Comparative effects of stereoisomers of psychotomimetic phenylisopropylamines

Life Sciences Vol. 13, pp. 885-898, 1973 . Printed in Great Britain

Pergamon Press

COMPARATIVE EFFECTS OF STEREOISOMERS OF PSYCHOTOMIMETIC PHENYLISOPROPYLAMINES Donald C . Dyer Department of Pharmacology and The Anesthesia Research Center School of Medicine, University of Washington Seattle, Washington 98195 and David E . Nichols, David B . Rusterholz and Charles F . Barfknecht Division of Medicinal Chemistry College of Pharmacy, The University of Iowa Iowa City, Iowa 52240

(Received 13 July 1973 ; in final form 24 August 1973) Summary Stereoisomers, R(-) and S(+), of five psychotomimetic phenylisopropylamines contracted isolated strips of sheep umbilical arteries . The compounds exhibited the following order of potency : 2,5-dimeth- oxy-4-bromo-amphetamine (DOB) > 2,5-dimethoxy-4-ethyl-amphetamine DOET) > 2,5-dimetho -4-methyl-amphetamine (DOM) > 2,5-dimethoxyamphetamine (2,5-DMA) > 4-methoxy-amphetamine (PMA) . Stereoselectivity was observed in that the R(-) isomers were more active than the S(+) isomers except for PMA . Evidence was obtained for the action of these hallucinogens on 5-hydroxytryptamine receptors . There was a general correlation of smooth muscle stimulating activity with known hallucinogenic activity . Isomers of biologically active compounds usually differ in activity (1) . Barfknecht and Nichols (2) found that the psychotomimetic activity of 3,4dimethoxyamphetamine (3,4-DMA) seemed to reside in the R(-) Isomer .

The devel

opment of methods for routine synthesis of isomers of various psychotomimetic phenylisopropylamines has given a new impetus for the study of their biologic activity and receptors mediating their action (3) .

Hallucinogens d-lysergic

acid diethylamide (LSD), mescaline, bufotenine, psilocin and psilocybin, produce pronounced contractions of umbilical vasculature (4,5) .

The vasocon-

striction induced by these hallucinogens appears to be mediated via 5-hydroxytryptamine receptors .

The purpose of this study was to compare the potency of

selected psychomimetic phenylisopropylamine isomers on umbilical vasculature and

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to ascertain if they exhibited stereoselectivity . Methods Sheep umbilical arteries were obtained as previously described (6) .

The

arteries were helically-cut into strips approximately 2 cm long and placed in a series of 10 ml isolated organ baths .

The tissues were allowed to equilibrate

for about 2 hr in the Krebs-Henseleit (Krebs) solution under 1 g tension before beginning the experiment .

The Krebs solution was maintained at 37°C and oxygen-

ated with oxygen-carbon dioxide (95/5) .

The contractions were recorded isoton-

ically on a kymograph drum with a tenfold magnification .

In all experiments

except those in which the protection technique was employed, the tissues were first maximally contracted with 5-hydroxytryptamine (5-HT, serotonin) .

All

comparisons of activity were, therefore, made against the common agonist, 5-HT . Following relaxation of the tissue, dose-response relationships were obtained to all agonists by adding them in cumulative amounts by micropipets to the bath . In the protection studies, phenoxybenzamine (POB, 3.3 x 10 - 'M) was added to the bath for 10 min alone or after an agonist (protecting drug) had been added.

All

protecting drugs were in contact with the tissue for a minimum of 15 min or until the contraction reached a plateau.

The tissues were repeatedly washed

over a 30-min period or until they relaxed before challenging the tissues with an agonist .

When antagonists other than POB were employed, they were equili-

brated with the tissue for 15 min and remained in the bath when an agonist was added . The formulas for the ten compounds studied are given in Fig . 1 . pounds are :

These com-

S(+)-4-methoxy-amphetamine (PMA+) ; R(-)-4-methoxy-amphetamine

(PMA-) ; S(+)-2,5-dimethoxy-amphetamine (2,5-DMA+) ; R(-)-2,5-dimethoxy-amphetam ine (2,5-DMA-) ; S(+)-2,5-dimethoxy-4-methyl-amphetamine-(DOM+) ; R(-)-2,5-dimethoxy-4-methyl-amphetamine (DOM-) ; S(+)-2,5-dimethoxy-4-bromo-amphetamine (DOB+) ; R(-)-2,5-dimethoxy-4-bromo-amphetamine (DOB-) ; S(+)-2,5-dimethoxy-4-ethyl-amphetamine ,(DOET+) ; R(-)-2,5-dimethoxy-4-ethyl-amphetamine (DOET-) . compounds were prepared by methods previously described (3) .

