Neurochem. Int. Vol. 20, Suppl., pp. 63S-68S, 1992

0197-0186/92 $5.00+0.00 Copyright © 1992 Pergamon Press plc

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ALAN HORN MEMORIAL LECTURE SELECTIVE PROBES FOR CHARACTERIZATION OF DOPAMINE Di A N D D2 RECEPTORS JOHNL. NEUMEYER,L2NANDKISHOREBAINDUR,2VENKATESALUBAKTHAVACHALAM,l JUN YUAN,2 BERTHAK. MADRAS,3NORAS. KULA,4ALEXANDERCAMPBELL4 and Ross J. BALDESSARINI 4 JResearch Biochemicals Inc., Natick, MA 01760 U.S.A.; 2scction of Medicinal Chemistry, Northeastern University, Boston, MA, U.S.A.; 3NERPRC, Harvard Medical School, Southboro, MA, U.S.A. and 'Mailman Research Center, Harvard Medical School, Belmont, MA, U.S.A.

Since the classification of dopamine (DA) receptors into D, and D2 subtypes (Kebabian and Calne 1979), there has been a great upsurge in the development of new ligands selective for each subtype. These developments have stimulated reevaluation of the functional roles of the subtypes of DA receptors in the brain as well as at peripheral sites in various species (Waddington and O'Boyle 1989). Currently, a principle difference between the two receptor subtypes lies in their transducer-effector relationships with the D~ receptor able to stimulate adenylate cyclase and the D2 receptor either not linked or able to inhibit adenylate cyclase, most likely through different cyclicGMP dependent proteins. The two DA receptor types may affect cell function and behavior either by cooperative or synergistic interactions or by opposing effects. The relative contributions of such actions to the overall control of behavior may differ between species (Waddington and O'Boyle 1989). Further investigation of the pharmacological and biochemical correlates of each receptor subtype would be facilitated by the development of potent and selective agonists and antagonists. Furthermore, clarification of the molecular structure and function of the receptors may be enhanced by new molecular probes such as photoaffiity, affinity, fluorescent and biotin labeled probes with a high affinity and selectivity for each receptor subtype. Some of our recent work in this direction is summarized here. !)2AGONISTS Of known chemical classes of DA receptor agonists and antagonists, the aporphines are particularly interesting since they represent rigid readily modifiable analogues of DA. Substituted aporphines can have high DA receptor affinity and agonist as well as antagonist activity (Neumeyer 1985; Gao et al. 1990;

Campbell et al. 1990). Further modifications of the aporphine ring system have led to the development of several novel chemical classes of dopaminergic ligands. Our current efforts are directed to improving the affinity and agonistic potency of a very active dopaminergic aporphine, R(-)-N-n-propylnorapomorphine [(R)NPA]. Substitution at the 2position of NPA has lead to derivatives possessing unusually high affinity and D2 receptor selectivity (Gao et al. 1990; Neumeyer et al. 1990). 2-Fluoro-NPA (Table 1) shows the highest D e affinity (Ki = 12 pM) and selectivity (potency D2/D~ - - 58000) of any aporphine evaluated in our laboratories. The 2-OHNPA congener is also potent and D2 selective with only slightly lower De affinity shown with other 2substituents as: Br > OCH 3> NH2 (Table 1). The very high affinity and in vivo potency of 2-F-NPA probably arises from the capacity of the fluorine atom at the 2position to act as a highly efficient hydrogen bond acceptor complementary to a donor group on the D2 receptor, evidently with superior bond-forming or steric properties than a bromine atom or a hydroxy group (Table 1). DI AGONISTSANDANTAGONISTS Substituted 1-phenyl-3-benzazepines include compounds with moderately high affinity and high selectivity for the DI receptor in brain tissue (Weinstock et al. 1985). In order to further characterize the structure activity relationship (SAR) of this class of compounds, we are investigating a series of 3- and 6-substituted phenyl-benzazepines with Da agonist activity and a series of 3- and 7substituted compounds with D~ antagonist activity. We found that 3-alkyl-substitution (methyl > allyl) enhanced affinity in both the agonist and antagonist series and that 3-methyl substituted compounds had

63S

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l)(~pamiue 9(1 l'ablc 1. At~nity of 2-substituted N-n-propylnoraporphines at dopamine receptors in rat ~'orpt~ ~.triaiurrt~

3

4

Hou-.U7 9

IC50

Compound

R

X

R( - )-APO R ( - )-NPA R ( - )-2-F-NPA R ( - )-2-OH-NPA R( - )-2-Br-NPA R(-)-2-OCH3-NPA R ( - )-2-NH~-NPA

CH 3

H H F OH Br OCH3 NH~

n-C~H7 n-C3H7 n-C3H7 n-C3H7 n-C3H7 n-C3H7

Potency ratio

D,

D,

444 640 1300 1720 970 3345 10000

66.7 4.80 0.071 0.32 0.87 1.02 5.5

D~,'D~ 6.7 133 18300 5380 1115 3280 1800

assays were carried out as follows: D~tigand [3H]SCH-23390 (0.3nM) with Na ÷ present (150 raM) and cis (Z)flupenthixol (300 nM) used to define specific binding. D2ligand, [3H]spiperone (0.15 nM) with ( + ) butaclamol (300 nM) used to define blank.

