 50% of the total catecholamine content of mammalian brain [70,71] with a

localization distinct from that of norepinephrine [72] led to the proposal of a discrete dopamine mediated nervous system [72], Considerable evidence since then has supported the proposition that dopamine midht have important central neurohormonal properties [68,69],

Studies of the central distribution of dopamine have shown it to be highly concentrated in the retina, hypothalamus, basal ganglia and cerebral cortex [69], In particular the basal ganglia or caudate nucleus is very rich in dopamine and has been a valuable tissue for studies of central dopaminergic mechanisms. The study of the properties of dopaminergic neurons has been hampered by the lack of suitable peripheral dopaminergic neurons. Thus, while adrenergic and cholinergic receptor mechanisms have been studied with good success on peripheral tissues, dopaminergic studies have relied on brain homoSenates for in vitro assay and behavorial studies for iri vivo assay.

With the study of the central role of dopamine a number of clinically important discoveries have been made, A 20

destruction of the nidrostriatal pathway with a concomitant

decrease of dopamine levels in the neostriatum has been

implicated in the etiolody of Parkinsonism and motor

dysfunctions l!69?73»743. Recently a number of studies have

related the efficacy of neuroleptic adents with their

ability to modify the dopaminerdic nervous system C68;75-77]

while other studies have shown that the dopaminerdic

tuberoinfundibular neurons are involved in

hypothalmic-pituitary control [69,78],

A model for the dopaminerdic synapse has been presented

which is ouite similar to that for the central adrenerdic

synapse C79], Tyrosine is actively concentrated into the

presynaptic dopaminerdic neuron where it is converted to

dopa by tyrosine hydroxylase and then to dopamine by

enzymatic decarboxylation. Unlike the adrenerdic neuron

there is an absence of dopamine-/î-hydroxylase and the

dopamine is seauestered directly into dranules.

Depolarization of the neuron results in the release of

dopamine into the synaptic cleft where it is available for bindind to the postsynaptic receptor. In deneral it has been

observed that dopamine mediates an inhibitory response and

results in decreased postsynaptic firind [69], The effects of dopamine as with norepinephrine are terminated either by metabolism or reuptake into the presynaptic neuron. As with the adrenerdic system there appears to be a presynaptic bindind site which controls dopamine release and synthesis

[80,81],

Pharmacolodical studies have confirmed the similarities 21

between the dopsmineraic and adrenerdic neurons, In deneral

both systems display a similar response to a variety of

pharmacolodical adents, varyind in Quantitative rather than

Qualitative response [69], With the recodnition of the

importance of the adrenerdic and dopaminerdic systems in

mediating central functions it has become particularly

relevant to study the differences between the systems so that more selective adents may be developed.

Presynaptic Uptake by Dopaminerdic Neurons

In vitro studies with tissue slices or synaptasomes from the corpus striatum have demonstrated the presence of a hidhly specific mechanism for the accumulation of dopamine

[81-83], The affinity of the uptake system for dopamine (2) is greater than for other biodenic amines? in particular the central neurotransmitters serotonin (35) and norepinephrine

<36) [83], Similar dopamine uptake processes have been identified in other dopamine innervated areas? the median eminence? olfactory tubercle? nucleus accumbens? limbic cortex and the retina [81],

The uptake system not only displays a greater affinity for dopamine than norepinephrine but most compounds which 22

are potent inhibitors of the adrenergic uptake system are

relatively poor inhibitors of the dopamine uptake system

[84]* In deneral it is observed that the tricyclic

antidepressants are dood inhibitors of the adrenerdic but

not dopaminerdic uptake system* Catecholamine derived uptake

inhibitors show a similar spectrum of activity as for

adrenerdic neurons but denerally with lowered potency and

less sensitivity to chirality [85,863*

The most potent dopamine uptake inhibitors have been

found to be those compounds which possess two aromatic

rinds, an amine function as well as a dedree of

conformational flexibility C81D* Benztropine (37), the

prototype of dopaminerdic uptake inhibitors, is a potent

inhibitor of the dopaminerdic but not adrenerdic uptake

systems by a mechanism unrrelated to its cholinerdic

activity C84,87]* More recently the tricyclic compound

CP-24,441 (38) has been shown to be a potent inhibitor of

dopamine uptake with the geometrical and optical isomers

displaying larde differences in activity [88]* Other potent

inhibitors include Mazindol and Nomifensine [89] which like

benzotropine and CP-24,441 possess two aromatic rinds with

the potential for a hidh dedree of conformational

flexibility [88]» Koe has presented a model for the uptake bindind site which attempts to correlate the structurally diverse compounds which are able to bind to an apparently

identical site [88]* 23

-CH,

A number of studies have addressed themselves to determination of the prefered conformation of dopamine at this uptake site* Dopamine is a conformationally flexible molecule with the possibility for any number of orientations of amine and the aromatic rind* Theoretical conformational enerdy calculations have predicted that a trans orientation of aromatic rind and amino droup is only slidhtly favoured over the dauche confomer [90-92]» Fidure 1* The enerdy difference between the conformera is only minimal and it has been observed with NMR studies that the two dauche and one trans conformera are almost eoually populated in aoueous solution at ambient temperatures [90-93]. It is impossible to predict from these studies what is the prefered conformation of dopamine at the uptake site because the enerdy barriers to rotation are too low to allow discrimination of a particular conformer* 24

Figure 1 - Conformations of Dopamine

H NHg A

«•■ 60 * e-«teo*

The deneral tactic used to dain information about the

conformation at the uptake site has been with analods which

are able to define only one or at worst a few of the

possible dopamine conformations [81], Optimal analods would

be those which would have minimal variations in phsical

properties yet rididly define a certain confomer of

dopamine, Conformationally restrained analods which have been tested for uptake inhibition include the cis- and

trans- 2-phenylcyclopropylamines (39a) and (39b) respectivly

[94 » 95]f 2-amino-6» 7-dihydroxy-1 » 2 » 3 r 4-tetrahydronapthalene

(6f7-ADTN) (_40) and 6»7-dihydroxytetrahydroisoauinoline <41 ) analod [81,95], In deneral it has been observed that the trans analods have a dreater ability to inhibit uptake than the correspondind cis analods [81], Thus, in the cyclopropyl series the trans-isomer 39b was more potent than the cis-isomer 39a and 6,7-ADTN (40) more potent than 41, Results wi th decal in analods of norepinephrine are at variance with this and it was observed that the dauche isomer was a more potent inhibitor than the corresponding trans isomer [96], Apomorphine (42 ) > a potent dopaminerdic adonist which has a trans disposition of of phenyl and amine functions is a poor inhibitor of dopamine uptake [97], This may be due to the unfavourable ridid and planar disposition of the two aromatic rinds rather than an unfavourable trans alignment,

Studies to date would indicate that a trans or pseudo-trans orientation of phenyl and amine groups defines the prefered conformation of dopamine at the pre-synaptic uptake site [81], q> Q .xa „

22a NHg

hoAJ ü

il The Pre-synaptic Dopamine Receptor

Only recently has there been recognized a dopamine

bindind site associated with the pre-synaptic dopaminerdic

neuron [80]* That dopamine bound here was oridinally

postulated when it was observed that apomorphine (42)r a

dopamine adonist reduced the stimulation overflow of

dopamine C99]* Additional studies showed that dopamine was

also capable of reducind the stimulation evoked overflow by

activation of a presynaptic dopamine receptor* Activation of

the presynaptic receptor has been observed not only to

reduce the release of dopamine but also inhibit the enzyme tyrosine hydroxylase and dopamine synthesis [100]*

It is not clear what clinical sidnificance the presynaptic receptor has* Low doses of apomorphine (42 )» which midht selectivly activate the presynaptic receptor were observed to increase locomotor activity in mice [101]*

Additionally it has been shown that the neuroleptics inhibited the presynaptic effects of dopamine and apomorphine [80»102]* Contrary to this has been the report of neuroleptic inhibition of stimulation evoked dopamine release beind directly related to jjn vivo antipsychotic activity [75]*

Initial reports suddested that the presynaptic mechanism midht be mediated by an adenylate cyclase [100] but studies have shown that c-AMP increases rather than decreases tyrosine hydroxylase activity [100]* It has been alternatively suddested that the receptor midht control calcium permiability [103]* 27

There have been no reported attempts to determine the

prefered conformation of dopamine at this presynaptic

receptor site. Rotation about the aromatic-carbon bond (bond b in Fidure 2) has been proposed to be of importance for pre- and postsynaptic bindind ability [98], This scheme

suddests that a trans-cisoid (trans with respect to bond a, cisoid with respect to bond b) conformation of dopamine should bind preferentially with the presynaptic receptor

[98], Fidure 2,

Fidure 2 - Rotational Considerations of Dopaminerdic Adents

HO NHg b a HO

HO

HOX j u , Irons- tronsoid trons-cisold III

HO

HOXXX

6,7-ADTN Apomorphine

IS. ÛZ

Postsynaptic Dopamine Receptor Models

A maJor problem with the study of dopaminerdic adonist and antadonist interactions with postsynaptic dopamine receptors has been the inability to define with confidence a postsynaptic dopamine receptor. As a result a variety of 28 postsynaptic dopaminerslic models have be?en presented and studied C104]* ^ vivo studies of the postsynaptic receptor have generally concentrated on central dopamine receptors and the behavorial effects of dopaminergic aSents [69],

Other assays or models of the p-ostsynaptic dopamine receptor studied include the dod renal artery, the central neurons of

Helix Aspersa, s dopamine sensitive adenylate cyclase from rat striatum and a dopamine specific bindind site from calf and rat striatum.

a. Renal Vasodilation Model

It was recognized by Goldberg that renal, mesenteric, coronary and cerebral vascular beds contained a specific dopamine receptor mediating vasodilation [105,1063, Studies of the dog renal artery have revealed that the dopaminerdic effect is not mediated by an alpha- or beta-adrenerdic mechanism but is selectively activated by dopamine as well as dopaminerdic agonists including apomorphine (42)

[106,1073.

A potency series for renal vasodilation has been constructed [106,1083, dopamine (^_) = epinine (N-methyl dopamine) > 6-n-propylapomorphine > apomorphine (42), The vasodilation is antagonized by the neuroleptic adent haloperidol although only under strictly defined conditions

[1083, Whether this system represents a true dopaminerdic receptor is not clear, several centrally active dopaminerdic adonists, notably piribedil and amantadine have no 29

vasodilating! properties.

b. The Central Neurons of Helix Aspersa

Studies with the snail Helix Aspersa» an evertabrate»

have revealed that it posseses neurons selectivly sensitive

to the effects of dopamine C104»1093, Two types of central

dopaminerdic neurons have been defined» one on which

dopamine acts as an adonist to elicit excitation and another

on which dopamine as an adonist has inhibititory properties

[109], The particular virtue of these neurons is that their

activity is retained after surdical removal allowind the in

vitro assay of dopaminerdic adents [109],

Dopamine is an adonist at both sites» inhibitind firind

of inhibitory neurons (desidnated DA-i) and excitatory on

the other (desidnated DA-e), Other dopaminerdic adents

denerally have actions on only one or other of the two neurons. Results obtained with the study of a number of dopaminerdic adonists and antadonists illustrate this»

Table 5 CllO-112],

Whether the dual nature of the dopamine receptors of the

Helix Aspersa can be extended to the mammalian brain is not clear» Cools has proposed the presence of two analodous dopamine receptors in the brain of rat and cat on the basis of behavioral and electrophysiolodical studies C113»114], 30

Table 5 - The Activity of Dopaminergic Agents on the Neurons of Helix Aspersa

Drug DA-i DA-e receptors receptors

Dopamine (2) Full Agonist Full Agonist Apomorphine (42) Antagonist Partial Agonist Piribedil (43) Inactive Inactive DPI (44) Full agonist Inactive Ergometrine (45) Antagonist Antagonist Haloperidol Inactive Antagonist Chlorpromazine Inactive Antagonist 5,6-ADTN (40) Agonist Inactive

DPI is 2(3'f4-dihydroxyphenylamino)imidazoline

C» Dopamine Sensitive Adenylate Cyclase Model

Among the first vitro assay systems presented for

dopaminergic activity was a dopamine sensitive adenylate

cyclase from rat basal ganglia and retina [68,115], That

this adenylate cyclase was associated with a postsynaptic

dopamine receptor was demonstrated by its localization

coincident with dopamine containing terminals, its absence

in norepinephrine innervated areas and its retention after presynaptic lesioning with 6-hydroxydopamine [68],

(-)-Norepinephrine is about 20 fold less active in

activating the adenylate cyclase than dopamine and alpha- but not beta-blockers are weak an agonists [68,116],

Among rigid analogs studied apomorphine (42) was found to 31

be 3 partial adonist stimulating c-AMP formation to only 45%

of the dopamine induced maximum Cl 173» Amonsi the closely

related ridid compounds studied? 6?7-ADTN (40) was found to

ha potent stimulator of adenylate cyclase while the correspondind cis-isomer 6 »7-dihydroxytetrahydroisoQuinoline

<41) was considerably less potent and led to the proposal that the prefered conformation of dopamine at the postsynaptic receptor was trans extended C68]«

