Neurochem. Int. Vol. 20, Suppl.,pp. 157S-160S,1992

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A68930: A POTENT AGONIST SPECIFIC FOR THE D O P A M I N E D1 RECEPTOR JOHN W. KEBABIAN, MICHAELP. DENIt,~o, ROBERT SCHOENLEBER, ROBERT MACKENZIE, DONALDR. BR1TTONand KAREN E. ASIN Nenroseience Research Division, Abbott Laboratories, Abbott Park, IL 60061, U.S.A.

Abstract-- A68930, [1R, 3S] l-aminomethyl-5,6-dihydroxy-3-phenylisochroman HCi, is a potent, partial agonist in the dopamine-sensitive adenylate cyclase model of the D~ dopamine receptor in fish retina. In the rat caudate-putamen model of the D, dopamine receptor, A68930 is a potent (ECS0 2,1 nM) full agonist. In contrast, A68930 is a much weaker (EC50 = 3,920 nM) full agonist in a biochemical model of the D 2 dopamine receptor. A68930 also displays weak 2 agonist activity but the molecule is virtually inactive at the 1 and fl-adrenoceptors. When tested in rats bearing a unilateral 6-OHDA lesion of the nigro-neostriatal neurons, A68930 elicits prolonged ( > 20 hr) contralateral turning.

The existence of the dopamine D~ and the dopamine D2 receptors is now generally accepted (Garau et al., 1978; Kebabian and Calne,1979; Waddington and O'Boyle, 1989). However, the role of the dopamine D~ receptor in the functioning of the extrapyramidal nervous system is not well understood. Biochemical studies indicate that the density of dopamine DL receptors is greater than is the density of D2 receptors in many brain regions including the caudate-putamen (Richfield et al., 1989). Preclinical investigations of the dopamine D~ receptor in rats bearing a unilateral 6-OHDA lesion of the nigro-neostriatal pathway show that stimulation of the receptor not only alters glucose utilization within the extrapyramidal nervous system but also produces the characteristic contralateral rotation response (Trugman and Wooten,1987; Arnt and Hyttel, 1985). The benzazepine SK&F 38393 is the most widely-used dopamine D~ receptor agonist (Setler et al., 1978). Recent publications have described non-benzazepine agonists that stimulate the dopamine D~ receptor (Lovenberg et a1.,1989; Andersen et al., 1987; Brewster et al. 1990). In this communication, we report that A68930, [1R, 3S] l-aminomethyl-5, 6dihydroxy-3-phenylisochroman HCI, is a potent and specific agonist in in vitro models the D, receptor. In addition, we show that the molecule is capable of stimulating the DL receptor in vivo for up to 20 hrs following a single administration. We believe that A68930 may be a useful experimental probe of the D~ receptor in the brain. EXPERIMENTALPROCEDURES The procedures used to test compounds described in this

manuscript have been described elsewhere (Kerkman et al., 1989; DeNinno et al., 1990). RESULTS A68930 and its two enantiomers, A70108 and A70360, were tested in several receptor binding assays. A68930 displayed the highest affinity towards the D~ dopamine receptor; the compound had a 250 fold lower affinity towards the D2 dopamine receptor (table 1). Similarly, A70108, the IR, 3S enantiomer, had highest affinity towards the D Ldopamine receptor and a 500-fold lower affinity towards the D2 dopamine receptor. In contrast, A70360, the 1S, 3Renantiomer, had a much lower affinity towards the D~ dopamine receptor than did either of the other two compounds. Neither A68930 nor A70108 displayed as high an affinity towards the adrenergic and serotonergic receptors against which they were tested as they did towards the DL receptor; likewise, A70360 did not display high affinity towards any of these receptors. A68930 was an agonist in biochemical models of the D~ dopamine receptor. The dopamine-sensitive adenylate cyclase of the carpretina is a frequently-used model of the D, receptor (Watling e t al., 1979). In accord with previous results, dopamine produced halfmaximal activation of carp retinal adenylate cyclase activity at a concentration of 2,100 nM (data not shown). A68930 was a potent, partial agonist when tested in this assay system (table 2). In contrast, A69270, the desphenyl analog of A68930 (Figure 1), displayed much lower affinity in this biochemical model of the D~ dopamine receptor.As was the case in the binding assays, only one of the enantiomers of

