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PergamonPharmacology Biochemistry and Behavior, Vol. 47, No. 3. pp. 667-673, 1994

Copyright © 1994 Elsevier ScienceLtdPrinted n the USA. All rights reserved

0091-3057/94 $6.00 + .00

D rug D iscr im inat ion and R eceptor BindingStudies of N Isopropyl Lysergamide Derivatives

X U E M E I H U A N G , t D A N U T A M A R O N A - L E W I C K A , ~ R O B E RT C . P FA FF : ~A N D D AV I D E . N I C H O L S * ~ l

Departments o f Medicinal Chemistry and Pharmacognosy, and t Pharmacology and Toxicology,School o f Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907

~Department of Chemistry, University of Nebraska at Omaha, Omaha, NE 68182-0109

Received 2 June 1993

HUANG, X., D. MARONA-LEWICKA, R. C. PFAFF AND D. E. NICHOLS. Drug discrimination and receptorbinding studies of N-isopropyl lysergamide derivatives. PHARMACOL BIOCHEM BEHAV 47(3) 667-673, 1994.-N-Isopropyl (IPLA), N-methyl-N-isopropyl (MIPLA), N-ethyl-N-isopropyl (EIPLA), and NjN-diisopropyl (DIPLA) lysergam-ides were evaluated for lysergic acid diethylamide (LSD)-like activity. In rats trained to discriminate 0.08 mg/kg LSD tartratefrom saline, each of the subject compounds completely substituted, with an EDso two to three times larger than that of LSDexcept for DIPLA, which had an ED~o about eightfold greater. Similarly, all the compounds displaced [nsI](R)-l-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane ([125I]DOI) from rat cortical homogenates and displaced [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) from rat hippocampal homogenates with KI values similar to those of LSD, againwith the exception of DIPLA, which had about nine- and fourfold lower affinities, respectively. Interestingly, all thecompounds had four- to fivefold lower affinities than LSD in displacing [3H]ketanserin from 5-HT2 binding sites. Molecularmodeling studies found that all the compounds had low energy conformations similar to LSD. No correlation between theactivity of the compounds and the preferred conformation of the amide substituents was apparent. In summary, N-alkyl-N-isopropyl analogs of LSD retain LSD-like activity in drug discrimination and 5-HT,^ and 5-HT2 agonist binding assays only

until the N-alkyl substitution is as large as ethyl; LSD-like activity dramatically drops when the second alkyl substituent isN-isopropyl.

Lysergic acid diethylamide (LS D) N-Isopropyl lysergamides Drug discrimination (DD)[12~I]R-DOI bind ing [3H]8-OH-DPAT inding Molecular modeling Hallucinogens

[~H]Ketanserin binding5-HT2 receptor

FROM studies relating structure and activity, it is well knownthat minor molecular modifications of ergot alkaloids producesurprisingly great changes in pharmacolo gical effect (43). I n-cluded among these com pound s are various amides of lysergicacid. Lysergic acid diethylamide (LSD) is one of the classichallucinogenic agents, but struct ure-ac tivity studies of lyser-gamide hallucinogens have largely been neglected since thelate 1950s (1,14,20,23). With in the past eight years or so, o urlaboratory has begun to devote some effort toward filling insome of the obvious gaps that exist in our under stan ding ofthis class of hallucinogen.

Because of its structural complexity, there are a number oflocations in the lysergamides (Fig. 1) that one could modifyfor str ucture-a ctivit y studies (11,23,40). Recently, we havereported that the stereochemical properties of the amide sub-

stituent o f ha llucinogenic lysergamides can exert a critical in-fluence on activity. (R)- and (S)-2-butylamides of lysergic acidcompletely substituted for LSD in rats trained to discrimi-nate LSD from saline. Both isomers possessed high affinityfor [125I] R)- 1 ( 2,5-dimethoxy-4-i odophenyl)- -amin oprop ane([125I](R)-DOI)- and [3H]g-hydroxy-2-(di-n-propylamino)tet-ralin ([3Hl8-OH-D PAT)-labeled bindin g sites. However, theamide o f (R)-2-butylamine possessed somewhat higher po-tency (37).

The focus of this study was to examine further the effectson LSD-like activity of subs tituti on on the amide group o flysergamide. The N-isopropylamides were appealing for atleast two reasons. First, the N-methyl-N-isopro pyl comp ound(MIPL A) is an isomer of LSD and would be presumed to havesimilar lipophilicity and p harmacokin etic parameters. Second,

Requests for reprints should be addressed to David E. Nichols, Ph.D., Department of Medicinal Chemistry and Pharmacognosy, School ofPharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907.

