Design and synthesis of trans-N-[4-[2-(6-cyano-1,2,3,4- tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide (SB- 277011): A potent and selective dopamine D3 receptor antagonist with high oral bioavailability and CNS penetration

Article abstract of DOI:10.1021/jm000090i

A selective dopamine D₃ receptor antagonist offers the potential for an effective antipsychotic therapy, free of the serious side effects of currently available drugs. Using clearance and brain penetration studies as a screen, a series of 1,2,3

Full text of DOI:10.1021/jm000090i

1878
J . Med. Chem. 2000, 43, 1878-1885
Design a n d Syn th esis of tr a n s-N-[4-[2-(6-Cya n o-1,2,3,4-tetr a h yd r oisoqu in olin -2-
yl)eth yl]cycloh exyl]-4-qu in olin eca r boxa m id e (SB-277011): A P oten t a n d
Selective Dop a m in e D3 Recep tor An ta gon ist w ith High Or a l Bioa va ila bility a n d
CNS P en etr a tion in th e Ra t
Geoffrey Stemp,*,† Tracey Ashmeade,‡ Clive L. Branch,† Michael S. Hadley,† A. J acqueline Hunter,‡
Christopher N. J ohnson,† David J . Nash,† Kevin M. Thewlis,† Antonio K. K. Vong,† Nigel E. Austin,§
Phillip J effrey,§ Kim Y. Avenell,‡ Izzy Boyfield,‡ J im J . Hagan,‡ Derek N. Middlemiss,‡ Charlie Reavill,‡
Graham J . Riley,‡ Carole Routledge,‡ and Martyn Wood‡
Departments of Discovery Chemistry, Neuroscience Research, and Drug Metabolism and Pharmacokinetics,
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, U.K.
Received February 29, 2000
A selective dopamine D3 receptor antagonist offers the potential for an effective antipsychotic
therapy, free of the serious side effects of currently available drugs. Using clearance and brain
penetration studies as a screen, a series of 1,2,3,4-tetrahydroisoquinolines, exemplified by 13,
was identified with high D3 affinity and selectivity against the D2 receptor. Following
examination of molecular models, the flexible butyl linker present in 13 was replaced by a
more conformationally constrained cyclohexylethyl linker, leading to compounds with improved
oral bioavailability and selectivity over other receptors. Subsequent optimization of this new
series to improve the cytochrome P450 inhibitory profile and CNS penetration gave trans-N-
[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide (24,
SB-277011). This compound is a potent and selective dopamine D3 receptor antagonist with
high oral bioavailability and brain penetration in the rat and represents an excellent new
chemical tool for the investigation of the role of the dopamine D3 receptor in the CNS.
In tr od u ction
by a lack of compounds (either agonists or antagonists)
with the required selectivity and pharmacokinetic pro-
files. The aim of our research was therefore to identify
orally bioavailable, CNS penetrant antagonists with
high affinity (pKi g 8) for the dopamine D3 receptor and
high selectivity (g100-fold) over other receptors and ion
channels in order to test the above hypothesis and to
further characterize the role of the dopamine D3 recep-
tor in the central nervous system.6
Dopaminergic neurotransmission is mediated by five
receptor subtypes (D1-D5), which can be grouped into
two receptor families. D1-like receptors include the D1
and D5 subtypes, whereas D2-like receptors include the
D2, D3, and D4 subtypes.
Clinically effective antipsychotic agents share the
property of dopamine D2 and D3 receptor antagonism.
At the doses used in the clinic, these drugs occupy D3
as well as D2 receptors, and their antipyschotic effects
could therefore be mediated via D2 and/or D3 receptors.
Blockade of dopamine D2 receptors in the striatum leads
to serious extrapyramidal side effects, which result in
poor patient compliance and consequently poor control
of the disease. However, dopamine D3 receptors are
preferentially located in limbic brain regions, such as
the nucleus accumbens, where dopamine receptor block-
ade has been associated with antipsychotic activity. A
selective dopamine D3 receptor antagonist therefore
offers the potential for an effective antipsychotic therapy,
free of the serious side effects of currently available
drugs.1-4 The presence of the dopamine D3 receptor in
projection regions of the mesocorticolimbic system also
suggests a potential therapeutic role in reinforcement
processes and drug abuse.5
Several dopamine agonists have been identified with
selectivity for the dopamine D3 receptor over the D2
receptor (Chart 1). Although 7-OH-DPAT (1), quinelo-
rane (2), and PD-128907 (3), show high selectivity in
radioligand binding assays, these agonists lack the
Ch a r t 1. Dopamine D3 Receptor Agonists
Pharmacological approaches to understanding the
role of the dopamine D3 receptor have been hindered
* To whom correspondence should be addressed. Tel: 44 279 622133.
Fax: 44 1279 622790. E-mail: Geoff•Stemp-1@sbphrd.com.
† Department of Discovery Chemistry.
‡ Department of Neuroscience Research.
§ Department of Drug Metabolism and Pharmacokinetics.
10.1021/jm000090i CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/18/2000

Potent and Selective Dopamine D3 Receptor Antagonist
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1879
Ch a r t 2. Dopamine D3 Receptor Antagonists
appropriate functional selectivity7 to allow clear dis-
crimination between D3 and D2 receptor function in vivo.
