9-Dihydroxy-2,3,7,11b-tetrahydro-1H-naph[1,2,3-de]isoquinoline: a potent full dopamine D1 agonist containing a rigid-beta-phenyldopamine pharmacophore.

Article abstract of DOI:10.1021/jm950707+

The present work reports the synthesis and preliminary pharmacological characterization of 8,9-dihydroxy-2,3,7,11b-tetrahydro-1H-naph[1,2,3-de] isoquinoline (4, dinapsoline). This molecule was designed to conserve the essential elements contained in our D

Full text of DOI:10.1021/jm950707+

J . Med. Chem. 1996, 39, 549-555
549
8,9-Dih yd r oxy-2,3,7,11b-tetr a h yd r o-1H-n a p h [1,2,3-d e]isoqu in olin e: A P oten t F u ll
Dop a m in e D1 Agon ist Con ta in in g a Rigid â-P h en yld op a m in e P h a r m a cop h or e
Debasis Ghosh, Scott E. Snyder, Val J . Watts,† Richard B. Mailman,† and David E. Nichols*
Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University,
West Lafayette, Indiana 47907, and Brain & Development Research Center Departments of Psychiatry, Pharmacology, and
Medicinal Chemistry, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7250
Received September 25, 1995X
The present work reports the synthesis and preliminary pharmacological characterization of
8,9-dihydroxy-2,3,7,11b-tetrahydro-1H-naph[1,2,3-de]isoquinoline (4, dinapsoline). This mol-
ecule was designed to conserve the essential elements contained in our D1 agonist pharma-
cophore model (i.e., position and orientation of the nitrogen, hydroxyls, and phenyl rings). It
involved taking the backbone of dihydrexidine [3; (()-trans-10,11-dihydroxy-5,6,6a,7,8,12b-
hexahydrobenzo[a]phenanthridine], the first high-affinity full D1 agonist, and tethering the
two phenyl rings of dihydrexidine through a methylene bridge and removing the C(7)-C(8)
ethano bridge. Preliminary molecular modeling studies demonstrated that these modifications
conserved the essential elements of the hypothesized pharmacopore. Dinapsoline 4 had almost
identical affinity (KI ) 5.9 nM) to 3 at rat striatal D1 receptors and had a shallow competition
curve (nH ) 0.66) that suggested agonist properties. Consistent with this, in both rat striatum
and C-6-mD1 cells, dinapsoline 4 was a full agonist with an EC50 of ca. 30 nM in stimulating
synthesis of cAMP via D1 receptors. The design and synthesis of dinapsoline 4 provide a
powerful test of the model of the D1 pharmacophore we have developed and provide another
chemical series that can be useful probes for the study of D1 receptors. An interesting property
of 3 is that it also has relatively high D2 affinity (K0.5 ) 50 nM) despite having an accessory
phenyl ring usually thought to convey D1 selectivity. Dinapsoline 4 was found to have even
higher affinity for the D2 receptor (K0.5 ) 31 nM) than 3. Because of the high affinity of 4 for
D2 receptors, it and its analogs can be powerful tools for exploring the mechanisms of “functional
selectivity” (i.e., that 3 is an agonist at some D2 receptors, but an antagonist at others).
Together, these data suggest that 4 and its derivatives may be powerful tools in the study of
dopamine receptor function and also have potential clinical utility in Parkinson’s disease and
other conditions where perturbation of dopamine receptors is useful.
In tr od u ction
The accepted classification of dopamine receptors was
originally based on a scheme dividing the receptors into
two families (D1 and D2) based on pharmacological and
functional evidence.1 D1 receptors preferentially rec-
ognize the phenyltetrahydrobenzazepines and lead to
stimulation of the enzyme adenylate cyclase, whereas
D2 receptors recognize the butyrophenones and benza-
mides and are coupled negatively (or not at all) to
adenylate cyclase. More recently, it is clear that at least
five genes code for subtypes of dopamine receptors: D1,
D2, D3, D4, and D5. The traditional classification,
however, remains useful, with the D1-like class compris-
ing the D1 (D1A) and the D5 (D1B) receptors, whereas
the D2-like class consists of the D2, D3, and D4
receptors.2-7
Although there has been a specific focus on the D2
The early work on selective D1 receptor ligands
primarily focused on molecules from a single chemical
class, the phenyltetrahydrobenzazepines, such as the
antagonist SCH 23390 (1).10 In general, the agonists
that derived from this class (e.g., SKF 38393, 216) were
of partial efficacy. Even SKF 82958, purported to be a
full agonist, recently has been shown not to have full
intrinsic efficacy in preparations with decreased recep-
tor reserve.17 Thus, our laboratories initiated efforts to
design ligands that were full agonists, indeed, were of
full intrinsic efficacy. This ultimately led to the syn-
thesis of dihydrexidine 3, a hexahydrobenzo[a]phenan-
thridine, designed as a hybrid of the aminotetralins and
the 4-phenyltetrahydroisoquinolines.12,18 The resulting
family for more than two decades, more recently the
critical role of the D1 receptor in nervous system
function has become clear.8,9 The availability of both a
selective antagonist10 and a high-affinity full D1 ago-
nist11,12 has led to increased awareness of the manifold
functional roles of D1 receptors. In addition, the poten-
tial utility of novel D1 ligands in various central nervous
system (CNS) disorders has also become clear.13-15
* Address all correspondence to: Dr. David E. Nichols, Department
of Medicinal Chemistry and Pharmacognosy, Robert Heine Pharmacy
Building, School of Pharmacy and Pharmacal Sciences, Purdue Uni-
versity, West Lafayette, IN 47907. (317) 494-1461 (office); (317) 494-
6790 (FAX). Internet: drdave@sage.cc.purdue.edu.
† University of North Carolina School of Medicine.
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0022-2623/96/1839-0549$12.00/0 © 1996 American Chemical Society

550 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Ghosh et al.
