Eur. J. Med. Chem. 23 (1988) 391-396 0 Elsevier, Paris

391

Short communication

Synthesis and dopamine receptor binding of bridged tricyclic dopamine analogues Haralambos E. KATERINOPOULOS”, Kyriaki THERMOS . **, Demetrios K. VASSILATIS and David I. SCHUSTER

New York University, Department of Chemistry, New York, NY 10003, U.S.A.

(Received December 5, 1986, accepted November 24, 1987)

Summary - The bridged tricyclic dopamine analogues exo-2-(N-n-propylamino)-6,7-dihydroxybenzonorbornene, exo-2-(N, N-di-n-propylamino)-6,7-dihydroxybenzonorI;,ornene, and exo-2-amino-6,7-dihydroxybenzobicyclo[2.2.2]octene have been synthesized and assayed for their effects on the binding of [‘Hlspiperone and [3H]apomorphine to rat striatal mem- branes. The inactivity of these compounds is rationalized on the basis of proposed topographical models of dopamine receptors.

R&urn6 - Synth&se et liaison aux rkepteurs dopaminergiques d’analogues dopaminergiques tricycliques pontks. Les ana- Iogues tricycliques pontb de la dopamine exo (N-n-propylamino)-2 dihydroxy-6,7 benzonorbornPne, exo- (N,N-di-n-propyl- amino)-2 dihydroxy-6,7 benzonorbor&ne, et exo-amino-2-dihydroxy-6,7 benzobicyclo[2.2.2]oct&e ont e’tk synthe’tisb et ana- Iysb pour leurs efSets sur la liaison de la [3H]spip&one et de 1’[3H]apomorphine avec Ies membranes du corps strie’du Rat. On explique I’inactivite de ces composPs en se basant SUP des modGles topographiques propos&s de re’cepteurs de la dopamine.

dopamine analogues / dopamine receptors

Introduction

The partial or total rigidification of the dopamine (DA) moiety, as a result of its incorporation within a larger carbon framework, is one of the approaches that has been followed to address the question of the conformation of DA and its agonists required for effective interactiob with recognition sites on its receptors in the central nervous system (CNS) and in the periphery.

There has been a considerable number of structure- activity studies using semi-rigid DA analogues such as aporphines, aminotetralins, aminoindans, benzoquinolines, 3-phenylpiperidines, ergolines and benzazepine derivatives, as well as cyclobutyl and cyclopropyl DA analogues (for reviews on structure-activity relationships see [l--4]). These structures, however, retain some conformational flexibility as a result of partially restricted rotation around single bonds or, in the case of cyclic structures, the various conformations that can be assumed by the non-aromatic rings.

A number of tricyclic DA analogues has been studied by the authors [5] and others [6--81 in which the DA moiety

is rigidly held as a part of the benzonorbornene framework (as in 1 and 2). We now report the synthesis of exo-2- (N-n-propylamino)-6,7-dihydroxybenzonorbornene 3, exo- 2-(N, N-di-n-propylamino)-6,7-dihydroxybenzonorbornene 4, and exo-2-amino-6,7-dihydroxybenzobicyclo[2.2.2]octene 5, and assays of in vitro binding of these potential DA agonists to receptor sites in rat corpus striatum.

The synthesis of the N-n-propyl and N,N-di-n-propyl analogues was undertaken because structure-activity relationship data indicated that the introduction of one N-n-propyl group generally increases the activity of dopa- minergic agonists, whereas the introduction of a second n-propyl group increases or at least maintains the level of activity of the monopropyl derivative [9---131.

Benzobicyclo[2.2.2]octene analogue 5 appeared to have a double advantage. While maintaining the favorable anti- periplanar conformation of the DA moiety, the lipophilic bulk of the bridge is increased in this structure. Thus, comparison of the dopaminergic activity of this system compared with that of the benzonorbornene derivatives could provide information about the DA receptor region located ‘above’ the site which binds to the generally planar

*Current address: Smith Kline & French Laboratories, Department of Medicinal Chemistry, King of Prussia, PA 19406, U.S.A. **Current address: University of Pennsylvania School of Medicine, Department of Pharmacology, Philadelphia, PA 19104, U.S.A.