Antagonists with varying degrees of selectivity for the
D3 receptor compared to the D2 receptor (Chart 2) have
been reported. The compounds identified to date include
U 99194 (4),8 nafadotride (5),9 aryl pyrrole 6,10 arylpip-
erazines 7,11 8,12 and 9,13 aminotetralins 1014 and 11,15
benzopyranopyrrolidine 12,16 and tetrahydroisoquino-
line 13.17 Of these antagonists, U 99194, nafadotride,
and arylpiperazine 8 are only 10-30-fold selective for
the D3 over the D2 receptor, and GR 103691 (7) is only
10-fold selective over the 5-HT1A receptor. The arylpyr-
role derivative 6 and aminotetralin 11 are metabolically
unstable. Neither functional activity nor metabolic
stability data have been reported for aminotetralin
derivative 10. NGB 2904 (9) and S 33084 (12) have been
identified as potent and selective dopamine D3 receptor
antagonists. Some evidence for pharmacological activity
in vivo has been described for S 33084;18 however, no
details of the pharmacokinetic profile of either 9 or 12
have been disclosed.
such, PD-128907 and nafadotride have been shown to
lack the necessary in vivo selectivity to define the role
of the dopamine D3 receptor19 and have highlighted the
need for compounds with an improved selectivity and
pharmacokinetic profile.
Recently, we described the design and synthesis of a
novel series of 1,2,3,4-tetrahydroisoquinolines, exempli-
fied by 3-indolyl-2-propenamide derivative 13, which
had high affinity, pKi 8.4 (Ki 4 nM), for the dopamine
D3 receptor and 150-fold selectivity against the D2
receptor.17 Studies in the rat showed that compound 13
had a low blood clearance of 27 mL/min/kg, with a
plasma half-life of 2.5 h. However, further studies
demonstrated that 13 had oral bioavailability of only
7% in the rat and moderate selectivity against 5-HT1B
(pKi 7.0) and 5-HT1D (pKi 7.2) receptors. Alternative
substituents to the 7-CF3SO2 group of 13 were then
investigated, with the aim of reducing lipophilicity and
deactivating the tetrahydroisoquinoline ring toward
metabolism. From this approach, the 7-cyano derivative
14 (Table 1) was identified with an improved oral
bioavailability of 11% in the rat, although D3 affinity
was reduced compared to 13 and selectivity over 5-HT1B
and 5-HT1D receptors remained modest.
Experiments using mice where the dopamine D3
receptor has been genetically deleted have allowed an
assessment of the selectivity of some compounds. As

1880 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Stemp et al.
Ta ble 1. Receptor Binding Profiles of 7-Substituted 1,2,3,4-Tetrahydroisoquinolines
a
b
Analyses for the elements indicated were within (0.4% of the theoretical values. pKi values at human cloned receptors represent
the mean of at least three determinations.
Ta ble 2. Receptor Binding Profiles of trans-1,4-Cyclohexylethyl Derivatives
a,b
d
See footnotes a, b in Table 1. c NT ) not tested. Calcd: C, 69.23; found: C, 68.81. e pKi values represent the mean of 15
determinations.
Inspection of molecular models of compounds 13 and
14 and comparison of these with known selective 5-HT1B
and 5-HT1D ligands20 suggested that the flexible butyl
linker present in 13 and 14 could be partly responsible
for the moderate selectivity against these receptors.
Using parallel solution phase chemistry, a range of more
conformationally constrained linkers was investigated.
From this exercise, the trans-1,4-cyclohexylethyl deriva-
tive 15 (Table 2) was identified with high D3 receptor
affinity (pKi 8.9) and 110-fold selectivity over the D2
receptor. In agreement with our hypothesis, compound
15 also had improved selectivity over 5-HT1B and 5-HT1D
receptors compared to 13 and 14. Using compound 15
as a new lead for further parallel chemistry, we have
now identified 24, trans-N-[4-[2-(6-cyano-1,2,3,4-tet-
rahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinoline-
carboxamide (SB-277011), as a potent and selective
dopamine D3 receptor antagonist with high oral bio-
availability and CNS penetration in the rat.
Ch em istr y
Compounds 15-24 (Table 2) were readily prepared
as shown in Scheme 1. Esterification of trans-(4-amino)-
cyclohexylacetic acid 25,21 followed by reaction with di-
tert-butyl dicarbonate, gave 26. Reduction of 26 with
DIBAL-H gave aldehyde 27, which was reductively

Potent and Selective Dopamine D3 Receptor Antagonist
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1881
Sch em e 1
aminated with the appropriately substituted 1,2,3,4-
tetrahydroisoquinolines 28 (R1 ) 7-CN) and 29 (R1 )
6-CN) to give the protected amines 30 (R1 ) 7-CN) and
31 (R1 ) 6-CN), respectively. Deprotection with TFA to
the corresponding amines 32 (R1 ) 7-CN) and 33 (R1 )
6-CN) followed by coupling to the appropriate acids gave
15-17 (R1 ) 7-CN) and 18-24 (R1 ) 6-CN).
quinolines was prepared with the initial aim of improv-
ing affinity for the dopamine D3 receptor. Cinnamide
19 had an improved D3 receptor affinity compared to
18 and also had 100-fold selectivity against 5-HT1B and
5-HT1D receptors. Substitution around the phenyl ring
of the cinnamide moiety was allowed, as exemplified by
compounds 20 and 21. Although neither compound was
100-fold selective against the D2 receptor, selectivity
against the 5-HT1B and 5-HT1D receptors remained high.
Interestingly, the 3-methylsulfonylcinnamide 20 had
an encouraging oral bioavailability of 45% in the rat,
but in CNS penetration studies, this compound showed
a negligible level (<2%) of brain penetration. However,
the 4-fluorocinnamide 21 had a similar oral bioavail-
ability to that of 20 in the rat and a brain:blood ratio of
1:1. Presumably the improved CNS penetration of 21
is a result of both increased lipophilicity and reduced
hydrogen-bond acceptor potential compared to 3-meth-
ylsulfonylcinnamide 20. Additional analogues with phys-
icochemical properties similar to those of 21 were
therefore prepared. Although the 2-naphthyl derivative
22 achieved 100-fold selectivity against the D2 and
5-HT1B receptors, selectivity against 5-HT1D receptors
was only 10-fold. The 1-naphthyl analogue 23 retained
D3 receptor affinity, although selectivity against D2
receptors was reduced compared to 22. Replacement of
the 1-naphthyl group of 23 by a 4-quinolinyl group, as
in 24, maintained D3 receptor affinity and restored
g100-fold selectivity against the D2, 5-HT1B, and 5-HT1D
receptors. Subsequent cross-screening showed 24 to be
g100-fold selective against a package of 63 other recep-
tors and ion channels.