"Radioreceptor

the h i g h e s t affinity in b o t h series. H a l o g e n s u b s t i t u t i o n at the 6 - p o s i t i o n ( f o r a g o n i s t s ) a n d 7 - p o s i t i o n ( f o r a n t a g o n i s t s ) also i n c r e a s e d D~ affinity a n d selectivity. T a k i n g these r e s u l t s into c o n s i d e r a t i o n , 7 - b r o m o - 8 h y d r o x y - 3 - a l l y l - l - p h e n y l - 2 , 3 , 4 , 5 - t e t r a h y d r o - 1H - 3 -

b e n z a z e p i n e (the N-allyl a n a l o g o f S K F - 8 3 5 6 6 ) is p r o p o s e d as a Dt a n t a g o n i s t f o r f u r t h e r d e v e l o p m e n t . Its 3-allyl s u b s t i t u e n t e n h a n c e s lipophilicity a n d s h o u l d increase C N S activity. Similarly, 6 - b r o m o - 3 allyl S K F - 3 8 3 9 3 ( B r - A P B ) is p r o p o s e d as a Dj a g o n i s t

Table 2. Affinity of 3- and 6-substituted l-phenyl-3-benzazepines at dopamine receptors in the striatal tissue"

X

ICs0(nM)

Potency ratio

Compound

R

X

Dj

D,

(S)( - )SKF-38393 (R)(+)SKF-38393 (S)( -)APB (R)(+)APB (S)(-)Br-APB (R)(+)Br-APB

H H CH2CH =CH~ CH2CH =CH: C H 2 C H= CH 2 CH2CH= CH:

H H H H Br Br

I0,700 50 2803 8.1 1876 4.3

> 2000 > 10000 ca.5000 1415 >5000 511

"

D,/D~ > 100 >200 ca.l.8 175 >2.7 119

D~ assaysmsame as described in footnote to Table 1. D2 assays--ligand [3H]YM-0915I-2 (55 pM) with Na ÷ present (150 raM) and ( + )butaclamol (!,u M) used to define blank.

Dopamine 90

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Table 3. Pharmacological characterization of DI affinity probes'

~

CH3

R

IC~nM)

Potency ratio

Compound

R

X

D,

D2

D,/D,

(R)SCH 23390 (RS)SKF 83566-MUS (RS)SKF 83566-BROM (RS)SKF 83566-ITC (RS)SKF 83566-FUM

H NHCH2CH2CI NHCOCH2Br NCS NHCCOCH = CHCO2Et

CI Br Br Br Br

0.23 1.1 15.6 20.1 28.5

1809 .5000 934 2746 808

7865 ca.4500 60 137 28

Br

34.5

> 5000

> 145

o,,

(RS)SKF 83566-MAL NHCOCH 2 - - N ~

/ f O • All assays were carried out as described in the footnote to Table 1.

for further development since 3-allyl substitution should retain D, efficacy and enhance penetration into the CNS (Table 2).

all D, agonists reported to date (IC50 = 4.3 nM) and has been targeted for further in vitro and in vivo studies.

STEREOSELECrlVITY OF DI AGONISTS

PHOTOAFFINITY PROBES

Substituted l-phenyl-3-benzazepine DA agonists and antagonists which we have evaluated all show stereoselectivity in binding to and agonisin of D, receptors (Weinstock et al. 1985). The asymmetric carbon at the phenyl-substituted l-position affords R(+) and S ( - ) enantiomers. R(+) SKF-38393 (Table 2) shows high D, binding affinity and agonist activity while S(-)SKF-38393 is virtually inactive (Kaiser et al. 1982) Since earlier work had established the 3-allyl analog of SKF-38393 (APB) as a high affinity Dt agonist with potent/n vivo effects and our current SAR studies suggested the further development of 6-bromo-3-allyl analog of SKF-38393 (Br-APB) as an agonist, we resolved both compounds and compared their D~ affinity and stereoselectivity with respect to racemic (RS)SKF-38393. The R(+) enantiomers of both APB and Br-APB again show improved stereogelective D, binding affinity over that of R(+)SKF-38393, while their S ( - ) antipodes had considerably lower affinity than the more active isomers. R( +)Ik-APB showed the highest affinity of