157S

D o p a m i n e 9(1

158S

HO

HO O

o

NH2 HBr

A-68930

A-69270

O~ NH2 HCI

A-70108

HO OH

NH2 HCI

A-70360

Fig. I. Compounds utilized in the present communication. Table 1. Affinity of A68930, A70108 and A70360 in receptor binding assays Affinity (-log Ki) of A68930

A70108A

A70360

8.79 + 0.18 6.09 5:0.24 INACTIVE 4.68 4- 0.05 7.40 4- 0.21 5.59 4- 0.20 INACTIVE 5.30 4- 0.07

5.14 + 0.04 INACTIVE INACTIVE 5.22 4- 0.02 5.60 4- 0.27 INACTIVE 5.41 4- 0.31 6.10 5:0.07

Receptor D, D2 beta Alpha-I Alpha-2 5HT,A 5HTIc 5HT 2

8.52 6.11 3.88 4.97 6.91 4.74 5.03 6.66

+ 0.15 ± 0.21 5:0.27 4- 0.01 4- 0.12 4- 0.08 4- 0.03 4- 0.23

Data were obtained in between 3 and 8 replicate experiments. INACTIVE signifies that less than 50% displacement of radioligand was achieved with the highest concentration tested (10,000 nM).

A68930 was a potent D, receptor agonist (table 2). Thus, the agonist activity resides primarily in the 1R,3S enantiomer, A70108. In contrast, A70360 was 4000-fold less potent than A70108 as a D, receptor agonist. Thus, in the carp retina model of the D, dopamine receptor, A68930 and A70108 were partial agonists, approximately 1000fold more potent than dopamine. The dopamine-sensitive adenylate cyclase of the rat caudate- putamen is another frequently-used model of the D, dopamine receptor. A68930 was an agonist in

this assay system, causing half-maximal stimulation of enzyme activity at a concentration of 2.1 ± 0.4 nM (mean + SEM, N = 4). This value is comparable to the EC50 of A68930 in the fish retina model of the D, dopamine receptor (see above) The maximal response to A68930, a 75 + 8 percent (mean + SE, N = 4) increase in enzyme activity, was comparable to the maximal response to dopamine, an 83 =: 9 percent (mean + SEM, n = 4) increase in enzyme activity. Thus, in this model of the D, dopamine receptor. A68930 was a potent, full agonist. A68930 was a weak agonist in a biochemical model of the D 2 dopamine receptor (table 2). The weak D2 agonist activity of A68930 resided in A70108: A70360 was inactive at this receptor (table 2). A68930 displayed little activity towards either the 1 or fl-adrenoceptors (data not shownl. In a model of the 2 adrenoceptor, A68930 was a full agonist with an EC50 of 111 + 30 nM. In dopamine uptake studies, A68930 was inactive at concentrations as great as 10,000 nM where as the uptake inhibitor GBR 12909 was active with an IC50 of 32 + 9 nM. A68930 elicited contralateral turning behavior in 6OHDA lesioned rats. A striking feature of A68930 was its duration of action. Figure 2 shows that following a single dose of A68930 (3.2 /~mole/kg, s.c.), turning behavior resulted for the following 20 hours. Paralleling the in vitro biochemical results, A70108 was

Dopamine 90

159S

Table 2. Affinityand intrinsic activity of isochromans in biochemicalmodels of the D~and D2 dopamine receptors Compound

D,

A68930 A69270 AT0108 A70360

Receptor

D2 Receptor

-log[EC50]

IA

-log[EC50]

IA

8.67 ± 0.09 6.31 4-0.13 8.70 ± 0.12 5.06± 0.07

71.0± 7.0 71.1 ±7.0 59.0 ± 6.4b 98.3 ± 8.6

5.40±0.11

1.9 96 98.3 ± 6.1 lOl ±

5.54 4.99 -4-0.27 inactive

Data for the D, rex~ptorrepresentmean ± S.E.M. for ob~rvations made in betweenthree and six separate experiments.Data for the D2receptor rcprc~nt mean of data obtained in one (A70360) or two (A69270) experiments or mean ± S.E.M. for three (A70108) or four (A68930) experiments. Abbreviations: IA, intrinsic activity expressed as a percentage of the maximal response to dopamine (100/aM) for the D~ receptor or apomorphine(100/aM) for the D2 receptor. CONTRALATERAL Z