Sch em e 1a
structure 3 was unique from other D1 agonists because
the accessory ring system is tethered, making the
molecule relatively rigid. Molecular modeling studies
of 3 have shown that it has a limited number of low-
energy conformations, in all of which the aromatic rings
are held in a relatively coplanar arrangement.19 The
recent elucidation of the configuration of the active
enantiomer of 320 was consistent with predictions from
this model. Unlike other high-affinity, high intrinsic
activity D1 agonists like 3-substituted (aminomethyl)-
isochromans,21 3 provided a semirigid template for
developing such models. The essential features of this
receptor model include the presence of a transoid
â-phenyldopamine moiety, an equatorially oriented
electron lone pair on the basic nitrogen atom, and near
coplanarity of the pendant phenyl ring with the catechol
ring. Interestingly, dopaminergic phenyltetrahydroben-
zazepines22 have a cisoid â-phenyldopamine conforma-
tion.
It is important to note that the differentiation be-
tween agonists of full and partial efficacy is not only of
narrow biochemical interest, but appears to have gen-
eral importance in terms of complex CNS-mediated
events. For example, dihydrexidine 3 and the isochro-
man full agonist A-77636 have exceptional anti-parkin-
sonian effects in the MPTP-treated monkey model,13,21
whereas partial agonists are devoid of, or have signifi-
cantly less, activity. More recent data suggest that full
and partial agonists differ in their effects on other
complex neural functions.14,15 For these reasons, un-
derstanding of the molecular mechanisms responsible
for D1 receptor activation are of both heuristic and
practical interest.
The present report describes the synthesis and phar-
macological characteristics of a new rigid â-phenyl-
dopamine analog, 8,9-dihydroxy-2,3,7,11b-tetrahydro-
1H-naphth[1,2,3-de]isoquinoline (4). Tethering the two
phenyl rings of dihydrexidine through a methylene
bridge, and removing the C(7)-C(8) ethano bridge, led
to this molecule that conserved the relative orientation
of all the essential elements (position and orientation
of the nitrogen, hydroxyls, and phenyl rings) of the
molecular pharmacophore that we had hypothesized
were necessary for high D1 affinity and full intrinsic
efficacy. It was found that 4 was, in fact, a high-affinity
ligand at rat striatal D1 receptors and has full intrinsic
activity in at least two preparations of D1 receptors.
Moreover, 4 also has significant affinity for the D2
receptor (K0.5 ) 31 nM) despite the presence of its
accessory phenyl ring. These data suggest that 4 will
have potential clinical utility in Parkinson’s disease and
in other conditions where perturbation of dopamine
receptors can be of importance. On the basis of all the
earlier studies of 3, it is clear that derivatives of 4 will
also be powerful tools in the study of dopamine receptor
function.
a
(a) (i) sec-Butyllithium, TMEDA, Et2O, -78 oC; (ii) 7; (iii)
TsOH, toluene, reflux; (b) (i) 1-chloroethyl chloroformate, (CH2Cl)2;
(ii) CH3OH; (c) TsCl, Et3N; (d) H2/Pd-C, HOAc; (e) BH3‚THF; (f)
concentrated H2SO4, -40 oC to -5 oC; (g) Na/Hg, CH3OH,
Na2HPO4; (h) BBr3, CH2Cl2.
lowed by methanolysis of the intermediate, afforded 8.26
This secondary amine was treated with p-toluenesulfo-
nyl chloride and triethylamine to give 9.
An early attempt to synthesize 9 directly involved
condensation of 2-(p-tolyl sulfonyl)-2,3-dihydro-4(1H)-
isoquinolone27 with lithiated 6 in THF or ether, followed
by lactonization with acid; this provided only minute
amounts (<5%) of 9. Enolization of 2-(p-tolylsulfonyl)-
2,3-dihydro-4(1H)-isoquinolone under the basic reaction
conditions is one obvious explanation for the poor
yield.28
Hydrogenolysis of 9 in glacial acetic acid in the
presence of 10% palladium on carbon gave 10,24 which
on reduction with diborane afforded the key intermedi-
ate 11. Cyclization of 11 with concentrated sulfuric acid
at low temperature provided 12. N-Detosylation of 12
with Na/Hg in methanol buffered with disodium hydro-
gen phosphate gave 13.29 Finally, 13 was treated with
boron tribromide to effect methyl ether cleavage yielding
4 as its hydrobromide salt.
P h a r m a cology
Bin d in g a n d F u n ction a l Effects a t D1 Recep tor s.
We first examined the affinity of 4 for D1 receptors in
rat striatal homogenates. As shown in Figure 1, 4
competed with high affinity at this receptor, having
almost identical affinity to 3, the first full D1 agonist.
As can be seen in Figure 1, both 4 and 3 had shallower
slopes for their competition curves than did the proto-
typical D1 antagonist 1. These binding data are sum-
marized in Table 1.
Ch em istr y
The key starting material for the synthesis of 4,
2-methyl-2,3-dihydro-4(1H)-isoquinolone (5), was pre-
pared following the route described by Klein et al.23
In Scheme 1, ortho-directed lithiation of 2,3-dimethoxy-
N,N′-diethylbenzamide (6) with sec-butyllithium/TME-
DA in ether at -78 °C and condensation of this lithiated
species with 5 followed by reflux with p-toluenesulfonic
acid, gave spirolactone 7 in modest yield.24,25 N-De-
methylation of 7 with 1-chloroethyl chloroformate, fol-
We next determined whether 4, as predicted by our
model, would be a full agonist at D1 receptors in the
rat striatum. As is shown in Figure 2, saturating

A Novel High-Affinity Full D1 Agonist
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 551
F igu r e 1. Affinity of compound (()-4 at striatal D1 receptors.
The high-affinity full agonist (()-3 and the prototype antago-
nist (+)-1 were included for comparison purposes.
F igu r e 3. Compound (()-4 is a high-potency (EC50 ca. 30 nM)
full agonist at the primate D1A receptor expressed in C-6 cells.