Covalent photoaffinity labeling (Chowdhry and Westheimer 1979) is a useful technique for the molecular characterization of ligand binding sites or subunits of enzymes and receptors. The D 2 receptor has been photoaffmity labeled with several photoaffinity ligands (Niznik 1987; Neumeyer et al. 1989), the most successful one being N-(4'-azido-3"-[mI] iodophenethyl) spiperone ([t25I]N3-NAPS) (Amlaiky and Caron 1985) which identified a polypeptide of Mr = 94,000 as the ligand binding subunit of the D2 receptor. A photoatfmity label for the D~ receptor was also recently developed in our laboratories. This ligand, (RS)7-[m25I]iodo-8-hydroxy-3- methyl-l- (4'azidophenyl)- 2,3,4,5-tetrahydro-lH-3-benzazepine ( (RS) [t'sI]I-MAB), identified a major poiypeptide of Mr 74,000 as a ligand binding subunit (Baimiur et al. 1988; Niznik et al. 1988). The R(+) enantiomer of [nsI]I-MAB was prepared and found to stereoselectively photolabel the D~ receptor at a polypeptide similar to that photolabeled by the corresponding racemic ligand (Neumeyer et al. 1990). Further

Dopamine 90

66S

Table 4. Pharmacological characterization of D2 affinity probes ~'

x

IC~0(nM) Compound

X

PPHT PPHT-FUM PPHT-BROM PPHT-ITC

H N H C O C H = CHCO2Et NHCOCH2Br N=C=S

Potency ratio

Dt

D2

Dz/D~

1623 269 143 1097

56 0.19 0.47 0.74

129 1416 304 1480

0.47 0.39 1.88 0.49

419 223 238 447

O

NAPS NAPS-FUM NAPS-BROM NAPS-ITC

NHz N H C O C H = CHCO2Et NHCOCHzBr N=C=S

0 194 87 447 219

" All assays were carried out as described in the footnote to Table 1.

proteolytic degradation studies of the photolabeled receptors should provide information about the amino acid composition of the ligand binding sites of the receptors. AFFINITY P R O B E S

The use of covalent affinity labeling as a tool for enzyme or receptor studies parallels that of photoaffinity labeling (Jakoby and Wilcbek 1977). The principle difference between the two methods is that photoaffinity labels are activated by exposure to UV light to generate a reactive nitrene intermediate while affinity labels possess a spontaneously reactive electrophilic group which alkylates proteins directly, including after administration in vivo. Until recently, the only reports of selective affinity labeling of DA receptors involved the use of the N-(2-ehloroethyl)analogs of apomorphine (NCA) and SKF-38393.

NCA showed considerably lower D2 affÉnity than apomorphine (APO) and while not highly selective for DA receptors, bound irreversibly to some D2 receptors (Costall et al. 1980; Balde~sarini et al. 1980). A corresponding N-(2-chloroethyl)-substituted probe based on SKF-38393 retained the high Dt affinity and selectivity of the parent molecule but showed only low level of covalent incorporation (Cross et al. 1983). Our current work led to D and D. receptor affinity probes with high affinity and selectivity. Representative D~ receptor probes are based on the selective and potent D~ antagonist S K F 83566 (Table 3) derivatized with electrophilic groups such as isothiocyanate (ITC), fumaramide (FUM), bromacetamide (BROM), maleimide (MAL) and 2chioroethylamine, (MUS). The mustard derivative showed high affinity (ICS0 = 1.1 nM) and Dr/D2 selectivity (ca 4500). Other probes showed considerably lower affinity and selectivity (Table 3).

Dopamine 90

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Table 5. Pharmacologicalcharacterization of D, fluorescentand biotin labeledprobes' I~(nM) Compound

Potency ratio

D,

SKF 83566 SKF 83566-NH2 SKF 83566-FLU SKF 83566-NBD SKF 83566-GLY-NBD SKF 83566-GLY-AMCA SKF 83566-BIOTIN SKF 83566-X-BIOTIN SKF 83566-XX-BIOTIN

0.3 2.3 16.0

D2

D~/D2

Fluorescence

1360 1600 > 5000

4500 700 > 300

--green yellow yellow blue ----

5.3

710

130

10.8 5.3 3.51 13.5 11.7

3930 1547 2107 6100 5580

360 290 600 450 480

° Data abstracted from Madras et al. (1990). Table 6. Pharmacologicalcharacterization of D2 fluorescentand biotin labeledprobes" Potency ratio

