Synthesis and pharmacological evaluation of thiopyran analogues of the dopamine D3 receptor-selective agonist (4aR,10bR)-(+)-trans-3,4,4a,10b- tetrahydro-4-n-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol (PD 128907)

Article abstract of DOI:10.1021/jm0000113

Benzopyranoxazine (+)-7 (PD 128907) is the most dopamine (DA) D₃ receptor-selective agonist presently known. The only structural feature which distinguishes 7 from the analogous nonselective naphthoxazines is an oxygen atom in the 6-position. T

Full text of DOI:10.1021/jm0000113

J . Med. Chem. 2000, 43, 2871-2882
2871
Syn th esis a n d P h a r m a cologica l Eva lu a tion of Th iop yr a n An a logu es of th e
Dop a m in e D3 Recep tor -Selective Agon ist (4a R,10bR)-(+)-tr a n s-3,4,4a ,10b-
Tetr a h yd r o-4-n -p r op yl-2H,5H-[1]ben zop yr a n o[4,3-b]-1,4-oxa zin -9-ol (P D 128907)
L. Alexander van Vliet,* Nienke Rodenhuis, Durk Dijkstra, and Håkan Wikstro¨m
Department of Medicinal Chemistry, University Centre for Pharmacy, University of Groningen, Antonius Deusinglaan 1,
NL-9713 AV Groningen, The Netherlands
Thomas A. Pugsley, Kevin A. Serpa, Leonard T. Meltzer, Thomas G. Heffner, and Lawrence D. Wise
Departments of Chemistry and Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research,
Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Mary E. Lajiness, Rita M. Huff, and Kjell Svensson
CNS Diseases Research, Unit 7251, Pharmacia & Upjohn, 301 Henrietta Street, Kalamazoo, Michigan 49001
Staffan Sundell and Max Lundmark
Department of Medical Biochemistry, University of Go¨teborg, Medicinaregatan 9A, S-41390 Go¨teborg, Sweden
Received J anuary 11, 2000
Benzopyranoxazine (+)-7 (PD 128907) is the most dopamine (DA) D3 receptor-selective agonist
presently known. The only structural feature which distinguishes 7 from the analogous
nonselective naphthoxazines is an oxygen atom in the 6-position. To extend this series of tricyclic
DA agonists we used a classic bioisoster approach and synthesized thiopyran analogues of 7,
which have a sulfur atom in the 6-position. We prepared trans-4-n-propyl-3,4,4a,10b-tetrahydro-
2H,5H-[1]benzothiopyrano[4,3-b]-1,4-oxazin-9-ol (9, trans-9-OH-PTBTO), its enantiomers ((+)-9
and (-)-9), the racemic cis-analogue (10), and the racemic trans-sulfoxide (11) and studied the
potency and selectivity for DA receptors of these compounds. As with other rigid DA agonists,
the highest affinity for DA receptors resided in one of the enantiomers, in this case the (-)-
enantiomer of 9. On the basis of a single-crystal X-ray analysis of a key intermediate, the
absolute configuration of (-)-9 was found to be 4aS,10bR, which is homochiral with (+)-(4aR,-
10bR)-7. In contrast to (+)-7 however, (-)-9 displayed no selectivity for any of the DA receptors.
In addition, it has affinity for 5HT1A receptors. (()-cis-4-n-Propyl-3,4,4a,10b-tetrahydro-2H,5H-
[1]benzothiopyrano[4,3-b]-1,4-oxazin-9-ol (10), which was expected to be inactive, displayed
affinity and selectivity for the DA D3 receptor, whereas the sulfoxide 11 displayed some DA D3
selectivity, but with a lower affinity. Further pharmacological evaluation revealed that (-)-9
is a very potent full agonist at DA D2 receptors and a partial agonist at DA D3 receptors. The
cis-analogue (()-10 displayed the same profile, but with lower potency. These findings were
confirmed in vivo: in reserpinized rats (-)-9 displayed short-acting activation of locomotor
activity (DA D2 agonism) and also lower lip retraction and flat body posture, (5HT1A agonism).
Compound (()-10 had no effect on locomotor activity. In unilaterally 6-OH-DA lesioned rats,
(-)-9 gave short-acting locomotor activation. Furthermore, in microdialysis studies in rat
striatum, (-)-9 potently decreased DA release, confirming its activation of presynaptic DA D2
receptors.
In tr od u ction
subtypes.1,2 The selective brain distribution of these
receptors, particularly the DA D33-5 and D46 subreceptor
types, makes these receptors potential targets for the
development of novel psychoactive drugs. New anti-
psychotics may be developed, because the DA D3 and
D4 receptors are predominantly located in brain areas
where antipsychotics are supposed to exert their action,
such as the limbic system. These brain areas are known
to be associated with cognition and emotion and are
thought to be altered in psychiatric disorders such as
psychosis and schizophrenia.
New molecular biological techniques have, until now,
accomplished the identification of five dopamine (DA)
receptor subtypes. According to a generally accepted
receptor classification, which is mainly based on mo-
lecular structural features, these dopamine receptor
subtypes are divided into two groups: the DA D1-like
family of receptors, which includes the DA D1 and D5
receptor subtypes, and the DA D2-like family of recep-
tors, which includes the DA D2, D3, and D4 receptor
The function of the DA D3 receptor has been the
* To whom correspondence should be addressed. Phone: +31-50-
363 3296. Fax: +31 50 363 6908. E-mail: l.a.van.vliet@farm.rug.nl.
subject of much research.7,8 Several studies, many of
10.1021/jm0000113 CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/29/2000

2872 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
van Vliet et al.