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catecholamine moiety, which is not addressed in recently proposed models of the DA receptor [14].

Chemistry

The mono- and di-n-propyl analogues of 1 were prepared by treating exo-2-amino-6,7-dimethoxybenzonorbonene [2] with n-propyl iodide in the presence of potassium carbonate as a base. The methyl ethers were cleaved by treatment with 48% hydrobromic acid to yield 3 and 4 as hydro- bromide salts in 80 % and 92 % yield, respectively (Scheme 1). The synthetic pathway to the benzobicyclo[2.2.2]octene analogue 5 is depicted in Scheme 2. The key step was the

K2CO3 n-prt C6H6

.H N,

7

Pr - N:p’: s

(35%) (41.5%)

,H N, . HBr

Pr (79.8%)

3 -

Scheme 1. Synthesis of dopamine analogues 3 and 4.

Me0

I.NaN3\Hg(ORd2 2.NaBH4 S.LiAIH4

A NxH

to 64%

N#” .HBt ‘H

preparation of 9 by the cycloaddition of 1,3-cyclohexadiene to 4,5-dimethoxybenzyne. A number of approaches have been followed for the synthesis of this intermediate. Gener- ation of 4,5-dimethoxybenzyne in the presence of cyclo- hexadiene using 1-bromo-2-iodo-4,5-dimethoxybenzene and n-butyl lithium or magnesium metal gave 9 in 1.4 % and zero yields, respectively. Crews and Beard prepared benzo- bicyclo[2.2.2]octadiene by heating benzenediazonium-2- carboxylate with 1,3-cyclohexadiene [15]. They report a 36% yield of a mixture of hydrocarbons. In our hands, heating 4,5-dimethoxybenzenediazonium carboxylate in the presence of the diene yielded only 2.2% of the Diels-Alder reaction product. Only when the decomposition of the diazonium carboxylate took place in a high pressure vessel (conditions that generally favor Diels-Alder reactions [ 16, 171) did the yield of the desired reaction product increase to 10.6%. Treatment of the hydrocarbon with mercuric azide and reduction of the resulting azide with lithium aluminium hydride [36] gave selectively the exo-amine 10. Finally, cleavage of the methyl ethers with 48% hydro- bromic acid furnished 5 in 87% yield.

Pharmacology

The pharmacological behavior of compounds 3, 4, 5, (-i)N,N-di-n-propyldopamine (( +)DP-DA), R-(-)-apo- morphine (R-(-)APO) and (+)-ADTN (2-amino-6,7-di- hydroxytetralin) against typical DA agonists and anta- gonists was examined by studying the ability of these agents to inhibit specific binding of [3H]apomorphine and [tH]- spiperone to rat striatal membranes. Compounds 3 and 4 were used in a range of concentrations from 50 to 20 000 nM and compound 5 from 5 to 10 000 nM. (+)DP-DA, R-(-)- APO and (+)ADTN were used in the range of 0.1-1000 nM in the [CHIAPO studies, and l-10 000 nM in the [3H]- spiperone studies.

Rpults and Discussion

The in vitro binding studies indicated that compounds 3 and 4 up to concentrations of 20 000 nM, and compound 5 up to concentrations of 10 000 nM, displayed no ability to displace the specific binding of [3H]spiperone or [3H]- apomorphine in rat striatal membranes. These results are in agreement with data reported previously on the binding characteristics of endo- and exo-2-amino-6,7-dihydroxybenzo- norbornene [5], which failed to displace either [3H]spiperone or [3H]apomorphine from DA-ergic sites in rat striatal membranes. To verify the validity of our results, compounds 3,4 and 5 were tested in parallel with the active DA agonists : (+)DP-DA, R-(-)APO and (+)ADTN. The ZCsO values of these active DA agonists against [3H]apomorphine and [3H]spiperone were comparable to literature values [I]. The results of these studies are presented on Table I. The

HO /H

N,H HO /H 4 ,.