667



6 68 H U A N G E T A L .

1 2 9 1 1 ~ ~ l r

1 4 ~ 4

I

LSD

R1 R

-CH2CH3 --CH2CH

IP LA - H - ~ I C H 3CH3

MIPI..A -CH 3 -~ C H 3C H3

EIPLA -C H 2 C H -~ C H 3C H

DIPLA -CHCH3 -CHCH3CH3 CH3

FIG. 1 . Schematic representat ion of the s tructure of LSD der iva-t ives , showing the atom numbe r ing used , the po si t ion of ~ , and theamide subst itu t ions repor ted in th is wo rk .

t h e i s o p r o p y l g r o u p i s o n ly o n e c a r b o n l a r g e r t h a n a n e t h y l ,b u t b e c a u s e o f i t s b r a n c h p r o v i d e s le s s c o n f o r m a t i o n a l m o b i l -i t y t h a n a n e t h y l . I t w a s a n t i c i p a t e d t h a t b i o l o g i c a l a s s a y a n dc o n f o r m a t i o n a l a n a l y s i s o f t h is s m a l l s e ri e s o f N - a l k y l - N -i s o p r o p y l c o m p o u n d s m i g h t a f f o r d a d d i t i o n a l i n si g h t i n t o t h er e a s o n s f o r t h e i m p o r t a n c e o f t h e a m i d e s u b s t i t u e n t i n l y s e r -g a m i d e s .

H a l l u c i n o g e n i c a c t i v it y in h u m a n s c a n b e m o d e l e d i n t h ed r u g d i s c r i m i n a t io n ( D D ) p a r a d i g m b y s t u d y i n g L S D - l ik e d is -c r i m i n a t i v e s t i m u l u s ( D S ) p r o p e r t i e s i n a n i m a l s ( 1 7 ,3 3 ) . I n at y p i c a l p r o c e d u r e , r a t s a r e t r a i n e d t o p r e s s l e v e rs f o r p o s i t iv er e i n f o r c e m e n t a n d t o r e c o g n i z e t h e i n t e r o c e p t i v e s ta t e a s s o c i-a t e d w i t h t h e a c t i o n s o f a p a r t i c u l a r d o s e o f L S D a t a p a r t i c u -l a r ti m e a f t e r a d m i n i s t r a ti o n . T h e D S p r o p e r t ie s o f L S D h a v eb e e n s t u d i e d e x t e n s i v e l y a n d s e e m t o b e m e d i a t e d c r i t ic a l l y b y

a g o n i s t a c t i v i t y a t 5 - H T 2 r e c e p t o r s ( 5 , 1 6 , 1 7 ,3 4 , 4 2 ) . T h e r e f o r e ,t h e D D p a r a d i g m w a s e m p l o y e d a s a m e a n s o f e v a lu a t i n gt h e s e L S D a n a l o g s i n r a t s . I n a d d i t i o n , r e c e p t o r b i n d i n g s t u d -i e s w e r e e m p l o y e d a s a n i n v i t r o a s s e s s m e n t o f t h e r e l a t i v e5 - H T 2 a n d 5 - H T j A r e c e p t o r i n t e r a c t i o n s f o r t h e f o u r N - a l k y l -N - i s o p r o p y l l y s e r g a m i d e s. T h i s w a s a c c o m p l i s h e d b y m e a s u r -in g a f f in i ty f o r th e [1 2 ~I ] (R) -D O I - lab e led an d th e [3 H lk e tan se r -i n - l a b e l e d 5 - H T z r e c e p t o r i n r a t c o r t i c a l h o m o g e n a t e s a n d t h ea f f i n i t y f o r [ 3 H ] 8 - O H - D PA T- I a b e l e d 5 -H T ~A re c e p t o r s i n r a th i p p o c a m p a l h o m o g e n a t e s . S i n c e [~ 25 I] (R )- DO I a l s o b i n d sw i t h h i g h a f f i n i t y t o t h e 5 - H T ~ c r e c e p t o r ( 4 , 3 0 , 3 8 ), i t is p o s s i-b l e t h a t t h i s r e c e p t o r s u b t y p e i s a l s o i n v o l v e d i n h a l l u c i n o g e n -

e s is . H o w e v e r , t h e f o c u s i n t h i s w o r k i s o n t h e 5 - H T 2 r e c e p t o rs u b t y p e .