The full agonist (()-3 and the partial agonist (+)-2 (SKF38393)
were included for comparison purposes. The ability of the test
compounds to stimulate cAMP accumulation was examined
in C-6-mD1 cell membranes (see Methods for details). Data
represent the average of two experiments conducted in dupli-
cate.
Ta ble 1. Summary of Affinities of (()-3 and (()-4 at
Dopamine Receptors in Rat Braina
D1 binding
K0.5 (nM)
D2 binding
K0.5 (nM) nH
compound
nH
(()-4
5.93 ( 0.45 0.66 ( 0.01 31.3 ( 4.4 0.71 ( 0.03
4.59 ( 0.28 0.65 ( 0.01 43.2 ( 3.2 0.72 ( 0.04
(()-3
(+)-2
(+)-1
chlorpromazine
17 ( 4
0.75 ( 0.08 NTb
0.30 ( 0.01 1.05 ( 0.01 NTb
b
-
-
0.92 ( 0.12 0.93 ( 0.01
a
Radioligand binding studies for dopamine receptors were
conducted in rat striatal homogenates, using 0.3 nM [3H]SCH23390
(D1 sites) and 0.07 nM [3H]spiperone in the presence of 50 nM
unlabeled ketanserin (D2 sites). Competition curves were analyzed
by non linear regression to determine estimates for the K0.5 and
Hill slope (nH). Data represent the mean and standard error from
b
three independent assays for each test compound. Not tested.
F igu r e 4. Affinity of compound (()-4 at striatal D2 receptors.
The high affinity full agonist (()-3 and the prototype antago-
nist chlorpromazine were included for comparison purposes.
shown, unexpectedly, to have significant D2 affinity
despite its accessory hydrophobic ring,12,19 the ability
of 4 to compete for D2 receptors in rat striatal homoge-
nates also was investigated. As is shown in Figure 4
and Table 1, the affinity of 4 for D2-like receptors in
rat striatal homogenates is actually higher than for 3.
As can also be seen in Figure 4, the slope of the
competition curves for both 4 and 3 was shallower than
for the prototypical D2 antagonist chlorpromazine.
F igu r e 2. Compound (()-4 is a full agonist at D1 receptors
in the rat striatum. The high-affinity full agonist (()-3 and
the partial agonist (+)-2 were included for comparison pur-
poses. The ability of the test compounds to stimulate cAMP
accumulation was examined in rat striatal homogenates (see
Methods for details). Data represent means ( SEM from at
least three experiments [4, 95.8 ( 4.7; 3, 91.3 ( 4.6; 2, 40.7 (
7.0].
Resu lts a n d Discu ssion
As shown clearly by the pharmacological data, com-
pound 4 has high affinity for dopamine D1 receptors
labeled with [3H]SCH23390, almost identical to that of
(()-3 (dihydrexidine). Moreover, (()-4, in both rat
striatal membranes and in cloned expressed primate
D1A receptors, was a full agonist relative to dopamine,
like (()-3 but unlike the partial agonist (+)-2 that
caused less than half the maximal response in this assay
(e.g., see Figures 3 and 4). On the basis of our
underlying model of the D1 pharmacophore, we would
predict that both the affinity and intrinsic activity of
racemic 4 reside in only one of its enantiomers, that with
the 11bR absolute configuration. Resolution of the
racemate would therefore yield one isomer with ap-
proximately twice the D1 affinity obtained here, thus
making its affinity for the D1 receptor similar to (+)-3.
While the D1 affinity and functional efficacy of 4 were
concentrations (10 µM) of both 4 and 3 caused the same
degree of increase in cAMP synthesis as did a maximally
effective concentration of dopamine (100 µM). Con-
versely, the partial agonist (+)-2 caused less than 50%
stimulation. These effects were blocked by the D1
antagonist SCH23390 (1 µM). The functional efficacy
of 4 was also tested in cloned primate D1A receptors
expressed in C-6 glioma cells. As is shown in Figure 3,
compound 4 also was of full efficacy in this preparation,
with an EC50 of ca. 30 nM. As has been previously
shown, (()-3 also was of full efficacy in this preparation,
whereas (+)-2 was only of partial efficacy.17
Bin d in g a t D2 Recep tor s. Because 3 had been

552 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Ghosh et al.
aromatic hydrogen H(1) in 3 projects above the catechol
ring. In 4, however, this position is used to tether the
pendent phenyl ring through a methylene unit to the
catechol ring; this forces the pendent phenyl ring to
twist in a clockwise direction, relative to 3, when viewed
from above. The amino groups, which are not readily
visible in this figure, are in similar positions, given the
degree of conformational flexibility of the heterocyclic
rings. In addition, both molecules can present an N-H
vector in an equatorial orientation, a feature of the
pharmacophore that we have proposed is important for
D1 receptor agonists.12,18 From these results, one would
predict that the pharmacological properties of these two
molecules would be similar, and this is exactly what is
observed. Furthermore, prior to completion of the
synthesis of this molecule, it was fit into a receptor
model that we have developed for dopamine D1 agonists
using the active analog approach.19 Compound 4 gave
an ideal fit to the model. The present results help to
validate the predictive ability of that model.
Con clu sion s
The predicted high affinity and full efficacy of 4
provide additional support for our hypothesis that
â-phenyldopamine, locked into a properly oriented rigid
framework, can serve as the pharmacophore for potent
dopamine D1 agonists.12 Clearly, on the basis of the
results presented here, the pharmacophore takes an
optimal conformation when it is disposed in the relative
orientations provided by both 3 and 4. We have
previously reported that 3 demonstrated profound anti-
parkinsonian effects in the MPTP model of Parkinson’s
disease, and we would anticipate that 4 should show
similar effects. Moreover, 4, like 3, should also have
utility both as a research tool and clinical drug in cases
where full intrinsic efficacy D1 ligands are useful. As
noted above, the high affinity of 4 for D2-like receptors
fortuitously provides additional important research
tools. Our recent work has shown that it is possible to
tailor 3 by appropriate molecular modifications such
that the resulting analogs can be targeted to specific
subpopulations of the dopamine receptor family.17,20 It
seems obvious that similar strategies with 4 should
result in compounds that can have either novel receptor
subtype selectivity or atypical functional profiles.