F igu r e 1. Chemical structures of (R)-(+)-7-OH-DPAT (1),
PNU-990194A (2), quinpirole (3), and quinelorane (4).
F igu r e 2. Chemical structures of tricyclic DA agonists: a
benzo[f]quinoline (5), a naphthoxazine (6), benzopyranoxazines
(7 and 8), and benzothiopyranoxazines (S-oxide) (9-11).
which used the DA D3 preferring ligands described
below, have given some insight into this question. The
partially DA D3-selective ligands (R)-(+)-7-hydroxy-2-
(N,N-di-n-propylamino)tetralin (1, (R)-(+)-7-OH-DPAT,
agonist) and 5,6-dimethoxy-2-(N,N-di-n-propylamino)-
indan (2, PNU-99194A, antagonist) (Figure 1) were
found to inhibit and stimulate locomotor activity, re-
spectively, at doses that do not affect dopamine release.
Based on these findings it was speculated that the
functional DA D3 receptor is postsynaptically located
where it mediates an inhibitory role on psychomotor
function.9,10 Another study, which employed the par-
tially DA D3-selective agonists quinpirole (3) and quinelo-
rane (4), suggested the existence of striatal presynaptic
DA D3 autoreceptors, having an inhibitory effect on DA
synthesis.11
(Table 1). Within the benzopyranoxazine series33,34 (+)-7
((4aR,10bR)-(+)-trans-3,4,4a,10b-tetrahydro-4-n-propyl-
2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol) was found
to be rather selective for the DA D3 receptor subtype
and to have virtually no affinity for other non-dopamine
receptors, although the DA D3 over D2 selectivity of
(+)-7 was found to be much smaller when the affinities
for these receptors were obtained with an agonist
instead of an antagonist radioligand.35,36 Furthermore,
(+)-7 is presumed to interact selectively with DA D3
receptors only at low doses.35 Therefore, agonists ex-
hibiting higher DA D3 selectivity are still needed to
study this receptor subtype in more detail.
Furthermore, DA D3 receptors are suggested to
enhance the reinforcing properties of cocaine (presyn-
aptically),12 to play a role in the subjective effects of
cocaine,13 to mediate hypothermia,14 and to play a role
in memory processes.15 However, due to the low DA D3
selectivity (especially for agonists) of the agents used
in these studies, a DA D2 component in their action
cannot be excluded and the results have to be inter-
preted cautiously.
The pharmacological investigation of the DA D3 and
D4 receptors has generated a need for pharmacological
tools that exhibit a high degree of selectivity and affinity
for these receptor subtypes. With regard to the DA D3
receptor subtype, several ligands with a varying degree
of selectivity were developed. DA D3 antagonists include
compounds such as 2,10 cis-(+)-(1S,2R)-5-methoxy-1-
methyl-2-(n-propylamino)tetralin ((+)-AJ 76),16 cis-(+)-
(1S,2R)-5-methoxy-1-methyl-2-(di-n-propylamino)tetra-
lin ((+)-UH232),17 (+)-7-(N,N-dipropylamino)-5,6,7,8-
tetrahydronaphtho[2,3-b]dihydro-2,3-furan ((+)-S14297),18
N-[(n-butyl-2-pyrrolidinyl)methyl]-1-methoxy-4-cyano-
naphthalene-2-carboxamide (nafadotride),19 and (E)-
1,1′-(2-butene-1,4-diyl)bis[2-[4-[3-(1-piperidinyl)propoxy]-
phenyl]-1H-benzimidazole] (PD 152255).20
By comparing the structures of 5, 6, and (+)-7 (Figure
2) and their affinities (Table 1) it appears that selectivity
for the DA D3 receptor is affected by introducing an
oxygen atom in the 6-position of the tricyclic structure,
as in (+)-7. This selectivity is hard to explain on the
basis of geometry, because 5, 6, and (+)-7 are structur-
ally very similar. However, the DA D3 selectivity of (+)-7
may be explained by the higher electronegativity of
oxygen, as compared to carbon. The oxygen atom at the
6-position of 7 gives the potential for hydrogen-bond
formation with the receptor, which may be favorable for
binding to the DA D3 receptor and not so much to the
DA D2 receptor.
In this study we used a classic bioisoster approach
and replaced the oxygen in the 6-position of 7 with a
sulfur, which gave us benzothiopyranoxazines 9 and
10. Such bioisosteric replacements of oxygen by sulfur
have been applied extensively to study structure-ac-
tivity relationships of various pharmacologically active
agents.37,38 Often, a relationship is found between the
electronegativity of the substituent and the biological
activity. For the compounds in this study this replace-
ment will have the following effects: the lower elec-
tronegativity of sulfur (as compared to oxygen) will
eliminate the ability of the ligand to form a hydrogen
bond with the receptor. On the other hand, the intro-
duction of a sulfur will increase the hydrophobic proper-
ties around the 6-position of the ligand. The balance
between hydrogen bonds and hydrophobic interactions
is very important for the tight binding of a drug to a
receptor,39 and this balance may influence the affinity
and selectivity of the benzothiopyranoxazines for the DA
D3 receptor as compared to 7.