METHOD

Materials

[ ' 2 5 I ] ( R ) - D O I , [ 3 H l k e t a n s e r i n , a n d [ 3 H ] 8 - O H - D PAT w e r ep u r c h a s e d f r o m N e w E n g l a n d N u c l e a r ( B o s t o n ) a t s p e c i f i c

a c t i v it i e s o f 2 2 0 0 , 6 1 , a n d 1 3 5 . 5 -2 1 6 C i / m m o l , r e s p e c t iv e l y.T h e f o u r N - a l k y l - N - i s o p r o p y l a m i d e s w e r e s y n t h e s iz e d in o u rl a b o r a t o r y f o l l o w i n g s t a n d a r d m e t h o d s [ e . g . , s e e ( 3 7) ]. A l ln e w c o m p o u n d s m e t t h e u s u a l c r i te r i a f o r p u r i t y in c l u d i n gt h i n l a y e r c h r o m a t o g r a p h y ( T L C ) , n u c l e a r m a g n e t i c r e so -n a n c e s p e c t r o s c o p y (N M R ) , a n d e l e m e n t a l a n a l ys i s. ( + ) - L S Dt a r t r a t e w a s o b t a i n e d f r o m t h e N a t i o n a l I n s t i tu t e o n D r u gA b u s e . C i n a n s e r i n w a s a g i f t f r o m t h e S Q U I B B I n s t i t u t e f o rM e d i c a l R e s e a r c h . 5 - H T w a s p u r c h a s e d f r o m S i g m a ( S t.L o u i s ) .

nimals

M a l e S p r a g u e - D a w l e y r a t s ( H a r l a n L a b o r a t o r i e s , I n d i a n -a p o l i s ) w e i g h i n g 1 7 5 - 2 0 0 g w e r e u s e d i n a l l e x p e r i m e n t s .A n i m a l s w e r e e it h e r g r o u p h o u s e d s ix p e r c a g e ( r a d i o l i g a n db i n d i n g e x p e r i m e n t s ) o r i n d i v i d u a l l y c a g e d i n a t e m p e r a t u r e -c o n t r o l l e d r o o m w i t h a 1 2 - h l i g h t - d a r k s c h e d u l e ( D D e x p e r i -m e n t s ) . Wi t h t h e e x c e p t i o n o f r a t s u s e d i n t h e D D e x p e r i -m e n t s , f o o d a n d w a t e r w e r e a v a i l a b l e a d l i b a t a l l t i m e s . I nD D e x p e r i m e n t s r a t s w e r e t r a i n e d t o d i s c r i m i n a t e L S D t a r t r a t e( 1 8 6 n m o l / k g , 0 . 0 8 m g / k g ) f r o m s a l i n e . L S D w a s a d m i n i s -t e r e d I P d i s s o l v e d i n s a li n e s u c h t h a t a v o l u m e o f 1 m l / k g o fb o d y w e i g h t w a s u s e d . N o n e o f t h e r a t s h a d p r e v i o u s l y r e -c e i v e d d r u g s o r b e h a v i o r a l t ra i n i n g . W a t e r w a s f r e e ly a v a i l a b l ei n th e h o m e c a g e s a n d a s u f fi c i e n t a m o u n t o f s u p p l e m e n t a lf e e d i n g ( P u r i n a , L a b B l o x ) w a s m a d e a v a i l a b l e a f t e r e x p e r i-m e n t a l s e s s i o n s s o a s t o m a i n t a i n t h e m a t a p p r o x i m a t e l y 8007oo f f r e e f e e d i n g w e i g h t , c o m p a r e d w i t h c o n t r o l r a t s ( h o u s e du n d e r t h e s a m e c o n d i t i o n s ) .

Drug DiscriminationS i x s t a n d a r d o p e r a n t c h a m b e r s ( C o u l b o u r n I n s tr u m e n t s ,

L e h i g h V a l l e y, PA ) c o n s i s t e d o f m o d u l a r t e s t c a g e s e n c l o s e dw i t h i n s o u n d - a t t e n u a t e d c u b i c l e s w i t h f a n s f o r v e n t i l a t i o n a n db a c k g r o u n d w h i t e n o is e . A w h i t e h o u s e l ig h t w a s c e n t e r e dn e a r t h e t o p o f t h e f r o n t p a n e l o f t h e c a g e , w h i c h w a s a l s oe q u i p p e d w i t h t w o r e s p o n s e l ev e r s s e p a r a t e d b y a f o o d h o p -p e r , a l l p o s i t i o n e d 2 . 5 c m a b o v e t h e f l o o r. S o l i d s t a t e lo g i c ina n a d j a c e n t r o o m , i n t e rf a c e d t h r o u g h a C o u l b o u r n I n s tr u -m e n t s D y n a p o r t t o a n I B M P C , c o n t r o ll e d r e i n fo r c e m e n t a n dd a t a a c q u i s i t i o n w i t h a l o c a l l y w r i t t e n p r o g r a m .