::EDNH2 n

Scheme 2. Synthesis of dopamine analogue 5. 1 2 AOTN

393

Table I. Inhibition of [aH]spiperone and [3H]apomorphine binding to rat striatal membranes.”

structural relationship of these compounds to the active DA analogue ADTN indicates that the methylene or ethylene

Drug ZGO (nM) bridge introduced to attain structural rigidity in 3 and 4 definitely interferes with the receptor topology, preventing

[3H]spiperone [aH]apomorphine the molecule from fitting into the DA-ergic receptor site. introduction of N-n-propyl groups, which usually increases

3 NA” NAb

z NAb NAb

the activity of DA analogues [g--13], did not improve the NAC NAC activity of these compounds.

(&)DP-DA 4300+300 130130 These results can be analyzed in terms of various topo- R-(-)APO 300+50 13xt2 graphical models for DA receptors proposed in the literature (I)ADTN 400&20 1.84ZO.l [l, 14, 18-331. As mentioned before [5], the carbon bridge

“Values represent means * SD. Experiments were performed 3 times in one of the enantiomeric forms of 3,4 and 5, would interfere

in duplicate. unfavorably with receptor regions of steric bulk, such as bNot active up to a concentration of 20 000 nM. ‘obstacle P’ described by Seeman [l], whereas the other CNot active up to a concentration of 10 000 nM. enantiomer would interact with ‘obstacle Q’ defined in

Nitrogen

Fig. 1. Illustration of the fit of DA-analogues to the DA-receptor model (A) proposed by Seeman et al. [18]; ADTN can adopt aconformation that allows a fit to the model (B), whereas compound 5 cannot (C). D illustrates the two enantiomers of 5 with their m-OH, C-6 and nitrogen groups superimposed. Structures were constructed using the fragment library of version 3.3 of the SYBYL molecular modeling program (registered trademark of Tripos Associates Inc., St. Louis, MO 63117, USA): MAXIMIN, a versatile energy minimization program of SYBYL was used for optimization of the structures.

394

the same receptor model. Inactivity of these compounds is also consistent with the later ‘tetrahedral’ DA receptor model proposed by Seeman [18]. As illustrated in Fig. IC, when the m-OH and nitrogen moieties of compound 5 interact with their respective binding sites on the receptor, the carbon bridge interferes with the ‘back wall’ of the receptor [I 81.

Fig. 1D illustrates the relative orientation of the structures of the two enantiomers of compound 5 when their respective m-OH, C-6 and nitrogen groups are superimposed. It can be seen that in both enantiomers the ethano bridges have similar orientations. Thus, the interactions of these enan- tiomers with the receptor binding sites would be expected to be quite similar, such that neither enantiomer would be expected to exhibit DA-ergic activity. Thus, the lack of activity of the racemic compound is not likely to be due to fortuitous cancellation of opposite effects of the two enan- tiomers at D, receptor sites, as seen with racemic 3PPP at post-synaptic receptor sites [34].

We are currently investigating the effect of the incorpor-

3H). 13C NMR (CDC13): S 146.57, 146.38, 140.43, 137.69, 105.89, 105.37, 60.34, 56.08, 49.88, 47.69, 45.74, 42.68, 37.09, 22.52, 11.41. MS: m/e 261 (M+, 24x), 176 (100x), 161 (30x), 133 (6.2x), 115 (7.4%). Absolute mass for C16H23N02: calcd.: 261.1729; foun& 261.1716.