While the adenylate cyclase model does possess many properties that would be expected for the postsynaptic dopamine receptor there are several discrepancies. It has been observed that the neuroleptics effectivly inhibit the effects of dopaminerdic adonists but the potency of the butyrophenones relative to the phenothiazines does not parallel their activity in other systems [117-1193, The phenothiazines are observed to be better inhibitors of the adenylate cyclase than the butyrophenones an observation which has been suddested to perhaps be due to differences in absorption? distribution or metabolism in the various systems C683,

d. Membrane Bindind Models

Snyder and coworkers C120-1233 and Seeman et al [1243 have recently reported isolation of a membrane from calf and rat brain which binds dopamine and haloperidol in a saturable fashion. Regional variations of this bindind parallels dopaminerdic innervation and does not appear to vary between limbic and striatal areas. The bindind site

shows a 10 fold dreater affinity for dopamine (2) than

norepinephrine (35) while y-aminobutyric acidr carbachol and

serotonin all bind poorly to this membrane receptor [123]*

This membrane preparation appears to possess two

dopaminerdic bindind sitesr one associated with the

selective bindind of dopaminerdic antadonists labeled the

haloperidol site? and an adonist bindind site labeled the

dopamine site. Most dopaminerdic adents appear to bind

selectivly to one or other of the sites. Dopaminerdic

antadonists, the phenothiazines and butyrophenones have a

hidh affinity for the haloperidol but not dopamine bindind

site, similarly dopaminerdic adonists such as 6,7-ADTN (40)

bind preferentially to the dopamine site. A third class of

compounds which includes apomorphine (42) have the ability

to bind well to both sites, in deneral these compounds are found to be mixed adonist-antadonists and include LSD and erdometrine. A larde number of adents not considered to be dopaminerdic adents have very little affinity for either site, these include morphine, diazepam, psilocybin,

isoproteranol and tranylcypromine. It has been suddested

that the haloperidol and dopamine bindind sites midht not be

Physically distinct but rather reflect two conformational states of the same receptor.

This bindind site shares many properties in common with other proposed dopamine receptors. Apomorphine (42) has mixed adonist-antadonist properties in this bindind assay as

it does in the dod renal artery, dopamine sensitive J3 adenylate cyclase and Helix Aspersa, Also, both this membrane bindind site and the adenylate cyclase sensitive

receptor bind LSD as a mixed aaonist-antadonist. Despite the somewhat similar structure activity relationships for both of these dopamine receptor models they do not appear to be the same receptor. Rather larde variations occur in the bindind of dopaminerdic antadonists with the membrane bindind model bindind the butyrophenones to a dreater extent than the phenothiazines while the reverse is true for the adenylate cyclase model.

No studies have been reported which have addressed themselves to a study of the prefered conformation of dopamine at this site, Snyder noted that 6r7-ADTN» a trans-analod of dopamine, was bound as well as was dopamine

[123], Quite recently another droup, usind 6,7-ADTN (40 ) as the dopaminerdic lidand has reported characterization of a dopamine receptor from rat striatum [125], Althoudh this model closely parallels that from calf brain there are some differences in bindind constants and it cannot be concluded whether this bindind site is the same as that reported from calf brain.

e. Behavioral Studies

The realization that dopamine midht have central neurohormonal proerties has prompted many investidators to study the effect of suspected dopaminerdic adents on behavior [69], Behavioral patterns variously ascribed to dopamineraic control include stereotypic behavior -

sniffind» lickind» bitinsl and repititious head movements

[126] as well as hyperactivity and dnawind behavior [69],

Most dopaminerdic adonists also are potent emetics and this

property has proven to be a valuable assay procedure for

dopaminerdic activity although it does not appear to be

mediated via a central mechanism [127],

Pharmacolodical assays generally consist of central

administration of the dopaminerdic adent and observation of

the induction of stereotypic behavior and/or hyperactivity,

Additionally» turning behavior after unilateral central

dopaminerdic lesionind» has proven to be a valuable assay of

dopaminerdic activity and allows discrimination of pre- and

postsynaptic mode of action [128],

Certainly the lardest number of studies of a wide variety

of dopaminerdic adonists and antadonists have been with such

in vivo assays, A direct comparison of results is not always

feasible due to variations in techniques» species studied» drud pretreatment and method of assay. In deneral it is observed that the central administration of dopamine (2_) and apomorphine (42) induces hyperactivity and stereotypy in

rats which is antagonized by the butyrophenones and phenothiazines [69»113], Both compounds have a rather short duration of activity which is potentiated by the monoamine oxidase inhibitor nialamide [129], More recently studies of the postsynaptic dopamine receptor have focused upon the use of dopamine analogs in an attempt to define the nature of the central receptor. 35

A larde number of studies with analods of dopamine have

allowed construction of a structure activity relationship

for behavioral effects C1303* Dopamine (2> appears to

contain the minimum requirements for dopaminerdic activity»

replacement of one or both of the hydroxy droups with

hydroden results in a larde decrease in central activity.

Méthylation or methylenedioxylation of the catechol function

also results in almost complete abolition of stereotypic and

hyperactive activity. The same is true for apomorphine (42)»

any modification of the catechol portion of the molecule

results in larde decreases in dopaminerdic activity

[131,132], The two carbon connection between the aromatic

and amine functions appears to be optimal for activity»

extension or contraction by one carbon unit abolishes

dopaminerdic activity [130],

As described earlier with respect to the presynaptic

uptake of dopamine» dopamine has the ability to assume a

number of conformations with little enerdy distinction between them» Fidure 1, A number of studies have attempted to determine the conformation of dopamine which binds at the postsynaptic receptor by the study of tha effect of conformationally restrained analods on behavior [133],

Studies with 6»7-ADTN <40) and the isoouinoline analod 41 have shown the trans-analod 40 to be more potent in stimulating! locomotor activity than the cis-analod ^ [134],

Studies with an interestind pair of apomorphine analods which define cis and trans conformations have shown the trans-analod to be more potent. Thus apomorphine (42) 36

induced stéréotypé and hyperactivity while the cis analoS

0;0-desmethylnuciferene <^) was devoid of dopaminerdic

activity [140],

A third set of analods which further confirm the

preference for a trans orientation of amine and aromatic

function at the postsynaptic receptor are the cyclopropyl

analods 47a and 47b« Only the trans-analod 47b was active in

stimulating hyperactivity and stereotypy C129]»

Whether the study of behavioral effects of dopaminerdic

adents truly reflects affinity for a dopamine receptor is

not clear. Recently it has been suddested independently by

two droups that there are perhaps two or more dopamine

receptors mediating behavior [113,135]* It would seem that the best method for investigation of this proposal and the nature of the dopaminerdic receptor is by the use of dopaminerdic analods and comparison of their activities in a variety of systems.

4Za lit 37

OBJECTIVES

The use of riaid and conformationally restrained analods has proved invaluable in the elucidation of conformational reouirements for the activity of a variety of pharmacolodical adents. By definition of the dihedral andle between important structural components in the molecule it is possible to mimic a conformation or conformations of the labile molecule studied. Optimal analods would be those which have a miminal deviation in molecular parameters and physical properties from the parent molecule,

A variety of molecular systems have been prepared and studied in an attempt to rididly define particular conformations. The success of these analods have been variable, A number of systems, includind the octahydrophenanthrene [136] and decal in analods [137-138] of adrenerdic adents have allowed some comment on conformational preferences for such activity but suffer from introducind an unnatural bulk and hydrophobic portion to the molecule, Analods which introduce smaller bulk, includind the cyclopropyl moiety have met with dood success [139-141],

They are capable however of definind only specific dihedral andles and various analods must be employed to define a whole spectrum of dihedral andles, Fidure 3, The cyclobutyl moiety offers an attractive method for the definition of a trans or cis disposition of droups vicinal to one another, A 38

cia -Lr2 substitution defines a variable dihedral andle of

-10 to +40 dedrees while a trans-1>2 substitution defines a

variable dihedral andle of +110 to +160 dedrees [142-143],

Fidure 3 - Newman Projections of Several Ridid Analods

In d o n ti Cyelobuianfi

*— 1:0 * ••-MO*

Cyclopropon» GtnioblcycloocKnat

►•-150* • •l«0

A number of cyclobutyl analods of pharmacolodically

active adents have been prepared and found to dive variable biolodical activity [144-146], In deneral: while cis and

trans isomers have displayed differential activity and allowed some comment to be made about prefered conformations of the systems studied they have been found to be less potent than the molecules studied.

The purpose of this research then was to 1) devise a stereoselective synthesis of 2-aryIcyclobutylamines» 2) usind this stereoselective scheme prepare cyclobutyl analods of 6-hydroxydopaminep 3) prepare cyclobutyl analods of dopamine, 4) prepare cyclobutyl analods of the hallucinodens

2,4,5-trimethoxyamphetamine and methylenedioxyamphetamine. 39

1. The preparation of 2-aryilcyclobutylamines has been

reported by Beard and Burden C145] and Miller et al C1473*

The recent report of a facile preparation of cyclobutanones

C148fl49] from the corresponding aldehyde and ketone offers an attractive route to 2-arylcyclobutylamines* Several experimental complications have precluded the stereoselective conversion of these cyclobutyloximes to the cis and trans amines. It was the aim of this research to develop a deneral stereoselective synthesis of cis and trans

2-arylcyclobutylamines from the correspondind aldehydes.

2. There is some Question as to the relative importance of indoline formation as a reouisite step in the neurotoxic properties of 6-OHDA. It has been shown that 6-OHDA can form an adrenochrome intermediate vitro and possibly also in vivo and additional studies have shown that adrenochrome is a dood alkylatind adent. To clarify its importance the conformationally restrained analods 48 and ^ were prepared, the trans analod ^ would not be expected to form an adrenochrome like structure. An assay of its neurotoxic properties midht then allow some comment on the relative importance of indoline or adrenochrome formation in the neurotoxic mechanism of 6-OHDA.

Additionaly the analods and ^ should allow some comment about the prefered conformation of adrenerdic adents at the pre- and postsynaptic receptor* 40

HO NH, Br ÛSL

3« The realization that dopamine miaht have important

neurohormonal properties has prompted studies of the

conformational reeuirements for dopamine at the uptake site

and postsynaptic receptor. A number of conformationally

restrained analods of dopamine and apomorphine have been

prepared to permit such studies. Dopamine analods includind

apomorphine» 6.7-ADTN» 5»6-ADTN» 0 »0-desmethyInuciferene and

2-amino-6»7-dihydroxytetrahydro isoouinoline have indicated a preference pre- and postsynaptically for a trans disposition of amine and aromatic moieties. To verify these conclusions with analods which have a minimal variance from the dopamine structure the cyclobutyl analods ^ and 5J^ were prepared and studied for pre- and postsynaptic activity.

Additionally the dimethyl analods ^ and ^ were prepared to study the effect of N-substitution.

HO. H(\

HO HO- \ = / NHR2 Br

NHRg B r'

5 2 R - H &1 R«H

5 2 , R ■ CH5 6 3 R'CH, 41

4. It was recognized that by a suitable choice of

synthetic route that the cyclobutyl analogs of the

hallucinogenic compounds 2»4r5-trimethoxyamphetamine (54)

C1501 and 3r4-methylenedioxyamphetamine (^> [151] would be

intermediates* By the study of a limited number of

conformationally restrained analogs of the hallucinogens it

has been suggested that there is a preference for a trans

orientation of amine and phenyl functions [152]» The analogs

56 1 5 7 f 58 and 59 should allow with suitable testing further comment to be made on the conformational preferences of the hallucinogens at their active site,

CHjO OCH

CHjp R| gfi Rj^NHjCf 5â R," NH, Cl' Rj-H 5Z R|-H fVNH, Cf 52 R,'H Rs^NHjCl"

5, In the course of these and related studies a novel rearrangement of 2f4,5-trimethoxypropiophenone was discovered* A partial study of its properties and a proposed mechanism are presented* 42

RESULTS AND DISCUSSION

This section has been divided into a presentation of the synthetic and biolodical portions of this study. The synthetic presentation is divided into three sections corresponding to 1) the preparation of the

6-hydroxydopamine analogs 2) the preparation of the dopamine analogs 3) the rearrangement of

2f4»5-trimethoxypropiophenone. The biological section which immediatly follows the synthetic presentation is similarly subdivided.