F igu r e 5. Front and edge-on views of space-filling models
comparing minimum energy conformations of the proposed
active enantiomer of molecule 4 (A, left, top and bottom) and
(+)-dihydrexidine (3, B).
as predicted by our model, of equal interest (although
less well rationalized) were the actions of 4 at the D2
receptor. As was shown in Figure 4 and Table 1, (()-4
has greater affinity for D2 receptors than does (()-3.
This property, we believe, will in itself be a useful
research tool. As was noted in the Introduction, when
3 was synthesized some years ago, it was anticipated
that it would have been fairly selective for the D1 vs
D2-like receptors. It was somewhat surprising, there-
fore, to find that 3 was only about 10-fold D1:D2
selective.12,19 This observation became more interesting
when it was found that 3, while expected to behave as
a typical dopamine agonist, had an unusual property
that we have termed “functional selectivity”. Specifi-
cally, in rats (in vivo or in vitro), 3 acts as an agonist at
D2-like receptors located postsynaptically, but as an
antagonist at D2-like receptors located presynapti-
cally.30,31 This phenomenon is now hypothesized to be
due to differences in the ligand-receptor-G protein
complex, depending on the specific G proteins in a given
cellular milieu. We have shown that these D2 properties
of 3 reside in the same enantiomer (i.e., 6aR,12bS) that
is the high-affinity full agonist at the D1 receptor.20 On
this basis, we would predict that both the D1 and D2
properties of 4 also reside in the same enantiomer. The
optical isomers of 4, and appropriate analogs, should
be powerful tools to study this “functional selectivity”
hypothesis.
Exp er im en ta l Section
Melting points were determined with a Thomas-Hoover
melting point apparatus and are uncorrected. 1H NMR spectra
were recorded with a Varian VXR 500S (500 MHz) NMR
instrument, and chemical shifts were reported in δ values
(ppm) relative to TMS. The IR spectra were recorded as KBr
pellets or as a liquid film with a Perkin-Elmer 1600 series
FTIR spectrometer. Chemical ionization mass spectra (CIMS)
were recorded on a Finnigan 4000 quadruple mass spectrom-
eter. High-resolution CI spectra were recorded on Kratos
MS50 spectrometer. Elemental analysis data were obtained
from the microanalytical laboratory of Purdue University,
West Lafayette, IN. THF was distilled from benzophenone-
sodium under nitrogen immediately before use; 1,2-dichloro-
ethane was distilled from phosphorous pentoxide before use.
2′,3′-Dih yd r o-4,5-d im eth oxy-2′-m eth ylsp ir o[isoben zo-
fu r a n -1(3H),4′(1′H)-isoqu in olin ]-3-on e (7). To a solution
of 6 (14.94 g, 63 mmol) in ether (1400 mL) at -78 °C under
nitrogen was added sequentially, dropwise, N,N,N′,N′-tetra-
methylenediamine (9.45 mL, 63 mmol) and sec-butyllithium
(53.3 mL, 69 mmol, 1.3 M solution in hexane) through a rubber
septum via syringe. After 1 h, freshly distilled 5 (10.1 g, 62.7
mmol) was added to the heterogeneous mixture. The cooling
bath was removed, and the reaction mixture was allowed to
warm to room temperature over 9 h. Saturated NH4Cl
Figure 5 presents space-filling representations of the
low-energy conformations for (+)-3 and the 11bR enan-
tiomer of 4 that is homochiral to (+)-3 at its 12bS chiral
center. Overall, the two compounds appear remarkably
similar in shape. Two major structural features are
readily evident. First, the steric bulk provided by the
C(7)-C(8) ethano bridge in 3 has been removed. Sec-
ond, the angle of the pendent phenyl ring with respect
to the plane of the catechol ring is changed slightly. In
the top, face-on views, this is most evident, where the