Resu lts a n d Discu ssion
Following the highly encouraging receptor binding
profile obtained with the cinnamide derivative 15, this
new series of compounds required further optimization
to reduce the cytochrome P450 inhibitory potential and
improve the pharmacokinetic profile. For comparison
with the previous series of butyl-linked compounds, the
3-indolyl-2-propenamide derivative 16 was prepared,
but this modification reduced D3 receptor affinity and
selectivity over the D2 receptor compared to 15. Inves-
tigation of the effect of conformational constraint of the
cinnamide moiety of 15 resulted in the synthesis of
2-indolyl derivative 17, which retained affinity for the
D3 receptor. Interestingly, 17 had improved selectivity
over the 5-HT1B receptor compared to 15, but selectivity
over the D2 receptor was reduced to 50-fold. However,
it was particularly encouraging that 17 was found to
have an excellent pharmacokinetic profile in the rat,
with an oral bioavailability of 43%, blood clearance of 6
mL/min/kg, and half-life of 6.3 h. A CNS penetration
study showed 17 to have a brain:blood ratio of 3:1.
Unfortunately, the 7-cyanotetrahydroisoquinoline de-
rivatives 15-17 were shown to be inhibitors of cyto-
chrome P450 2D6, and 17 was the most potent with an
IC50 of 0.8 µM. However, the corresponding 6-cyanotet-
rahydroisoquinoline derivative 18 had greatly reduced
liability to inhibit 2D6 (17% inhibition at 10 µM), but
D3 receptor affinity was also reduced. Following this
important observation, a range of 6-cyanotetrahydroiso-
An in vitro functional assay using microphysiometry
in CHO cells expressing human cloned dopamine D3 and
D2 receptors7 demonstrated that 24 was devoid of
agonist-like activity and was a potent and selective
antagonist (D3, pKb 8.4; D2, pKb 6.5).

1882 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Stemp et al.
Compound 24 (SB-277011) was shown to have an
excellent pharmacokinetic profile in the rat (oral bio-
availability 43%, half-life 2.0 h, plasma clearance 19 mL/
min/kg) and to be highly brain-penetrant (brain:blood
ratio of 3.6:1), with a clean P450 profile.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Buchi
melting point apparatus and are uncorrected. 1H NMR spectra
were recorded on a Bruker AMX 400, a J EOL GX 270, or a
Bruker AC 250. High-resolution mass spectra were recorded
using a J EOL DX303 at 70 eV. Low-resolution mass spectra
(API+) were recorded using a Fisons VG Platform. LC-mass
spectra (LCMS) were recorded using a Hewlett-Packard HP
1100 System attached to a Waters Micomass ZMD electrospray
ionization mass spectrometer. A SORBAX SB C18 3.5 µm (30
× 2.1 mm) column was used with elution using the following
conditions: eluant A, 0.1% TFA/H2O v/v, eluant B, 0.1% TFA/
CH3CN v/v; flow rate, 0.75 mL/min. The eluation gradient was
5-95% B increased over 1.5 min and then held for 0.5 min. A
detection wavelength of 215 nm was used. Karl Fischer
analysis was used to confirm the presence of water in analyti-
cal samples submitted for combustion analysis. Merck Kiesel-
gel 60 was used for column chromatography. All solvent
evaporation was performed under vacuum.
Using in vivo microdialysis in the rat, the dopamine
agonist quinelorane has been shown to reduce dopamine
levels in the nucleus accumbens and the striatum.22
Compound 24 dose dependently reversed the effects of
quinelorane in the nucleus accumbens, with complete
reversal at a dose of 3 mg/kg po. In contrast, the effects
of quinelorane in the striatum were not reversed by 24
at a high dose of 93 mg/kg. The regional selectivity of
the effect of 24 on the reversal of the quinelorane-
induced reduction in dopamine efflux is in good agree-
ment with the regional distribution of D3 receptors in
rat forebrain.23
tr a n s-2-(1-(4-(N-ter t-Bu t yloxyca r b on yl)a m in o)cyclo-
h exyl)a cetic Acid , Meth yl Ester (26). A mixture of trans-
(4-amino)cyclohexylacetic acid hydrogen sulfate21 (27.0 g, 106
mmol), concentrated H2SO4 (3 mL), and methanol (300 mL)
was stirred at reflux for 5 h. The resulting solution was filtered
and the filtrate evaporated in vacuo to give a brown oil (36 g).
A mixture of this material, triethylamine (36 mL, 26.1 g, 259
mmol), dichloromethane (600 mL) and di-tert-butyl dicarbonate
(25.5 g, 117 mmol) was stirred at 20 °C for 18 h. The resulting
solution was partitioned between saturated aqueous NaHCO3
(500 mL) and dichloromethane (3 × 200 mL), and the combined
extracts were dried (Na2SO4) and evaporated in vacuo to give
The low level of dopamine D3 receptors in the dorsal
striatum and the pituitary gland has led to the hypoth-
esis that selective D3 receptor antagonists would have
reduced liability to induce extrapyramidal movement
disorders and would not induce hyperprolactinaemia.
In agreement with this hypothesis, 24 dosed up to 80
mg/kg po displayed no cataleptic activity in the rat and
did not elevate prolactin levels. In contrast, the typical
antipsychotic agent haloperidol produced a complete
cataleptic response and significantly elevated serum
prolactin levels at a dose of 3 mg/kg po.
