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Isomeric selectivity at dopamine D3 receptors
European Journal of Pharmacology, 239 (1993) 269-270
269
© 1993 Elsevier SciencePublishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 21312 Rapid communication
Isomeric selectivity at dopamine
D 3
receptors
Ross J. Baldessarini, Nora S. Kula, Christopher R. McGrath, Venkatesalu Bakthavachalam, J o h n W. Kebabian and John L. N e u m e y e r The Mailman Research Center, McLean Division of Massachusetts GeneralHospital and Department of Psychiatry and Neuroscience Program, Harvard Medical School, and Research Biochemicals, International (RBI), Natick, MA, USA
Received 24 June 1993,accepted6 July 1993 Racemic 7-hydroxy-N,N-dipropylaminotetralin(7-OH-DPAT) shows greater affinity for limbic-selective dopamine D 3 receptors than for more ubiquitous dopamine D 2 receptors. R( + )-7-OH-DPAT was prepared and evaluated in radioreceptor assays using membranes of fibroblasts expressing the human dopamine D 3 receptor as well as rat striatal membranes containing dopamine D 2 receptors. This enantiomer had 2-fold greater D 3 affinity than the racemate and similarly greater D 3 vs. D2 selectivity (64-fold). The results may facilitate development of D 3 selective agents and evaluation of functions of these receptors. Aminotetralins; Dopamine D 3 receptors; 7-OH-DPAT (7-hydroxy-2-(N,N-di-n-propylamino)tetralin) Discovery of the dopamine D 3 receptor, a unique gene product with dopamine D 2 receptor-like characteristics but preferential distribution to limbic areas in mammalian forebrain, encourages hope that limbicselective dopamine agents can be developed (Sokoloff et al., 1990). These might include drugs having clinically useful psychotropic properties but limited extrapyramidal neurological side effects. Preferential affinity for dopamine D 3 over dopamine D E receptors has been reported in transfected cells selectively expressing dopamine D 3 receptors, including dopamine and several of its agonists, particularly the monohydroxyaminotetralin R,S( __)-7-hydroxy-N,N-di-n-propyl2-amino- 1,2,3,4-tetrahydronaphthalene (7-OH-DPAT; L6vesque et al., 1992) and its N-iodopropyl analog (Foulon et al., 1993). McDermed and Freeman (1982) previously reported stereoselectivity of hydroxyaminotetralins for dopamine receptors in striatal tissue of calf brain labeled with [3H]apomorphine or [3H]spiperone. There was a strong (200-1000-fold) preference for the R ( + ) enantiomer of 7-OH-DPAT, but a more moderate (25-fold) preference for the S ( - ) antipode of 5-OH-DPAT. These findings, consistent with D E selectivity for R(+)-7-OH-DPAT, led us to predict similar stereoselectivity at dopamine D 3 receptors.
Correspondence to: R.J. Baldessarini, Mailman Research Center, McLean Hospital, 115 Mill Street, Belmont, MA 02178, USA. Tel. (617) 855-3203, fax (617) 855-3479.
Accordingly, we prepared R( + )-7-OH-DPAT, starting from R(+)-2-amino-7-methoxy-l,2,3,4-tetrahydronaphthalene, which was separated from its racemate by liquid chromatography on a chiral column, N-acylated with propionyl chloride with triethylamine, and reduced with diborane to obtain the N-n-propylamino derivative. Further N-alkylation with propargyl bromide in ethanol containing potassium carbonate, and cleavage of the methoxyl with boron tribromide afforded the R(+)-[N-propargyl-N-n-propyl]-2-amino-7hydroxytetralin. Catalytic hydrogenation of the propargyl moiety over palladium-on-carbon gave R(+)-7OH-DPAT, which was purified as the hydrochloride and characterized for chemical and enantiomeric integrity. Neuropharmacologic assessment used previously reported radioreceptor assay methods (Baldessarini et al., 1992) modified to employ cell membranes (50 /~g protein/assay) prepared from cultured mouse fibroblasts stably transfected to express human dopamine D 3 receptor protein selectively, [3H](R,S)-YM-09151-2 as radioligand, and S(-)-eticlopride (50 nM; RBI) as blank, incubated for 60 min at 30°C in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCI. Concentrations (N = 14; 0 and 0.1 nM to 10/~M, in triplicate) of R(+)-7-OH-DPAT and its racemate or the isomers and racemate of eticlopride were added to determine IC50. For comparison, membranes from rat striatal tissue were used as a source of dopamine D 2 receptors. D 3 cell membranes and test agents were from Research Biochemicals International (RBI, Natick, MA).