Synthetic

A) Preparation of the cyclobutyl analogs of

6-hydroxydopamine»

cis- and trans-2(2't A ' »5'-trihydroxyphenyl)cyclobutylamine

hydrobromide and (48)

The synthesis of the analogs of 6-hydroxydopamine and

2r4r5-trimethoxyamphetamine is outlined in Scheme 6. The initial step in the scheme» preparation of

2(2'»4'»5'-trimethoxyphenyl)cyclobutanone (61) was attempted by the method of Trost and Bogdanowicz C148»149]. Although

Miller et al C147] and Beard and Burger [145] have prepared

2-arylcyclobutylamines by different methods» it seemed that intermediacy of a 2-arylcyclobutanone offered the the best application to a variety of cyclobutyl analogs. 43

Scheme 6

OCH CH

H

K0fBu/-l5°/THF CH,0 OCH; SO 61

NH OH-HCI NaOH/EtOH

NOH 62 Ha/ptOg DNo/iPrOH \cHCI,/EtOH 2)HCI CH,0 OCH OCH

NH

S6 57

HO. OH HO OH

NH HO HO NHNH, Br + ^ 49 44

Cyclopropyldiphenylsulfonium fluoroborate (64) was

readily prepared by the method of Trost and Boddanowicz»

Scheme 7» It was found that methylene chloride was a more

convenient solvent for the preparation of ^ than

nitromethane with no decrease in yield noted [153], The

cyclization of 63 to 64 with sodium hydride in THF Save dood

results while cyclization with potassium tjbutoxide in

dimethyl sulfoxide [154] dave rise to many products and the

ylid precursor 64 was obtained in very poor yield.

Scheme 7

PhgS/AgBF* CHjClj

^ [ > - S P h 2 r * A bV §3 I______KOlBu/DMSO______y ^ Sd

Trost and Boddanowicz found that the best yields of a variety of cyclobutanones could be achieved by reaction of an aldehyde or ketone with the ylid denerated by reaction of

6^ with potassium hydroxide in dimethyl sulfoxide [149],

This reversible ylid they found dave better yields than the ylid denerated in an irreversible fashion with the sodium dimsylate anion in DMSO C149], Use of either of these conditions to denerate the cyclobutanone from the aldehyde 60 dave poor yields of cyclobutanone. Even after 45

extended periods of reaction there remained larde amounts of

starting material as well as a number of unidentified side

products♦

To optimize the yield of ketone for academic as well as

economic reasons» a number of other methods for ylid

generation were studied and are listed in Table 6,

Table 6 - Effect of Reaction Conditions on Yield of 61

Readent Temperature Results

K0H(10ea)/DMS0 25°C poor yield

Dimsyl anion/DMSO 25°C poor yields

NaH/THF 25°C no reaction

K0H(2ea)/THF 0°and 25°C no reaction

K0H(2ea)/THF/DMS0 0°arid 25°C slow» poor yield

NaOCH3/THF 0 and 2 5 °C no reaction

KOtBu/THF 25°C many products

KOtBu/THF 0°C variable yield

KOtBu/THF 15°C dood yield

It was not possible to study temperature effects with

DMSO as it solidifies at 18°C and precludes lower

temperatures. When THF was used as a cosolvent and the

temperature lowered to 0°C no difference in yield was noted

as when the reaction was run at 25°C, The use of KOH or

sodium methoxide alone in THF dave no reaction. Good yields of the cyclobutanone were obtained however when the ylid was denerated by the slow addition of Potassium t-butoxide in

THF, While the ketone M could be obtained in dood yield by 46

Generation of the ylid at 0°C the reaction was extremely sensitive to the rate of base addition, Coolind the reaction to -15°C resulted in consistently Good yields of the ketone and allowed the reaction to be scaled up to 30mmol, Attempts to synthesize the ketone directly from the acyclic ylid precursor by reaction with two equivalents of KOtBu failed to yield the ketone although it has been reported that treatment of 63 with potassium ^ b u t o x i d e yields the cyclized precursor 64 C1543f Scheme 8»

Scheme 8

"■XCCH5O - ea £1

The oxime could be readily prepared from the cyclobutanone ^ by refluxind the ketone with hydroxylamine hydrochloride in ethanol at neutral p H, It was not possible to prepare the 0-methyl oxime ether from the ketone by its reaction with methoxyamine hydrochloride,

BurSer in his studies of the 2-phenylcyclobutylamines

[145] reported the preparation of the trans isomer by reduction of the corresponding oxime with sodium in ethanol,

FiGure 4, When the oxime ^ was reacted with sodium in ethanol it was found that no amine was produced and startinG 47

material was recovered. However when the reduction with

sodium was run in anhydrous isonroeanol the trans-amine 56

could be obtained in fair yields (50-60%).

Fisure 4 - Reactions of 2-phenyIcyclobutyloxime

N o/EIO H

NOH

An alternative route to the trans amine that was

investigated is outlined in Scheme 9. It has been reported

that alkyl phenylhydrazones readily rearrange in pentane or

benzene to the corresponding azo compound C1553. If the

hydrazone ^5 was to rearrange it was expected that the azo

derivative that formed would be the more thermodynamically

stable trans-isomer 66 and reduction of this azo derivative

catalytically should then yield the trans-amine 56. Although

the hydrazone ^ could be formed it would not and could not

be rearranged to the azo-derivative 66. Whether phenyl

hydrazones rearrange to the azo derivatives is not clear

although a more recent study concluded that the hydrazones were more energetically stable and had little tendency to

rearrange to the azo isomer C156D. 48

Scheme 9

CHjO ICH. OCH.

Ph-NH-NH. >

O N -N H -P h

CH)< Hj/catalyst

N 'N -P ti SS

The stereoselective synthesis of the cis-amine 57 was not

readily accomplished. As was noted by Beard and Border in

their synthesis of the isomeric 2-phenylcyclobutylamines

Fidure 4» 2-phenylcyclobutyloxime could not be reduced to

the cis-amine under a wide variety of conditions [145], Thus

in a similar manner attempted reduction of 62 with platinum

or palladium catalysts under conditions effective for the

reduction of aliphatic oximes to amines [157,158] dave

decomposition products or startind material. Reduction with

diborane C159] was found to dive in poor yield a mixture of

the cis and trans amines _57 and 56 which could only be

separated by careful chromatodraphy, Attempted reductions

with Red-Al and lithium aluminum hydride returned startind

material.

Stereoselective reduction of the oxime to the cis-amine

57 was finally achieved in 70% yield by the method of

Secrist and Lodue [160], Hydrodenation of the oxime 62 with platinum oxide in anhydrous ethanol with chloroform as the 49 proton source afforded in 70% yield the cis-amine 57 as the hydrochloride salt. This reduction method appears to have wide applicability as a mild technioue for the preparation of amine hydrochlorides from oximes y azides, nitriles and nitro compounds [160],

Although the stereochemistry of the two amines was evident from the synthetic route their assignments were confirmed by the preparation of the N-acetyl derivatives.

Reaction of the amine hydrochlorides ^ and ^ with pyridine and acetic anhydride or sodium acetate and acetic anhydride

Save Sood yields of the corresponding amides 67 and 68,

Scheme 10,

Scheme 10 CHjO OCH

NHCOCH- 61

CHjO OCH,

Acfi /Pyridine 5Z NHCOCH or AcgO/NoOAc

66

It has been reported that with 2-arylcyclobutylacetamides the methyl Sroup of the acetamide shows a shift in the NMR which is dependant on its orientation relative to the aryl

Sroup [147,161-163], While the trans acetamides have a shift corresponding to that of an acyclic acetamide the methyl resonance of the cis-acetamides is shifted upfield due to 50

an aromatic shield!nd p|lenomena « Such a shielding effect was

noted for the cis-acetamide 68 and confirms the

stereochemical assignments. Table 7, Similar shifts are

observed for the amide proton of the cis-acetamide which

would be expected to be shielded by the aromatic rind while

the trans-amide proton would be unaffected. The trans-amide

67 the vicinal methine proton was observed to be shifted

upfield by a similar mechanism.

Table 7 - Chemical Shifts of Acetamides 67 and 68

-CO-CH -NH-CQ -CH-N-CO 3 Trans-acetamide (67) 1,93 6,30 4,48

Cis-acetamide (68) 1,75 5,35 4,70

Isomeric purities of the cis and trans amines 57 and ^ was assayed by NMR spectroscopy and das chromatodraphic

analysis. In deneral NMR methods were not applicable for the

analysis of isomeric impurities of less than 2% both with

the amines and acetamides. Gas chromatodraphy of the amines

35 the hydrochloride salt and free base on a number of

different column and temperature combinations did not effect

separation. It was possible however to separate the cis and

trans amines as the correspondind trifluoroacetamides on a 4 foot OV-101 column at 170°C, The trifluoroacetamides were prepared by the reaction of the amine hydrochlorides with trifluoroacetic anhydride [164], Scheme 11, with no detectable amounts of the amines present. Analysis of the cis and trans amines by this method with flame ionization 51 detection showed the amines to have an isomeric purity of greater than 99.5%,

Scheme 11

C H .0 OCH OCH

EtOAc NHg NHCOCF,

The conversion of the methoxylated amines to the corresponding 6-hydroxydopamine derivatives could be effected by two methods, Senoh and Witkop originally prepared 6-OHDA from the corresponding monomethoxy amine by refluxing the amine with HBr in acetic acid C2], Using the same procedure the amines ^ and ^ were converted to the hydroxy derivatives 48 and 49, A maJor problem arose with the isolation of these,amines » they tended to be ouite hygroscopic and could not be isolated in a state suitable for manipulation and characterization. Additionally the use of reflux temperatures appeared to cause some decomposition of the product,

A more facile conversion of the methoxylated compounds to the corresponding hydroxy derivatives was affected by reaction of the methoxylated amines with boron tribromide in methylene chloride C58rl65], Thus it was possible to prepare the amines 48 and 49_ in good yield at room temperature. The trans amine 48 fell out of solution directly on hydrolysis of the borate ester while the cis amine was crystallized

from diethyl ether after borate ester hydrolysis.

The structure of the two amines ^ and ^ was verified by

spectral and chemical means. Mass spectral analysis by

electron ionization Save a molecular ion at an m/e of 195

for both the cis and trans amines corresponding to the loss

of HBr, The chemical ionization spectra Save a molecular ion

at an m/e of 196 corresponding to the loss of bromide ion?

the base peak was at m/e 177 accounted for by the loss of

ammonia.

Proton magnetic resonance analysis yielded spectra for

the two hydroxy amines which were ouite similar to those for

the methoxylated amines 56 and _^7 with the notable absence

of methoxy resonances at 3,8-3,9 ppm. Infrared analysis

showed a broad 0-H absorption similar to that reported for

6-hyroxydopamine [166], The reaction of the amines 48 and 49

with ferric chloride save a very distinctive colour

reaction. Whereas ferric chloride reacts with catechols to

yield a green precipitate the 6-hydroxydopamine analogs ^

and 42 ëave a deep red colour which is probably the ouinone anion 69 [37f38], Under the same conditions

6-hydroxydopamine gave a similar colour reaction.

An acceptable analysis for ^ was not obtained,

" 0 o

sa 53

B) Preparation of the cyclobutyl analogs of dopamine» trans-

and cis-2(3'»4'-dihydroxyphenyl)cyclobutylamine hydrobromide

50 and 51 and trans- and cis-N »N-dimethyl-

2(3'»4'-dihydroxyphenyl)cyclobutylamine hydrobromide ^ and

53 «

The synthetic route used for the preparation of the

amines ^0» 51» 52^ and 5^ is outlined in Scheme 11 « It was

found that the procedure developed for the stereoselective

synthesis of the 6-hydroxydopamine analods was directly

applicable to the preparation of these compounds. In deneral

the dopamine analods could be prepared in better yields and

were easier to work with.

The conversion of piperonal (70) to the cyclobutanone 7JL was accomplished by its reaction with the ylid denerated

from 64 with potassium t-butoxide in THF at ice bath

temperature. This reaction was not as temperature sensitive as that for the preparation of ^ , It was possible to prepare the ketone 71 by reaction of piperonal with ^4 in

DMSO with KOH C149] thoudh in poorer yields.

The preparation of the oxime 72 could be effected directly from the oily ketone/diphenyl sulfide mixture without purification of _71* By the use of two eauivalents of base the oxime 72. remained in the aoueous layer and the

Phenyl sulfide removed with a lidroin wash. Acidification of

the aoueous layer followed by chloroform extraction and concentration afforded the oxime in dreater than 70% yield from piperonal. 54

Scheme 11

KO tBu/ THF

7 0

NoO H /E ton

Ha/PtOe 1)Na/iPrOH \E t O H /C H C I 2) MCI NOH 12

5 9 58 BBr, 8Br, HO.

HO HO

NH, Br 5 0

HO, HO

HO HO The preparation of the trans- and cis-amines 58 and 59

was accomplished ba the same methods used for the preparation of the 6-hadroxadopamine analods ^6 and 57♦

Reduction of the oxime 72 with sodium in isopropanol wielded the trans-amine free base which was converted to the salt ^ ba treatment with ethereal hadroden chloride. The cis-amine hadrochloride was obtained directla from the oxime in excellent wield ba reduction over platinum oxide in ethanol with chloroform as the proton source [160].

The isomeric purities of the amines was determined ba conversion of the amines to the triflouroacetamides and das chromatodraphic analysis [164], Scheme 12. Analysis on a 4 ft OV-101 column at 150°C dave dood separation of the acetamides and showed them to be of dredter than 99.5% isomeric purita.