1
26 (24.6 g, 86%) as a colorless solid. H NMR (CDCl3) δ: 1.08
(4H, m), 1.43 (9H, s), 1.76 (3H, m), 2.00 (2H, m), 2.20 (2H, d,
J ) 7 Hz), 3.37 (1H, m), 3.66 (3H, s), 4.39 (1H, br s).
Con clu sion
tr a n s-2-(1-(4-(N-ter t-Bu t yloxyca r b on yl)a m in o)cyclo-
h exyl)a ceta ld eh yd e (27). To a stirred solution of trans-2-
(1-(4-(N-tert-butyloxycarbonyl)amino)cyclohexyl) acetic acid,
methyl ester (46.0 g, 170 mmol), in dry toluene (920 mL) at
-78 °C under argon was added a solution of diisobutylalumi-
num hydride (1 M, 285 mL, 285 mmol), dropwise over 0.5 h.
The resulting solution was stirred for a further 0.3 h and
quenched with a mixture of methanol (28 mL) in toluene (50
mL) and then poured into saturated aqueous potassium
sodium tartrate (1.2 L). The resultant mixture was extracted
with ether (4 × 1 L). The combined organic extracts were dried
(Na2SO4) and evaporated in vacuo to give a waxy solid which
was purified using silica gel, eluting with 10-50% ethyl
acetate/hexane to give 27 (21.77 g, 53%) as a colorless solid.
1H NMR (CDCl3) δ: 1.12 (4H, m), 1.44 (9H, s), 1.78 (3H, m),
2.00 (2H, m), 2.33 (2H, dd, J ) 7, 2 Hz), 3.37 (1H, m), 4.40
(1H, m), 9.75 (1H, m).
tr a n s-2-(2-(1-(4-(N-ter t-Bu tyloxyca r bon yl)a m in o)cyclo-
h exyl)eth yl)-6-cya n o-1,2,3,4-tetr a h yd r oisoqu in olin e (31).
A mixture of trans-2-(1-(4-(N-tert-butyloxycarbonyl)amino)-
cyclohexyl) acetaldehyde (6.0 g, 24.9 mmol), 6-cyano-1,2,3,4-
tetrahydroisoquinoline (3.93 g, 24.9 mmol), and sodium triace-
toxyborohydride (7.7 g, 36.3 mmol) in 1,2-dichloroethane (270
mL) was stirred at 20 °C for 16 h. The resulting solution was
partitioned between aqueous K2CO3 (200 mL) and dichlo-
romethane (100 mL), and the combined extracts were washed
with brine (200 mL), dried (Na2SO4), and evaporated in vacuo
to a minimum volume and filtered through a pad of silica (100
g), washing with ethyl acetate. The filtrate was evaporated in
vacuo to give 31 (8.33 g, 87%) as a yellow solid. 1H NMR
(CDCl3) δ: 1.08 (4H, m), 1.28 (1H, m), 1.44 (9H, s), 1.48 (2H,
m), 1.78 (2H, m), 1.99 (2H, m), 2.52 (2H, m), 2.72 (2H, t, J )
7 Hz), 2.91 (2H,,m), 3.37 (1H, m), 3.63 (2H, m), 4.40 (1H, m),
7.12 (1H, d, J ) 8 Hz), 7.39 (2H, m).
The tetrahydroisoquinoline derivatives 13 and 14,
designed as dopamine D3 receptor antagonists with
improved metabolic stability compared to the N-propyl-
2-aminotetralin derivative 11, were the starting point
for a chemical program aimed at further improving oral
bioavailability and selectivity over other receptors within
this class of compound. Molecular modeling studies
suggested that conformational constraint of the flexible
butyl linker present in 13 and 14 would improve
selectivity, and this approach led to the identification
of cinnamide derivative 15 containing a trans-cyclo-
hexylethyl linker. Further optimization of 15 to improve
the cytochrome P450 inhibitory potential and pharma-
cokinetic profile resulted in the identification of 24 (SB-
277011). This compound is a potent and selective
dopamine D3 receptor antagonist with excellent oral
bioavailability and brain penetration in the rat. Pre-
liminary studies using in vivo microdialysis in the rat
suggest that the regional selectivity of the effect of 24
on the reversal of the quinelorane-induced reduction in
dopamine efflux is in good agreement with the regional
distribution of D3 receptors in rat forebrain. The lack
of induction of catalepsy or elevation of serum prolactin
with high doses of 24 provides further support for the
hypothesis that a selective dopamine D3 antagonist
would have reduced liability to induce extrapyramidal
movement disorders and would not induce hyperpro-
lactinaemia. Further details of these encouraging pre-
liminary findings, together with a more extensive
evaluation of 24 in a range of pharmacological models
designed to probe the biological relevance of the dopa-
mine D3 receptor, will be reported.
tr a n s-2-(2-(1-(4-(N-ter t-Bu tyloxyca r bon yl)a m in o)cyclo-
h exyl)eth yl)-7-cya n o-1,2,3,4-tetr a h yd r oisoqu in olin e (30)
1
was prepared in a manner similar to compound 31. H NMR
(CDCl3) δ: 1.06 (4H, m), 1.28 (1H, m), 1.44 (9H, s), 1.47 (2H,
m), 1.77 (2H, m), 1.99 (2H, m), 2.52 (2H, m), 2.72 (2H, t, J )

Potent and Selective Dopamine D3 Receptor Antagonist
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1883
7 Hz), 2.94 (2H, m), 3.37 (1H, m), 3.60 (2H, s), 4.37 (1H, m),
7.18 (1H, d, J ) 8 Hz), 7.32 (1H, s), 7.39 (1H, d, J ) 8 Hz).