Synthesis of exo-2-(N,N-di-n-propylamino)-6,7-dihydroxybenzonorbor- nene hydrobromide 4 A solution of 8 (36 mg, 0.12 mmol) in 6 ml of 48 % hydrobromic acid was heated under reflux for 3 h; the solvent was then removed in vacua to yield a dark green solid which was treated with charcoal in absolute methanol to yield an off-white solid. Recrystallization from absolute methanol-ether in a dry ice-acetone bath gave 39 mg (92%) of the product, Rf = 0.52 (silica, methylene chloridc:methanoc 5 :l). The highest melting point observed for this hvnroscouic comuound was 117--120°C. Spectral data: IR: 3375, 32iO; 2956, 2120,^1625, 1490, 1460, 1300, 1165, 1045, 1010, 830, 790 cm-l. lH NMR (D20): 8 6.65 (s, 1H); 6.60 (s, 1H); 3.51-2.40 (m, 7H); 1.97-1.10 (m, SH); 1.06-0.90 (m, 6H). W NMR: S 141.48, 141.10, 139.94, 135.51, 110.01, 109.51, 66.37, 52.86, 51.84, 44.81, 44.00, 42.12, 33.58, 16.37, 15.30, 9.65. MS: In/e 275 (M+, 58x), 148 (49x), 128 (100x), 114 (30%). Anal. (C17H26NOzBr): calcd.: C: 57.31 ; H: 7.36; N: 3.93; found: C: 56.76; H: 7.39; N: 3.83. (C17Hz6NOaBr)*0.2 Hz0 calcd. : C: 56.73; H: 7.39; N: 3.89; found: C: 56.76; H: 7.39; N: 3.83.

ation of a heteroatom i&o the bridge of analogues of 3 Synthesis of exo-I?-(N-n-propylamino)-6,7-dihydroxybenzonorbornene

on DA-ergic activity. Such studies Gould proviYde useful hydrobromide 3

information concerning the lipophilicity or hydrophilicity A solution of 7 (25 mg, 0.1 mmol) in 5 ml of 48% hydrobromic acid

of the proposed obstacles on the DA receptor surface. was heated under reflux for 3 h. The product was isolated and purified with the same method described for 4. The yield of the reaction product was 25 mg (79.8”/,). The mu of the Droduct was IOS-1llOC and the

Experimental protocols

Chemistry

Melting points were taken on a Thomas-Hoover apparatus and are uncorrected. Infrared snectra were obtained using a Perkin-Elmer Model 735 spectrophotometer. Proton nuclear magnetic resonance (lH NMR) spectra were recorded on Hitachi Perkin-Elmer R-20 and Varian EM 360 60 MHz spectrometers; 13C NMR spectra were obtained on a Varian KL-100 Fourier transform sDectrometer. Proton chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane. Mass spectra were taken on a DuPont 21-492 double-focusing mass spectrometer. All the chemicals used were of reagent grade Duritv and all solvents were ourified according to standard purification methods prior to use. Analyses indicated by elemental symbols were within &0.4’% of the theoretical values and were performed by the Analytical Department of Rockefeller University, New York, NY, U.S.A.

Synthesis of exo-2- (N,n-propylamino) -6,7-dimethoxybenzonorbornene 7 andexo-Z(N,N-di-n-propylamino)-6,7-dimethoxybenzonorbornene 8 A solution of 6 hydrochloride (128 mg, 0.5 mmol) and n-propyl iodide (340 mg, 2.0 mmol) in 2.0 ml of benzene was heated under reflux with 0.5 ml of saturated potassium carbonate solution for 7 days. The crude mixture, after removal of the solvent, was purified by column chromatography on neutral alumina on elution with 1 :l methylene chloride:hexane. Two liauid comnonents were isolated. The first component (63 mg, 41.5%) with Rr = 0.12 was identified as 8 via spectral analysis. IR: 2940, 2920, 2860, 2820, 1610, 1495, 1460, 1315, 1220, 1125, 1075, 850, 790 cm-l; lH NMR (CDC13): 6 6.74, 6.72 (overlapping singlets, 2H) ; 3.80, 3.79 (overlapping singlets, 6H) ; 3.32-3.01 (m,2H);2.66-2.26(m,4H);2.14-1.90(m, 1H); 1.80-1.01 (m, 8H); 1.00-0.60 (t, J = 7 Hz, 6H). 13C NMR (CDC13): 6 147.33, 147.06, 141.48, 139.20, 106.55, 106.06, 65.95, 56.57, 54.21, 47.07, 46.48, 44.10, 36.18, 20.08, 12.25; MS: m/e 303 (M+, 19x), 176(100%), 161 (23 %), 115 (6.4%). Anal. (ClgH29NOz) calcd. C: 75.21; H: 9.63 ; N: 4.62; found: C: 75.32; H: 9.67; N: 4.56. The second component (46 mg, 35%) with an Rf = 0.03 was identified as 7. Spectral data: IR: 2950. 2860. 2825. 1610. 1585. 1490. 1460. 1310. 1210. 1115. 1070. 1020, 846, 780; 720 cm-l;’ IH tiMR <CDd,) B 6.70 (;, 1Hj; 6.68 (s, 1H); 3.74, 3.72 (overlapping singlets, 6H); 3.14 (br. s, 2H) ; 2.90-2.40 (m, 4H); 1.98-1.60 (m, 2H); 1.60-1.06 (m, 4H); 0.84 (t, J = 7 Hz,