Relatively few DA D3 preferring agonists were devel-
oped. The 2-aminotetralin (+)-7-OH-DPAT (1) was
already known as a DA agonist and was later found to
be DA D3 preferring.4,21-23 As an extension of the
2-aminotetralin series, rigid tricyclic dopamine agonists
were developed. Tricyclic agonists such as the octahydro-
benzo[f]quinolines24-30 (e.g. 5, Figure 2) and the hexahy-
dronaphthoxazines31,32 (e.g. 6) display nanomolar af-
finities for dopamine receptors but are nonselective

Thiopyran Analogues of PD 128907
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2873
Sch em e 1a
a
Reagents: (a) 3-bromopropionic acid, NaOH, H2O; (b) PPA; (c) NH2OH‚HCl, pyridine, EtOH, reflux; (d) 1. p-toluenesulfonyl chloride,
pyridine, 2. KOtBu, EtOH, toluene, 3. HCl (concd); (e) 1. NaBH4, CH2Cl2/iPrOH (1/1), 35 °C, 2. HCl (aq, 10%); (f) chloroacetyl chloride,
EtOAc, H2O, NaHCO3; (g) NaOH, H2O, iPrOH; (h) LiAlH4, Et2O, reflux; (i) (S)-(+)-R-methoxyphenylacetic acid chloride, CH2Cl2, NaOH
(aq, 5%); (j) KOtBu, THF, H2O; (k) 1-iodopropane, CH3CN, Cs2CO3, reflux; (l) AlCl3, EtSH, 0 °C.
The replacement of oxygen by sulfur may (also) be
favorable from a pharmacokinetic point of view: Be-
cause sulfides are more lipophilic than their correspond-
ing oxygen compounds, passage through the blood-
brain barrier may be enhanced. This is illustrated by a
log D calculation with the computer program Pallas
1.2.40 Compound 7 has a calculated log D of 1.13, where-
as for 9 log D ) 1.62. (A log D-value of 2.5 is regarded
optimal for blood-brain penetration of a drug.41)
We also decided to study the effects of oxidation of
benzothiopyranoxazine 9 to its corresponding sulfoxide
11, thereby increasing the electron density around the
6-position. This restores the ability of the ligand to form
a hydrogen bond with the receptor at this position.
However, the lipophilicity is reduced quite dramatically
(log D ) -0.15 as predicted with Pallas 1.240).
This study describes the synthesis and pharmacologi-
cal evaluation of (()-trans-, (+)-trans-, (-)-trans-, and
(()-cis-4-n-propyl-3,4,4a,10b-tetrahydro-2H,5H-[1]ben-
zothiopyrano[4,3-b]-1,4-oxazin-9-ol ((()-9, (+)-9, (-)-9,
and (()-10, respectively) and the sulfoxide (()-trans-4-
n-propyl-3,4,4a,10b-tetrahydro-2H,5H-[1]benzothiopy-
rano[4,3-b]-1,4-oxazin-9-ol 6-oxide ((()-11). We expected
the highest affinity to reside in the isomer which has a
trans configuration with respect to the fusion of oxazine
to the thiopyran ring, as with the other tricyclic DA
agonists. Therefore, we optimized the formation of the
trans product, and only the trans isomer was resolved.
thiol, 12 (compound 12 is commercially available or can
be prepared by chlorosulfonylation of anisole42 and
subsequent reduction of 4-methoxybenzenesulfonyl chlo-
ride with zinc and sulfuric acid43 (not in scheme)).
Alkylation with 3-bromopropionic acid gave 13,44 which
was ring-closed with polyphosphoric acid45 to benzothio-
pyranone 14. Condensation of 14 with hydroxylamine
gave oxime 15, which was reacted with p-toluenesulfo-
nyl chloride to the corresponding tosyl oxime and
subsequently converted to amino ketone 16 in a Neber
rearrangement reaction. The amino ketone 16 was
reduced to trans- and cis-amino alcohols 17 and 18,
respectively, with sodium borohydride, in a 1/1 solvent
mixture of methylene chloride and 2-propanol at 35 °C,
which gave a trans:cis ratio of 93:7 (determined with
GC analysis of the chloroacetamide mixture (19 and 20)
formed in the next reaction step). Lower reaction
temperatures and more polar solvents (methanol, etha-
nol) gave more of the cis product. Amino alcohols 17 and
18 were difficult to purify and were first acylated with
chloroacetyl chloride to afford trans-chloroacetamide 19
and cis-chloroacetamide 20. The trans-chloroacetamide
19 was obtained pure by recrystallization, whereas the
cis-chloroacetamide 20 could be purified by column
chromatography. trans-Chloroacetamide 19 can be dis-
tinguished from its cis isomer 20 by comparing the
coupling constants of their respective benzylic protons,33
which are in the diaxial position for the trans-chloro-
acetamide 19. For the chloroacetamides this difference
is quite small (J ) 3.7 Hz (δ 4.62 ppm) and J ) 2.7 Hz
(δ 4.69 ppm) for trans- and cis-chloroacetamide, respec-
tively). However, for the conformationally more re-
Ch em istr y
The syntheses of 9 (and its enantiomers) and 10 are
outlined in Scheme 1 starting from 4-methoxybenzene-

2874 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
van Vliet et al.
Sch em e 2a
F igu r e 3. Stereoview of the molecular structure of (+)-23‚
HCl, determined with single-crystal X-ray crystallography.
a
stricted tricyclic structures this difference is more
pronounced (see Experimental Section, compounds 27
and 28). trans- and cis-chloroacetamide were separately
reacted to final products.
Reagents: (a) NaIO4, MeOH, H2O.
receptors.35 In the agonist binding studies, the affinity
for the D2L DA receptor was determined using [3H]NPA
([3H]-N-propylnorapomorphine) as the radioligand. The
affinity data obtained with [3H]NPA are comparable to
those obtained with [3H]N-0437, the agonist radioligand
which was used for the reference compound 7. In vitro
affinity for rat hippocampal serotonin 5HT1A and corti-
cal 5HT2 receptors was determined with [3H]-8-hydroxy-
2-(N,N-di-n-propylamino)tetralin ([3H]8-OH-DPAT) and
[3H]ketanserin as radioligands, respectively.35 Receptor
affinities are presented in Table 1.