Scheme 12

(CF.CO-O EtO A c N H -C CF, NHg

The same technioue was used to determine the stereoselectivity of the reduction methods. Table 8. The crude amines 58 and ^ were reacted directla with triflouroacetic anhydride and chromatodraphed without purification. It can be seen that the catalytic reduction afforded dood selectivity in preparation of the cis-amine 59 56

while reduction with sodium ancS isopropanol dave poorer

selectivity in preparation of the trans-isomer 58« Reduction

with diborane in didlume dave almost eoimolar amounts of the

two amines which could only be separated by column

chromatodraphy on silica del*

Table 8 - Stereoselectivity in the Reduction of 72

Method trans / cis

H2/Pt02 1/99

BH3/Didlyme 54 / 46

Na/i-PrDH 92/8

The stereochemical assignments for the two amines 5^ and

59 were based on the route of preparation and confirmed by a

study of the N-acetyl derivatives. Scheme 13* Reaction of .58.

and 2 ? with pyridine/acetic anhydride yielded the amides 73

and 74 respectively* The NMR data for two acetamides are

summarized in Table 9* It was observed that the

cis-acetamide 74 methyl resonance was shifted upfield 0*2 ppm relative to the trans-acetimide 73* The amide protons

and vicinal methine protons showed a similar shielding effect consistent with the stereochemical assignments*

Table 9- Chemical Shifts of the Acetamides 73 and 74

-COCHg -NH-CO -CH-N-CO

Trans-acetamide 73 1*95 5*75 4*46

Cis-acetamide 74 1*76 5*15 4*72 57

Scheme 13

HN-COCH

II

*-

NH, Cf HN-COCH

The stereochemical assignments were further verified chemically by cyclization of the cis-amine 59 to the tetrahydroisoouinoline analod 7^* The conversion was affected by reaction of 59 with formic acid/formaldehyde

[167] followed by méthylation with methyl iodide. Scheme 14*

The NMR spectra of 75, Fidure 5, was consistent with the structure assigned, only two methyl resonances were observed and the aromatic portion was tetra-sustituted. Cyclization to the 6-position was indicated by the two sindlet resonances for the aromatic protons, cyclization to the

2-Position should have aiven rise to an AB ouartet. It was also interesting to note that one of the cyclobutyl protons was shifted upfield 0*7 ppm relative to the other protons by an aromatic shielding phenomenon* I - C H , I

CM, 7 5

I . L I 1. L . L t I I I I I I I ■ I ■_ I 1 t . , . I . . . I , . . i , . . , I . . . . I * » ■ ‘ * J * t I I t I ■ I ■ I J-li

cn 00 Fiaure 5-90 MHz NMR Spectrum of 75 ay

Scheme 14

DHCOgH/HCHO ^ ShjCi' "'XJ N» I

Conversion of ^ and ^ to the dopamine analogs 50 and ^ was readily accomplished with boron tribromide in methylene chloride C165], The cis-amine 51 was characterized as the hydrobromide salt while the trans-amine 50 was characterized as the hydroscopic hemihydrate hydrobromide salt. The NMR spectra of 50 and ^ were similar to the spectra of the protected amines 58 and ^ with the notable absence of the the -CH^- resonance of the methylenedioxy function. Both compounds save a molecular ion with chemical ionization at m/e 180 corresponding to the loss of a bromide ion.

Additionally, reaction of the amines ^0 or ^ with ferric chloride dave a positive test for a catechol function.

The preparation of the dimethylamines ^ and 5^ was effected from the protected amines rather than the catechols

50 and Scheme 11, Attempted dimethylation of the cis- and trans-amines 51^ and _50 with formic acid/formaldehyde C1671 resulted in intermolecular and intramolecular condensation reactions. It was possible however to prepare the dimethyl analods by reductive alkylation with formaldehyde in the presence of hydroden and platinum oxide [168] followed by deprotection with boron tribromide. Thus 5^ was converted to

52 and 59 to 53 in dood yield. 60

Mass spectral chemical ionization spectra save a molecular ion for both amines ^2 and ^3 at m/e 208 corresponding to the loss of a bromide ion* The NMR spectra of the trans-amine 52 showed the two methyls as a sindlet while the cis-amine 53 slave two distinct resonances* That this was due to a hindered rotation of the dimethwlamino droup was shown by a temperature study» Fidure 6* On warmind the sample the methyl resonances were observed to slowly collapse and coalesce at 55°C* What biolodical significance this hindered rotation midht have is not clear but further confirms the cis assignment for the amine*

Figure 6 Temperature Study of 53

HO

HO

•335*K

I 0^^ 61

C) Rearrangement of 2r4f5-trimethoxapropiof»henone

to N-propionyl-21> 4 » 5-trimethoxaani 1 ine

The synthesis of a variety of phenethalamines has been

readily accomplished ba condensation of the corresponding

aromatic aldehyde with a nitroparaffin and the resulting

nitrostarene reduced to the amine catalaticalla or with metal hydrides» Scheme 15. A variety of conditions have been used to effect the condensation of an aldehyde with the nitroparaffin including ammonium acetate in acetic acid

[169], amine catalysis [170] and more recently trimethalorthoformate in methanol [171],

Scheme 15

NOz L A H

The condensation reaction has been limited however to the reaction of nitroparaffins with aromatic aldehydes [172].

The corresponding reaction of nitroparaffins with aromatic ketones to yield ^-substituted nitrostyrenes and l-substituted phenethylamines has not been reported [172].

The reaction of nitromethane with cyclohexanone has been reported to yield the nitroolefin [173] as well as a rather complex tricyclic oxime [174,175]. In an attempt then to prepare /3-ethyl-2»4»5-trimethoKy-

phenethylamine the reaction of 2f4,5-trimethoxypropiophenone

(76) with nitromethane was studied. Usina conditions

effective for the preparation of nitrostyrenes [1693 from

aromatic aldehydes, alacial acetic acid and ammonium

acetate, the ketone was found to be slowly converted to a

second component of lower Rf$ Purification by column

chromatoaraphy yielded a white solid (A) which was not the

startina ketone 76 and had the spectral characteristics

listed in Table 10. It was found that if the nitromethane or

the ammonium acetate was omitted from the reaction only

startina material could be recovered. Replacement of the

nitromethane with nitroethane yielded two products. The

spectral characteristics of the two components labeled (A)

and (B) are listed in Table 10«

Table 10 - Spectral Data for A and B

Compound (A) (B)

Source CHgNOg or CHgCH^NO^ CH^CHgNOa

NMR (CDC13) 8.16 (s,lH) 8.10 (s,lH) 7.6 (br,lH) 7.6 (br,lH) 6.54 (S,1H> 6.54 (s,lH) 3.86 (s,9H) 3,86 (s,9H) 2.41 (Q,2H,J=8.5Hz) 2.18 (s,3H) 1.24 (t,3H,J=8.5Hz)

IR (KBr) 3240 cm-1 sharp 3210 cm-1 sharp 1650 cm-1 strona 1660 cm-1 strona

Mass Spectra(EI) M+=239 (base peak) M+=225 (base peak) 183 183 168 168 63

Analysis of the spectral data suggests a number of

possible structures for the two compounds* The NMR data

strondly suggests that the two compounds are identical

except that one contains a methyl and the other an ethyl

functionr this is further validified by the mass spectral

data* Possible structures for (A) are listed in Table 11 *

Table 11 - Possible Structures for A

OCH, OCH, ^ÿ^v^ONHCHjCH,

OCH3 OCH, 7 8 12

CMCH NHCOCH

CH,0" OCH, OCH, 29 Si

Assignment of structure _77 to A seemed unlikely on the

basis of the NMR and mass spectral data* Compound 7 7 would

be expected to show a coupling of the amide proton with the

vicinal methylene group, which was not observed as well as a

major mass spectral fragment corresponding to the loss of

-NHCH CH at 195. It seemed that the structure 78 acounted

best for the spectral data though the structure 79 arising

from an aromatic migration process could not be excluded*

To prove the structural assignment of (A) and verify the aromatic substitution pattern the synthesis of (A) was undert 'ken in an unambiguous fashion. Scheme 16* The 64

nitration of 1 »2f4-triiîiethoMybensene with nitric acid in

dlacial acetic acid afforded the previously reported nitro

compound 80 C1763 in low yield. The conversion of ^ to 78

was accomplished by reduction with palladium on charcoal and

acétylation with propionic anhydride. The solid so obtained

possessed spectral characteristics identical to that for the

amide isolated from the reaction mixture and the mixed

meltind point showed no depression. Thus the structure 7 8

was assigned to the product (A) isolated from the nitrostyrene reaction. On the basis of the above synthesis

and the correlation of spectral characteristics the reaction product (B) w>s assigned the structure 81 and probably arises from 78 by an acid catalyzed amide exchange.

Scheme 16

OCH OCH,

iCH CH SQ za

The mechanism of this oxidative rearransement does not have any direct precedent in the literature. The reaction

involves a net oxidation and insertion of nitroden ouite similar to a Beckmann type of product a1thoudh there does not appear to be a suitable leavind droup as in the Beckmann reaction. A possible mechanism for the rearrendement is outlined in Scheme 17 and some evidence For its plausibility Scheme 17

OCH

NH4PAC / HOAc

CH^ RChyjOg

H y CH3 0 ♦ V 0 + H I ^"«^lO-NCHgR

C H f -H CH^' OCH, OCH, SSL m

’' ■ h iNCHgR -H

CH3 0 C H f

M.

H ^ N ( ^ N C H ^ OCH3 ^ggk^NHCOCHgCHa

+ ONCf^R

OCH, OCH, 7 8 66

is discussed below.

The initial formation of the imine 82 from the ketone

would seem ouite likely under the dehydrating conditions of

the reaction and probably is the initial step in the

formation of nitrostyrenes from aromatic aldehydes. A

similar catalysis of nitrostyrene formation by butylamine

has been reported C170I1»

The presence of the nitroparaffin is critical» when it is

omitted from the reaction there is no product isolated and

starting material is recovered. There have been reports that

the nitro group can add in a l»3-diPolar manner [177-178] to

olefins and this concept might be extended to an imine in

this case, Alternativly addition might be via an anionic

attack at the imine carbon to form intermediate 83 which

then undergoes N attack at the oxygen atom to give the

cyclic intermediate JH. Similar addition products have been

reported for the addition of nitroparaffins to protonated

acetylenes and ynamines [ 1 79-180],

The decomposition of the common cyclic intermediate ^ to

the product 7 ^ could then proceed with the participation by

resonance donation of the methoxy group. This has very

strong precidence in the literature where it was observed that a 2 »4»b-trimethoxyorganoborane could have undergone

such a decomposition» Scheme 18 [181]. 67

Scheme 18 ïk"?'

CH5O NHgOSOjH CH.0

OCH OCH3 OCH3

? N H -B -C H -E t -fc. R' yKcH, OCH,

It can not he proven by these studies that the mechanism

presented is the actual mechanism for the reaction. Whether

the source of the nitrosen is the ammonium acetate and not

the nitroparaffin can not be conclusivly stated although the use of a labeled nitroSen misht allow such conclusions,

Alternativly the study of the reaction of imines and other dipolar reasents midht allow further comment. It is also not clear on the basis of these studies whether the

rearrangement midht be of general preparative synthetic value. 68

Biological

4' A ) Pharmacological Assay of the Neurotoxic Properties

of the 6 -Hydroxydopamine Analogs 48 and 49

Neurotoxicity was assayed by assessing the ability of heart tissue to accumulate ^H-(-)-norepinephrine 2 0 hr after treatment with 6 -hydroroxydopaminep the trans-analog 48 or cis-analog ^ [61], The results are presented in Figure 7»

While 50 mg/kg 6 -hydroxydopamine produced destruction as evidenced by a greater than 90% decrease in

3H-norepinephrine accumulation, the trans- and cis-isomers

48 and ^ at doses u p to 200 mg/kg and 300 mg/kg i«p, produced no such effect. The use of higher doses of the isomers 48 and 49 were precluded due to their lethality. To ensure that the compounds were not being metabolized and were available at the heart the compounds were tested by i,v, administration. As can be seen in Figure 7 neither compound ^ or 49 caused sympathectamy,

The relative affinities of compounds ^ and ^ for the membrane associated amine pump were determined by assaying the abilities of the trans-analog 48 and the cis-analog 49 to inhibit the uptake of ^H-(-)-norepinephrine into heart tissue. The results, presented in Figure 8 show that the trans but not cis analog caused an approximate 50% inhibition of uptake at 50 mg/kg. It would appear that the lack of sympathectant properties of the trans-analog 48 is not due to lack of access to the heart. HQ OH HO. OH

f = ( NH» Br HO

48 ââ -I r

100

8 0

6 0

9 : 4 0

8 0

S aline SOmg/kg SOmg/kg ZOOmg/kg lOOmg/kg 50mg/kg 200mg/kg 300mg/kg lOOmg/kg LP. LRX3 I.P. I.V. I.P .X 3 IJ> I.V. 6 -O H D A

Figure 7- Results of Neurotoxicity Studies of 6 -HydroxydoP3 mine; 48 and 49 in vivo O' 70

Figure 8 - Inhibition of ^H-Norepinephrine Uptake in VIVO by 48 and 49

100

3 80 o DC H z o Ü 60

w

=) LÜ Z I X to 20

CONTROL 50mg/kg I.V. SOm^kg I.V. C IS + ^ N E TRANS + ^H-NE (4 9 ) (48) 71

The lack of neurotoxicity of both the cis and trans

analogs was dissappointind. It is not clear whether this

lack of neurotoxicity allows any comment of the relative

importance of indoline formation in the neurotoxicity of

6 -hydroxydopamine, It does not seem that the nuerotoxic

properties of this compound were overlooked, doses of 1 0 0

md/kai i,v, of the trans- and cis-isomers were ineffective in

causing neurodestruction while doses of 50 md/kg of the

trans-isomer were shown to have affinity for the nueronal

uptake mechanism. Although the trans-amine 48 had affinity

for the uptake mechanism it may be that addition of the

steric bulk of the cyclobutyl moiety prevents uptake into

the neuron, an event reouisite for 6 -hydroxydopamine like

activity.