Rf = 0.15 (silica; &ethanoi:CH&%; 4 :l). Spectral data : lH NMR (DaO): 6 6.65 (s, 1H); 6.59 (s, 1H); 3.28 (s, 1H); 3.14 (s, 1H); 2.89-2.73 (m, 3); 1.76-1.18 (m, 6); 0.72 (t, J = 7 Hz, 3H). MS: m/e 233 (M+, 25x), 148 (100x), 130 (16x), 102 (18%). Absolute mass for C14H19- NOa : calcd. : 233.1416; found: 233.1394. Anal. (C14H2oNO2Br)* 1.5 HzO: calcd.: C: 49.28: H: 6.79: N: 4.10: found: C: 49.12: H: 5.84; N: 3.78. (C14H~,Nd2)*1.3 Hgr: calcd.: C: 49.28; H: j.91; N: 4.11; found: C: 49.12; H: 5.84; N: 3.78.

Synthesis of 6,7-dimethoxybenzobicyclo[2.2.2]octadiene 9 Method A. A solution of 1-bromo-2-iodo-4,5-dimethoxybenzene [35] (8.75 g, 25 mmol) in 25 ml of ether was placed in a 120 ml three- necked round-bottomed flask equipped with a thermometer and a magnetic stirrer. 1,3-Cyclohexadiene (4.05 g, 50 mmol) was added to the solution and the system was cooled down to -70°C under nitrogen. A 1.5 M solution of n-butyllithium in hexane (16.7 ml, 25 .mmol) was slowly added to the solution. The system was stirred at -70°C for 2 h and then allowed to warm slowly to 10% over a 3 h period, at which temperature the mixture was added to 50 g of dry ice. The mixture was stirred for 15 min. 25 ml of water was then added to the system; the organic layer was separated, washed with 10% NaOH and water and dried over MgS04. Removal of the solvent gave a yellow oil which was chromatographed on a silica gel column eluted by 2:3 hexane:CHCb. The product was identified via spectral analysis (see below) as the desired product 9. The yield of the product was 75 mg (1.4%). Method B. Into a three-necked flask equipped with a magnetic stirrer and a dropping funnel was placed magnesium (1.21 g, 50 mmol) and the system was flame-dried and flushed with nitrogen for 45 min. A solution of 1-bromo-2-iodo-4,5-dimethoxybenzene (17.5 g, 50 mmol) and 1,3-cyclohexadiene (9.53 g, 50 mmol) was placed in the dropping funnel. A part of the solution from the funnel was added to cover the ,magnesium and was heated to boiling. The rest of the solution wa$ adaed dropwise over a period of 1 h. The system was then heated at ieflux for an additional 0.5 h. The solvent was removed in vacua and the residue was poured into a mixture of ether and a saturated solution of ammonium chloride. The organic layer was separated, washed with a 5 M solution of sodium bisulfite, and dried over Na2S04. The solvent was removed in vacua and the residue was chromatographed on a silica gel column eluted by 1 :l hexane :CH#&. No trace of the desired compound could be identified among the isolated compo- nents according to gas chromatography (GC)/MS analysis. Method C. Into a 250 ml three-necked round-bottomed flask equipped with a magnetic stirrer, a thermometer and a reflux condenser, were