A f i x e d r a t i o 5 0 (F R 5 0 ) s c h e d u le o f f o o d r e i n f o r c e m e n t( B i o s e r v 4 5 -m g d u s t l e ss p e l l e ts ) in a t w o - l e v e r p a r a d i g m w a su s e d . T h e d r u g d i s c r i m i n a t i o n p r o c e d u r e d e t a i ls h a v e b e e n

d e s c r i b e d e l s e w h e r e (3 5 , 3 6 ). H a l f o f t h e r a t s w e r e t r a i n e d o nd r u g - L ( l e ft ) , sa l i n e - R (r i g h t) , a n d t h e o t h e r h a l f o n d r u g - R ,s a l i n e - L t o a v o i d p o s i t i o n a l p r e f e r e n c e . T r a i n i n g s e s s i o n s la s -t e d 1 5 m i n a n d w e r e c o n d u c t e d a t th e s a m e t i m e e a c h d a y.Tr a i n i n g s e s s io n s w e r e c o n t i n u e d u n t i l a n a c c u r a c y o f a t l e a s t8 5°7 0 ( n u m b e r o f c o r r e c t p r e s se s x 1 0 0 / n u m b e r o f t o t a lp r e s s e s ) w a s a t t a i n e d f o r 8 o f 1 0 c o n s e c u t i v e s e s s i o n s . O n c ec r i t e r i o n p e r f o r m a n c e w a s a t t a i n e d , t e s t s e s s i o n s w e r e i n t e r -s p e r s e d b e t w e e n t r a i n i n g s e s si o n s , e i th e r o n e o r t w o t i m e s p e rw e e k . A t l e a s t o n e d r u g a n d o n e s a l i n e s e s s i o n s e p a r a t e d t e s ts e s s io n s . R a t s w e r e r e q u i r e d to m a i n t a i n th e 8 5 c o r r e c t r e -s p o n d i n g c r i t e r i o n o n t r a i n i n g d a y s t o b e t e s t e d . I n a d d i t i o n ,



ISOPROP YL ANAL OGS OF LSD 669

test da ta (less tha n 5070) were discard ed when t he ac curacycriterion of 85% was not achieved on the two tra ining sessionsfollowing a test session. Test drugs were administered IP 30rain prior to the sessions; test sessions were run under condi-tions of extinction, with rats removed from the operant cham-ber when 50 presses were emitted on one lever. If 50 presseson one lever were not completed wit hin 5 min the session wasended and scored as a disruption. Treatments were random-

ized at the begin ning of the study.

Radio l igand Bind ing S tud ies

The procedure of Johnson et al. (25) was employed.Briefly, the frontal cortex or hippocampal brain regions from20-40 male Sprague-Dawley rats (175-199 g, Harlan) werepooled and homogenized (Brinkman Polytron, setting 6 for 2x 20 s) in four or eight volumes of 0.32 M sucrose for fron talcortex or hippocampus, respectively. The homogenate wascentrifuged at 36 000 x g for 10 min, a nd the resulting pelletswere resuspended in the same volume of sucrose. Separatealiquots of tissue were then frozen at - 70°C un til assay.

For each separate experiment, a tissue aliquot was thawedslowly an d dilute d 1 to 25 with 50 mM Tri s HCI (pH = 7.4).

The homogenate was then incubated at 37°C for 10 min andcentrifuged twice at 36 500 x g for 10 rain, with an intermit-tent wash. The resulting pellet was resuspended in 50 mM TrisHCI with 0.5 mM Na2EDTA, 0.1 070 Na ascorb ate, and 10 #Mpargy line HCI (pH = 7.4). In experi ments with [125I](R)-DOIand [3H]ketanserin, either 10 mM MgCI 2 or 5.7 mM CaClzwere included, respectively. A second preincubation for 10min at 37°C was conducted, and the tissues were then cooledin an ice bath.