The results would indicate a preference for a trans

disposition of amine and aromatic moiety at the uptake site.

This may however be a reflection of differences in

disposition or metabolism so a firm conclusion is not valid.

Clearly, an investigation of the in vitro properties of

the trans- and cis-analoSs 48 and ^ would allow further

comment on the neurotoxic properties of the two analogs as

well as conformational preferences at the uptake site. It

would also be of interest to test both compounds in

neuroblastoma cell cultures which appear to concentrate

6 -hydroxydopamine by a diffusion rather than an active uptake mechanism C183T, 72

B) Presynaptic Uptake of the Dopamine Analogs ^ and 51

The ability of the trans- and cis-dopamine analods ^ and **■5 “X 51 to inhibit H-dopamine and '^H-norepinephrine uptake was assayed with striatal and cortex swnaptosomes in a manner similar to that reported by Ferris et al [184], The uptake of striatal synaptosomes has been ascribed to a presynaptic dopamine uptake mechanism while uptake into cortex synaptosomes has been associated with a presynaptic adreneraic mechanism C81],

The results of inhibition of H-dopamine uptake into striatal synaptosomes by the trans- and cis-analods 50 and

51 are illustrated in FiSure 9, It can be seen that although both compounds were reasonably poor inhibitors of uptake the trans-isomer 50 was more than three times as potent as the cis-isomer 51♦

The ability of compounds 50 and ^ to inhibit

^H-(-)norepinephrine into cortex synaptosomes is given in

Figure 10, As was noted for the striatum the trans-isomer 50 was more potent than the cis-isomer 51 in inhibiting uptake by a factor of approximately ten. The Ki for both cortex and striatum are listed in Table 12,

Table 12 - Uptake Ki Values for ^ and 51

Compound n Cortex Striatum ID50 ID50

(50) 5 8 , 6 X 10-6 1,41 x 10-4 (51) 5 7,3 X 10-5 >3,0 x 10-4

The results of the studies indicate a preference for the Cortex Striatum 140 HO

120 O L C O c 100 u o2

80 C o o 60 3 E 3 V u HO < 40 < Û I I NH^Br 20 trans 50

r4

Inhibitor (M)

Fiaure 10- Fiâure 9-

Inhibition of ^H-NoreFinephrine Inhibition of ^H-Dopaniine Uptake ba 50 and 51 Uptake ba 50 and 51 N i w 74 trans-isomer 50 at both the cortex and striatal uptake sites. It seems reasonable to extrapolate these results and predict that dopamine and norepinephrine are preferentially bound to the adrenersic and dopaminerdic uptake mechanisms in a trans extended form 1126 «S III, 75

C) Dopamine Binding Studies of the Dopamine Analods 22' 21

52 and 53

The dopamine sensitive membrane binding model was used as

an assay for dopaminergic activity. A number of

investigators have shown the membrane components from the

corpus striatum to selectively bind dopamine and dopaminergic agents C123-125].

The method presented by Burt et al [123] for this assay

in calf brain was used directly for the corpus striatum from

rat. Roberts et al [125] and Seeman et al [124] have

reported characteristics for a dopamine receptor from rat brain which differ somewhat from those reported by Burt et al for calf brain. The membrane preparation used from rat brain in this study had properties auite similar to those

reported by Burt et al [123].

Using this preparation the compounds 50» 51» 52 and 53 were studied for their ability to inhibit specific

^H-dopamine binding. The results are given in Table 13 and

Figure 11.

Dopamine was observed to have a Ki of 25.6 riM which compares well to the value of 17.5 nM reported by Burt et al

[123] for their calf brain studies. There was observed a difference in activity between the trans 2 2 and cis 2 ^^ analogs» of approximately twenty fold with the trans analog more active. It was dissappointing to note that the trans-isomer 50 was almost 320 fold less active than dopamine. 76

Table 13- Ki Values for 50» 51» 52 and 53

Structure «_Kj i ------±SEM Loi HO OH

25.62 nM± 4.45 (7)

NH

HO OH

8.16 jjM ± 1.71 (6 )

2 m HO OH

NH 160.19 jjM ±50.11 (6)

51

HO OH

529.13 nM ±56.35 (7) N(CH,), 52 HO OH

I8 .3 4 jjM± 2.19 (6)

53 95

3 O Dopamine trans (5 2 ) trans(50) cis (53) cis(51) m 80 w m 60 o 5 50 ce 40 £L

20 II 52 5 u . 5 ^ üj 0_ to

-9 ,-8 7 -6 5 - 4 r3

CONCENTRATION

3 Fiaure 11- Inhibition of H-UoFaraine Binding in vitro

by 50» 51» 52 and 53 ~ ---- N i NJ 78

Based on the observation that the NfN-dimethyl analods of

6»7-ADTN and 2-amino-4rS-dihwdroxyindane were far more potent than the nonmethylated analogs in causind turnind in unilaterally lesioned rats C1283» the dimethyl analods ^ and ^ were prepared. The results as summarized in Table 13 and Fidure 12 indicate a similar preference for a trans disposition of amine and aromatic function for inhibition of dopamine bindind. The trans-analod 52 was 30 times more active than the cis-analod 5 3 . Additionally? the trans- and cis-analods 52 and 53 were more potent than the correspondind nonmethylated analods 50 and ^ respectivly? with 52 beind only 20 fold less active than dopamine.

On the basis of these studies it midht be concluded that the addition of a two carbon fradment correspondind to the cyclobutyl moiety does not severely inhibit dopamine bindind. While it does reduce bindind it also clearly illustrates a prefered bindind of a trans form of the cyclobutyldopamine analods and possibly also dopamine at the postsynaptic dopamine site. 79

EXPERIMENTAL

Meltina points were determined in open capillaries on a

Thomas Hoover Uni-Melt apparatus and are uncorrected.

Infrared spectra were obtained on a Beckman 4230

spectrophotometer as potassium bromide discs (KBr) or nuJol

mulls and are specified for the spectra reported.

Proton magnetic resonance spectra (NMR) were recorded

either on a Varian A-60A NMR spectrometer <60MHz) or a

Broker MX 90E NMR spectrometer (90MHz) in a pulsed fourier

transform mode, The samples were prepared for NMR analysis

in either deuterochloroform (CDCl^) with tetramethylsilane

as internal standard or deuterium oxide (BO) with

2 »2 -dimethyl-silapentane-S-sulfonate as internal standard o and run at approximatly 25 C , The spectra are reported here

usina the notation s-sinalet, d-doublet, t-triplet,

Q-ouartetf p-pentet and br-broad.

Mass spectra were obtained with a BuPont Model 21-491 double focusind mass spectrometer. Electron impact spectra were obtained at a source potential of 70 eV, chemical

ionization spectra were obtained at the same potential with

isobutane as the carrier das. In many cases molecular ions could be observed only in the chemical ionization mode.

Gas chromatodraphic analyses were run on a

Hewlett-Packard 5710A das chromatodraph and 7130A recorder.

Detection was by flame ionization with a helium carrier at

ZO ml/min, 80

Elementa.I analyses were performed by Galbraith

Laboratories Inc*y Knoxville Tenn,

Organic chemicals were obtained from Aldrich Chemical

Company and were used without further purification unless otherwise noted, Inordanic chemicals» including silver tetrafluoroborate were obtained from the Alfa Products division of Ventron Corporation and were used without further purification, Deuterated solvents for NMR studies were obtained from the Aldrich Chemical Company or Stohler

Isotope Chemicals,

All solvents were readent sfrade and were used without further purification except for those listed below.

Anhydrous isopropanol was obtained by the distillation of commercial anhydrous isopropanol from madnesium and storade over 3A molecular sieves, Tetrahydrofuran was distilled from lithium aluminum hydride and stored over 4A molecular sieves,

Column chromatodraphy was with 70-230 mesh Silica Gel 60 obtained from E , Merck» Darmstadt and was used without resedimentation or activation. Thin layer chromatodraphy was with pre-coated silica del F-254 Plates» E,Merck» Darmstadt and were used without activation. B1

Preparation of trBns-2(2'f4'y5'-trihydroxyphenyl)

cyclobutylamine hydrobromide (48)

To a stirred suspension of trans-2(2' fA',5-trimethoxyphenyl>

cyclobutylamine hydrochloride <300md»1♦Immol) in methylene

chloride (10ml) cooled to 0°C under arson there was added

dropwise boron tribromide (l,2lS,4*8mmol) in methylene chloride

(5ml), After the addition was complete the reaction was allowed to warm to room temperature and stir an additional 16 hr. The

reaction was then cooled to 0°C and Quenched cautiously with methanol (1ml) and the solid amine filtered under arson. The amine was characterized as a white powdery solid (253mS, 83%) mp 220“224°C (discolours),

NMR (D^O) (90MHz) 6,71 (s,lH) Ar-H 6,49 (s,lH) Ar-H 3,5-3,8 (m,2H) Ar-CH-CH-N 2,0-2,3 (m,4H) -CH^CH^-

IR (KBr) 2800-3600 cm-1 0-H and C-H stretch

Mass Spec, (Cl) Calc, M+l=196 Found M+l=196

Preparation of cis-2(2',4'r5'-trihydroxyphenyl) cyclobutylamine hydrobromide (49)

To a stirred suspension of cis-2(2',4',5'-trimethoxyphenyl) cyclobutylamine hydrochloride (300mS,1♦Immol) in methylene chloride (10ml) cooled to 0°C under arson there was added dropwise boron tribromide (l,21s,4,8mmol) in methylene chloride

(5ml), After the addition was complete the reaction was allowed to warm to room temperature and stir an additional 16 hr. The reaction was then cooled to 0°C and the reaction Quenched with methanol (3ml), Concentration under hish vacuum yielded a solid 82

which was filtered from ether to yield the amine hydrobromide

as a tan solid (234ma, 77%) mp 210-212°C (dec),

NMR (B 0) (90MHz) 6.83 (s,lH) Ar-H 2 6.52 (srlH) Ar-H 3,9-4,2 (m,2H) Ar-CH-CH-N 2,2-2,7 (m,4H) -CH_CH_-

IR (KBr) 2800-3600 cm-1 0-H and C-H stretch

Mass Spec, (Cl) Calc, M+l=196 Found M+l=196

Analysis for C^^H^^NO^Br, H^O

C H N 0 Br Calculated 40,83 5,48 4,76 21,76 27,20 Found 41,15 5,20 5,05

Preparation of trans-2(3'*4'-dihydroxyphenyl)cyclobutylamine hydrobromide (50)

To a suspension of trans-2(3',4'-methylenedioxyphenyl) cyclobutylamine hydrochloride (210md,0,925mmol) methylene chloride (15ml) cooled to -78°C there was added dropwise boron tribromide (695 md, 2,78 mmol). After addition was complete the solution was allowed to warm to room temperature and stir 18 hr under ardon. The reaction was Quenched with methanol (3ml) and the solvent removed in vacuo. The white solid was filtered from methylene chloride/dlyme to dive the dihydroxyamine hydrobromide as a hydroscopic solid (180 md» 75%), Extended dryind dave a tan hydroscopic solid mp, 123-125°C, which analyzed as the hemihydrate, and dave a positive ferric chloride test. 83

NMR (D^O) (90MHz) 6 ,7-7,0 (m,3H) Ar-H 3,3-4,0 <(rif2H> N-CH- and Ar-CH- 1,5-2,5 (m,4H) -CH^-CH^- *U J,

IR (KBr) 3600—2600 cm— 1 N—HrC-HfO—H stretch

Mass Spec, (Cl) Calc, M+l=180 Found M+l=180

Analysis for C^^H^^NO^Br, 1/2 H^O

C H N 0 Br Calculated 44,62 5,62 5,20 14,86 29,69 Found 44,34 5,56 4,94

Preparation of cis-2(3',4'-dihydroxyphenyl)cyclobutylamine

hydrobromide (51)

To a suspension of cis-2(3',4'-methylenedioxyphenyl)

cyclobutylamine hydrochloride (l,10d,4,86mmol) in methylene

chloride (60ml) cooled to -78°C under arson there was added

with stirring boron tribromide (3,5s»14,Ommol), After the

addition was complete the reaction was allowed to warm to room

temperature and stir an additional 1 2 hr. Addition of anhydrous

methanol and removal of solvent in vacuo Save a white solid

which was taken u p in methanol-methylene chloride and

crystallized in an ether chamber. Filtration under arson save

the catechol (1,10 s, 87%) mp, 248°C (dec,)» which save a

positive ferric chloride test,

NMR (irO)(90MHz) 6.7-7,1 (m»3H) Ar-H 3,7-4,2 (m»2H) -CH-N & Ar-CH- 1,9-2,7 (m»4H) -CH_-CH_-