270 TABLE 1 Dopamine D 3 receptor isomeric selectivity. Assay conditions are described in the text. Compound
IC50 5-S.E.M. (nM)
D 3 assay
S( - )-Eticlopride R,S( 5-)-Eticlopride R( + )-Eticlopride R,S( 5-)-7-OH-DPAT R( + )-7-OH-DPAT
0.058 + 0.005 0.13 5-0.01 10.75:1.2 " 11.5 + 0.8 6.6 5-0.6 a
D 2 assay
R,S( 5-)-7-OH-DPAT R( + )-7-OH-DPAT
380 _+53 420 5-58
d o p a m i n e D 3 r e c e p t o r s for a d o p a m i n e r e c e p t o r a n t a g onist a n d agonist, a n d i n d i c a t e t h a t t h e R ( + ) e n a n t i o m e r o f 7 - O H - D P A T is highly 0 3 vs. O 2 selective. This p r o p e r t y might b e e x p l o i t e d in t h e f u r t h e r develo p m e n t o f c o m p o u n d s with selectivity for d o p a m i n e D 3 receptors and exploration of the actions of dopamine D 3 receptors.
Acknowledgments Supported in part by USPHS (NIMH) grants MH-34006, MH47370, and a grant from the Bruce J. Anderson Foundation.
a Significantly lower than for the racemate (t = 4.9, P < 0.01); results at D 2 are not different.
References
P r e l i m i n a r y e v a l u a t i o n s o f t h e assay s h o w e d high affinity ( K d -- 0.2 nM), a c c e p t a b l e specificity ( 6 0 % ) a n d s a t u r a b l e , r e v e r s i b l e b i n d i n g (Bm~x = 1 p m o l / m g p r o t e i n ) o f t h e r a d i o l i g a n d , as well as high s t e r e o s e l e c t i v i t y for e t i c l o p r i d e . S ( - ) - E t i c l o p r i d e s h o w e d t w i c e - g r e a t e r D 3 affinity t h a n t h e r a c e m a t e , a n d 185-fold h i g h e r affinity t h a n its R( + ) a n t i p o d e ( T a b l e 1). A s p r e d i c t e d , R(+)-7-OH-DPAT also s h o w e d a b o u t t w i c e - g r e a t e r D 3 affinity t h a n its r a c e m a t e , i n d i c a t i n g t h a t it is m u c h m o r e avid t h a n t h e S ( - ) i s o m e r ( T a b l e 1). I n a d d i t i o n , r a c e m i c 7 - O H - D P A T s h o w e d high D 3 - o v e r - D 2 selectivity (33-fold) t h a t was even g r e a t e r (64-fold) with t h e R ( + ) i s o m e r ( T a b l e 1). T h e s e results a d d to uses o f g e n e t i c a l l y t r a n s f e c t e d cells as test systems for p h a r m a c o l o g i c a s s e s s m e n t o f a g e n t s with selective i n t e r a c t i o n s with d o p a m i n e D 3 o r o t h e r r e c e p t o r s , a d d e v i d e n c e for s t e r e o s e l e c t i v i t y o f
Baldessarini, R.J., N.S. Kula, A. Campbell, V. Bakthavachalam, J. Yuan and J.L. Neumeyer, 1992, Prolonged D 2 antidopaminergic activity of alkylating and nonalkylating derivatives of spiperone in rat brain, Mol. Pharmacol. 42, 856. Foulon, C., M. Kung and H. Kung, 1993, Synthesis of (R,S)-2'-trans7-hydroxy-2[N-n-propyl-N-(3'-iodo-2'-propenyl)]-aminotetralin (trans-7-OH-PIPAT): a new D 3 dopamine receptor ligand, J. Med. Chem. 36, 1499. L~vesque, D., J. Diaz, C. Pilon, M.-P. Martres, B. Giros, E. Souil, D. Schott, J.-L. Morgat, J.-C. Schwartz and P. Sokoloff, 1992, Identification, characterization, and localization of the dopamine D 3 receptor in rat brain using 7-[3H]hydroxy-N,N-di-n-propyl-2aminotetralin, Proc. Natl. Acad. Sci. USA 89, 8155. McDermed, J.D. and H.S. Freeman, 1982, Stereochemistry of dopamine receptor agonists, in: H. Kohsaka, T. Shohmori, Y. Tsukada and G.N. Woodruff (eds.), Advances in Dopamine Research (Pergamon Press, New York) p. 179. Sokoloff, P., B. Giros, M.-P. Martres, M.-L. Bouthenet and J.C. Schwartz, 1990, Molecular cloning and characterization of a novel dopamine receptor (D 3) as a target for neuroleptics, Nature 347, 146.