All experiments were performed with triplicate determina-tions using the appropriate buffer, to which 200-400 #g ofprotein was added, giving a final volume of 1 ml. The tubeswere allowed to equilibrate for 15 min at 37°C befor e filteringthrough Whatman GF/C filters using a cell harvester (Bran-del, Gaithersburg, MD), followed by two 5-ml washes using

ice-cold Tris buffer. Specific bindi ng was defined as t hat dis-placeable with 10 #M cinanse rin in both the [3H]ketanserinand [125I](R)-DOI binding study and with 10/~M 5-HT in the[3H]8-OH-DPAT binding study. Filters were air-dried andplaced into scintillat ion vials with 10 ml of Ecolite scintillat ioncocktail and allowed to sit overnight before counting at anefficiency of 37°70 for t ritiu m, and directly counted in agam ma coun ter for [~25I]-ligand at an efficie ncy of 79.4070.

[~25I](R)-DOI sat ura tio n experi ments were car ried out byvarying the concentration of unlabeled ligand with a constantamount (0.1 nM) of radioligand, according to the proceduredescribed previously (22,24,25,28). This method is accuratewhen the unla beled and labeled ligands have identical physio-chemical properties (32). Und er the above experimental condi-tions, ['25I](R)-DOI was found to bind to a single site (Hill

coeffic ient of 0.91 ± 0.03) with a Bm~ of 44.0 + 5.67 fmol /mg protein and a KD of 1.88 ± 0.24 nM. The abilit y of 8-10con cent rati ons of test d rug to displace 0.1 nM o f [~25I](R)-DOIbinding was measured in drug displacement studies. Four tofive concentrations of radioligand were used in both [3H]-ketanserin and [3H]8-OH-DPAT saturation experiments.[3H]Ketanserin bo un d to a single site (Hill coefficient 1.08 ±0.06) with a Bm~ of 297.3 ± 39.6 fmol/ mg protei n and a Koof 1.37 ± 0.20 riM. [3HI8-OH- DPAT bo un d to a single site(Hill coeffici ent 1.00 ± 0.01) with a Bma x of 119 ± 8 fmol /mg protein and a KD of 2.49 ± 0.23 nM. The abil ity of 8-10concent rations of test drug to displace 0.75 nM o f [3H]ketan-

serin or [3H]8-OH-DPAT was determined in drug displace-ment studies.

Molecu la r Model ing

Although lysergamides are relatively rigid, they do haveconformational flexibility in both the amide substituent andthe D-ring. In particular, the D-ring of LSD, and of mostrelated ergolines, has been found to exist in the C(7) flap-upconformation in CDCI3 solution (7,21,26) as well as in thecrystal structure of LSD iodobenzoate monohydrate (8,9).The D-ring of LSD can undergo a conformational flip intowhat has been called the flap-down conf ormat ion, with theC(7) methylene group below the D-ring plane. The C(7)-upand C(7)-down conformations differ in energy by only about2-4 kcal/mol and have been investigated by Andrews et al.(3). In addition, Pierri et al. (39) have reported that ergota-mine exists in the C(7) flap-down con forma tion in CDCI 3 solu-tion. For the current work, however, the D-ring was assumedto be in the conventional flap-up conformation. The amidesubstitue nt has a mu ch greater degree of con forma tiona l flexi-bility than the D-ring and is the focus of the conformationalstudies in this article.

Classical molecular mechanics were performed on LSD andits isopropyl amide derivatives. The molecular flexibility ofthe com pound s is principally described by one dihedral angle,rl (identified in Fig. 1), formed by the O(19)-C(18)-C(8)-C(7)atom set. The dihedral angle was investigated by performinggeometry searches, or grid scans, with minimiza tion at eachstep. The structure as drawn illustrates the compounds with rj-~ 180 °. The a ngles were searched over t he r ange - 180 ° to4-180 ° in 5 ° increments. T o avoid sterically controlled falsemini ma the searches were performed twice, once in the clock-wise direction ( -1 80 ° to + 180 °) an d once in the counter-clockwise direction (+ 180 ° to -1 80 °) , with the smaller en-ergy at each angle being used in the analysis.