In tr in sic Activity. The intrinsic activity (IA) of
compounds (-)-9 and (()-10 was determined with a
functional test, the mitogenesis assay.47,48 In CHO-L6
cells transfected with the rat DA D2L or D3 receptor, [3H]-
thymidine uptake was determined as a measure of
agonism at these receptors (Table 2). The IA was
compared with that of quinpirole, which is a full agonist
at both receptors (IA ) 100%). In the same cells the
ability of (-)-9 and (()-10 to inhibit quinpirole-
stimulated [3H]thymidine uptake was determined, which
is a measure of antagonism (Table 2).
Locom otor Activity a n d Beh a vior in Reser p in -
ized Ra ts. In vivo, postsynaptic agonistic effects of
compounds (-)-9 and (()-10 were determined in rats,
which had been treated with reserpine 18 h beforehand.
The reversal of the reserpine-induced akinesia is a
measure of DA D2 agonism. In addition, (stereotypic)
behavior was observed, such as sniffing (a marker of
DA D2 agonism), flat body posture and lower lip retrac-
tion (markers of agonism at serotonin 5HT1A receptors)
and yawning (a putative marker of activation of DA D3
receptors21,49). The time course of locomotor activity is
depicted in Figure 4; a summary of behavior is pre-
sented in Table 3.
Con t r a la t er a l Tu r n in g in 6-OH -DA Lesion ed
Ra ts. Compound (-)-9 was further evaluated in rats
unilaterally lesioned with 6-OH-DA (Figure 5). In this
model, the DA neurons of one side (left or right) of the
nigrostriatal DA system are selectively and completely
degenerated by intracerebral injection of the neurotoxin
6-OH-DA. This causes a postsynaptic supersensitivity
to develop on the lesioned side.50 Upon systemic admin-
istration of a DA agonist, the rat will start to turn
contralaterally, i.e., toward the nonlesioned side.51 The
evoked turning behavior is a measure of the DA (D1 and/
or D2) agonist properties of a compound.
Cyclization of chloroacetamides 19 (trans) and 20 (cis)
was achieved with sodium hydroxide in 2-propanol,33
affording oxazinones 21 (trans) and 22 (cis), respec-
tively, which were reduced with LiAlH4 to give oxazines
23 (trans) and 24 (cis), respectively. The trans-oxazine
23 was resolved by coupling with (S)-(+)-R-methoxyphen-
ylacetic acid chloride and column chromatographic
separation of the thus formed diastereomeric amides 25
(eluting first) and 26 (eluting second).30 Cleavage of the
amides 25 and 26 by potassium tert-butoxide and water
in THF afforded (+)-trans-oxazine (+)-23 and (-)-trans-
oxazine (-)-23, respectively. The hydrochlorides of these
secondary amines yielded crystals suitable for single-
crystal X-ray crystallography (Figure 3). On the basis
of the X-ray analysis of (+)-23, amines (+)-23 and
(-)-23 could be assigned the absolute configurations
4aS,10bR and 4aR,10bS, respectively.
trans-Oxazines 23, (+)-23 and (-)-23, and the racemic
cis-oxazine 24 were alkylated with 1-iodopropane in
acetonitrile using cesium carbonate as the base, giving
the tertiary amines 27, (-)-27, (+)-27, and 28, respec-
tively. Upon propylation, the optical rotation of the
enantiomers of 23 changed both sign and amplitude.
To be able to assign all hydrogen atoms in the 1H
NMR spectrum of the trans-benzothiopyranoxazine 27,
a COSY experiment was performed. Interestingly, the
two methylene hydrogen atoms (of the propyl group)
attached to the oxazine nitrogen (hydrogens H1′) show
a high degree of nonequivalency resulting in two
separate signals (2 multiplets, at δ ) 2.35 and 2.78
ppm). These hydrogens are nonequivalent because they
are diastereotopic. However, for the cis-benzothiopyra-
noxazine 28 this nonequivalency is much less pro-
nounced, and no separate signals are observed. This
phenomenon has been described previously for trans-
N-propylhexahydronaphthoxazine (6)46 and also for
trans-N-benzyloctahydrobenzo[f]quinolines, for which
the benzylic protons are nonequivalent.25,26
Finally, demethylation of 27, (-)-27, (+)-27, and 28
was achieved cleanly by applying aluminum chloride in
EtSH, giving the final products (()-9, (-)-9, (+)-9, and
(()-10, respectively. The sulfoxide 11 was prepared by
oxidizing the (()-trans-sulfide (()-9 with NaIO4 in
MeOH/H2O (Scheme 2).
P h a r m a cology
Micr od ia lysis in Ra t Str ia tu m . The effects of (-)-9
on in vivo DA release in rat striatum were assessed with
microdialysis methods (Figure 6).