269
© 1993 Elsevier SciencePublishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 21312 Rapid communication
Isomeric selectivity at dopamine
D 3
receptors
Ross J. Baldessarini, Nora S. Kula, Christopher R. McGrath, Venkatesalu Bakthavachalam, J o h n W. Kebabian and John L. N e u m e y e r The Mailman Research Center, McLean Division of Massachusetts GeneralHospital and Department of Psychiatry and Neuroscience Program, Harvard Medical School, and Research Biochemicals, International (RBI), Natick, MA, USA
Received 24 June 1993,accepted6 July 1993 Racemic 7-hydroxy-N,N-dipropylaminotetralin(7-OH-DPAT) shows greater affinity for limbic-selective dopamine D 3 receptors than for more ubiquitous dopamine D 2 receptors. R( + )-7-OH-DPAT was prepared and evaluated in radioreceptor assays using membranes of fibroblasts expressing the human dopamine D 3 receptor as well as rat striatal membranes containing dopamine D 2 receptors. This enantiomer had 2-fold greater D 3 affinity than the racemate and similarly greater D 3 vs. D2 selectivity (64-fold). The results may facilitate development of D 3 selective agents and evaluation of functions of these receptors. Aminotetralins; Dopamine D 3 receptors; 7-OH-DPAT (7-hydroxy-2-(N,N-di-n-propylamino)tetralin) Discovery of the dopamine D 3 receptor, a unique gene product with dopamine D 2 receptor-like characteristics but preferential distribution to limbic areas in mammalian forebrain, encourages hope that limbicselective dopamine agents can be developed (Sokoloff et al., 1990). These might include drugs having clinically useful psychotropic properties but limited extrapyramidal neurological side effects. Preferential affinity for dopamine D 3 over dopamine D E receptors has been reported in transfected cells selectively expressing dopamine D 3 receptors, including dopamine and several of its agonists, particularly the monohydroxyaminotetralin R,S( __)-7-hydroxy-N,N-di-n-propyl2-amino- 1,2,3,4-tetrahydronaphthalene (7-OH-DPAT; L6vesque et al., 1992) and its N-iodopropyl analog (Foulon et al., 1993). McDermed and Freeman (1982) previously reported stereoselectivity of hydroxyaminotetralins for dopamine receptors in striatal tissue of calf brain labeled with [3H]apomorphine or [3H]spiperone. There was a strong (200-1000-fold) preference for the R ( + ) enantiomer of 7-OH-DPAT, but a more moderate (25-fold) preference for the S ( - ) antipode of 5-OH-DPAT. These findings, consistent with D E selectivity for R(+)-7-OH-DPAT, led us to predict similar stereoselectivity at dopamine D 3 receptors.
Correspondence to: R.J. Baldessarini, Mailman Research Center, McLean Hospital, 115 Mill Street, Belmont, MA 02178, USA. Tel. (617) 855-3203, fax (617) 855-3479.
Accordingly, we prepared R( + )-7-OH-DPAT, starting from R(+)-2-amino-7-methoxy-l,2,3,4-tetrahydronaphthalene, which was separated from its racemate by liquid chromatography on a chiral column, N-acylated with propionyl chloride with triethylamine, and reduced with diborane to obtain the N-n-propylamino derivative. Further N-alkylation with propargyl bromide in ethanol containing potassium carbonate, and cleavage of the methoxyl with boron tribromide afforded the R(+)-[N-propargyl-N-n-propyl]-2-amino-7hydroxytetralin. Catalytic hydrogenation of the propargyl moiety over palladium-on-carbon gave R(+)-7OH-DPAT, which was purified as the hydrochloride and characterized for chemical and enantiomeric integrity. Neuropharmacologic assessment used previously reported radioreceptor assay methods (Baldessarini et al., 1992) modified to employ cell membranes (50 /~g protein/assay) prepared from cultured mouse fibroblasts stably transfected to express human dopamine D 3 receptor protein selectively, [3H](R,S)-YM-09151-2 as radioligand, and S(-)-eticlopride (50 nM; RBI) as blank, incubated for 60 min at 30°C in 50 mM Tris buffer (pH 7.4) containing 150 mM NaCI. Concentrations (N = 14; 0 and 0.1 nM to 10/~M, in triplicate) of R(+)-7-OH-DPAT and its racemate or the isomers and racemate of eticlopride were added to determine IC50. For comparison, membranes from rat striatal tissue were used as a source of dopamine D 2 receptors. D 3 cell membranes and test agents were from Research Biochemicals International (RBI, Natick, MA).