395

placed 2-carboxy-4,5-dimethoxybenzenediazonium chloride [36] (12.9 g, 53 mmol) and 150 ml of 1,2-dichloroethane. The mixture was stirred and gradually heated under nitrogen up to 85oC when evolution of gas was observed. Cyclohexadiene (13.1 g, 164 mmol) was added to the mixture followed by 10 ml of propylene oxide. The system was stirred and heated under reflux for 3 h until the evolution of gas ceased. The solution was neutralized with 10 ‘A NaOH and the product was steam distilled and purified by flash chromatography on a silica gel column eluted by CHCls :pentane, 1 :l. The yield of the reaction product 9, which, was identified by spectral analysis, was 250 mg (2.18 %). Method D. Into a 50 ml high pressure reaction vessel equipped with a magnetic stirrer were placed 2-carboxy-4,5-dimethoxybenzene- diazonium chloride (6.70 g, 29 mmol), cyclohexadiene (6.57 g, 83 mmol), 5.0 ml of propylene oxide and 30 ml of 1,2-dichloroethane. The vessel was sealed and the system was stirred at 80--85OC for 72 h. The system was then cooled down to room temperature. the nressure was released and the solvent was removed in v&o. The product was purified by flash chromatography on a silica gel column eluted by 1 :I CHaClz : hexane. The yield of the reaction product 9 was 600 mg (10.6%); mo : 82--84%.

‘Spectral data for 9: IR: 3040, 3000, 1610, 1590, 1505, 1450, 1420, 1330, 1300, 1260, 1225, 1190, 1115, 1050, 1000, 880, 860, 810, 760, 700 cm-l. IH NMR (CDCla) : 6 6.67 (s, 2H); 6.5 (dd, 4.5 Hz and 3.0 Hz, 2H): 3.83 (s, 8H): 1.45 (br. s, 4H). 13C NMR (CDCla) : 6 146.49;136.65, 135.44, 107.81, 75.97, 46.25, 26.36. MS: m/e 216 (M+, 27x), 188 (100x), 173 (13x), 145 (22x), 127 (14%). Anal. (C14- H1602): calcd.: C: 77.75; H: 7.46; found: C: 78.03; H: 7.38.

Synthesis of exo-2-amino-6,7-dimethoxybenzobicyclo[2.2.2]octene 10 [37] Sodium azide (490 mg, 7.5 mmol) was added to 20 ml of 1: 1 Hz0 :THF (tetrahvdrofuran). The mixture was cooled to O°C and mercuric acetate (800 mg; 2.5 mmol) was added in one portion. The system was stirred for 20 min and 9 (540 mg, 2.5 mmol) was added. After having been stirred at 75OC for 24 h, the system was diluted with 5 ml of a 15 “A solution of KOH and treated with a solution of sodium borohydride’f50 mg, 1.3 mmol) in 15 ml of 15% KOH. The THF layer was separated and the aqueous layer was extracted with ether. The combined organic layers were washed with water and dried over MgS04. The ether solution was slowly added to a suspension of lithium aluminium hydride (190 mg, 5 mmol) at O’C. After the addition was comnlete. the mixture was stirred at room temnerature for 1 h. It was -then quenched by the subsequent addition bf 0.5 ml of water, 0.5 ml of 15’/, KOH solution and 1.5 ml of water. The mixture was filtered and the precipitate was washed with ether. The filtrate was extracted with ether and the organic layer was washed with water. saturated sodium chloride solution and dried over MgS04. Removal of the solvent gave 374 mg (64 ‘A) of crude material which was purified on an alumina column eluted by 1:4 CHzClz :methanol, to give 210 mg (36%) of pure product Rf = 0.08 (silica gel-G,CHa Clz: methanol, 4:l). The hydrochloride salt was formed by treating a solution of the liquid amine in ether with gaseous HCI. The mp of the hydrochloride was 133-135OC. Spectral data for 10: IR: 3320, 3260, 2910, 2840, 1600, 1495, 1460, 1280, 1120, 1110, 1020, 850, 800, 745 cm-l. lH NMR (CDCla): 6 6.6 (s, 2H); 3.80 (s, 6H); 3.20-2.90 (m, 3H); 2.53-1.13 (m, 8H). MS: m/e 233 (M+, 20x), 190 (100x), 175 (28x), 147 (12x), 128 (11%).