All computations were performed on a CAChe Scientificworksystem runn ing CAChe proprietary software, includi ngtheir implemen tations of Extended Hiickel, ZINDO v3.0, andMOPA C v6.10 (19). CAChe molecular mechanics uses Alling-er's MM2 force field (2) as augmented by CAChe Scientific.With MOPA C, the AMI Hamil toni an (18) was used. Themolecular mechanics were investigated on the CAChe system(12) by mini mizing the tot al molecular energy according tothe m olecular mechanics expression

Etotai ~- Ebonding 4- go 4- E~, Eimprop 4- Eelec 4- Evdw + Ebb

Further, molecular mechanics calculates energies relative to ahypothetical perfect geometry, rather than an absolute en-ergy, and uses a temperature of 0 K. The structures citedhere are those obtained from molecular mechanics, and thecalculated structure of LSD is in good agreement with thepublished X-ray results (9).

Calculations were performed in vacuo, and no a ttempt wasmade to consider the effect of solvation. In additi on, the con-formations of the lysergamides where the N(6)-methyl wasequatorial were of lower energy than those in which the methylwas axial.

Stat is tical Ana lysis

In the DD study, data were scored in quantal fashion withthe lever on which the rat first emitted 50 presses in a testsession scored as the selected lever. The percentage of ratsselecting the dr ug lever (070SDL) for ea ch dose of test com-pound was determined. If that drug was one that completely



670 HUAN G ET AL.

substituted for the training drug (at least one dose resultedin 070SDL = 80°70 or hi gher), the met hod of Litchf ield an dWilc oxon (27) was used t o d eter mine th e EDs0 and 95070 confi -dence interval (CI). In the receptor bindi ng studies, data wereanalyzed using the computer programs EBDA and Ligand asdescribed by McPherson (28). The values from three to fourseparate experiments were combined. Protein determinationswere made using the procedure of Bradford (10).

RESULTS

Drug Discrimination

The results of the drug discrim inatio n testing are given inTable 1, and the radioligand displacement data are in Table2. N-Isopropyl (IPLA), MIPLA, and N-ethyl-N-isopropyl(EIPLA) all substituted in LSD-trained rats with a potencyabout two- to threefold less than LSD. DD potencies mostclosely parallel the bindi ng data for [~25I]DOI displacement.N,N-Diis opropyl (DIPL A) is the onl y one in the series far lesspotent (about eightfold) than LSD in the DD paradigm.

Radioligand inding Studies

At [3H]8-OH-DPAT binding sites, IPLA, MIPLA, andEIPLA all have affinities similar to LSD. However, the affin-

ity of DIPLA for [3H]8-OH-DPAT sites is about four timeslower than LSD. At [3H]ketanserin bindin g sites, all four LSDanalogs have about four- to eightfold lower affinities thanLSD. At [nSI]DOI sites, IPLA and MIPLA have affinitiessimilar to LSD, EIPLA has slightly less affinity, and DIPLAhas the lowest affinity of the series, about ninefold less thanthat of LSD.

Molecular Modeling

The outcome of the geometry searches of z~ for the com-pound s is given in Fig. 2. All of the co mpound s have similarminim um-ene rgy conforma tions. The relative energy at unf a-vored conformati ons (e.g., r = 1800) varies considerably, butdoes not correlate with the potencies of the compounds. It isperhaps likely that only one particular conformation of theamide group allows binding at each of the receptors consid-ered here; however, the present series of compounds possessesenough conformationa l mobility in the amide region that thepreferred conformation can be easily reached by any of thecompounds. Given the present data, the variation in potencyamong these five compounds can best be explained by thepresence of a hydrophobic pocket in the receptor which best

accommodates about a four-carbon hydrophobic alkyl substi-tution on the amide nitrogen; the most potent c ompounds in

TABLE 1DRUG DISCRIMINATION DATA FROM LSD-TRAINED RATS

Drug Dose (mg/kg) N D SDL

EDs0(95 CI)

(mg/kg) (nmol/kg)

1. LSD 0.01 9 0 110.02 14 0 360.04 13 1 83

0.06 10 3 1000.08 13 0 100

0.021 (0.014-0.032) 48 (32-73)

2. IPLA 0.04 8 2 370.06 9 1 780.08 10 1 900.10 10 2 100

0 . 0 4 6 0 . 0 3 6 - 0 . 0 5 8 ) 1 1 0 8 6 - 1 4 0 )

3. MIPLA 0.02 14 3 90.04 9 2 560.06 9 2 890.08 10 1 900.I0 10 1 100

0 . 0 3 6 0 . 0 2 4 - 0 . 05 6 ) 8 5 5 5 - 1 3 0 )