A Novel High-Affinity Full D1 Agonist
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 553
solution (400 mL) was then added a,nd the mixture was stirred
for 15 min. The ether layer was separated, and the water layer
was extracted with dichloromethane (4 × 100 mL). The
organic layers were combined, dried (MgSO4), and evaporated
to a brown oil. The oil was dissolved in toluene (500 mL),
heated at reflux for 8 h with 3.0 g of p-toluenesulfonic acid,
cooled, and concentrated under vacuum. The residue was
dissolved in dichloromethane, washed with dilute aqueous
NaHCO3 and water, then dried (Na2SO4), filtered, and evapo-
rated to a gummy residue. On trituration with ethyl acetate-
hexane (50:50), a solid precipitated. Recrystallization from
ethyl acetate-hexane afforded 12.75 g (63%) of 7: mp 193-
7.63 (d, 2H, J ) 8.5 Hz, ArH); MS (CI) m/ z 468 (16), 450 (63),
296 (100); HRCIMS calcd for C25H25NO6S 468.1481, found
468.1467. Anal. (C25H25NO6S) C, H, N.
2-N-(p-Tolylsulfonyl-4-(2-(hydroxymethyl)-3,4-dimethoxy-
p h en yl)-1,2,3,4-t et r a h yd r oisoq u in olin e (11). To a solu-
tion of 10 (1.4 g, 2.99 mmol) in dry tetrahydrofuran (30 mL)
was added 1.0 M borane-tetrahydrofuran (8 mL) at 0 °C under
nitrogen. After the addition was complete the mixture was
stirred at reflux overnight. Additional diborane (4 mL) was
added, and stirring was continued for another 30 min. After
cooling and evaporation under reduced pressure, methanol (30
mL) was carefully added, and the solvent was removed at low
pressure. The process was repeated three times to ensure the
methanolysis of the intermediate borane complex. Evapora-
tion of the solvent gave 1.10 g (81%) of crude 11. An analytical
sample was purified by flash chromatography (silica gel,
EtOAc-hexane) followed by recrystallization from ethyl
acetate-hexane: mp 162-164 °C; 1H NMR (CDCl3) δ 2.38 (s,
3H, CH3), 3.18 (dd, 1H, J ) 7.5, 11.9 Hz), 3.67 (dd, 1H, J )
4.5, 11.8 Hz), 3.81 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.27 (d,
1H, J ) 15 Hz), 4.40 (d, 1H, J ) 15 Hz), 4.57 (t, 1H, J ) 6 Hz,
CHAr2), 4.71 (s, 2H, CH2OH), 6.58 (d, 1H, J ) 8.5 Hz, ArH),
6.74 (d, 1H, J ) 8.6 Hz, ArH), 6.84 (d, 1H, J ) 7.7 Hz, ArH),
7.08 (t, 2H, J ) 7.6 Hz, ArH), 7.14 (t, 1H, J ) 6.6 Hz, ArH),
7.27 (d, 2H, J ) 8 Hz, ArH), 7.65 (d, 2H, J ) 8 Hz, ArH); MS
(CI) m/ z 454 (2.57), 436 (100). Anal. (C25H27NO5S) C, H, N.
8,9-Dim eth oxy-2-(p-tolylsu lfon yl)-2,3,7,11b-tetr a h yd r o-
1H-n a p th [1,2,3-d e]isoqu in olin e (12). To 50 mL of cold
concentrated sulfuric acid (50 mL) at -40 °C under nitrogen
was added with vigorous mechanical stirring powdered 11 (427
mg, 0.98 mmol) in several portions. After the addition, the
reaction mixture was warmed to -5 °C over 2 h and then
poured onto crushed ice (450 g) and left stirring for 1 h. The
product was extracted with dichloromethane (2 × 150 mL),
washed with water (2 × 150 mL), dried (MgSO4), filtered, and
evaporated to afford an oil that on trituration with ether at 0
°C yielded 12 (353 mg, 82%) as a white solid that was used
for the next step without further purification. An analytical
sample was prepared by centrifugal rotary chromatography
using 50% ethyl acetate-hexane as the eluent, followed by
recrystallization from EtOAc-hexane: mp 204-206 °C; 1H
NMR (CDCl3) δ 2.40 (s, 3H, CH3), 2.80 (m, 1H, H-1a), 3.50
(dd, 1H, J ) 4.5, 17.5 Hz, H-1b), 3.70 (dd, 1H, J ) 7, 14 Hz,
H-3a), 3.828 (s, 3H, OCH3), 3.832 (s, 3H, OCH3), 3.9 (m, 1H,
H-11b), 4.31 (d, 1H, J ) 17.6 Hz, H-7a), 4.74 (ddd, 1H, J )
1.7, 6.0, 11.2 Hz, H-7b), 4.76 (d, 1H, J ) 14.8 Hz, H-3b), 6.77
(d, 1H, J ) 8.3 Hz, ArH), 6.87 (d, 1H, J ) 8.4 Hz, ArH), 6.94
(d, 1H, J ) 7.6 Hz, ArH), 7.13 (t, 1H, J ) 7.5 Hz, ArH-5), 7.18
(d, 1H, J ) 7.2 Hz, ArH), 7.33 (d, 2H, J ) 8.1 Hz, ArH), 7.78
(d, 2H, J ) 8.2 Hz, ArH); MS (CI) m/ z 436 (55), 198 (86), 157
(100); HRCIMS calcd for C25H25NO4S 436.1583, found 436.1570.
Anal. (C25H25NO4S) C, H, N.
8,9-Dim eth oxy-2,3,7,11b-tetr ah ydr o-1H-n apth [1,2,3-de]-
isoqu in olin e (13). Following the procedure of Pyne et al.,29
a mixture of 12 (440 mg, 1.01 mmol), dry methanol (10 mL),
and disodium hydrogen phosphate (574 mg, 4.04 mmol) was
stirred under nitrogen at room temperature. To this mixture
was added 6.20 g of 6% Na/Hg in three portions, and the
reaction mixture was heated at reflux for 2 h. After cooling,
water (200 mL) was added and the mixture was extracted with
ether (3 × 200 mL). The ether layers were combined, dried
(MgSO4), filtered (Celite), and evaporated to give an oil that
solidified under vacuum. After rotary chromatography, 142
mg (50%) of 13 was obtained as an oil. The oil quickly
darkened on exposure to air and was used immediately for
the next step. A small portion of the oil was treated with
ethereal HCl, and the hydrochloride salt of 13 was recrystal-
lized from ethanol-ether: mp (HCl salt) 190 °C dec; 1H NMR
(CDCl3, base) δ 3.13 (dd, 1H, J ) 10.8, 12 Hz, H-1a), 3.50 (dd,
1H, J ) 3.4, 17.4 Hz, H-1b), 3.70 (m, 1H, H-11b), 3.839 (s, 3H,
OCH3), 3.842 (s, 3H, OCH3), 4.03 (dd, 1H, J ) 6, 12 Hz, H-7a),
4.08 (s, 2H, H-3), 4.33 (d, 1H, J ) 17.4 Hz, H-7b), 6.78 (d, 1H,
J ) 8.24 Hz, ArH), 6.92 (m, 2H, ArH), 7.11 (t, 1H, J ) 7.5 Hz,
ArH), 7.18 (d, 1H, J ) 7.5 Hz, ArH); MS (CI) m/ z 282 (100);
HRCIMS calcd for C18H19NO2 282.1494, found 282.1497.