270 TABLE 1 Dopamine D 3 receptor isomeric selectivity. Assay conditions are described in the text. Compound
IC50 5-S.E.M. (nM)
D 3 assay
S( - )-Eticlopride R,S( 5-)-Eticlopride R( + )-Eticlopride R,S( 5-)-7-OH-DPAT R( + )-7-OH-DPAT
0.058 + 0.005 0.13 5-0.01 10.75:1.2 " 11.5 + 0.8 6.6 5-0.6 a
D 2 assay
R,S( 5-)-7-OH-DPAT R( + )-7-OH-DPAT
380 _+53 420 5-58
d o p a m i n e D 3 r e c e p t o r s for a d o p a m i n e r e c e p t o r a n t a g onist a n d agonist, a n d i n d i c a t e t h a t t h e R ( + ) e n a n t i o m e r o f 7 - O H - D P A T is highly 0 3 vs. O 2 selective. This p r o p e r t y might b e e x p l o i t e d in t h e f u r t h e r develo p m e n t o f c o m p o u n d s with selectivity for d o p a m i n e D 3 receptors and exploration of the actions of dopamine D 3 receptors.
Acknowledgments Supported in part by USPHS (NIMH) grants MH-34006, MH47370, and a grant from the Bruce J. Anderson Foundation.
a Significantly lower than for the racemate (t = 4.9, P < 0.01); results at D 2 are not different.
References
P r e l i m i n a r y e v a l u a t i o n s o f t h e assay s h o w e d high affinity ( K d -- 0.2 nM), a c c e p t a b l e specificity ( 6 0 % ) a n d s a t u r a b l e , r e v e r s i b l e b i n d i n g (Bm~x = 1 p m o l / m g p r o t e i n ) o f t h e r a d i o l i g a n d , as well as high s t e r e o s e l e c t i v i t y for e t i c l o p r i d e . S ( - ) - E t i c l o p r i d e s h o w e d t w i c e - g r e a t e r D 3 affinity t h a n t h e r a c e m a t e , a n d 185-fold h i g h e r affinity t h a n its R( + ) a n t i p o d e ( T a b l e 1). A s p r e d i c t e d , R(+)-7-OH-DPAT also s h o w e d a b o u t t w i c e - g r e a t e r D 3 affinity t h a n its r a c e m a t e , i n d i c a t i n g t h a t it is m u c h m o r e avid t h a n t h e S ( - ) i s o m e r ( T a b l e 1). I n a d d i t i o n , r a c e m i c 7 - O H - D P A T s h o w e d high D 3 - o v e r - D 2 selectivity (33-fold) t h a t was even g r e a t e r (64-fold) with t h e R ( + ) i s o m e r ( T a b l e 1). T h e s e results a d d to uses o f g e n e t i c a l l y t r a n s f e c t e d cells as test systems for p h a r m a c o l o g i c a s s e s s m e n t o f a g e n t s with selective i n t e r a c t i o n s with d o p a m i n e D 3 o r o t h e r r e c e p t o r s , a d d e v i d e n c e for s t e r e o s e l e c t i v i t y o f
Baldessarini, R.J., N.S. Kula, A. Campbell, V. Bakthavachalam, J. Yuan and J.L. Neumeyer, 1992, Prolonged D 2 antidopaminergic activity of alkylating and nonalkylating derivatives of spiperone in rat brain, Mol. Pharmacol. 42, 856. Foulon, C., M. Kung and H. Kung, 1993, Synthesis of (R,S)-2'-trans7-hydroxy-2[N-n-propyl-N-(3'-iodo-2'-propenyl)]-aminotetralin (trans-7-OH-PIPAT): a new D 3 dopamine receptor ligand, J. Med. Chem. 36, 1499. L~vesque, D., J. Diaz, C. Pilon, M.-P. Martres, B. Giros, E. Souil, D. Schott, J.-L. Morgat, J.-C. Schwartz and P. Sokoloff, 1992, Identification, characterization, and localization of the dopamine D 3 receptor in rat brain using 7-[3H]hydroxy-N,N-di-n-propyl-2aminotetralin, Proc. Natl. Acad. Sci. USA 89, 8155. McDermed, J.D. and H.S. Freeman, 1982, Stereochemistry of dopamine receptor agonists, in: H. Kohsaka, T. Shohmori, Y. Tsukada and G.N. Woodruff (eds.), Advances in Dopamine Research (Pergamon Press, New York) p. 179. Sokoloff, P., B. Giros, M.-P. Martres, M.-L. Bouthenet and J.C. Schwartz, 1990, Molecular cloning and characterization of a novel dopamine receptor (D 3) as a target for neuroleptics, Nature 347, 146.