Recep tor Bin d in g. Sulfides (()-9, (-)-9, (+)-9, and
(()-10 and the sulfoxide 11 were tested for their in vitro
binding affinity for human dopamine (DA) D2L, D3, or
D4.2 receptors, expressed in Chinese hamster ovary
(CHO) K-1 cells. In the antagonist binding studies, the
affinity of the compounds was determined by their abil-
ity to displace [3H]spiperone from D2L, D3, or D4.2 DA
Resu lts a n d Discu ssion
Recep tor Bin d in g. As has been discussed previ-
ously, for DA agonists DA D2 affinity, and consequently
DA D3 selectivity, is most reliably measured using an

Thiopyran Analogues of PD 128907
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2875
Ta ble 1. DA (human D2L, D3, and D4) and Serotonin (rat 5HT1A and 5HT2) Receptor Affinities of the 9-OH-benzothiopyranoxazines
and Several Known Tricyclic DA Agonists
Kia (nM)
D2L [3H]spip D3 [3H]spip
ratio
Kia (nM)
compd
D
2L [3H]NPA
D4 [3H]spip
D2A/D3
5HT1A [3H]8-OH-DPAT
5HT2 [3H]ketanserin
9-OH-benzothiopyranoxazines
(()-9
NDb
1.14
854
74
533
ND
ND
ND
ND
1.56
2.87
3003
3.58
29
44.4
17.6
ND
30
ND
>5000
>5000
NA
(-)-9
0.4
(+)-9
(()-10
(()-11
>3300
>1000
193
21
6.8
NAc
197
IC50 > 10000
NA
NA
Other Tricyclic DA Agonists
6
ND
ND
42f,g
ND
ND
6.24d
0.21d
78
1.4g
>10000
>10000
ND
ND
169g
ND
ND
(()-7e
(+)-7
(-)-7
(()-8
>10000
1180h
30
1670g
NA
>10000
>10000
a
b
Ki and IC50 values are means of three separate experiments, the results of which did not vary by more than 25%. ND, not determined.
c NA, not active. Unpublished results. e Compound 7 is PD 128907. f Determined with the agonist radioligand [3H]N-0437. Ref 35.
Ref 36.
d
g
h
Ta ble 2. Agonist and Antagonist Effects of Selected Compounds as Measured in the Mitogenesis Assay at the Rat D2L and D3
Receptorsa
[3H]thymidine uptake, EC50, nM (IAb)
inhib of quinpirole (30 nM)-stimulated [3H]thymidine uptake
compd
DA D2L
DA D3
DA D2L
DA D3
(-)-9
0.013 ( 0.004 (80% ( 7)
122 ( 39 (87% ( 12)
5.7 ( 1.2 (81% ( 1.2)
2.2 (100%)
0.007 ( 0.001 (45% ( 7)
36 ( 16 (57% ( 8)
0.36 ( 0.12 (54% ( 2)
1.7 (100%)
NAc
NA
NA
NA
Ad g0.1 nM
NA
NA
NA
(()-10
(+)-7e
quinpirole
a
b
d
All values are means of three determinations ( SEM. IA, intrinsic activity. c NA, not active. A, active. e (+)-PD 128907.
F igu r e 4. Locomotor activity (LMA). Each point is the mean
F igu r e 5. Effect on turning behavior of (-)-9 in unilaterally
( SEM of four determinations (saline, n ) 8).
6-OH-DA lesioned rats. Each point is the mean ( SEM of five
(3.8 µmol/kg) or three (11.3 µmol/kg) rats. The total number
of full contraversive rotations ( SEM is indicated at the top.
Ta ble 3. Behavior in Reserpinized Ratsa
behavior
Sni
In this series of benzothiopyranoxazines, the trans
compound 9 (as compared to its cis analogue 10) has
the highest affinity for dopamine receptors, like in the
series of the before mentioned benzo[f]quinolines,25,28,30
naphthoxazines,31 and benzopyranoxazines (Table 1).
Concluding from the X-ray analysis (Figure 3) of its
intermediary secondary amine (+)-23, the configuration
of the chiral centers in the (-)-trans-benzothiopyran-
oxazine (-)-9 is 4aS,10bR. Although (-)-9 has a nega-
tive optical rotation, it is homochiral (i.e., has an
identical spatial orientation at its chiral centers) with
(4aR,10bR)-(+)-7, the active enantiomer of the trans-
benzopyranoxazines. (The designation 4aS for (-)-9 is
the result of a change in substituent priority, as
compared to (+)-7, upon introduction of a sulfur.) Of
both trans enantiomers, (-)-9 has the highest affinity
for dopamine receptors (Table 1). Thus, with regard to
the stereochemistry of the trans-9-OH-benzothiopyra-
noxazines the structure-affinity relationship is ana-
logous to that of the before mentioned benzo[f]quino-
compd
FB
LL
Ya
saline
(-)-9
0/8
4/4
0/4
0/8
3/4
0/4
0/8
4/4
0/4
0
0
(()-10
11 ( 3
a
Flat body posture (FB), lower lip retraction (LL), and sniffing
(Sni): shown is the number of rats displaying the indicated
behavior during the first 30 min after injection. Yawning (Ya):
shown is the mean ( SEM of total yawns/rat during the first 30
min after injection.
agonist radioligand.22,35 Accordingly, the calculated DA
D3 selectivity for (+)-7 was substantially reduced when
DA D2 affinity was measured with [3H]N-0437 (agonist
radioligand) instead of [3H]spiperone (antagonist radio-
ligand).35 To obtain a proper selectivity ratio for the
newly synthesized benzothiopyranoxazines 9 and 10 and
the sulfoxide 11, also for these compounds the DA D2
affinity was measured with an agonist radioligand, in
this case [3H]NPA. (See Table 1, the affinity of racemic
trans-9 was in a preliminary binding assay only deter-
mined with [3H]spiperone.)

2876 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
van Vliet et al.
However, by optimizing the trans:cis ratio, we did not
obtain enough of the cis secondary amine 24 to resolve
this compound.