ICso(nM) Compound (RS)PPHT (R)PPHT (S)PPHT (RS)PPHT-FLU (R)PPHT-FLU (S)PPHT-FLU (RS)PPHT-NBD (R)PPHT-NBD (S)PPHT-NBD Spiperone NAPS-NBD NAPS-GLY-NBD NAPS-Biotin

D,

D2

DI/D2

Fluorescence

710 6500 230 340 24000 230 170 3700 130 323 99 120

6.8 60 2.1 7.0 11 0 2.1 0.45 3.2 0.3 0.058 1.3 2.57 0.58

100 110 I10 50 220 59 380 1200 430 5600 150 50 190

---green green green yellow yellow yellow -yellow yellow --

I l0

• Data abstracted from Madras et al. (1990). D 2 receptor probes were based on the selective agnnist N-(phenethyl)-N-n-propyl-5-hydroxy-2aminotetra-line (PPHT) and the selective antagonist spiperone (Table 4). P P H T and N A P S (the paminophenethyl derivative o f spiperone) again were derivatized with electrophilic groups such as isothiocyanate (ITC), fumaramide ( F U M ) and bromoacetamine (BROM). The N A P S derivatives retained high D2 affinity (e.g., the I T C derivative IC50 = 0.47 nM) while the P P H T derivatives showed considerable enhancement, over P P H T , o f both D2 affinity and selectivity (e.g., fumaramide IC50 = 0.19 nM, D2/D~ selectivity = 1416; I T C IC50 = 0.74 nM, selectivity = 1480). FLUORESCENT AND BIOTIN LABELED PROBES

The technique o f fluorescent labeling of receptors, enzymes and other m e m b r a n e components has been successfully exploited to localize them at the histological to subcellular levels and also monitor their conformations, mobility and fluidity (Waggoner 1986). In contrast to radioligand-based techniques

such as autoradiography, which permit resolution only at the tissue level, the use o f techniques such as fluorescence microscopy permits a good resolution at the cellular and even sub-cellular levels. Biotin labeled probes have an added advantage of providing a multitude of diagnostic applications (Bayer and Wilchek 1988). Biotin binds virtually irreversibly to avidin with a high affinity, and avidin derivatized with a variety of indicators or "reporter groups" are available. These moieties include fluorescent groups (for U V microscopy), electron-dense groups (for electron microscopy), enzymes (for immunoassays) and immobilized matrices (for affinity chromatography). We have developed several fluorescent and biotin labeled probes for the D~ and D2 receptors (Madras e t aL 1990). The probes were based on the D~ antagonist SKF-83566, the D2 agonist P P H T , and the D2 antagonist spiperone. Amino-functionalized analogues o f I P P H T and SKF-83566 were coupled to fluorescein and 4-nitro-benzo-2-oxa-l,3-diazol-4-yl (NBD). In the case of spiperonc, its N-(p-aminophenethyl) derivative (NAPS) was coupled to N B D . All o f these fluorescent ligands retained high D~ or D2 receptor

68S

I)opamine 90

affinity and selectivity, and the NBD-coupled P P H T derivative exhibited higher D2 affinity and selectivity than the parent ligand P P H T (Tables 5 & 6). However, the fluorescence characteristics of these probes did not permit specific histological localization of labeled receptors. Probes with better fluorescence characteristics were developed by coupling N B D to glycyl spacer chain-linked derivatives of SKF--83566 and N A P S to obtain probes emitting an intense yellow fluorescence. Such a chain-linked derivative of S K F 83566 also was coupled to 7-amino-4-methylcoumarin-3-acetic acid ( A M C A ) to obtain a probe emitting blue fluorescence. These fluorescent probes all retained high receptor D~ or D 2 affinity and selectivity. The non-overlapping N B D and A M C A probes may permit simultaneous multi-color mapping of the D, and D 2 receptors in the same tissue specimen. D~ and D2 antagonists also were coupled to biotin. Both SKF-83566-biotin and NAPS-biotin retained high affinity and selectivity. Spacer-arm linked biotin derivatives of S K F 83566 also were developed to permit more efficient simultaneous binding of coupled ligand to avidin and the DL receptor. SKF-83566-[C7]biotin and SKF-83566-[C712-biotin (each [C7] representing a 7-atom hexanoylamino spacer moiety) were prepared. The spacer-arm linked biotin probes showed a slightly lower affinity than the directly coupled biotin probe. Further chemical and cellular studies are now in progress to further extend the utility of these D, and D2 fluorescent and biotin labeled probes.

Acknowledgements--This work was supported by USPHS grants MH-34006, MH-45692, MH- 47370, and by National Science Foundation Grant ISI-8760870. We also wish to acknowledge the assistance of Diane LeBlanc in the preparation of this manuscript. REFERENCES

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