All of the above These compounds

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887

were employed as their hydrochloride salts and were dissolved in normal saline .

Isomers of Substituted Phony lisopropylaminas 2

2

Compound

Isomer

aryl substitution

PMA

S (+)

4-inethoxy

PMA

R (-)

4-inethoxy

2,5-DMA

S (+)

2,5-dimethoxy

2,5-DMA

R (-)

2,5-dimethoxy

DOM

S (+)

2,5-dimethoxy-4-methyl

DOM

R (-)

2,5-dimethoxy-4-methyl

DOB

S (+)

2,5-dimethoxy-4-bromo

DOB

R (-)

?,5-dimethoxy-4-bromo

DOEt

S (+)

2,5-dimethoxy-4-ethyl

DOEt

R (-)

2,5-dimethoxy-4-ethyl

FIG . 1

Structural formulas of isomers of substituted phenylisopropylamines . See the text for details . Results The smooth muscle stimulating activity of the isomers of PMA and 2,5-DMA is presented in Fig. 2 .

Both isomers of PMA possessed a small amount of

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A...... A

PMA (+) PMA (-) *--- " 2,5-DMA (+) 0---0 2,5-DMA(-) R-a 2,5- DMA (-)+80L

FIG. 2 A comparison of the isomers of PMA and DMA to contract sheep umbilical arteries . is represent points on the 2,5-DMA(-) curve which are significantly different from 2,5-DMA(+) (P<0 .05) . 2-Bromolysergic acid diethylamide (BOL, 10 - °M) readily blocked 2,5-DMA(-) . Vertical bars are + S .E .M . of four experiments . activity ; whereas, 2,5-DMA(-) was able to elicit a sizeable contraction.

This

contraction was readily blocked by 2-bromolysergic acid diethylamide (BOL) . Both DOB and DOET contracted the tissues at low concentrations (Figs . 3 and 4) . The activity of the R-(-) isomer of both compounds was greater than that of the

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889

Contractions to all four isomers were antagonized by BOL .

100 90= 80~

Zv h m ó

7060

h

g 50

10

t

0L/ -9

'

I

/ ~

r

0--0 DOB (-) """"""" DOB(-) +BOL DOB (+) A,"" - A DOB(+)+SOL

-8 -7 -6 -5 Log agonist concentration ( M

-4

FIG . 3 Dose-response curves to DOB(-) and DOB(+) alone and in the presence of BOL (10-° M) . DOB(-) was more active than DOB(+) at all points on the dose-response curve and BOL significantly blocked contractions to all agonists . Vertical bars are + S.E .M . of four to five experiments .

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100, 90~

4>--* 0- "-0 Ir --A AP , ...,o

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DOET(-) DOET(-) +BOL DOET (+) DGET(+) +BOL

80~

m

é 50~ .E 40~

20 10 -9

-8

-7

-6

-5

Log agonist concentration ( M )

-4

FIG . 4

Dose-response curves to DOET(-) and DOET(+) alone and in the presence of BOL (10 - BM) . DOET(-) was more active than DOET(+) at all points on the dose-response curve and BOL significantly blocked contractions to all agonists . Vertical bars are + S .E .M . .of four to five experiments . As with the latter two hallucinogens, DOM readily contracted sheep umbilical arteries .

The DOM(-) isomer proved to be more active than the DOM(+)

isomer, and both were blocked by BOL (Fig . 5) .

Neither atropine nor piperoxan

antagonized DOM(-) mediated contractions (Fig . 5) .

The time needed to

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" -" DOM (-)

DOM (-) + BOL Gp.. . ..d DOM (-)+Atropine à"- -à DOM (-)+Piperoxan

of -9

-8 7 -6 -5 Log agonist concentration (M)

-4

FIG. 5 Dose-response curves to DOM(-) and DOM(+) alone and in the presence of BOL (10 -8 M) . DOM(-) was more active than DOM(+) at all points on the dose-response curve and BOL significantly blocked contractions to all agonists . Atropine (1 .2 x 10 -7 M) and piperoxan (3 x 10 - 'M) did not significantly alter responses to DOM(-) . Vertical bars are + S .E .M . establish a complete dose relationship for DOM, DOB, and DOET (approximately 2 hr) was about twice as long as that needed for 5-HT .