The affinity for dopamine receptors of the sulfoxide
compound 11 was substantially reduced (Table 1), as
compared to compound 9. However, the affinity for the
DA D3 receptor was much less reduced than the affinity
for the DA D2 receptor, suggesting that an electroneg-
ative (hydrogen-bonding) site in the 6-position of the
molecule may be allowed by the DA D3, but not by the
DA D2 receptor. The lower lipophilicity of compound 11
(log D ) -0.15, as predicted with Pallas 1.240), or the
electron-withdrawing effect of the sulfoxide group on the
aromatic nucleus and the resulting higher acidity of the
phenolic hydroxy group, may contribute to its lower
affinity.
In tr in sic Activity. The functional assay revealed
that (-)-9 is very potent (EC50 ) 0.013 nM, Table 2)
and a full agonist (IA ) 80%) at DA D2L receptors. This
was confirmed by its inability to inhibit quinpirole
stimulated [3H]thymidine uptake. At DA D3 receptors
(-)-9 displayed partial agonism, unlike (+)-7, which is
reported to be a full agonist at DA D2 and D3 receptors.35
The cis analogue (()-10 displayed full agonism at DA
D2 and partial agonism at D3 receptors, but with lower
potency, reflecting its affinity for these receptors. No
inhibition of quinpirole stimulated [3H]thymidine up-
take was detected for this compound, supporting its
agonism.
Locom otor Activity a n d Beh a vior in Reser p in -
ized Ra ts. The in vivo effects in reserpinized rats
confirmed the receptor binding profile of (-)-9 (Figure
4). This compound shows actions at both DA and 5HT1A
receptors: activation of locomotor activity and sniffing
behavior (reflecting postsynaptic DA D2 (and/or D1)
agonism), besides lower lip retraction and flat body
posture (reflecting 5HT1A agonism), were observed. The
onset of these effects can be observed almost im-
mediately after injection, which reflects a good absorp-
tion, but the maximum effect only lasts for about 30
min.
Compound (()-10 had no effect on locomotor activity
in reserpinized rats, probably due to its relatively low
DA D2 affinity (Ki ) 74 nM). Behaviorally, (()-10
appeared to increase yawning, which may reflect its
preference for DA D3 receptors.21,49 However, this is
controversial, and yawning may be mediated by a
number of other neurotransmitters and neuropeptides
(for a review see ref 53). Yawning elicited by (+)-PD
128970 ((+)-7) has been observed at lower doses (for
example, 0.35 µmol/kg (+)-7 causes 12 ( 2 yawns/rat,
monitored during 20 min).54
Con tr a la ter a l Tu r n in g in 6-OH-DA Lesion ed
Ra ts. Also in rats with a unilateral lesion of the
nigrostriatal system, (-)-9 turned out to be a potent
agonist at DA receptors, as is obvious from the evoked
rotation behavior (Figure 5). The time course of the
rotation was similar to the time course of the effects of
(-)-9 observed in reserpinized rats: an immediate onset
of action after injection but a short duration of the
maximum effect.
F igu r e 6. Effect of (-)-9 on DA release in rat striatum. Each
point is the mean ( SEM of three determinations. The arrow
indicates the time of injection. DA decreases were significant
(p < 0.05) for 0.1, 0.3, and 10 µmol/kg.
lines, naphthoxazines, and benzopyranoxazines. In each
of these tricyclic dopamine agonists the (+)-trans com-
pound (which for all three compound classes is homo-
chiral with (-)-9) is the enantiomer with the highest
affinity for DA receptors.30,31,34
Compound (-)-9 binds to the DA D3 receptor with
similar affinity as to the DA D2 receptor (Table 1), and
it also shows good affinity (Ki ) 17.6 nM) for the DA D4
receptor. With this combined DA D2 and DA D3 affinity,
(-)-9 shows more similarity with the corresponding
trans-benzo[f]quinoline 5 and trans-naphthoxazine 6,
which are also nonselective, than with (+)-7. This
similarity may be explained by the charge distribution
around the sulfur atom, because the electronegativity
of sulfur is closer to carbon than to oxygen. (The
electronegativity values for carbon, oxygen, and sulfur
are 2.75, 3.65 and 2.96, respectively, according to the
Sanderson scale.52) Apparently, with the introduction
of more hydrophobic properties around the 6-position,
the affinity for DA D2-like receptors is enhanced, but
with the loss of a hydrogen-bonding site the selectivity
for the DA D3 receptor is lost.
To our surprise, during the locomotor activity and
microdialysis experiments with (-)-9, we observed the
5HT1A behavioral syndrome (flat body posture, lower lip
retraction, see Figure 4). Therefore, we decided to
determine binding to serotonin (5HT1A and 5HT2) recep-
tors as well. Unlike (+)-7, compound (-)-9 displayed
moderate affinity for rat hippocampal 5HT1A receptors
(Ki ) 30 nM). 5HT1A receptor binding has been reported
for 10-hydroxy-substituted octahydrobenzo[f]quino-
lines.28 However, for 9-hydroxy-substituted tricyclic DA
agonists, affinity for 5HT1A receptors has not been
reported previously.
In contrast with the corresponding cis-benzopyran-
oxazine 8, which has no affinity for DA D2 or D3
receptors, the difference in affinity between trans-9 and
its cis analogue 10 is much less pronounced (Table 1).
On the contrary, 10 shows a remarkable affinity and
selectivity for the DA D3 receptor, which is unexpected
for a cis tricyclic dopamine agonist structurally related
to the benzo[f]quinolines, naphthoxazines, and benzopy-
ranoxazines. One of the enantiomers of 10 may display
an even higher selectivity for the DA D3 receptor.