IR (KBr) 3400-2900cm-l N-H»C-H»0-H stretch 1605 cm-1 Ar-H bend

Mass Spec, (Cl) Calc, M+l=180 Found M+l=180 84

Analysis for

C H N 0 Br Calculated 46,17 5,42 5,38 12,30 30,72 Found 46,05 5,60 5,41

Preparation of trans-N,N-dimethyl-2(3'»4'-dihydroxyphenyl)

cyclobutylamine hydrochloride (52)

A suspension of trans-2(3'’r4'-methylenedioxyphenyl )

cyclobutylamine hydrochloride (600md,«459mmol),

Platinum oxide (lOmd) and 37% formalin solution

(2,0ml,25,3 mmol) in ethanol (50ml) was hydroSenated

at 3 atm of hydroden for 3hr, The catalyst was removed by filtration throudh celite and the solvent removed in vacuo. The amine was taken u p in 6 N HC1» washed with methylene chloride, basified with 40% NaOH and extracted with methylene chloride. The methylene chloride layer was dried with madnesium sulfate, filtered and concentrated to dive the amine as an oily free base. The oil was taken up in the minimal amount of chloroform and converted to the hydrochloride salt by addition of ethereal

HCl, Filtration dave the protected amine hydrochloride

(520md, 77%) as a white solid mp, 177^C,

To a solution of the protected amine (90md,,35mmol) in methylene chloride (5ml) cooled to -78°C there was added dropwise under an ardon atmosphere boron tribromide (264iiid, 1,Ommol ) in methylene chloride

(3ml), After the addition was complete the solution was allowed to warm to room temperature and stir an additional 12 hr. The reaction was then cooled to 0°C 85

and cautiously treated with methanol (3ml)p the solvent was removed in vacuo to yield the amine as a tan oil»

Addition of cyclohexanone caused solidification and the pure white solid was filtered and dried in vacuo to yield the dihydroxyamine hydrobromide (92mdp91%) mp. 210~211°C.

NMR (D^O) (90MHz) 6.93 (m,3H) Ar-H 3,3-4.0 (m,2H) Ar-CH-CH-N 2.69 (s,6 H) N(CH^) 1.6-2, 6 (m,4H) -5hSCH„- «:! 4L IR (KBr) 3240 cm-1 0-H stretch 2800-3000 cm-1 C-H stretch 2600-2800 cm-1 NHR_+ Cl- •L Analysis for C^giHigNOgBr

C H N 0 Br Calculated 50.01 6.30 4.86 11.10 27.73 Found 50.05 6.44 4.78

Preparation of cis-NpN-dimethyl-2(3'p4'-dihydroxyphenyl) cyclobutylamine hydrobromide (53)

A solution of cis-2(3'?4'-methylenedioxyphenyl) cyclobutylamine hydrochloride (200mdp.782mmol)p 37% formalin

(.7ml) and platinum oxide (30ma) in ethanol (15ml) was hydrodenated at 3 atm of hydroden for 4 hr. The catalyst was removed by filtration with celitep concentrated ^ vacuo and taken up in chloroform. The chloroform was washed with

10% NaOHp brine solution and dried over madnesium sulfate.

Filtration and concentration dave an oil which was taken

U P in methylene chloride (5ml)p cooled to -78°C under ardon and to which was added boron tribromide (600mdp2.39mmol)

The reaction was allowed to warm to room temperature and stir an additional 12 hr. The reaction was Quenched with 86

methanol (3ml) and the solvent removed in vacuo to yield

the amine as an oil. The amine salt was crystallized

from isoeropanol/acetone to yield the amine as a tan solid

<140mSf62%) mp « 214-215°C. The amine Save a positive ferric

chloride test,

NMR (D^O) (90MHz) 6,99(s,3H> Ar-H 3.7-4.1 (m,2 H> Ar-CH-CH-N 2.68 (s,3H) N-CHv 2.59 (s,3H) N-CHg 2.0-3.0 (m,4H) -CHgCHg-

IR (KBr) 2600—3600 cm-1 0-H and C-H stretch 1615 cm-1 Ar-H bend 1600 cm-1 N-H bend

Analysis for Cj^2Hl8^*^2®’'

C H N 0 Br Calculated 50.01 6.30 4.86 11.10 27.73 Found 49.81 6,49 4.67

Preparation of trans-2(2',4',5'-trimethoxyphenyl)cyclobutylamine

hydrochloride (56)

To a refluxins solution of anhydrous isopropanol (120ml) and

2 (2 'f4',5-trimethoxyphenyl)cyclobutyloxime (1.2S,4.8mmol) was

added sodium (4.3d »187mmol) in small pieces over a 30 min

period. Note: the reaction Proceeded poorly with ethanol or if

the isopropanol was not scrupulously anhydrous. After the

addition was complete the reaction was refluxed an additional

hour. Water was added to solubilize the salt and excess

isopropanol was removed under reduced pressure. The aoueous

layer was extracted with methylene chloride which was back extracted with 10% HCl. Basification of the acidic extract with

40% NaOH caused the amine to oil out and was extracted with methylene chloride. The ordanics were washed with water, brine 87

solution and dried over madnesium sulfate. Filtration and

concentration dave the amine as an oil. This oil was converted

to the hydrochloride salt with ethereal HCl, Two

recrystallizations from ethanol-ether dave pure trans amine

hydrochloride (710md,55%) mp 196-197°C (dec,),

(Isomeric purity >99.5%)

NMR (DnO) (90MHz) 6,82 (srlH) Ar-H 6,72 (s,lH) Ar-H 3,89 (s,3H) O-CH3 3.85 (s,3H) O-CH3 3,5—3,8 (m»2H) Ar—CH—CH-N 2.0-2,5 (m,4H) -CH^rCHg-

IR (KBr) 3100 cm-1 N-H stretch 2700-3000 cm-1 C-H stretch 2000 cm-1 NH3 +CI- 1610 cm-1 Ar-H bend

Analysis for C^3 H2 qN0 3 C 1

C H N 0 Cl Calculated 57,04 7,36 5,12 17,53 12,95 Found 57,23 7,51 5,18

Preparation of cis-2(2'rA',5'-trimethoxyphenyl)cyclobutylamine

hydrochloride (57)

A solution of 2(2'f4',5'-trimethoxyphenyl)cyclobutyloxime

(470mdfll,87mmol)f platinum oxide (50md), chloroform (1ml) in

absolute ethanol (50ml) was hydrodenated at 3 atm of hydroden on 3 Parr Apparatus for 12 hr. The colourless suspension was filtered through a celite pad which was washed with ethanol.

Concentration under reduced pressure yielded a white solid.

Recrystallization from ethanol-ether dave pure amine hydrochloride (320md» 6 6 %) mp 219-220°C (dec,),

(Isomeric purity >99,5%) 88

NMR (DgO) (90MHz) 6.92 (s,lH> Ar-H 6,74 (s,lH) Ar-H 3.9-4,3 (m,2H) Ar-CH-CH-N 3,89 (s,3H) O-CH3 3.85 (s,3H) O-CH3 3.84 (s,3H) O-CH3 1.9-2, 8 (m,4H) -CH^-CH?-

IR (KBr) 3150 cm-1 N-H stretch 2600-3000 cm-1 C-H stretch 1980 cm-1 NH3 +CI- 1610 cm-1 Ar-H bend 1595 cm-1 N-H bend

Analysis for C 1 3 H2 0 NO3 CI

C H N 0 Cl Calculated 57,04 7,36 5,12 17,53 12,95 Found 56,88 7,42 5,09

Preparation of the trifluoroacetamides of (56) and (57)

The amine (56) or (57)» (lOmd) was taken u p in ethyl acetate

(1ml) to which was added trifluoroacetic anhydride (,5ml). The

reaction was allowed to stand at room temperature for 15 min

and then concentrated under a stream of arson. The amide was

taken u p in ethyl acetate and injected onto a 4 ft 00101 column» injection port at 250°C and flame ionization detector at 300°C, With a flow rate of 30 ml/min of helium and the column at 170°C the retention times for the acetamide derived

from the trans amine was 1 0 , 2 min and that from the cis amine

7,4 min. Quantitation was by cuttins» weishins and calibration of the peaks. wv

Preparation of trans 2(3'»4'-methylenedioxyphenyl)

cyclobutylamine hydrochloride (58)

To a solution of 2(3'p4'-methylenedioxyphenyl)

cyclobutyloxime <2*0Si>9.75mmol) in anhydrous isopropanol

(150ml) heated to reflux was added sodium (8*0a,350mmol) in

small Pieces over a one hour period. After the addition was

complete the solution was refluxed an additional hour and

cooled to room temperature. The resulting solid was acidified

with 20% HCl and the solvent removed in vacuo. The residue was

taken up in water, extracted with ether, basified with 40%

sodium hydroxide and extracted with methylene chloride. The

methylene chloride was washed with brine and dried over

madnesium sulfate. Filtration and concentration dave a

colourless oil which was taken up in a minimal amount of

anhydrous ether and treated with ethereal HCl to dive the salt.

Filtration dave the amine HCl (1.55d, 70%) as a white solid.

Three recrystallizations from ethanol-ether dave puretrans

amine 099.5% isomeric purity) mp. 207-208°(dec).

NMR (DoO) (90MHz) 6.93 (s,lH) Ar-H 6.87 (s,2H) Ar-H 5.96 (s,2H) -0CH?0- 3.4-4.0 (m,2H) Ar-CH and N-CH 1.7-2.5 (m,4H) -CHg-CHg-

IR (KBr) 3300-2600 cm-1 N-H and C-H stretch 2030 cm-1 NH3 +CI- 1610 cm-1 aromatic C-H bend

Analysis for Ci^H^^NO^Cl

C H N 0 Cl Calculated 58.03 6.20 6.15 14.05 15.57 Found 57.97 6.30 6.06 90 Preparation of ci5-2(3',4'-methylenedioxyphenyl)

cwclobutwlamine hydrochloride (59)

A suspension of 2(3',4'-methylenedioxyphenyl)cyclobutyloxime

(2»0d»9.74mmol)» platinum oxide <250mdr1♦Immol) and chloroform

(2 »0 mlflémmol) in 180 ml of anhydrous ethanol was hydrodenated

at 3 atm of hydroden for 24 hr. The solution wass filtered

throudh a celite pad to remove the suspended platinum and

concentrated to yield a white solid. Recrystallization from

ethanol/ether yielded pure cis-amine (1.8 dr 79%) mp. 247-248°C

(dec),

(Isomeric purity > 99.5%)

NMR (D?0)(90MHZ) 6.90 (s,3H) Ar-H 5 .9 9 (s ,2 H ) -OCHgO- 3.8—4.2 (mr2H) Ar—CH— r N—CH— 2.2—2.7 (mr4H) —CH^^CH?—

IR (KBr) 3200—2600 cm—1 C-H and N-H stretch 2000 cm-1 NH 3 +CI- 1600 cm-1 Ar-H bend

Analysis for C^j^H^^NO^Cl

C H N 0 Cl Calculated 58.03 6.20 6.15 14.05 15.57 Found 57.94 6.20 6.13

Preparation of the trifluoroacetamides of (58) and (59)

The amine ( ^ ) or (^)r (lOmd), was takenu p in ethyl acetate (1 ml) to which was added trifluoroacetic anhydride

(,5ml). After standind 15 min at room temperature the sample was concentrated under a stream of ardon. The amide was taken

U P in ethyl acetate and injected onto a 4 ft OVIOI column, injection port at 250°C and flame ionization detector at 300°C.

With a flow rate of 30 ml/min of helium and the column temperature at 150°C the trans-acetamide derivative had a 91

retention time of 9,0 min while the cis-acetamide had a

retention time of 6 , 8 min. Quantitation was by cutting; weighing and calibration of the peaks.

