I 7:

EJP 57645

ynthesis and pharmacology of irreversible affinity labels cocaine antagonists: aryl 1,4_dialkylpiperazines related to Howard M. Deutsch. Margaret M. Schweri “, Christopher

Received

AS p;irl of ;I program aimed at designing both slimulant

binding

and dopaminc

IO June 1992. accepted 23 June I992

irrcvcrsiblc

containing clcctrophilic

dcrivativcs of GBR-12783

antagonists

suhstitucnts

transport,

was modified

to incorporate

in one phcnyl ring of the gcminal diphcnyl

well as their rcspcctivc

amino-

ranging from tcchniquc

radiorcccptor

11.0 (m-nitro)

(rcpcatcd

assay. Under

washing with 100 mM compounds

site. The m-malcimido

the isothiocyanatc

precursors,

compourids.

derivative Ncithcr

on stimulant

the parent

found

rcmovcd

to irrcvcrsihly

also irrcvcrsihly

the p-malcimido,

and reinforcing

GBR-12783,

cithcr

isothiocyanatc

of the molecule.

inhibit

inhihitcd

of cocaine,

or malcimido

groups at the

The effect of these compounds,

binding to rat striatal GBR-12783.

propcrtics

a potent and sclcctivc inhihitor of

used. the compounds

compound.

KC!) which complctcly wcrc

portion

the assay conditions

to lh77 (p-malcimido);

the m- and p-isothiocyanatc recognition

or nitro-substituted

of the stimulant

wcrc synthcsizcd.

mcta- or para-positions [‘Hlmethylphcnidatc

T. Culbertson I and Leon H. Zalkow

as

tissue was studied using the

were found to have IC5,,s (nM)

had an IC,,,

of 12.0. Using a washout

the tightly bound, but rcvcrsihlc GBR-11783, binding of [‘Hlmcthylphcnidatc

both

to the stimulam

binding, albeit with lower efficacy than was observed with

nor the amino or nitro intcrmcdiatcs. wcrc capable of irrcvcrsihlc

inhibition. Cocaine: GBR-1278.3:

[‘H]Mcthylphcnidatc;

1. Introduction Stimulant

crisis

ahusc

proportions

(primarily

in

the

of cocaine) is rcachin; United States (National

Household Survey on Drug Abuse, 1985). This work aims to contribute to the solution of this problem by developing antagonists directed towards the stimulant recognition site on the dopaminc transport complex, whcrc the reinforcing effect of thcsc agents is thought to be mediated (Ritz et al., 1987). Our goal is to synthcsizc irrcvcrsihlc affini!y labels conlaining clcctrophilic groups which can react covalently with nuclcophilcs present in the region of the dopaminc carrier rccognizcd by psychomotor stimulants. This approach is based in part on reports that mctaphit, a compound which in vitro irreversibly inhibits binding of [‘HI cocaine (Bcr,gcr et al., 19861, [‘HJmcthylphenidatc

I’orrcspondcnce

to:

chcmislry, Georgia

tt.M. Dcu~sch.School

Institute

of Technology.

of Chemistry Atlanta.

and Bio-

GA 3(133?~04W

USA.

’

NSF Undergr;ldu;lk

of Tcchnolo>gy.

Rcwxrch

P;lrticip;lnl ;rI the Gcorgi:l

Inslilule

Stimulants:

Dopaminc

transporters

(Schwcri et al., 1987, lY8Y) and [‘H]mazindol (Zimanyi et al.. 1989) to stimulant recognition sites in rodent brain tissue preparations. attenuates the increased locomotor activity observed in mice foilowing the administration of cocaine and other compounds belonging to the mcthylphcnidatc class of stimulant drugs (Scrshen et al., fO88). This approach is also iupported by recent findings that GBR-12909, a compound considered to bc a ‘pseudo-irreversible’ inhibi% because it dissociates very slowly from this site. antagonizes the increase in cxtraccllular dopaminc levels caused by cocaine (Rothman ct al., 1989, 1990). Althrmgh the behavioral

results

ohtaincd

with

mctaphit wcrc encouraging, its low potency (IC,,,s of S-IO PM) (Bcrgcr et al., 1986: Schwcri ct al., 19%‘: Zimanyi et al., 1980) and lack of sclcctivity (it also binds irreversibly to the phcncyclidinc receptor. Raffcrty ct al., 1985) detract from its potential as a clinically useful cocaine antagonist. In order to circumvent these problems, WC have synthcsizcd isothiocyanatc and malcimido dcrivativcs of GBR-12783. This comof a series

pound,

one

stituted

bcnzbydryl

Zcc

et al., (l980),

cthcrs

of

aryl

first

alkyl

piperazinc-sub-

synthesized

was shown

by Van dcr

to bc a potent

and

The precursors

for the potential irrcvcrsihlc

ligands

were synthesized using tho general methods of Van der Zee tt al. (t%W, as shown in fig. 1. A minor, but significant

rn~~dificati~~n,was the elinlin~tio~

of toiuene

st&~~t in the ctherification step (i.e. step b). The nitro group in 3 was reduced to amintt using the sulfohorul~ydrid~ method (~l~n~~tt~ and Brindle, 19711 in ordcr to preserve the douhlc bond in the final product. The

amino group was convcrted to the isothiocyanatc

and maleimido grtqs using the methods of Burke et itI. (IftX4) and Portc>ghesc et al., (1977), respcctivcly. ~~~nip~~unds 3c-3h wcrc not crystalline and wcrc isolated and/or charxtcrized as maleate salts. Tho purity and structures

of all new compounds were confirmed

hy thin layer ~hr(~rn~it(~~r~phy, proton nuclear magnetic resonance spectroscopy, infrared spcctmscopy imd where appropriate mass spectroscopy. A complctc description

of thcsc synthcscs

will

he publisbcd

clsc-

whcrc.