The rate of contraction

seemed to be slowest in the lower concentration range and resembled that previously seen with the ergot alkaloids (6) .

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To explore in more detail the possibility that DOM(-) was acting on 5-HT receptors, we decided to employ the protection technique .

If DOM(-) were acting

on 5-HT receptors, then it should be possible to show that 5-HT will protect DOM(-) receptors against phenoxybenzamine block and DOM(-) should also protect 5-HT receptors against phenoxybenzamine . tractions to DOM(-) (Fig . 6) .

1001

Phenoxybenzamine readily blocked con-

However, when the 5-HT receptors were occupied

.._ . F --+ Poe. DOM(-)

â---ô 5-HT-POS . DOM(-)

FIG. 6 Demonstration of protection by 5-HT of DOM(-) receptors . All tissues were maximally contracted with 5-HT . The 5-HT underscored with phenoxybenzamine (POB) indicates the drug used to protect against POB. If there is no underscoring, .the POB was added after the tissue had relaxed (not protected . A cumulative dose-response relationship was then obtained to DOM(-J on all three tissues . When POB (3 .3 x 10 - 'M) was used, it was left in contact with the tissue for 10 min . Tissues were then washed until they relaxed, or for at least 30 min in the case of the nonprotected strips . The concentration of 5-HT used to protect against POB was 1 x 10 -4 M. Points on the curves are ± S .E .M . of four experiments .

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before exposure of the tissue to POB, the DOM(-) receptors were protected, and the tissue continued to give good responses to DOM(-) .

We have previously

shown that 5-HT protects its own receptors against POB block and not those of norepinephrine or acetylcholine (7) .

Also, neither acetylcholine nor norepin-

ephrine protected 5-HT receptors in sheep umbilical arteries (7) . To ascertain if the protection experiment could be run in the reverse direction, the tissues were first maximally contracted with DOM(-) .

While the

tissues were contracted, POB was added to selected tissues for 10 min . and all tissues in the study were then repeatedly washed .

These

It took 5 to 6 hr

for the tissues to relax to baseline, even with repeated changes in the bath solution .

This is opposed to the 1 .5 to 2 hr needed for the tissues to relax

following a maximal contraction to 5-HT .

In this series of experiments, POB

again blocked contractions to a subsequent administration of DOM(-) (Fig . 7, left panel) .

However, in those tissues in which DOM(-) protected the receptors,

the DOM(-) induced contraction was retained .

The response of the DOM(-) pro-

tected tissues did not differ significantly from the DOM(-) "time control" tissues .

It was necessary to study the effect of time on the response of the

arteries to DOM(-) since these experiments lasted about 4 hr longer than those in which the tissue was first maximally contracted with 5-HT (Figs . 2-6) . Phenoxybenzamine blocked contractions to 5-HT (Fig . 7, right panel) .

The

response of those tissues to 5-HT, which were protected by DOM(-), could not be distinguished from the 5-HT control . Discussion It is apparent from this investigation that isomers of psychotomimetic phenylisopropylamine differ in their potency in stimulating umbilical vasculature .

For every compound (except PMA) the more potent isomer proved to have the

R(-) configuration .

Isomers with the R(-) configuration produced contractions

at lower concentrations and their maximum effect was greater than those with the S(+) .

Our study allowed us to obtain a complete dose-response relationship

to the isomers which is not practical or possible when their CNS effects are

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FIG. 7

Demonstration that DOM(-) protects its own receptors and those of 5-HT . All tissues were maximally contracted with DOM(-) . The DOM(-) underscored with the phenoxybenzamine (POB) indicates the drug used to protect against POB . If there is no underscoring, the POB was added after the tissue had relaxed (not protected) . A cumulative dose-response relationship was then obtained to either (DOM(-) (left panel) or to 5-HT (right panel) . When POB (3 .3x10-'M) was used it was left in contact with the tissue for 10 min . Tissues were then washed until they relaxed, or for at least 30 min in the case of the nonprotected strip . The concentration of DOM(-) used to protect against POB was 4 .06 x 10 -S M. Points on the curves are ± S .E .M . of five to six experiments . studied .

When the smooth muscle stimulating activity of the R(-) isomers is

compared to mescaline the following order of potency is found : DOM>Mescaline>2,5-DMA>PMA (Table 1) .

DOB>DOET>

This same general relationship exists for

their hallucinogenic potency with the exception of 2,5-DMA and PMA .