Synthesis of exo-2-anzino-6,7-dilzydroxybenzobicyclo[2.2.2]octane 5 Compound 10 (27 mg, 0.1 mmol) was treated with ‘4 ml of 47 % HBr and purified according to the general method described above. The yield of the purified product 5 was 18 mg (87%) ; mp : 158- 160°C; RF = 0.15 (silica, methanol:CH~C&, 4:l). Spectral data for 5: NMR (DzO): 6 7.60 (m, 2H); 3.80-3.40 (m, 1H); 3.17-2.77 (m, 2H); 2.53-0.80 (m, 6H). MS : nzje 205 (Mf 32x), 162 (100x), 116 (16%). Anal. (ClzHrsNOz)*1.5 HBr: calcd.: C: 44.13; H: 5.09; N: 4.29; found: C: 44.23; H: 5.45; N: 4.30.

Pharmacology

Binding studies were carried out three times in duplicates of rat striatal membranes. Male Sprague-Dawley rats, 200-250 g, were sacrificed by decapitation and the striata were removed. Pooled striata were

homogenized in 50 mM Tris-HCl buffer, pH 7.4, using a Tissue- mizer (Tekmar, Inc.) at setting 45 for 15 s. The resultant homogenate was centrifuged at 45 000 xg for 10 min in a Sorvall OTD-2 ultra- centrifuge at 4OC. The supernatant was discarded and the pellet was resuspended in ice-cold Tris-HCI buffer and rehomogenized with the Tissuemizer. The homogenization and washing process was repeated a total of 4 times and the final pellet was resuspended in TEAN buffer (15 mM Tris-HCl, 5 mM NazEDTA, 1.1 mM ascorbate, 12.5 mM nialamide, pH 7.4) at 4OC at 2.5 mg of wet weight/ml. This preparation is subsequently referred to as ‘striatal membranes’.

13HlSuiuerone assay Compounds 3, 4 and 5 were dissolved in a minimum volume of ethanol and TEAN. (&)DP-DA, R-(-)APO, and (&)ADTN (obtained from Research Biochemicals Inc.) were dissolved in TEAN. Into 13 x 100 mm glass test tubes were placed [3H]spiperone (New England Nuclear, 25.7 Ci/mmol), test ligands, TEAN buffer or control ligand and striatal membranes (800 ,ul) in a total volume of 1 ml. The final concentration of [3H]spiperone was 0.5 nM. The tubes were incubated at 25OC for 45 min and the reaction was terminated by filtration over GF/B filters using a Brandell Harvester under vacuum. The filters were’ washed with three 5 ml aliquots of TEAN and then counted for tritium radio- activity in a Tracer Analytic scintillation counter in vials containing 4 ml of Budget Solve. Specific binding was operationally defined as the total [3H]spiperone activity bound minus the amount bound in the presence of 1 PM (+)-butaclamol in the incubation medium.

[aH]Apomorphine (New England Nuclear, 27 Ci/mmol) was used as described above at a final concentration of 1 nM. Specific binding was operationally defined as the total [sH]apomorphine bound minus that bound in the presence of 1 pM unlabeled apomorphine.

References

1 2 3 4

5

6

7

8

9 10

11

12

13

14

42 17

18

19 20 21

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