8,9-Dih ydr oxy-2,3,7,11b-tetr a h yd r o-1H-n a pth [1,2,3-d e]-
isoqu in olin e (4). To a solution of 13 (25 mg, 0.089 mmol) in
1
194 °C; IR (KBr) 1752 cm-1 (CdO); H NMR (CDCl3) δ 2.47
(s, 3H, NCH3), 2.88 (d, 1H, J ) 11.6 Hz), 3.02 (d, 1H, J ) 11.7
Hz), 3.76 (d, 1H, J ) 15.0 Hz), 3.79 (d, 1H, J ) 15.1 Hz), 3.90
(s, 3H, OCH3), 4.17 (s, 3H, OCH3), 6.83 (d, 1H, J ) 8.4 Hz,
ArH), 7.03 (d, 1H, J ) 8.2 Hz, ArH), 7.11 (m, 3H, ArH), 7.22
(m, 1H, ArH); MS (CI) m/ z 326 (100). Anal. (C19H19NO4) C,
H, N.
2′,3′-Dih yd r o-4,5-d im e t h oxysp ir o[isob e n zofu r a n -1-
(3H),4′(1′H)-isoqu in olin ]-3-on e (8). Following the method
of Olofson et al.,26 to a suspension of 7 (6.21 g, 19.2 mmol) in
100 mL of 1,2-dichloroethane was added dropwise 1-chloro-
ethyl chloroformate (5.1 mL, 46.3 mmol) at 0 °C under
nitrogen. The mixture was stirred for 15 min at 0 °C and then
heated at reflux for 8 h. The mixture was cooled and
concentrated under reduced pressure. To this mixture was
added 75 mL of methanol, and the reaction was heated at
reflux overnight. After cooling, the solvent was evaporated
under reduced pressure to afford the hydrochloride salt of 8
in nearly quantitative yield. It was sufficiently pure to use in
the next step without further purification: mp (HCl) 220-
222 °C; mp (base) 208-210 °C; IR (film) 1754 cm-1 (CdO); 1H
NMR (CDCl3, base) δ 3.18 (d, 1H, J ) 13.5 Hz), 3.30 (d, 1H, J
) 13.5 Hz), 3.84 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 4.02 (s,
2H, CH2N), 6.67 (d, 1H, J ) 7.5 Hz, ArH), 7.12 (m, 2H, ArH),
7.19 (d, 1H, J ) 7.5 Hz, ArH), 7.26 (t, 1H, J ) 7.5 Hz, ArH),
7.41 (d, 1H, J ) 8.5 Hz, ArH); MS (CI) m/ z 312 (100); HRCIMS
calcd for C18H17NO4 312.1236, found 312.1198. Anal. (C18H17
NO4) H, N; C: calcd, 69.44, found, 68.01.
-
2′,3′-Dih yd r o-4,5-d im et h oxy-2′-(p -t olylsu lfon yl)sp ir o-
[isoben zofu r a n -1(3H), 4′(1′H)isoqu in olin ]-3-on e (9). To
a mixture of p-toluenesulfonyl chloride (3.6 g, 18.9 mmol), 8
(as the HCl salt, obtained from 19.2 mmol of 7), and chloroform
(100 mL) was added 7 mL of triethylamine, dropwise, at 0 °C
under nitrogen. After the addition was complete, the ice bath
was removed and the reaction mixture was stirred at room
temperature for 1 h. It was then acidified with 100 mL of cold
aqueous 0.1 N HCl and extracted with dichloromethane (2 ×
100 mL), and the organic extract was dried (MgSO4), filtered,
and evaporated under vacuum to afford a viscous liquid that
on trituration with ethyl acetate-hexane at 0 °C gave a solid.
Recrystallization from ethyl acetate-hexane afforded 8.74 g
(97%, overall from 7) of 9: mp 208-210 °C; IR (KBr) 1767
cm-1 (CdO); 1H NMR (CDCl3) δ 2.43 (s, 3H, CH3), 3.22 (d, 1H,
J ) 11 Hz), 3.88 (d, 1H, J ) 11 Hz), 3.90 (s, 3H, OCH3), 3.96
(d, 1H, J ) 15 Hz), 4.17 (s, 3H, OCH3), 4.81 (d, 1H, J ) 15
Hz), 6.97 (d, 1H, J ) 7.7 Hz, ArH), 7.16 (m, 3H, ArH), 7.26
(m, 1H, ArH), 7.38 (d, 2H, J ) 8 Hz, ArH), 7.72 (d, 2H, J ) 8
Hz, ArH); MS (CI) m/ z 466 (100). Anal. (C25H23NO6S) C, H,
N.
3,4-Dim et h oxy-6-(2-(p -t olylsu lfon yl)-1,2,3,4-t et r a h y-
d r oisoq u in olin )-4-yl)b en zoic Acid (10). Using a proce-
dure modified from de Silva and Snieckus,24 a solution of 9
(2.56 g, 5.51 mmol) in glacial acetic acid (250 mL) with 10%
palladium on activated carbon (6.30 g) was shaken on a Parr
hydrogenator at 50 psig for 48 h at room temperature. The
catalyst was removed by filtration, and the solvent was
evaporated to afford 2.55 g (99%) of 10 that was sufficiently
pure to carry into the next step. An analytical sample was
recrystallized from ethanol-water: mp 182-184 °C; IR (KBr)
1
1717 cm-1 (COOH); H NMR (DMSO-d6) δ 2.35 (s, 3H, CH3),
3.12 (m, 1H), 3.51 (dd, 1H, J ) 5, 11.5 Hz), 3.71 (s, 6H, OCH3),
4.10 (m, 1H, Ar2CH), 4.23 (s, 2H, ArCH2N), 6.52 (d, 1H, J )
7.5 Hz, ArH), 6.78 (d, 1H, J ) 7.5 Hz, ArH), 6.90 (m, 1H, ArH),
7.07 (t, 1H, J ) 8 Hz, ArH), 7.14 (t, 1H, J ) 6.5 Hz, ArH),
7.20 (d, 1H, J ) 7.5 Hz, ArH), 7.38 (d, 2H, J ) 8 Hz, ArH),

554 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2
Ghosh et al.