4. EIPLA 0 . 0 4 I I 3 1 3

0 . 0 6 I I I 6 0

0 . 0 8 I I I 8 0

0 . I 0 I I I 9 0

0 . 0 5 9 0 . 0 4 7 - 0 . 07 3 ) 1 3 3 I 0 8 - 1 6 4 )

5. DIPLA 0 . 0 8 7

0 . I 0 I 0 3 1 3

0 . 2 0 I 0 I 6 7

0 . 4 0 I 0 2 I 0 0

0 . 1 6 1 0 . 1 5 - 0 . 2 2 6 ) 3 5 1 2 5 0 - 4 9 3 )

The EDs0 values for substitution were calculated according to the method of Litchfield and Wilcoxon2 7 ) . For all dose-dependent curves, the slopes are not significantly different (all are parallel). N =

number of animals tested, D = number of animals disrupted (50 presses not completed in 5 min),SDL = percentage of nondisrupted animals that selected drug level.



ISOPR OPYL ANAL OGS OF LSD 671

LSDIPLAMIPLAEIPLADIPLA

TABLE 2RESULTS OF LSD AND ITS ANALOGS DISPLACING [3H]8-OH-DPAT,

[~H]KETANSERIN AND [ml]DOI BINDING

[3H]8_OH_DPA T [3 HlKetanserin [~23I]DO1

Kt(nM) n Kl(nM) n K~(nM) n

4.41 ± 0.77 4 4.78 ± 0.53 3 1.43 ± 0.13 45.23 ± 0.15 3 26.2 ± 0.23 3 1.73 ± 0.26 44.60 ± 0.35 3 28.1 ± 4.5 4 1.75 ± 0.27 43.67 ± 0.48 3 16.8 ± 2.0 4 3.26 ± 0.19 3

17.9 ± 2.8 3 17.2 ± 2.0 3 12.5 ± 1.8 4

the group (MIPLA and LSD) each have four carbons compris-ing the am±de alkyl chains. Pot ency declines as the total n um-ber of ca rbons deviates from this ideal.

DISCUSSION

All the N-isopropyl analogs of LSD displaced [3H]8-OH-DPAT and [t25I](R)-DOI binding with affinities similar toLSD except for DIPLA, which had affinity about four- andnin efo ld lower tha n LSD for [~2~I](R)-DOI and [3H]8-OH-DPA T bin ding sites, respectively. All of the four compo undsbou nd to [3H]ketanserin-labeled sites with aff inities similar toeach other but all had lower affinity than LSD at this site.Curiously, LSD is unique amon g these five compo unds in thatit alone has high affinity both at [3H]ketanserin sites and at[~2SI]DOI-labeled sites; the N-isopropyl analogs have four- toeightfold lower affinit y at [3H]ketanserin sites than LSD. Per-haps this factor is related to its unique behavior al potency.

The results of the drug di scrimin ation studies show that allthe isopropyl analogs tested except for DIPLA fully substi-tuted in LSD-tra ined rats with EDs0s two- to threefold higherthan that of LSD. The degree of decrease in potency ofDIPLA in the DD paradigm seems to correlate best with theextent of drop in the affinity at the 5-HT2 agonist bindingsite. This would be consistent with the cue's being mediated

by a 5-HT 2 agoni st effect (4,16,17,34,42). Howeve r, a rolefor the 5-HT1^ receptor cannot be excluded, particularly ifa 5-HTIA effect is synergistic with the 5-HT 2 agonist action6 , 3 3 ) .

The role of the lone pair electrons of the tertiary nitrogenatom in the ergoline D-ring has been discussed extensivelywith respect to binding forces of the drug-receptor interaction(15,21,29,31,44). For the purposes of the work report ed here,it is assumed that the electron pair on N(6) points toward thec~ (lower) face of the molecule, corresponding to the C(7)flap-up conformation.

On the basis of energy and conformational considerationsalone, one would expect in vivo experiments to show little, ifany, reduction in the potencies of IPLA, MIPLA, EIPLA,and DIPLA relative to LSD if the compounds adopt a confor-mati on at the receptor similar to their min imum-e nergy con-formation in vacuo. Similarly, if the receptor constrains thecompounds to adopt a higher energy conformation, onewould expect their potencies to be correlated with the relativeease with which they adopt such a nonprefer red confor mati on(e.g., decreasing potencies of IPLA > MIPLA > LSD >EIP LA > DIP LA if a configurati on of r = - 180 ° must beadopted). The fact that neither such prediction is confirmedpoints to the conclusi on that specific steric and hydrophob icconstraint s in the am±de-binding region of the receptor out-

14 t ----O---- IPLA ]A

1 2 o Z L A . b

10 ~ ~ DIP LA I~t~

ua 4

2

0-180 -150 -120 -90 -60 -30 0 30 60 90 120 150 180

O(19)-C(18)-C(8)-C(7) dihedral

FIG. 2. Potential energy of the subject compounds as a function of rl. Each compound'senergy has been taken relative to its global minimum (energy of the global minimum is 0kcal/mol). From top to bottom, rl = 180% 105% 45 , - 15 °, and -60 °, respectively.