Micr od ia lysis in Ra t Str ia tu m . A decrease of DA
release was observed after administration of (-)-9,
which was to be expected for a DA D2 agonist. At 0.3

Thiopyran Analogues of PD 128907
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2877
Single-crystal X-ray analysis was performed by S. Sundell
at the Department of Medical Biochemistry, University of
Go¨teborg.
µmol/kg (-)-9 showed a DA reduction of 75% (which is
almost the maximum effect which can be observed with
microdialysis, a reduction of 80%), which is more potent
than (+)-7, which displays a 55% DA reduction at the
same dose.35 Furthermore, the reduction of DA release
lasted longer for (-)-9, which may reflect its higher
lipophilicity, as compared to (+)-7. As was also observed
with the experiments with reserpinized rats, the rats
were activated and showed lower lip retraction and flat
body posture.
Concluding from the results described above, it is
clear that the pharmacological profile of the thiopyran
analogue (-)-9 is quite different from that of (+)-7.
Compound (-)-9 displays a mixed receptor binding
profile, having affinity for DA D2 and D3 and 5HT1A
receptors. A small change in the structure of a tricyclic
DA ligand turns out to have a large effect on its receptor
binding profile. Although sulfur is often used as a
bioisosteric replacement of oxygen, within this series of
tricyclic DA agonists, sulfur substitution on the 6-posi-
tion has a dramatic effect.
In contrast with (-)-9, the cis analogue (()-10 dis-
plays DA D3 (over D2) selectivity. Study of the enanti-
omers of 10 could certainly be worthwhile, as one of the
enantiomers may display twice the affinity for DA D3
receptors, as compared to the racemate. The selectivity
for DA D3 receptors of one of the enantiomers of 10 may
even be higher, because the DA D2 affinity which is
measured for the racemate may reside fully in the other
enantiomer.
Finally, the DA D3 preference of the sulfoxide (()-11
suggests that an electronegative (hydrogen-bonding) site
on the 6-position of the tricyclic system is allowed by
the DA D3 but not by the DA D2 receptor. However,
other factors, such as the influence of the sulfoxide on
the nitrogen or aromatic hydroxy pKa value, or on the
aromatic ring, may play a role.
â-(4-Meth oxyp h en ylm er ca p to)p r op ion ic Acid (13). A
solution of sodium carbonate (19.1 g, 0.18 mol) and 3-bro-
mopropionic acid (27.5 g, 0.18 mol) in 90 mL of ice-cold water
was added to a solution of sodium hydroxide (7.2 g, 0.18 mol)
and 4-methoxybenzenethiol 12 (24.8 g, 0.18 mol) in 90 mL of
water. This mixture was refluxed for 1.5 h, and after cooling
to room temperature it was washed once with ethyl acetate
(100 mL). The water layer was then acidified with 10%
hydrochloric acid, and the product was extracted with ethyl
acetate (2 × 100 mL). These combined organic layers were then
washed once with brine, dried over MgSO4, and concentrated
under reduced pressure, which yielded an off-white crystalline
mass (25.9 g, 68%): mp 71 °C; EIMS m/e 212 (M+); IR (KBr,
cm-1) 2839, 1701, 1595, 1496; 1H NMR (CDCl3, 60 MHz) δ 2.4
(t, 2H), 2.8 (t, 2H), 3.7 (s, 3H), 6.6 (d, 2H), 7.15 (d, 2H), 9.8 (s,
1H). Anal. (C10H12O3S‚1/4H2O) C,H.
6-Met h oxy-3,4-d ih yd r o-2H -[1]b en zot h iop yr a n -4-on e
(14). Polyphosphoric acid (70 g) was heated to 90 °C and
mechanically stirred. Melted (ca. 80 °C) â-(4-methoxyphenyl-
mercapto)propionic acid (13) (25.0 g, 0.12 mol) was added in
one portion, and the mixture was stirred for 3 min. Then
another amount of polyphosphoric acid (120 g), which was
heated to 90 °C in advance, was added in one portion, and the
mixture was stirred for 4 min. After cooling to 60 °C, ice (180
g) was added and the reaction mixture was stirred until the
polyphosphoric acid had hydrolyzed (ca. 10 min). Subsequently,
the product was extracted with ethyl acetate (4 × 100 mL).
The combined ethyl acetate layers were washed once with
water, once with NaOH (5%, aq), then again with water until
neutral pH. After washing the combined organic layers once
with brine they were dried over MgSO4 and concentrated
under reduced pressure. The resulting brown oil was purified
by distillation under reduced pressure which yielded 14 as a
yellow oil (15.5 g, 68%): bp 109 °C (0.03 mmHg); IR (neat,
1
cm-1) 2833, 1674, 1598, 1471; H NMR (CDCl3, 200 MHz) δ
2.94-3.00 (m, 2H), 3.19-3.25 (m, 2H), 3.83 (s, 3H), 7.01 (dd,
J 1 ) 8.7, J 2 ) 2.9, 1H), 7.19 (d, J ) 8.7, 1H), 7.61 (d, J ) 2.9,
1H); 13C NMR δ 26.8, 39.7, 55.5, 111.3, 122.3, 128.9, 131.6,
133.6, 157.4, 194.1; HRMS calcd (obsd) for C10H10O2S 194.0401
(194.0400).