tr a n s-2-(2-(1-(4-Am in o)cycloh exyl)eth yl)-6-cyan o-1,2,3,4-
tetr a h yd r oisoqu in olin e (33). A mixture of trans-2-(2-(1-(4-
(N-tert-butyloxycarbonyl)amino)cyclohexyl)ethyl)-6-cyano-1,2,3,4-
tetrahydroisoquinoline (8.3 g, 21.7 mmol), trifluoroacetic acid
(15 mL), and dichloromethane (180 mL) was stirred at 20 °C
for 2 h. The resulting solution was evaporated in vacuo and
the residue partitioned between saturated aqueous K2CO3 (200
mL) and dichloromethane (2 × 100 mL). The combined organic
extracts were washed with brine (100 mL), dried (Na2SO4),
and evaporated in vacuo to give 33 (4.99 g, 81%) as a brown
oil. 1H NMR (CDCl3) δ: 0.91-1.16 (4H, m), 1.22-1.40 (3H,
m), 1.47 (2H, m), 1.72-1.91 (4H, m), 2.52 (2H, m), 2.59 (1H,
m), 2.72 (2H, t, J ) 7 Hz), 2.92 (2H, m), 3.64 (2H, s), 7.11 (1H,
d, J ) 8 Hz), 7.39 (2H, m). Mass spectrum (API+): Found 284
(MH+). C18H25N3 requires 283.
(2H, m), 2.87 (2H, m), 3.29 (3H, s), 3.63 (3H, m), 6.81 (1H, d,
J ) 16 Hz), 7.31 (1H, d, J ) 8 Hz), 7.52 (1H, d, J ) 16 Hz),
7.61 (2H, m), 7.72 (1H, t, J ) 8 Hz), 7.93 (2H, m), 8.02 (2H,
m). Mass spectrum (API+): Found 492 (MH+). C28H33N3O3S
requires 491.
The following compounds were prepared in a manner similar
to compound 20.
tr a n s-N-[4-[2-(6-Cya n o-1,2,3,4-tetr a h yd r oisoqu in olin -
2-yl)eth yl]cycloh exyl]-1H-in d ole-2-ca r boxa m id e (18). 1H
NMR (DMSO-d6) δ: 0.88 (2H, m), 1.03-1.32 (5H, m), 1.60 (4H,
m), 2.29 (2H, m), 2.46 (2H, m), 2.65 (2H, m), 3.42 (2H, m),
3.56 (1H, m), 6.82 (1H, m), 6.95 (2H, m), 7.09 (1H, d, J ) 8
Hz), 7.23 (1H, d, J ) 8 Hz), 7.38 (3H, m), 8.01 (1H, d, J ) 8
Hz), 11.34 (1H, s). Mass spectrum (API+): Found 427 (MH+).
C
27H30N4O requires 426.
tr a n s-(E)-N-[4-[2-(6-Cya n o-1,2,3,4-tetr a h yd r oisoqu in o-
lin -2-yl)eth yl]cycloh exyl]-3-p h en yl-2-p r op en a m id e (19).
1H NMR (DMSO-d6) δ: 0.83-1.22 (5H, m), 1.34 (2H, m), 1.70
(4H, m), 2.38 (2H, m), 2.54 (2H, m), 2.73 (2H, m), 3.50 (3H,
m), 6.49 (1H, d, J ) 16 Hz), 7.17 (1H, d, J ) 8 Hz), 7.29 (4H,
m), 7.45 (4H, m), 7.88 (1H, d, J ) 8 Hz). Mass spectrum
(API+): Found 414 (MH+). C27H31N3O requires 413.
tr a n s-(E)-N-[4-[2-(6-Cya n o-1,2,3,4-tetr a h yd r oisoqu in o-
lin -2-yl)et h yl]cycloh exyl]-3-(4-flu or op h en yl)-2-p r op en -
a m id e (21). 1H NMR (CDCl3 + CD3OD) δ: 1.03-1.40 (5H,
m), 1.54 (2H, m), 1.84 (2H, m), 2.05 (2H, m), 2.55 (2H, m),
2.75 (2H, t, J ) 7 Hz), 2.94 (2H, m), 3.66 (2H, s), 3.82 (1H, m),
5.90-6.15 (1H, m), 6.30 (1H, d, J ) 16 Hz), 6.97-7.17 (3H,
m), 7.35-7.61 (5H, m). Mass spectrum (API+): Found 432
(MH+). C27H30 FN3O requires 431. Purity was determined as
>98% by LC-MS, retention time 1.05 min.
tr a n s-N-[4-[2-(6-Cya n o-1,2,3,4-tetr a h yd r oisoqu in olin -
2-yl)eth yl]cycloh exyl]-2-n a p h th a len eca r boxa m id e (22).
1H NMR (DMSO-d6) δ: 1.10 (2H, m), 1.24-1.53 (5H, m), 1.88
(4H, m), 2.52 (2H, m), 2.67 (2H, m), 2.86 (2H, m), 3.63 (2H,
m), 3.82 (1H, m), 7.30 (1H, d, J ) 8 Hz), 7.61 (4H, m), 8.00
(4H, m), 8.42 (2H, m). Mass spectrum (API+): Found 438
(MH+). C29H31N3O requires 437.
tr a n s-N-[4-[2-(6-Cya n o-1,2,3,4-tetr a h yd r oisoqu in olin -
2-yl)eth yl]cycloh exyl]-1-n a p h th a len eca r boxa m id e (23).