Preparation of 2<2'»4'r5'-trimethoxyphenyl)cyclobutanone (61)

To a stirred solution of 2 r4 »S-trimethoxybensaldehyde

(3,68g>18,6mmol) and cyclopropyldiphenyIsulfonium fluoroborate

(5,80g»18,6mmol) in anhydrous THF (100ml) cooled to -15°C there was added potassium t-butoxide (2,803,25,Ommol) in small portions over a twenty minute period, Alternativly the base was slowly added as a slurry in THF, Note: if the base was not added slowly the yields were considerably reduced. After addition was complete the reaction was stirred an additional 30 min and then «uenched with IN fluoroboric acid (20ml,20mmol),

The reaction was allowed to warm to room temperature, taken u p in ether and washed with successive portions of saturated bicarbonate, water, brine solution and dried over magnesium sulfate. Filtration and concentration Save an oil which was adsorbed onto silica del and washed with lidroin to remove the diphenyl sulfide. The ketone was eluted with ether which on concentration gave an oil that slowly crystallized on standing to yield the ketone (3,3d, 75%), Recrystallization from isopropanol afforded an analytical sample mp 69-70,5°C,

NMR (CDClj) (60MHz) 6,70 (s,lH) Ar-H 6,54 (s,lH> Ar-H 4,45 (t,lH, J"9Hz) Ar-CH-CQ 3,87 (s,3H) O-CH3 3,82 (s,3H> 0 -CH3 3,77 (s,3H) O-CH3 2,9-3,3 (m,2H) CO-CH? 2.1-2,6 (m,2H) Ar-C-ÊHo 92

IR (KBr) 2800-3000 cm-1 C-H stretch 1780 cm-1 C=0 stretch 1609 cm-1 Ar-H bend

C H 0 Calculated 66,08 6.83 27.09 Found 65.93 7.00

Preparation of 2(2'f4'f5'-trimethoxyphenyl)cyclobutyloxime (62)

A solution of 2(2'f4'f5'-trimethoxyphenyl)cyclobutanone

<3.3df14.0mmol>» hwdroxylamine hydrochloride (8.03» llSmmol) and 5% sodium hydroxide (90ml »112mmol) in ethanol (75ml) was refluxed for 2 hr. The solution was cooled» adjusted to pH 6 and extracted with chloroform. The ordanics were washed with brine solution and dried over madnesium sulfate. Filtration and concentration dave an oil which crystallized on standind.

Recrystallization from ethanol-water dave the oxime as a crystalline solid (2.77d» 79%) mp 144-145°C.

NMR (CDCI3 ) (60MHz) 8.1 (s»lH) N-OH (broad) 6.98 (s»lH) Ar-H 6.55 (s»lH) Ar-H 4.57 (t»lH) Ar-CH- 3.87 (s»3H) O-CH3 3.83 (s»3H) O-CH3 3.80 (s»lH) O-CH3 1.8-3. 2 (in»4H) -CH.2 -CH.2 ~

IR (NuJol) 3440 cm-1 0-H stretch 2800-3100 cm-1 C-H stretch 1690 cm-1 C=N stretch (weak) 1610 cm-1 Ar-H bend

Analysis for CiaH^yNO^

C H N 0 Calculated 62.14 6.82 5.57 27.47 Found 62.03 6.97 5.41 93

Preparation of trans-N-acetyl-2(2'r4'»5'-trimethoxyphenwl)

cwclobutylamine (67)

Trans-2(2'rA'r5'-trimethoxypheny1)cyclobutylamine

hydrochloride (200mdf,74mmol) was taken u p in 5% NaOH (10ml)

and the turbid solution was titrated with IN HCl until Just

clear. To this stirred solution was added ice (5s) and acetic

anhydride (1ml) followed by sodium acetate (Id) in water (5ml).

The solution was stirred an additional 30 min and then

extracted with chloroform. The chloroform extract was washed

with 5% NaOH» brine solution and dried over madnesium sulfate.

Filtration and evaporation yielded the acetamide as a solid

which was recrystallized from chloroform-ether mp 126-127°C.

NMR (CDCI3 ) (60MHz) 6.98 (s»lH) Ar-H 6.50 (s»lH) Ar-H 6.35 (SflH) N-H (broad) 4.45 (m»lH) N-CH- 3.84 (s»6 H) O-CH3 3.78 (s»3H) O-CH3 3.45 (m»lH) Ar-CH- 1.3—2.5 (m»4H) —CH?—CH^— 1.93 (s»3H) C0 -CH3 ^

IR (KBr) 3340 cm-1 N-H stretch 2800-3000 cm-1 C-H stretch 1655 cm-1 C=0 stretch

Analysis for C 1 5 H 2 1 NO4

C H N 0 Calculated 64.50 7.58 5.01 22.91 Found 64.56 7.73 4.80

Preparation of cis-N-acetyl-2(2'»4'»5'-trimethoxyphenyl) cyclobutylamine (6 8 )

A solution of cis-2(2'»4'»5'-trimethoxyphenyl) cyclobutylamine hydrochloride (200md».74mmol)» pyridine (2ml) and acetic anhydride (2 ml) were stirred todether at room 9A

temperature for 24 hr « The solution was concentrated in vacuo

and and the residue taken u p in chloroform* The chloroform

layer was washed 3 times with 10% NaOHi- 3 times with 10% HCl

once with brine solution and dried over madnesium sulfate*

Filtration and concentration dave the amide which was

recrystallized from isopropanol mp 157-158°C*

NMR (CDClg) (60MHz> 6*83 (s,lH) Ar-H 6*55 (s,lH) Ar-H 5*25 (br»lH) N-H 4*70

IR (KBr) 3340 cm-1 N-H stretch 2800-3000 cm-1 C-H stretch 1650 cm-1 C=0 stretch 1610 cm-1 Ar-H bend

Analysis for Ci5 h2 iN0 q

C H N 0 Calculated 64.50 7*58 5*01 22.91 Found 64.26 7*74 4.91

Preparation of 2(3'f4'-methylenedioxyphenyl)cyclobutanone (71)

To a solution of piperonal (2.0d»13.3 mmol) and cyclopropyldiphenylsulfonium flouroborate (4*60d»14*6 mmol) in THF (50ml) cooled to 0°C there was added dropwise with stirrind potassium t-butoxide (1 *80d» 16*Immol) in a minimal amount of THF, After addition was complete the reaction was stirred an additional .5 hr and Quenched with 10 ml of IN fluoroboric acid. The solution was taken up in ether, washed with saturated bicarbonate, brine solution and dried over anhydrous madnesium sulfate. Filtration and concentration vs

afforded an oil consisting of ketone and dir-'henyl sulfide,

Chromatodraphy over silica del with a 7% ether/1idroin mixture

in a cold room afforded the ketone as an oil which solidified

on coolind and was dried under reduced pressure (1,8 dr 71%);

mp « 44-46^C,

NMR (CDCIt ) (90 MHz) 6,72 (S;3H) Ar-H 5,92 (s,2H) -OCHnO- 4,42 (t;lH;J=9Hz)“ Ar-CH-C=0 2,8 - 3 , 4 (m,2H) -CO-CH?- 2 ,0 -2 ,8 (m,2 H) -CO-C-ÙH?-

IR (KBr) 2800-3050 cm-1 C-H stretch 1781 cm-1 C=0 stretch 1608 cm-1 Ar-H bend

Analysis for

C H 0 Calculated 64,38 5,30 25,24 Found 69,46 5,40

Preparation of 2-(3';4'-methylenedioxypheny1)cyclobutyloxime (72)

The oil obtained from the reaction of piperonal

(8 ,0d;53,3mmol)with cyclopropyldiphenyIsulfonium flouroborate before chromatodraphy was refluxed for 1 hr with hydroxylamine hydrochloride (24,Od;345mmol) in ethanol (200ml) and 10% aoueous sodium hydroxide (300ml ;750mmol), The solution was cooled and extracted twice with 1 0 0 ml portions of lidroin which were back extracted with 50 ml of a 10% sodium hydroxide solution. The aoueous layers were combined; cooled to about 1 0 °

C with an ice bath; acidified to p H 5 with concentrated HCl and extracted four times with 150 ml portions of chloroform which were dried over anhydrous madnesium sulfate. Filtration and concentration dave a heavy oil that crystallized on standind.

Recrystallization from ethanol-water dave the oxime (8,3d; 77%) 96

mp. i 0 9 ~ U 0 ”c.

NMR (CDCI3 ) (90MHz) 8*29 (s*lH) =N-Q-H (broad) 6,72 (sr 3H) Ar-H 4*32 (tplHfJ= 8 Hz) Ar-CH- 3*7-3*1 (m,2H) 0=C-CH?- 2*7—3*6

IR (KBr) 3260 cm-1 0-H stretch 2800-3000 cm-1 C-H stretch 1705 cm-1 C=N stretch 1615 cm-1 Ar-H bend

Analysis for CiiHiiNOg

C H N 0 Calculated 64*38 5*40 6*82 23*39 Found 64*49 5*46 6*85

Preparation of trans-N-acetyl-2(3',4'-methylenedioxyphenyl) cyclobutylamine (73)

A solution of trans-2-(3',4'-methylenedioxyphenyl) cyclobutylamine hydrochloride (100map0*44mmol), acetic anhydride (2 ml) and pyridine (2 ml) was stirred at room temperature for 16 hr* The sample was concentrated in vacuo, taken up in chloroform, washed three times with 10% HCl, twice with 10% NaOH, once with brine solution end dried over madnesium sulfate* Filtration and concentration dave an oil which crystallized on standind* Recrystallization from ether-lidroin dave the amide as a white solid mp* 132-133°C*

NMR (CDCI3 ) (90MHz) 6*71 (s,3H) Ar-H 5*91 (s,2H) -OCH2 O- 5*75 (s,lH) N-H (broad) 4*46 (p,lH,J=9Hz) C-CH-N 3*23 (q ,IH,J=9Hz) Ar-CH- , 1*5—2.5 (m,4H) —CHo—CHo— 1*95 (s,3H) 0 =C-CH3 97

IR (KBr) 3295 cm-1 N-H stretch 2800-3000 cm-1 C-H stretch 1650 cm-1 0=0 stretch 1605 cm-1 Ar-H bend

Analysis for C 1 3 H 1 5 NO3

C H N 0 Calculated 66,94 6,48 6,00 20,58 Found 66,79 6,50 5,93

Preparation of cis-N-acetyl-2(3',4'-methylenedioxyphenyl>

cyclobutylamine (74)

A solution of

cis-2(3',4'-methylenedioxyphenyl)cyclobutylamine hydrochloride

(100mdf0,44 mmol)» acetic anhydride (2ml) and pyridine (2ml)

was stirred at room temperature for 16 hr. The sample was concentrated ^ vacuo» taken up in chloroform» washed three

times with 10% HCl» twice with 10% NaOH» once with brine solution and dried over madnesium sulfate. Filtration and concentration dave an oil which slowly crystallized on standind. Recrystallization from ether-lidroin dave the amide as a white solid mp, 90-91°C,

NMR (CDCI3 )(90MHz) 6 ,6 -6 ,9

IR (KBr) 3300 cm-1 N-H stretch 2800-3000 cm-1 C-H stretch 1640 cm-1 C=0 stretch 98

Analysis for C 1 3 H 1 5 NO3

C H N 0 Calculated 66,94 6,48 6,00 20,58 Found 66,95 6,50 5,97

Preparation of 6r7-methylenedioxy-cis-3,4-cyclobutyl-

l,2p3f4-tetrahydro-NrN-dimethylisoQuinolinium iodide (75)

To a cooled solution of cis-2(3'>4^-methylenedioxyphenyl)

cyclobutylamine (200mdf0»88mmol) in 8 8 % formic acid (3,0ml) was

added dropwise a 28% formalin solution (3,0ml), After addition was complete the solution was heated to 70°C for 12 hry cooled? diluted with 6 N HCl and washed with chloroform. The aeueous

layer was basified with 10% NaOH» extracted with methylene chloride» washed with brine and dried over madnesium sulfate,The solution was filtered» concentrated and taken up in a small Quantity of anhydrous THF to which was added methyl iodide, The resulting solid was filtered and dried to dive a pale yellow solid (ISOmd? 57%), Recrystallization from ethanol-ether dave a white solid mp, 252°C (dec),

NMR (DoO)(90MHz) 6,81 (s»lH) Ar-H 6.77 (s»lH) Ar-H 6,00 (s»2H) -QCHoO- 4.67 (1H»AB doublet»J=14 Hz) Ar-CH-N 4,34 (1H,AB doublet»J=14Hz) Ar-CH-N 4,24 (m»lH) -N-CH- 3,79 (m»lH) Ar-CH-C 3,13 (s»3H) -N-CH, 2,97 (s»3H) -N-CH3 2,42 (m»3H) -CH-CHo- 1.77 (m, 114) -CH-C-"

IR (KBr) 2900-3000 cm-1 C-H stretch 1620 cm-1 Ar-H bend (weak)

Analysis for Ci4 HigN0 2 l

C H N 0 I Calculated 46,81 5,05 3,90 8,91 35,33 Found 46,75 5,30 3,85 y 9

Preparation of' 2 r 4 f5~triiiiethoxypropiopherione (76)

To 3 solution of propionyl chloride (4.63dr♦471mol>r

aluminum chloride (63.Od f »S22mol) in 200ml of carbon disulfide

cooled to O^C there was added dropwise 1 f2 f4 -trimethoKuben2 ene

(70.OdF.417mol) with vigorous mechanical stirrind. Shortly

after the addition was complete stirrind was no londer possible

and the mixture was allowed to stand at room temperature

overnidht. The liouid portion was filtered throudh a dlass wool

plud and the aluminum salts decomposed by the addition of ice

and water. The product was extracted with methylene chloride

washed with 2N sodium hydroxidef water f brine and dried over

madnesium sulfate. Filtration and concentration dave a solid

which on recrystallization from 95% ethanol afforded

2i<4F5-trimethoxypropiophenone (60.5dF 64%) mp. 107-108°C. (lit. o mp. 108 C) [182], Concentration of the mother liouids afforded an additional 4.0d of product.