dichloromethane (5 mL) at -78 °C was added boron tribromide
(0.04 mL, 0.106 g, 0.42 mmol). After stirring at -78 °C under
nitrogen for 2 h, the cooling bath was removed and the reaction
mixture was left stirring at room temperature for 5 h. It was
then cooled to -78 °C, and methanol (2 mL) was carefully
added. After stirring for 15 min at room temperature, the
solvent was evaporated under reduced pressure. More metha-
nol was added, and the process was repeated three times. The
resulting gray solid was recrystallized from ethanol-ethyl
acetate to yield a total of 12 mg (41%) of the hydrobromide
and D2 receptor binding assays, respectively. As an internal
standard, a competition curve with six concentrations of
unlabeled SCH23390 (D1 binding) or chlorpromazine (D2
binding) was included in each assay. Triplicate determinations
were made for each drug concentration. Assay tubes were
incubated at 37 °C for 15 min, and binding was terminated
by filtering with ice cold buffer on a Skatron 12-well cell
harvester (Skatron, Inc., Sterling, VA) using glass fiber filter
mats (Skatron no. 7034). Filters were allowed to dry, and 1.0
mL of Optiphase HI-SAF II scintillation fluid was added.
Radioactivity was determined on an LKB Wallac 1219 Rack-
Beta liquid scintillation counter (Wallac, Gaithersburg, MD).
Tissue protein levels were estimated using the BCA protein
assay reagent (Pierce, Rockford, IL).
1
salt of 4: mp 258 °C dec; H NMR (HBr salt, CD3OD) δ 3.43
(t, 1H, J ) 12 Hz, H-1a), 3.48 (dd, 1H, J ) 3.5, 18 Hz, H-1b),
4.04 (m, 1H, H-11b), 4.38 (dd, 2H, J ) 5.5, 12 Hz, H-7), 4.44
(s, 2H, H-3), 6.58 (d, 1H, J ) 8.5 Hz, ArH), 6.71 (d, 1H, J )
8.5 Hz, ArH), 7.11 (d, 1H, J ) 7.5 Hz, ArH), 7.25 (t, 1H, J )
7.5 Hz, ArH), 7.32 (d, 1H, J ) 7.5 Hz, ArH); MS (CI) m/ z 254
(100); HRCIMS calcd for C16H15NO2 254.1181, found 254.1192.
P h a r m a cologica l Meth od s. Su bjects. Adult male Spra-
gue-Dawley rats (200-250 g) were obtained from Charles
River Breeding Laboratories (Raleigh, NC). Rats were killed
by decapitation, and the whole brains removed and chilled
briefly in ice-cold saline. Brains were sliced with the aid of a
dissecting block,32 and central striata were then dissected from
two coronal sections containing the majority of this region.
Tissue was frozen immediately on dry ice and stored at -70
°C until the day of the assay. The animal protocols used in
the present experiments were consistent with current NIH
principles of animal care and were approved by the University
of North Carolina Institutional Animal Care and Use.
Da ta An a lysis for Ra d ior ecep tor Assa ys. Binding data
from each assay were analyzed separately. Data were nor-
malized by expressing the average dpm at each competitor
concentration as a percentage of total binding. These data
were then subjected to nonlinear regression analysis using the
algorithm for sigmoidal curves in the curve-fitting program
InPlot (Graphpad Inc.; San Francisco, CA) or EBDA and
LIGAND software, as adapted for the IBM-PC by McPherson,33
to generate K0.5 values and a Hill coefficient (nH) for each curve.
Analysis of the residuals indicated an excellent fit; r values
were above 0.99 for all curves in the present experiments.
Ad en yla te Cycla se Activity in Ra t Str ia tu m . The
automated HPLC method of Schulz and Mailman34 was used
to measure adenylate cyclase activity by separating cAMP from
other labeled nucleotides. Briefly, striatal tissue from rat was
homogenized with eight manual strokes in a Wheaton Teflon-
glass homogenizer in 5 mM HEPES buffer (pH 7.5) containing
2 mM EGTA (50 mL/g tissue). Following the addition and
mixing of 50 mL/g of 50 mM HEPES buffer (pH 7.5) containing
2 mM EGTA, a 20 µL aliquot of this tissue homogenate was
added to a prepared reaction mixture (final volume of 100 µL)
containing 0.5 mM ATP, 0.5 mM isobutylmethylxanthine, [32P]-
ATP (0.5 µCi), 1 mM cAMP, 2 mM MgCl2, 100 mM HEPES
buffer, 2 µM GTP, 0-100 µM dopamine, DHX, or SKF38393,
10 mM phosphocreatine, and 5 units of creatine phosphoki-
nase. Triplicate determinations were performed for each drug
concentration. The reaction proceeded for 15 min at 30 °C and
was terminated by the addition of 100 µL of 3% sodium dodecyl
sulfate (SDS). Proteins and much of the noncyclic nucleotides
were precipitated by addition of 300 µL each of 4.5% ZnSO4
and 10% Ba(OH)2. Samples were centrifuged (10000g for 8-9
min), and the supernatants were injected on an HPLC system
(Waters Z-module or RCM 8 × 10 module equipped with a C18,
10 micron cartridge). The mobile phase was 150 mM sodium
acetate (pH 5.0) with 23% methanol. A UV detector (254 nm
detection) was used to quantify the unlabeled cAMP added to
the samples to serve as internal standard. The radioactivity
in each fraction was determined by a flow-through radiation
detector (Inus Systems, Tampa, FL) using Cerenkov counting.
Sample recovery was based on UV measurement of total
unlabeled cAMP peak areas quantified using PE Nelson
(Cupertino, CA) Model 900 data collection modules and Tur-
boChrom software. Tissue protein levels were estimated using
the BCA protein assay reagent (Pierce, Rockford, IL).