1H NMR (DMSO-d6) δ: 1.08 (2H, m), 1.30 (3H, m), 1.47 (2H,
m), 1.83 (2H, m), 1.97 (2H, m), 2.52 (2H, m), 2.67 (2H, m),
2.86 (2H, m), 3.63 (2H, s), 3.81 (1H, m), 7.30 (1H, d, J ) 8
Hz), 7.54 (6H, m), 7.99 (2H, m), 8.17 (1H, m), 8.42 (1H, d, J )
8 Hz). Mass spectrum (API+): Found 438 (MH+). C29H31N3O
requires 437.
tr a n s-N-[4-[2-(6-Cya n o-1,2,3,4-tetr a h yd r oisoqu in olin -
2-yl)eth yl]cycloh exyl]-4-qu in olin eca r boxa m id e (24). A
mixture of trans-2-(2-(1-(4-amino)cyclohexyl)ethyl)-6-cyano-
1,2,3,4-tetrahydroisoquinoline (4 g, 14.1 mmol), quinoline-4-
carboxylic acid (2.45 g, 14.1 mmol), 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide hydrochloride (2.71 g, 14.1 mmol),
1-hydroxybenzotriazole (0.251 g. 1.86 mmol), and dichlo-
romethane (150 mL) was stirred at room temperature for 3 h.
Further dichloromethane (50 mL) was added and stirring
continued for 17 h. Dichloromethane (200 mL) was added and
the mixture washed with saturated aqueous sodium hydrogen
carbonate (500 mL). The aqueous phase was extracted with
dichloromethane (2 × 250 mL). Combined organic extracts
were dried (Na2SO4) and evaporated in vacuo. The residue was
purified by chromatography on silica gel (350 mL) using 10-
100% ethyl acetate-hexane and then a 1-5% methanol-ethyl
acetate gradient elution to give 24 (3.06 g, 49.4%) as a colorless
solid. A sample recrystallized from ethyl acetate-dichlo-
romethane, mp 207-210 °C. 1H NMR (CDCl3) δ: 1.17-1.45
(5H, m), 1.53 (2H, m), 1.89 (2H, m), 2.20 (2H, m), 2.55 (2H,
m), 2.73 (2H, t, J ) 6 Hz), 2.91 (2H, t, J ) 6 Hz), 3.65 (2H, s),
4.07 (1H, m), 5.83 (1H, d, J ) 8 Hz), 7.10 (1H, d, J ) 8 Hz),
7.38 (3H, m), 7.60 (1H, m), 7.76 (1H, m), 8.12 (1H, m), 8.19
(1H, m), 8.90 (1H, d, J ) 4 Hz). Mass spectrum (API+): Found
439 (MH+). C28H30N4O requires 438.
tr a n s-2-(2-(1-(4-Am in o)cycloh exyl)eth yl)-7-cyan o-1,2,3,4-
tetr a h yd r oisoqu in olin e (32) was prepared in a manner
1
similar to compound 33. H NMR (CDCl3) δ: 0.91-1.16 (4H,
m), 1.18-1.40 (3H, m), 1.47 (2H, m), 1.73-1.92 (4H, m), 2.53
(2H, m), 2.62 (1H, m), 2.72 (2H, t, J ) 7 Hz), 2.94 (2H, m),
3.60 (2H, s), 7.19 (1H, d, J ) 8 Hz), 7.32 (1H, s), 7.41 (1H, d,
J ) 8 Hz). Mass spectrum (API+): Found 284 (MH+). C18H25N3
requires 283.
tr a n s-N-[4-[2-(7-Cya n o-1,2,3,4-tetr a h yd r oisoqu in olin -
2-yl)eth yl]cycloh exyl]-1H-in d ole-2-ca r boxa m id e (17). A
mixture of trans-2-(2-(1-(4-amino)cyclohexyl)ethyl-7-cyano-
1,2,3,4-tetrahydroisoquinoline (350 mg, 1.24 mmol), indole-2-
carboxylic acid (200 mg, 1.24 mmol), 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide (238 mg, 1.24 mmol), 1-hydroxybenzo-
triazole (catalytic amount), and dichloromethane (8 mL) was
shaken for 16 h. Saturated sodium bicarbonate (4 mL) was
then added and the mixture shaken for 0.25 h. Chromatog-
raphy of the organic layer on silica with 50-100% ethyl acetate
in hexane and 0-10% methanol in ethyl acetate gradient
elution gave 17 as a yellow solid (90 mg, 17%). 1H NMR
(CDCl3) δ: 1.08-1.36 (4H, m), 1.50-1.70 (4H, m), 1.86 (1H,
m), 2.12 (2H, m), 2.55 (2H, m), 2.73 (2H, t, J ) 7 Hz), 2.94
(2H, m), 3.60 (2H, s), 3.95 (1H, m), 5.97 (1H, d, J ) 8 Hz),
6.81 (1H, m), 7.17 (2H, m), 7.34 (2H, m), 7.42 (2H, t, J ) 8
Hz), 7.64 (1H, d, J ) 8 Hz), 9.22 (1H, br s). Mass spectrum
(API+): Found 427 (MH+). C27H30N4O requires 426.
The following compounds were prepared in a manner similar
to compound 17.
tr a n s-(E)-N-4-[2-(7-Cya n o-1,2,3,4-tetr a h yd r oisoqu in o-
lin -2-yl)eth yl]cycloh exyl]-3-p h en yl-2-p r op en a m id e (15).
1H NMR (DMSO-d6) δ: 1.00-1.60 (7H, m), 1.90 (4H, m), 2.54
(2H, m), 2.74 (2H, t, J ) 6 Hz), 2.95 (2H, t, J ) 6 Hz), 3.65
(2H, s), 3.67 (1H, m), 6.69 (1H, d, J ) 16 Hz), 7.38 (1H, d, J )
8 Hz), 7.46 (4H, m), 7.65 (4H, m), 8.07 (1H, d, J ) 8 Hz). Mass
spectrum (API+): Found 414 (MH+). C27H31N3O requires 413.