[

600-

O o3 nr W 0,. oo Z

_o

IJJ O3 +l Z

...I

ROTATION PRODUCED

I

I

I o ~ o e ~ e &~a

BY A68930 I

I

0.32 u m o l / K g , sc 1.00 u m o l / K g , sc 3.20 u m o l / K g , sc

3O0

< LU

/

ILl < ft. )-Z 0 0

0 -50,'

~{)o°oooooooooooooooooooooooooooooooo~ :

0

:

:

:

', 5

:

:

:

:

', 10

:

:

TIME POST-INJECTION

:

~

I 15

~

~

~ ~

I 20

(HRS)

Fig. 2. A68930 stimulates contralateral turning in 6-OHDA lesioned rats. Rats bearing 6-OHDA-induced lesions in the substantia nigra were treated with the indicated doses of A68930 and rotational behavior was recorded for the following 20 hours. Data represent moan SEM of the responses obtained from 5 or 6 animals for each dose tested.

effective in eliciting turning behavior in the 6 - O H D A lesioned rats while A70360 did not elicit this behavioral response (not shown). After subcutaneous administration, A68930 produced seizure-like activity in rats• The forelimb clonus was a series o f rapid, rhythmic movements o f the forelimbs (table 3). The forelimb clonus was not seen until between 45 and 60 minutes after drug administration at doses as high as 12•2/zmole/kg. This delay contrasted with the rapid onset of turning behavior in response to A68930. DISCUSSION The data reported in this communication show that A68930 is a potent and specific agonist for the D~

dopamine receptor• It is surprising that A68930 is a partial agonist for the D, receptor in the carp retina but is a full agonist for this receptor in the rat striatum. The reason(s) for this discrepancy are not immediately obvious. The results obtained in vivo with A68930 are consistent with the molecule acting as an agonist upon the Di dopamine receptor. Because A68930 is a potent, specific and long-acting D, dopamine receptor agonist, we believe that it may be a useful addition to the pharmacological tools available for studying the D~ dopamine receptor (Setler et al., 1978, Andersen et al., 1987, Lovenberg et al., 1989, Brewster et al., 1990). The Dt dopamine receptor has been implicated in lowering the threshold for pilocarpine-induced seizures. Thus, treating rodents with the D~ agonist S K & F 38393 renders the animals more susceptible to pilo-

160S

Dopamine90

Table 3. Effectsof A68930 on seizure-likeactivity in the rat Dose (/lmole/kg, so)

Animals showing seizure-like behavior

1.0

10% (10)

1.5 2.0 4.0 12.2

33% (6) 100% (6) 100% ( 16t 100% (29)

Rats (number used in parentheses) were treated with A68930 as indicated and then observed for the presence or absence of seizure-like activity described in Experimental Procedures. carpine-induced seizures (AI-Tajir et al., 1990a, b, B a r o n e et al., 1990, Turski et al., 1990). The ability o f A68930 as well as A70108 (but not A70360) to elicit forelimb clonus in otherwise drug-free rats is c o m p a t ible with a n i n v o l v e m e n t o f the D t d o p a m i n e receptor in regulating seizure threshold, since it parallels the interaction between the c o m p o u n d s a n d the receptor. The clonus induced by these i s o c h r o m a n s can be blocked with a n t a g o n i s t s selective for the D~ receptor but n o t by D2 receptor a n t a g o n i s t s (KE Asin and D Britton, in preparation). The e n a n t i o m e r i c selectivity o f the i s o c h r o m a n s in eliciting seizures contrasts with one report t h a t S - S K & F 38393, the inactive enantiomer, is capable o f p o t e n t i a t i n g pilocarpine-induced seizures (Barone et al., 1990). Because A68930 a n d A70108 elicit clonus f r o m otherwise drug-free animals, they prove to be useful tools for investigating the role of the D, receptor in seizure p r o p a g a t i o n . REFERENCES