Malt Sprrtguc-Dawlcy rats (Harlan Spnlguo-DawIcy, Indianapolis. IN) weighing ISO-. g wcrc used in thcsc cxperimcnts. After killing, the brains wcrc quickly rcmovcd and placed in Lx-cold 0.X M sucrose. Striatal tissue was disscctcd nut and asseycd for [‘Hlmcthylphcnidate

binding

Ph

iiccclrding to a modification

ef a

175

previously described method (Schweri et al., 1987). Briefly, a Pz fraction was prepared by diffcrcntial centr~fugat~on and resuspended in 50 volumes (original wet weight) of assay buffer (SO mM Tris-Cl, IO0 mM NaCI, pH 7.3 at O”Ck Samples containing 400 ~1 of the tissue suspension, 450 ~1 assay buffer, 50 &I water or test compound (dissolved in 1.2% dimethylsulfoxid~, or less), SO @I water or 200 PM amfonelic acid (to define nonspecific binding), and 50 yf ~~H~m~thylphenidate ( f ~-three-[methyl--~HJmcthylphenidate~ specific activity, 70.7 Ci/mmol; DuPont/New England Nuclear, Boston, MA) in assay buffer were incubated for 30 min at 0°C then filtered under vacuum through GF/B fillers mounted in a Milliporc fi!!ra!l:m manifold. A 5 ml aliquot of assay buffer was addea to the sample immediately before filtering it, and a second 5 mt aliquot of assay buffer was used to wash the fifter. The filters were then shaken for 30 min with Beckman Ready-Protein ‘I’ scintillation fluid and counted in a liquid scintillation counter. In most cxperimcnts. tissue was incubated in the presence of 5-10 nM [“Hlmethylphenidate. Washout stud& to cvaluatc the irreversibility of the iRhibiti~~nof binding were conducted by suspending a Pz fra~ti~~nfrom the striatal tissue of four rats in 19 ml of assay buffer and combining it with I ml of test compound dissolved in 0.15% or less dimcthylsulfoxide for 30 min at 0°C. Control samples were combined with I ml of vehicle Afler removal of an aliquot for dctcrmi~ation of ~~H]metll~~phenid~~tc binding, the rcmainder of the sample was diluted 1: I with 1011mM KC1 and ccn~rifuged at 27ttttO x g for 5 min at 0°C to t~rminatc the reaction. The sup~rnatant was discardLad

to remove any unreacted ~omp~~~nd. the petlet was rcsusp~nd~d in SO ml of 1tK) mM KCI, aliowcd to set for 10 min to promote dissociation. and centrifuged again, as above. The centrifugation and resuspension steps were repeated on the remaining tissue suspension for a total of five washes. After the second. fourth. and fifth washes, part of the tissue pellet was resuspended in assay buffer, and inhibitic~~ of ~~H~m~thylpl~e~idate binding was determined by comparison with identically treated control samples. Binding was normalized for protein content, determined according to the method of Miller (195%

3. Results

GBR-12783 and its derivatives inhibited ~~H~m~thylphenidate binding in a dose-dependent manner (fig, 2). The ICI,,, and apparent Hill coefficient values arc shown in table 1. The parent compound and its 3-nitro dcriv;ttive were most potent (IC5,, = I2 nM; table 1). Addition of an isothiocyanate group at the meta- or paraposition led to a 3% and 4tl-fold reduction in potency, respectively. ~ntrodu~ti~~nof a ma~~jmido group at these positions led to X0- and 14%fold redu~tj~~ns in potency, rcspcctivcly. The It-nitro- and the 3- and 4-amino-substituted intermediates showed only small decreases in potency, compared to the parent compound. With the exception of the amino adducts, the mcta-substituted compounds appcarcd more potent than the para-suhstitutcd ones. The apparent Hill ~~~~ffj~i~ntsfor all compounds were greater than unity, ranging from I.32 + 0. I8 (~(~rnp~~und3g) to 3.03 _t ft.05 for GBR-I 2783.

ETHYLPHENIDATE

% INHIBITION

BINDING

OF

(‘H[METHYLl’HENIDATE tQ0

90'

A

BINDING

~~~~~~--~~---

I’

81)

-*.. _

60 _

60 -

‘4.

40 .

Y

Fig. 3. Effect membrane

of KCI

on the dissociation

preparations

were incubated

for 30 min at 0°C in standard I(H) mM

NaCI).

daterminatinn

A portion

01

tm

10

1

[INHIBITOR].

Fig. 2. Inhibition d&vat&s.. varying

of

[ ‘Hjmrthylphenidate

concentrations

of

the

test

for 30 min :II II