The purity was determined as >98% by LC-MS, retention time
1.15 min.
tr a n s-(E)-N-[4-[2-(7-Cya n o-1,2,3,4-t et r a h yd r oisoq u in -
olin -2-yl)e t h yl]cycloh e xyl]-3-(3-1H -in d ole )-2-p r op e n -
1
a m id e (16). H NMR (DMSO-d6) δ: 0.85-1.50 (7H, m), 1.80
(4H, m), 2.35 (2H, m), 2.61 (2H, m), 2.84 (2H, m), 3.52 (3H,
m), 6.56 (1H, d, J ) 16 Hz), 7.13 (2H, m), 7.27 (1H, d, J ) 8
Hz), 7.41 (1H, d, J ) 8 Hz), 7.52 (3H, m), 7.69 (2H, m), 7.84
(1H, m), 11.50 (1H, br s). Mass spectrum (API+): Found 453
(MH+). C29H32N4O requires 452. The purity was determined
as >98% by LC-MS, retention time 1.07 min.
tr a n s-(E)-N-[4-[2-(6-Cya n o-1,2,3,4-tetr a h yd r oisoqu in o-
lin -2-yl)eth yl]cycloh exyl]-3-[3-(m eth ylsu lfon yl)p h en yl]-
2-p r op en a m id e (20). A mixture of trans-2-(2-(1-(4-amino)-
cyclohexyl)ethyl)-6-cyano-1,2,3,4-tetrahydroisoquinoline (0.10
g, 0.35 mmol), (E)-3-(3-methylsulfonyl)phenylpropenoic acid
(0.079 g, 0.35 mmol), 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (0.067 g, 0.35 mmol), 1-hydroxyben-
zotriazole (catalytic amount), and dichloromethane (5 mL) was
treated in a manner to similar to compound 5 to give 20 (0.065
g, 38%) as an off-white solid. 1H NMR (DMSO-d6) δ: 0.97-
1.38 (5H, m), 1.48 (2H, m), 1.84 (4H, m), 2.52 (2H, m), 2.68
Treatment of a solution of the free base of 24 (1.84 g, 4.3
mmol) in methanol (40 mL) and dichloromethane (20 mL) with
hydrochloric acid (2 M, 2.15 mL), followed by evaporation in

1884 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Stemp et al.
vacuo, gave a solid. Recrystallization from ethanol (250 mL)
gave the monohydrochloride salt (0.86 g) as an off-white solid,
mp 216-219 °C. 1H NMR (DMSO-d6) δ: 1.03-1.25 (2H, m),
1.25-1.45 (3H, m), 1.67-1.90 (4H, m), 2.01 (2H, m), 3.09 (1H,
m), 3.26 (4H, m), 3.72 (1H, m), 3.82 (1H, m), 4.37 (1H, m),
4.65 (1H, m), 7.45 (1H, d, J ) 8 Hz), 7.51 (1H, d, J ) 4 Hz),
7.64-7.87 (4H, m), 8.10 (2H, m), 8.70 (1H, d, J ) 8 Hz), 8.97
(1H, d, J ) 4 Hz), 11.01 (1H, br s).
Biologica l Assa ys. In vivo studies were conducted in
compliance with the Home Office Guidance on the operation
of the UK Animals (Scientific Procedures) Act 1986 and were
approved by the SmithKline Beecham Procedures Review
Panel.
Clon ed Cell Lin es Exp r essin g D2 a n d D3 Recep tor s.
Cloned human D2 (long) receptors expressed in CHO cells were
obtained from the Garvan Institute of Medical Research,
Sydney, Australia. Human D3 receptors expressed in CHO or
NG108-15 cells were obtained from Unite de Neurobiologie et
Pharmacologie (U.109) de l′INSERM, Paris, France.
Ra d ioliga n d Bin d in g Assa ys. Radioligand binding assays
at hD2 and hD3 receptors were carried out using membranes
from CHO cells. Membranes (5-15 µg of protein) were
incubated with [125I]iodosulpride (0.1 nM) in buffer containing
50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and
1 mM MgCl2 (pH 7.4) for 30 min at 37 °C in the presence or
absence of competing ligands. Nonspecific binding was defined
with 0.1 mM iodosulpride. Binding to a wide variety of
monoamine receptors was performed as described in ref 20.
Radioligand binding assays were also performed on 55 recep-
tors, ion channels, and enzymes by Cerep, Le Bois l′Eveque,
B. P. 1, 86000 Celle L′Evescault, France.
Cell Cu ltu r e. CHO cells expressing hD2 receptors were
grown in 50:50 Dulbecco’s modified Eagles Medium (DMEM;
without sodium pyruvate, with glucose):Ham’s F-12 containing
10% (v/v) foetal bovine serum (FBS). For hD3 CHO, clones the
growth medium was DMEM (without sodium pyruvate, with
glucose) containing 10% FBS, 100 nM methotrexate, 2 mM
glutamine, 500 nM (-)-sulpiride, and 1% (v/v) essential amino
acids. Cells were removed from confluent plates by scraping
and were harvested by centrifugation (200g, 5 min, room
temperature). Following resuspension in 10 mL of fresh culture
medium, an aliquot was counted and the cells passaged at
12 500 or 25 000 cells cm2. Cultures between passages 5 and
30 were used for functional studies.
required to shift the quinpirole concentration-effect curve.
Antagonist affinity was expressed as pKb (-log Kb).
In Vivo Micr od ia lysis. Male Sprague Dawley rats (250-
350 g) were anaesthetized with medetomidine HCl (0.4 mg/
kg, sc) and fentanyl (0.45 mg/kg, ip), and a guide cannula (BAS,
Congleton, UK) was implanted in either the nucleus accum-
bens (AP + 2.7 mm from bregma, L + 1.6 mm from midline,
V - 5.6 mm from dura), striatum (A/P + 0.0 mm, L + 2.8 mm,
V - 3.5 mm), or frontal cortex (A/P + 3.2 mm, L + 2.0 mm, V
- 1.2 mm) according to the atlas of Paxinos & Watson.