Al-Tajir, G., C.J. Chandler, B.S. Starr and M.S. Starr. (1990a) Opposite effects of stimulation of D~ and D 2 dopamine receptors on the expression of motor seizures in mouse and rat. Neuropharmacology 29, 657. Al-Tajir, G., M.S. Starr and B.S. Starr. (1990b) Proconvulsant effect of SKF 38393 mediated by nigral D, receptors. Eur. J. Pharmacol. 182,245. Andersen, P.H., E.B. Nielsen, J. Scheel-Kruger, J.A. Jansen, and R.Hohlweg. (1987) Thienopyridine derivatives identified as the first selective, full efficacy dopamine D~ receptor agonists. Eur. J. Pharmacol. 137. 291. Arnt, J. and J. Hyttel (1985) Differential involvement of dopamine D, and D 2 receptors in the circling behavior induced by apomorphine, SK&F38393, pergolide and LY 171555 in 6-hydroxydopamine-lesioned rats. Psychopharmacology 85, 346

Barone, P., S.S. Parashos, V. Palma, C. Matin, (i Campanella and T.N.Chase. (1990) Dopamine-D~ receptor modulation of pilocarpine-induced convulsions Neuroscience 34, 209. Brewster, WK., D.E. Nichols, R.M. Riggs, D.M. Mottola, T.W. Lovenberg,M.H. Lewis, and RB. Mailman. (1990) trans- 10,1 l-Dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine: A highly potent selective dopamine D, full agonist. J Med Chem 33, 1756. DeNinno, M.P., R. Schoenleber, K.E. Asin, R. MacKenzie, and J.W. Kebabian (1990) (1R,3S)-l-(Aminomethyl)3,4-dihydro-5,6-dihydroxy-3-phenyl- 1H-2-benzopyran: A potent and selective D~ agonist. J Med Chem 33. 2948. Garau, L., S. Govoni, E. Stefanini, M. Trabucchi and P.F. Spano. (1978)Dopamine receptors: pharmacological and anatomical evidences indicate that two distinct dopamine receptor populations are present in rat striatum. Life Sci. 23, 1745. Kebabian J.W. and D.B. Calne. (1979) Multiple receptors for dopamine. Nature 1979; 277: 93. Kerkman D.J., M. Ackerman. L.D. Artman, R.G. MacKenzie, M.C. Johnson, L.Bednarz, W. Montana, K.E. Asin, H. Stampfli and J:W. Kebabian. (1989) A69024: a non-benzazepine antagonist with selectivity for the dopamine D~ receptor. Eur. J. Pharmacoi. 166, 481. Lovenberg, T.W., W.K. Brewster, D.M. Mottota, R.C. Lee, R.M. Riggs, D.E.Nichols, M . H Lewis and R.B. Mailman (1989) Dihydrexidine, a novel selectivehigh potency full dopamine D, receptor agonist. Eur. J Pharmacol 166,111. Richfield, E.K., J.B. Penney and A.B. Young. (1989) Anatomical and affinitystate comparisons between dopamine D, and D, receptors in the rat central nervous system. Neuroscience 30, 767. Setler P.E,, H.M. Sarau, C.L. Zirkle and H.L. Saunders. (1978) The central effects of a novel dopamine agonist. Eur. J Pharmacol, 50, 419. Trugman J.M., and G.F, Wooten. (1987) Selective D~ and D2 dopamine agonists differentially alter basal ganglia glucose utilization in rats with unilateral 6-hydroxydopamine substantia nigra lesions. J Neuroscience 7, 2927. TurskL W.A., E. Cavalheiro, C. lkonomidou, Z.A. Bortolotto, T. Klockgether and L. Turski. (1990) Dopamine control of seizure propagation: Intranigral dopamine D, agonist SKF-38393 enhances susceptibility to seizures. Synapse 5, 113. Waddington, J.L. and K.M. O'Boyte. (1989) Drugs acting on brain dopamine receptors: A conceptual re-evaluation five years after the first selective D~ antagonist. Pharmacol. Ther 43:1. Watling, K.J., J.E. DoMing and L.L. Iversen (1979) Dopamine receptors in the retina may all be linked to adenylate cyclase. Nature 281,578.