NMR (CDCI3 ) (60MHz) 7.43 (sfIH) Ar-H 6.51 (s f IH) Ar-H 3.93 (sf3H) O-CH3 3.90 (sf3H) O-CH3 3.87 (sf3H) 0 -CH3 3.00 (o f 2H fJ=7Hz ) CO-CHo- 1.16 (tf3HFJ=7Hz) CO-C-CH3

IR (NuJol) 2800—3000 cm-1 C—H stretch 1655 cm-1 C=0 stretch

Rearrangement of 2 f4 f5-trimethoxypropiophenone (76)

A solution of the ketone (1.Odf4.46mmol)f ammonium acetate

(1.25dF16.2mmol)F and nitroethane (2.Odf26.Ommol) in dlacial acetic acid (10ml) was heated to 105°C for 12 hr. After this period there was no ketone present by thin layer chromatodraphic analysis but two lower Rf components were 100

noted» silica ael plates diethyl ether as solvent. The solution

was cooled» diluted with water and extracted with chloroform.

The chloroform was washed with dilute sodium hydroxide» brine

solution and dried over magnesium sulfate.

Filtration and concentration dave an oil which was column chromatographed on silica del with diethyl ether as eluent. Two products were obtained» a higher Rf component A (600md) and a

lower Rf component B (300md) which solidified on standing and were recrystallized from ether/1idroin, The spectral characteristics of these compounds may be found in Table 10,

Compound A mp, 8 8 °C» compound B mp, 101°C,

Preparation of 2 »4 »5-trimethoxynitrobenzene (80)

To a solution of 1»2»4-trimethoxybenzene (10,0d»60,2 mmol) in glacial acetic acid (1 0 0 ml) cooled with an ice bath was added dropwise nitric acid (4,14d»66,2 mmol) and two drops of sulfuric acid in glacial acetic acid (10ml), After the addition was complete the dark mixture was stirred an additional 0,5 hr » allowed to warm to room temperature and Quenched with water.

The mixture was extracted with methylene chloride'which was washed twice with water» twice with 10% NaOH» again with water» then brine solution and dried over magnesium sulfate.

Filtration of the dessicant and concentration gave an oil which was chromatographed over silica gel with ether. The product eluted first as a bright yellow fraction. Recrystallization from ethanol gave the nitro compound as a bright yellow solid

(1,4 g» 11%) mp,126-127°C (lit, mp, 129°C) [176], 101

NMR (CDCI3 )(60MHz) 7,59 (s»lH> ortho Ar-H 6,62 (SflH) iTieta Ar-H 4,00 (s,6 H) Ü-CH3 3 , 9 0 (s,3H) O-CH3

Preparation of N-propionyl-2f4,5-trimethoxyaniline (78)

A solution of 2 , 4 » 5-t riniethoxyni trohenzene (l,2df5,64mmol) in ethanol (50ml) with 10% palladium on charcoal (50md) was hydroSenated at 2 atm of hwdroden for 1 hr. The catalyst was removed by filtration and concentrated in vacuo. The residue was taken u p in pyridine (1 0 ml) to which was added propionic anhydride (10ml) and stirred at room temperature for 16 hr. The sample was concentrated ^ vacuo, taken u p in methylene chloride and washed successivly with 10% HCl, saturated sodium bicarbonate, brine solution and dried over madnesium sulfate.

The sample was filtered, concentrated and chromatodraphed on silica del with diethyl ether. The amide eluted as the first component and solidified on standind. Recrystallization from ether/lidroin yielded the amide as a flaky solid (520md,39%) mp 87-88°C,

NMR (CDCI3 ) (90MHz) 8,16 (s,lH) Ar-H 7,6 (br,lH) -NH-CO- 6,54 (s,lH) Ar-H 3,86 (s,9H) O-CH3 2,41 (a,2H,J=8,5Hz) CO-CHo 1,24 (t,3H,J=8,5Hz) CO-C-CH3

IR (KBr) 3240 cm-1 N-H stretch 1650 cm-1 0=0 stretch

Mass Spec, (El) M+=239

Analysis for C 1 2 H 1 7 NO4

C H N Calculated 60.24 7,17 5,85 26,7; Found 60.43 7,22 5,87 102 Assay for Syiripathectant Properties of 48 and 49

Male CD-I mice weiahina 18-233 were used in all

experiments. To test for ability to produce chemical

sympathectomy» mice were treated i,v, or i,p, with saline»

the test compound or 6 -hydroxydopamine (as an internal

standard)» in a volume of 0,lml/203» 2 0 hr prior to bolus

i,v, administration of ^H-norepinephrine (0«5uCi/3na/203),

The mice were sacrificed after an additional 60 min by

cervical dislocation and the hearts dissected free from

pericardium. The hearts were rinsed for 1 min in 5m1

ice-cold sodium phosphate buffer (0,13 M»p H 7,4)» blotted dry on filter paper and weiahed. Total radioactivity was extracted by homoaenization in 2m1 of 0,4 N perchloric acid

in a alass homoaenizina tube fitted with a teflon pestle.

The homoaenizina apparatus was washed with an additional 1ml of perchloric acid wh ich was then added to the oriainal homoaenate. The entire homoaenate was centrifuaed at 30»000

X a for 40 min at 4°C and 1ml of the resultina supernatant assayed for tritium by dissolvina in 10ml ACS liouid scintillation cocktail (Amersham-Searle) and countina for 20 min in a Beckman LS 345 lieuid scintillation spectrometer.

Quench was monitered by automatic external standardization and countina efficiency for tritium was 20-25%, No corrections were made for norepinephrine metabolites.

To test for ability to inhibit nueronal uptake» the test compounds were injected i,v, simultaneously with

^^-norepinephrine in a volume of 0,2ml/20a, The animals were sacrificed after 1 0 min and the tritium content of the heart 103 determine d <;i s d e s c i' I b e d b c? v e.

Norepinephrine uptake is expressed as a percent of control and differences were tested for significance

(p<0,05) by student's t-test for unpaired observations.

Uptake Assay for 50 and 51

In each experiment, three male albino rats of the

Sprasue-Dawley strain (200-3003) were killed by decapitation. The corpus striatum (including caudate nucleus, putamen and dlobus pallidus) and cortex were dissected from the brain as described by Glowinski and

Iversen [185], Synaptosomes were isolated according tothe procedure used by Ferris et al [184], The brain tissues were weighed and homogenized in 20 volumes of 0,32 M sucrose by using 10 u p and down strokes with the pestle revolving at

840 rpm. The teflon pestle was modified so that the clearance was 0,25mm, The cell debris and nuclei were separated by cetrifugation at 1000 x g for 10 minutes in a

Sorvall RC2-B centrifuge, A 0,2ml aliouot of the supernatant containing the synaptosomes and other cellular components was added to 1,79ml of the modified Krebs buffer. The composition of the modified Krebs buffer used in the incubations was (mM): NaCl,118) KC1,4,7J CaCl^,l,26>

MgCl2,0»54î NaHC02,25f NaH^PO^,i; glucose,11) and ethylene diamine tetraacetic acid (EDTA),0,13, Iproniazid phosphate, lOuM, was added to inhibit monoamine oxidase.

The modified Krebs solution was bubbled with 95% Qg - 5% 1Ü4 CO2 das mixture for at least 10 minutes before addition of

the synaptosomal preparation to ensure complete oxygenation

of the medium. After a 10 minute preincubation with or

without drugs at 37°C in a metabolic shaker under 95% O 2 - 3 3 5 % COgif H—DA (600f000 dpm) or H—NE (600f000 dpm) was added

to the incubation medium in lOul providing a final

concentration of 0,luM, Thus» the final volume of the medium

was 2,0ml, The incubations were continued for an additional

5 minutes at 37°C, Each sample was then collected on a

Celolate membrane filter 25mm An diameter of 0,5 u pore size

(Millipore) by vacuum filtration using a 3025 Multiple

Sampling Manifold, The filters were washed twice with 5m1 of

ice-cold incubation medium, transferred to counting vials, and dissolved in 2m1 of dioxane. Ten ml of scintillation fluid (ACS, Amersham-Searle) was added and the radioactivity in the samples was measured in a Beckman LS 345 liouid scintillation spectrometer, 3 o In all experiments, the H-amine accumulated at 0-4 C was subtracted from the uptake At 37°C to give the amount of

^H-amine accumulated by an active uptake process.

The molar concentration of a drug which produced 50% 3 3 inhibition of accumulation of H-NE and H-DA in the cortex and corpus striatum, respectively, was obtained from the semi logarithmic plot of the data. 105 Dopamine Binding Assay for 5 0 r 51r 52 and 53

Binding assays were performed similar to the method of

Burt et al C1233» The corpus striatum of four Spradue-Bawley

rats <200-300d) were homogenized in 40 volumes of cold 50mM

Tris buffer» p H 7*7 at 25°C» with a Brinkman Polytron PT-10

(setting 6f 5 sec twice). The homogenate was centrifuged

twice at 50»000 x g for 10 minutes with rehomogenization

(setting 6r 5 sec) of the intermediate pellet fresh buffer.

The final pellet was homogenized in 90 volumes of cold 50mM

Tris buffer containing 0,1% ascorbic acid, lOuM iproniazid,

120mM NaCl, 5mM KCl, 2mM CaCl„ and ImM MgCl^, to give a

final p H of 7,1 at 37°C, This homogenate was placed in a 37°

C hath for 5 minutes and returned to ice, o Incubations (10 minutes, 37 C ) were carried out in a 2m1 3 final volume containing 10 nM H-dopamine (16,41 or

21,42Ci/mmol, New England Nuclear) which was added in 100 ml of 0,1% ascorbic acid, 1,3 ml of tissue suspension, and 100 ul of various concentrations of drugs dissolved in 0,1% ascorbic acid. Incubations were terminated by filtration under vacuum through Whatman GF/B filters with two 5m1 rinses of cold buffer. The filters were counted by liouid scintillation spectrometry. All the drugs in higher concentrations decreased binding of ^H-dopamine to the glass fibre filters, so an eoual number of samples were prepared identical to those described above, except the membranes were omitted. The ^H-dopamine retained by the filters under these conditions was subtracted from the corresponding sample containing the membranes. 106

SUMMARY

To investigate the mechanism of 6-hydroxydopamine induced neurotoxicity the cyclobutyl analodsf cis- and trans-2(2'» 4'r5'-trihydroxyphenyl)cyclobutylamine were prepared. The two analods were prepared in a stereoselective manner from 2(2'p4'f5-trimethoxyphenyl) cyclobutyloxime,

Reduction of the oxime with sodium in isopropanol followed by déméthylation with boron tribromide afforded the trans-analod. The cis-analod was obtained by reduction of the oxime with hydrosen/platinum oxide/chloroform in ethanol followed by déméthylation with boron tribromide. The oxime was obtained by reaction of 2>4»5-trimethoxybensaldehyde with cyclopropyldiphenylsulfonium flouroborate and potassium t-butoxide followed by reaction with hydroxylamine hydrochloride «

Pharmacological testing of the two analogs» cis- and trans-2(2' »4' »5 -trihydroxylphenyl ) cyclobutylamine» revealed that neither compound possessed nuerotoxic properties. The trans-analog had affinity for the uptake mechanism although it is not clear whether it is taken into the neuron.

The dopamine analogs cis- and trans-

2(3'»4^-dihydroxyphenyl)cyclobutylamine were prepared in a manner analogous to that reported for the 6-hydroxydopamine analogs, Piperonal was converted in two steps to the corresponding cyclobutyloxime which was reduced stereoselectively to cis- and 107

trans-2(3',4'-methylenedioxyphenyl) cyclobutylamine» Cleavage

of the methylenedioxy function with boron tribromide afforded

the corresponding cis- and trans-dihydroxyamines♦

The trans-amine had greater affinity than the cis-analog

for the uptake mechanisms of adrenergic and dopaminergic

synaptosomes* Assay at a membrane bound postsynaptic receptor

showed the trans-analog to be better bound than the cis-analog* Based on these observations it would appear that

3 trans disposition of amine and aromatic functions defines the prefered conformation of dopamine at the postsynaptic dopamine receptor and presynaptic uptake site.

The dimethyl analogs» cis- and trans-

N»N-dimethyl-2 ( 3 *' » 4 " -di hydroxy phenyl > cyclobutyl ami ne were prepared and assayed for their ability to bind to the postsynaptic dopamine receptor* The dimethyl analogs had a greater affinity for the binding site than the corresponding cis- and trans- primary amines* The trans-dimethyl analog was more active than the cis-dimethyl analog in the binding assay confirming a prefered trans- orientation of amine and aromatic functions at this postsynaptic dopamine receptor*

A novel rearrangement of 2»4»5-trimethoxypropiophenone to

N-acetyl and N-propiony1-2r4 r5-trimethoxyaniline occurred when the ketone was reacted with nitroethane and ammonium acetate in glacial acetic acid* The reaction appears to involve addition of nitroethane to an imine derivative of the ketone. 108

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