Anaesthesia was reversed with atipamezole HCl (2.5 mg/kg,
sc) and nalbuphine HCl (2 mg/kg, sc). Rats were housed singly,
and at least 2 weeks were allowed for postoperative recovery.
The rats were allowed food and water ad libitum up to
approximately 400 g in weight, when their diet was restricted
to 20 g/day. On the day of an experiment, rats were anaes-
thetized with isoflurane to facilitate insertion of the microdi-
alysis probe (BAS Congleton, UK; 4 mm membrane for
striatum, 2 mm membrane for nucleus accumbens and cortex)
into the guide cannula and allowed to recover for 1 h. Probes
were perfused with artificial cerebrospinal fluid (NaCl, 125
mM; KCl, 2.5 mM; MgCl2, 1.18 mM; CaCl2, 1.26 mM; pH 7.4)
at a flow rate of 1 µL/min. Perfusate from the first 2 h was
discarded and subsequent samples were collected at 1 h
intervals for 6 h. Each sample was collected into 10 µL acetic
acid (0.3% w/v) to prevent degradation of dopamine.
After the first hourly fraction had been collected, either SB-
277011 (0.28-2.8 mg/kg, po; nucleus accumbens) or SB-277011
(93 mg/kg, po for striatum) or vehicle (1% methylcellulose, 2
mL/kg) was administered. Two hours later either quinelorane
(30 µg/kg, sc) or vehicle (saline, 1 mL/kg, sc) was administered.
Samples were collected for a further 3 h.
Samples were analyzed for dopamine using HPLC-ECD. A
J asco (PU-980) HPLC pump (flow rate 0.3 mL/min) pumped
mobile phase (0.07 M KH2PO4, 1 mM octane sulfonic acid, 0.1
mM EDTA, 10% methanol, pH 2.5) through a Symmetry C18
analytical column (3.5 µm, 2.1 × 150 mm plus guard column).
Eluates were detected using an ANTAC Decade detector set
at a voltage of 0.8 V. The amount of dopamine in the first 1 h
collection sample was used as the baseline, and all subsequent
values were calculated as a percentage of this for each
individual rat. A standard sample was included every six
samples to enable quantification and check for reproducibility.
Deter m in a tion of Extr a cellu la r Acid ifica tion Ra tes in
Micr op h ysiom eter . Cells were seeded into 12 mm transwell
inserts (Costar) at 300 000 cells/cup in FBS-containing growth
medium. The cells were incubated for 6 h at 37 °C in 95%O2/
5%CO2, before changing to FBS and sulpiride-free medium.
After a further 16-18h, cups were loaded into the sensor
chambers of the Cytosensor microphysiometer (Molecular
Devices, California). The chambers were perfused with run-
ning medium (bicarbonate-free Dulbecco’s modified Eagles
medium containing 2 mM glutamine and 44 mM NaCl) at a
flow rate of 100 µL/min and temperature of 37 °C. Each pump
cycle lasted 90 s. The pump was on for the first 60 s and the
acidification rate determined between 68 and 88 s. Cells were
exposed (4.5 min for hD2, 7.5 min for hD3) to increasing
concentrations (at half log unit intervals) of quinpirole at half
hourly intervals. For antagonist studies, a control concentra-
tion-response curve to quinpirole was conducted, and the cells
were then exposed to antagonist for at least 42 min prior to
construction of a further quinpirole concentration-effect curve
in the presence of antagonist. Each chamber therefore acted
as its own control. Drug additions were performed using the
Cytosensor autosampler (Molecular Devices) from deep well
blocks.
Ca ta lep sy. Catalepsy was assessed by positioning rats with
their hindpaws on the bench and their forelimbs rested on a
1 cm diameter horizontal bar, 10 cm above the bench. The
length of time in this position was recorded to a maximum of
120 s. Vehicle (1% methylcellulose, 2 mL/kg po) or SB-277011
(2.5, 7.9, 25.2 or 78.8 mg/kg po) or haloperidol (2.8 mg/kg po)
was injected (2 mL/kg). Catalepsy was assessed 180 and 210
min (for habituation purposes) and 240 min after drug
administration. Rats were judged cataleptic and assigned a
score of 1 if they maintained an immobile attitude for 30 s or
more at the 240 min time point; otherwise, they were given a
score of 0. A logistic regression analysis (SAS-RA, version 6.11;
SAS Institute Inc.) was used to analyze the data at the 240
min time point. In a separate experiment, vehicle (1% meth-
ylcellulose, 2 mL/kg po) or SB-277011 (2.5, 7.9, 25.2 or 78.8
mg/kg po) was injected in a volume of 2 mL/kg, followed 150
min later by saline or haloperidol (1.13 mg/kg ip) in a volume
of 1 mL/kg. Catalepsy was assessed 180, 210, and 240 min
after SB-277011 administration.
P la sm a P r ola ctin Levels. Animals were pretreated with
either, haloperidol (3 mg/kg po), SB-277011 (93 mg/kg po) or
vehicle (1% methylcellulose, 2 mL/kg po). After 2 h the animals
were decapitated and the blood collected into glass vials.
Samples were kept at 4 °C overnight, and then the serum was
separated and stored at -70 °C until subsequent assay. Serum
prolactin was assayed by radioimmunoassay (Amersham Life
Sciences). Serum prolactin measures were transformed (log)
prior to analysis by analysis of variance and Dunnett’s t-test
(Statistica Version 6.0).
Da ta An a lysis a n d Sta tistics. Radioligand binding studies
were analyzed using an iterative four-parameter logistic model
to generate IC50 values and from these pKi values were
determined. Concentration-effect curves from microphysio-
metry experiments were constructed from the peak acidifica-
tion response and analyzed using a four-parameter logistic
equation. Antagonist data were analyzed as the concentration

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