6 72 H U A N G E T A L .

w e i g h r o t a t i o n a l e n e r g y d i f f e r e n c e s a b o u t t h e C ( 8 ) - C ( c a r-b o n y l ) b o n d .

O n e n o t e w o r t h y p o i n t t o c o n s i d e r i s t h e f a c t t h a t t h e c l in i c a ll i te r a t u r e o n h a l lu c i n o g e n s c o n t a i n s n o r e p o r t o f a c o m p o u n dw i t h p o t e n c y c o m p a r a b l e t o L S D . E v e n t h e c l os e l y r e l a te d N -m e t h y l - N - e t h y l a n d N - e t h y l - N - a l l y l l y s er g a m i d e s , w h i c h o n em i g h t e x p e c t n o t t o d i f f e r s i g n i f i ca n t l y in r e c e p t o r b i n d i n g a n dp h a r m a c o k i n e ti c s f r o m L S D , h a v e m e r e l y 3 0 o f t h e p o t e n c y

o f L S D i n h u m a n s ( 4 1) . T h e r a t b e h a v i o r a l d a t a i n t h e c u r r e n ts t u d y a l so i n d i ca t e s a u n i q u e p o t e n c y f o r L S D . A s w a s n o t e dp r e v i o u s l y, p e r h a p s t h i s f i n d i n g i s r e l a t e d t o t h e p a r t i c u l a r l yh i g h a f f i n i t y o f L S D f o r t h e [ 3 H ] k e t a n s e r i n - l a b e l e d s i t e . W h a ts u b j e c t iv e d i f f e r e n c e s i n e x p e r i e n c e s u ch p h a r m a c o l o g i c a lc h a n g e s m a y p r o d u c e m u s t a w a i t h u m a n t r ia l s .

I n s u m m a r y, N - i s o p r o p y l a n a l o g s o f L S D , e x c e p t f o rD I P L A , r e t a in L S D - l i k e b e h a v i o r a l e f f e c t s i n D D e x p e r i m e n t sa n d a p h a r m a c o l o g i c a l p r o f i l e g e n e r a l ly s i m i l a r t o t h a t o f L S D

i n [ 3 H ] 8 - O H - D PA T, [ 3 H ] k e t a n s e ri n , a n d [ J2 5I] DO I b i n d i n g e x -p e r i m e n t s . T h e D D p o t e n c y p a r a l l e l s a f f i n i t y f o r [ lz s I ]D O I -l a b e l e d 5 - H T 2 s i te s , i n d i c a t i n g t h a t a l l o f t h e c o m p o u n d s a r ep o t e n t 5 - H T 2 a g o n i s ts . T h o u g h t h e c o m p o u n d s a r e l ik e l y t oh a v e h a l l u c i n o g e n i c e f fe c t s in m a n , t h e q u a l i t a t i v e d i f f e r e n c e si n th e s e e f f e c t s p r o d u c e d b y t h e o b s e r v e d v a r i a t i o n s i n r e c e p -t o r a f f i n i t ie s w i l l n o t b e k n o w n u n t i l h u m a n s t u d i e s c a n b ec o n d u c t e d .

ACKNOWLEDGEMENTS

This research was suppor ted b y U.S. Publ ic Heal th S ervice grantDA02189 f rom the Nat iona l Ins t i tu te on Drug Abuse . R.C.P . thanksCA Ch e Scient if i c , Inc . for par t i a l cont r ibut ions of model ing hardwa reand sof tware . We thank Stewar t Frescas for synthes is of the IPLA ,M I P LA, EI PLA , a nd DI PL A; A r t h i Ka n tha s a m y f o r e x c e l le n t t e c hn i -ca l ass is t ance wi th the b inding exper iments ; and Ma t t Parker for he lp-fu l comm ents dur ing the prepara t ion of th i s a r ti c le .

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