Cell Cu ltu r es. C-6 glioma cells expressing the rhesus
macaque D1A receptor, (C-6-mD1A; Machida et al., 1992) were
grown in DMEM-H medium containing 4500 mg/L glucose,
L-glutamine, 5% fetal bovine serum and 600 ng/mL G418 or 2
µg/mL puromycin. Cells were maintained in a humidified
incubator at 37 °C with 5% CO2.
Mem br a n e P r ep a r a tion . Cells were grown in 75 cm2
flasks until confluent. The cells were rinsed and lysed with
10 mL of ice cold hypoosmotic buffer (HOB) (5 mM Hepes, 2.5
mM MgCl2, 1 mM EDTA; pH 7.4) for 10 min at 4 °C. Cells
were then scraped from the flasks using a sterile cell scraper
from Baxter (McGaw Park, IL). Flasks received a final rinse
with 5 mL of HOB. The final volume of the cell suspension
recovered from each flask was ca. 14 mL. Scraped membranes
from several flasks were then combined. The combined cell
suspension was homogenized (10 strokes), 14 mL at a time,
using a 15 mL Wheaton Teflon-glass homogenizer. The cell
homogenates were combined and spun at 43000g (Sorvall RC-
5B/SS-34, Du Pont, Wilmington, DE) at 4 °C for 20 min. The
supernatant was removed, and the pellet was resuspended (10
strokes) in 1 mL of ice cold HOB for each original flask of cells
homogenized. This homogenate was then spun again at
43000g at 4 °C for 20 min. The supernatant was removed,
and the final pellet was resuspended (10 strokes) in ice cold
storage buffer (50 mM Hepes, 6 mM MgCl2, 1 mM EDTA; pH
7.4) to yield a final concentration of ca. 2.0 mg of protein/mL.
Aliquots of the final homogenate were stored in microcentri-
fuge tubes at -80 °C. Prior to their use for adenylate cyclase
assays, protein levels for each membrane preparation were
quantified using the BCA protein assay reagent (Pierce,
Ad en yla te Cycla se Assa y in C-6m -D1A Cells. Frozen
membranes were thawed and added to assay tubes (10 µg of
protein/tube) containing a prepared reaction mixture [100 mM
Hepes, (pH 7.4), 100 mM NaCl, 4 mM MgCl2, 2 mM EDTA,
500 µM isobutylmethylxanthine (IBMX), 0.01% ascorbic acid,
10 µM pargyline, 2 mM ATP, 5 µM GTP, 20 mM phosphocre-
atine, 5 units of creatine phosphokinase (CPK), 1 µM propra-
nolol] and selected drugs. The final reaction volume was 100
µL. Basal cAMP activity was determined by incubation of
tissue in the reaction mixture with no drug added. Tubes were
assayed in duplicate, and after a 15 min incubation at 30 °C,
the reaction was stopped by the addition of 500 µL of 0.1 N
HCl. Tubes were vortexed briefly and then spun in a BHG
HermLe Z 230 M microcentrifuge for 5 min at 15000g to
precipitate particulates.
Rockford, IL) adapted for use with
(Molecular Devices; Menlo Park, CA).
a microplate reader
Dop a m in e Recep tor Bin d in g Assa ys. Frozen rat striata
were homogenized by seven manual strokes in a Wheaton
Teflon-glass homogenizer in 8 mL of ice cold 50 mM HEPES
buffer with 4.0 mM MgCl2 (pH 7.4). Tissue was centrifuged
at 27000g for 10 min, the supernatant was discarded, and the
pellet was homogenized (five strokes) and resuspended in ice
cold buffer and centrifuged again. The final pellet was
suspended at a concentration of 2.0 mg wet weight/mL. The
amount of tissue added to each assay tube was 1.0 mg, in a
final assay volume of 1.0 mL. D1 receptors were labeled with
[3H]SCH23390 (0.30 nM); D2 receptors were labeled with [3H]-
spiperone (0.07 nM); and unlabeled ketanserin (50 nM) was
added to mask binding to 5-HT2 sites. Total binding was
defined as radioligand bound in the absence of any competing
drug. Nonspecific binding was estimated by adding unlabeled
SCH23390 (1 µM) or unlabeled chlorpromazine (1 µM) for D1
Ra d ioim m u n oa ssa y (RIA) of cAMP . The concentration
of cAMP in each sample was determined with an RIA of
acetylated cAMP, modified from that previously described.35
Iodination of cAMP was performed using a method reported

A Novel High-Affinity Full D1 Agonist
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 2 555
by Patel and Linden.36 Assay buffer was 50 mM sodium
acetate buffer with 0.1% sodium azide (pH 4.75). Standard
curves of cAMP were prepared in buffer at concentrations of
2-500 fmol/assay tube. To improve assay sensitivity, all
samples and standards were acetylated with 10 µL of a 2:1
solution of triethylamine-acetic anhydride. Samples were
assayed in duplicate. Each assay tube (total volume 300 µL
contained 25 µL of each sample, 75 µL of buffer, 100 µL of
primary antibody (sheep, anti-cAMP, 1:100000 dilution with
1% BSA in buffer) and 100 µL of [125I]cAMP (50 000 dpm/100
µL of buffer). Tubes were vortexed and stored at 4 °C
overnight (ca. 18 h). Antibody-bound radioactivity was sepa-
rated by the addition of 25 µL of BioMag rabbit, anti-goat IgG
(Advanced Magnetics, Cambridge, MA), followed by vortexing
and incubation at 4 °C for 1 h. To these samples was added
1 mL of 12% polyethylene glycol/50 mM sodium acetate buffer
(pH 6.75), and tubes were centrifuged at 1700g for 10 min.
Supernatants were aspirated, and radioactivity in the pellet
was determined using an LKB Wallac gamma counter (Gaith-
ersburg, MD).
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Ack n ow led gm en t. This work was supported by
PHS Grants MH42705 and MH40537, Center Grants
HD03310 and MH33127, and Training Grant GM07040.
The authors gratefully acknowledge the gift of C-6-mD1
cells from Dr. Kim Neve, Oregon Health Sciences
University.
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