The latter

two compounds have relatively more hallucinogenic than smooth muscle activity in relation to mescaline .

The results also indicate that substitution in the

4-position (bromo>ethyl>methyl>hydrogen) affects the hallucinogenic and smooth muscle activity in a similar direction .

Benington et al . (8) found both the

R(-) and S~+) isomers of DOB to be more active than the corresponding isomers of

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DOM in behavioral studies in rats but for each compound the R(-) was the more active isomer .

The R(-)-DOM isomer is also the most active in humans (9) . TABLE 1

Sheep Umbilical Artery : A Comparison of the Concentration of Various Agonists Required to Produce a Contraction Equal to that Obtained at the ED 3o of 5-Hydroxytryptamine Dose-Ratio at ED3o Mescaline er goni -T

ED 3o DOET(-) DOET(+) DOM(-) DOM(+) DOB(-) DOB(+) 2,5-DMA(-) PMA(-) 5-HT Mescaline

4 .8 21 .5 24 .0 139 .7 2 .4 18 .0 800 .0 100,000 .0 205 .0 610 .0

x x x x x x x x x x

10 -9 - M 10 s 10- s 10- s 10 s 10 -- s 10 s 10 -I - approx . 10 s 10-9 b

127 .0 28 .0 25 .0 4 .4 254 .0 34 .0 0 .6 0 .006 3 .0 1 .0

Hallucinogenic Potencya Mescaline aATT RET nogen 200 80-100

Ref . 10 11,12

400 8 5

c 6 6

1

a Potency based on the use of racemic mixtures in reference to mescaline (Ref-12) . b Based on data extracted from previous work (Ref .5) . C Potency was assigned as 400 based on the work of Dr . A .T . Shulgin, who found DOB to be more active than DOM . Private communication from Dr . Shulgin to C .F .B . All of these compounds, with the exception of PMA which was not studied in detail, were antagonized by BOL .

In addition, protection and cross-protection

against phenoxybenzamine block was found to occur between 5-HT and DOM(-) . Contractions to DOM(-) were not blocked by atropine or piperoxan .

This gives

strong support for the notion that DOM, DOB and DOET act via 5-HT receptors in umbilical vasculature similarly to that previously found for mescaline, LSD, bufotenine and .psilocin (5) . Using the terminology suggested by Portoghese (1) 2,5-DMA, DOM, DOET and DOB exhibit "stereoselectivity" in their action on umbilical vasculature and not "stereospecificity" which would imply that only one of the isomers possessed activity .

The close relationship between the hallucinogenic action and the

ability of hallucinogens to contract umbilical vasculature suggests this smooth muscle may be useful in continued evaluation of the mechanism of action of hallucinogens and in screening for compounds with possible hallucinogenic activity .

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Acknowledgments We thank Barbara Osborne for her technical assistance and Dr . W .A . Hodson for his cooperation in obtaining the sheep tissue .

This study was supported in

part by U .S . Public Health Service Grant GM15991, the Washington State Heart Association, a Salsbury Foundation fellowship (D .B .R .) and a grant from Ortho Research Foundation . References 1.

P .S . PORTOGHESE, Ann . Rev . Ph a rmacol . 10, 51 (1970) .

2.

C .F . BARFKNECHT and D .E . NICHOLS, J . Med . Chem . 15, 109 (1972) .

3.

D .E . NICHOLS, C .F . BARFKNECHT, D .B . RUSTERHOLZ, F . BENINGTON and R .D . MORIN, J . Med . Chem . 16, 480 (1973) .

4.

D .W . GANT and D .C . DYER, Life Sci . 10, 235 (1971) .

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D .C . DYER, J . Pharmacol . Exp . Ther . 184, 366 (1973) .

6.

D .C . DYER, J . Pharmacol . Exp . Ther . 175, 565 (1970) .

7.

D .C . DYER, Submitted for publication .

8.

F . BENINGTON, R .D . MORIN, J . BEATON, J .R . SMYTHIES and R .J . BRADLEY, Nature New B iology 242, 185 (1973) .

9.

A .T . SHULGIN, J . Ph arm . Pharmacol . 25, 272 (1973) .

10 .

S .H . SNYDER, L . FAILLACE and H . WEINGARTNER, Amer . J . Psychiat . 125, 113 (1968) .

11 .

S .H . SNYDER and L . FAILLACE, Science 158, 669 (1967) .

12 .

A .T . SHULGIN, T . SARGENT and C . NARANJO, Nature 221, 537 (1969) .