6-Meth oxy-3,4-d ih yd r o-2H-[1]ben zoth iop yr a n 4-oxim e
(15). A solution of 6-methoxy-3,4-dihydro-2H-[1]benzothiopy-
ran-4-one (14) (12.9 g, 66.5 mmol), hydroxylamine hydrochlo-
ride (9.9 g, 142 mmol) and pyridine (9.8 mL) in 100 mL of
ethanol was heated to reflux under a nitrogen atmosphere for
35 min. After cooling to room temperature, about 2/3 of the
ethanol was evaporated under reduced pressure. The resulting
liquid was dissolved in 150 mL of 3% HCl, and the product
was extracted with ethyl acetate (2 × 150 mL). The combined
organic layers were washed with brine, dried over MgSO4, and
concentrated under reduced pressure which yielded 15 as a
white crystalline solid (13.7 g, 98%): mp 121 °C; EIMS m/e
Exp er im en ta l Section
Ch em istr y, Gen er a l. Melting points were determined in
open glass capillaries on an Electrothermal digital melting-
point apparatus and are uncorrected. Optical rotations were
determined at 589 nm, at 20 °C on a Perkin-Elmer 241
1
polarimeter. H NMR spectra were recorded at 60 MHz on a
Hitachi Perkin-Elmer R-24 B spectrometer, at 200 MHz on a
Varian Gemini 200 spectrometer, or at 500 MHz on a Varian
Unity 500 spectrometer. Chemical shifts are given in δ units
(ppm) and are relative to TMS for 60 MHz spectra or rela-
tive to the solvent for 200 and 500 MHz spectra. Coupling
constants are given in hertz (Hz). The spectra recorded were
consistent with the proposed structures. IR spectra were
obtained on an ATI-Mattson spectrophotometer, and only the
important absorptions are given. Electronic ionization (EI)
mass spectra were obtained on a Unicam 610-Automass 150
GC-MS system. Elemental analyses were performed by the
Analytical Chemistry Section at Parke Davis, Ann Arbor,
MI, and were within 0.4% of the theoretical values, except
where noted. Compounds that were obtained as an oil, or as a
solid in a very small amount, were analyzed by high-resolution
mass spectrometry (HRMS), performed on a J EOL MSroute
J MS-600H by the Department of Chemistry, University of
Groningen.
1
209 (M+); IR (KBr, cm-1) 2830, 1593, 1481; H NMR (CDCl3,
200 MHz) δ 2.91-2.98 (m, 2H), 3.13-3.19 (m, 2H), 3.82 (s,
3H), 6.86 (dd, J 1 ) 8.6, J 2 ) 2.9, 1H), 7.18 (d, J ) 8.6, 1H),
7.46 (d, J ) 2.9, 1H); 13C NMR δ 26.1, 26.4, 55.4, 109.7,
117.2, 129.4, 153.8, 157.5, 172.0, 192.5. Anal. (C10H11NO2S)
C,H,N.
3-Am in o-6-m eth oxy-3,4-dih ydr o-2H-[1]ben zoth iopyr an -
4-on e Hyd r och lor id e (16). A solution of 15 (13.7 g, 65.2
mmol) in 50 mL of pyridine was cooled to 0 °C, and treated
with p-toluenesulfonyl chloride (54.0 g, 283 mmol) which was
added slowly in 5 g portions. After complete addition, the
reaction mixture was stirred 1 h at 0 °C and 2 h at room
temperature. Ice (320 mL) was added, and after the ice had
melted the mixture was extracted with ethyl acetate (3 × 100
mL). The combined organic layers were washed once with 1
N HCl, once with brine, and dried over MgSO4. Concentration
under reduced pressure yielded the tosyloxime as a brown
powder (26.4 g, ca. 100%), which was used without further
purification.
If necessary, compounds were purified with medium-pres-
sure chromatography (MPLC) on silica, starting with an apolar
eluent (usually 100% hexane) and gradually increasing its
polarity (usually by adding ethyl acetate), finishing with an
eluent mixture that gave a Rf value of approximately 0.3 on
TLC.

2878 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
van Vliet et al.
A suspension of the tosyl oxime (23.8 g, ca. 65 mmol) in 260
mL of toluene was added slowly to a cooled (0 °C) solution of
potassium tert-butoxide (12.6 g, 113 mmol) in 90 mL of ethanol.
The reaction mixture was stirred at 0 °C for 1 h, then stirred
at room temperature for 1.5 h, and finally stirred at 35 °C for
30 min to complete te reaction. The precipitate (potassium
tosylate) was removed by filtration, and concentrated hydro-
chloric acid (26.5 mL) was slowly poured to the filtrate, which
turned dark purple-red. Ether was added to promote crystal-
lization of the product, which was filtered off as a yellow
powder and dried in an desiccator (12.8 g, 80%): mp 174 °C
dec; IR (KBr, cm-1) 2830, 1685, 1560, 1476; 1H NMR (D2O,
200 MHz) δ 3.39 (dd, J 1 ) 12.8, J 2 ) 4.7, 1H), 3.59-3.74 (m,
1H), 3.85 (s, 3H), 4.68-4.77 (m, 1H), 7.16 (m, 1H), 7.29 (d, J
) 10.3, 1H), 7.51-7.53 (m, 1H); 13C NMR δ 31.1, 45.0, 71.8,
72.1, 128.8, 139.6, 145.3, 145.5, 149.5, 173.3; HRMS calcd
(obsd) for C10H11NO2S 209.0510 (209.0512).
tr a n s- a n d cis-3-Am in o-6-m eth oxy-3,4-d ih yd r o-2H-[1]-
ben zoth iop yr a n -4-ol Hyd r och lor id e (17 a n d 18). Amino
ketone 16 (8.5 g, 34.7 mmol) was dissolved in a mixture of
135 mL of dichloromethane and 135 mL of 2-propanol. The
solution was heated to 35 °C, and sodium borohydride (4 g,
105 mmol) was added at a rate of 0.5 g per 10 min. After
complete addition, the reaction mixture was stirred 1 h at 35
°C and allowed to cool to room temperature. The reaction
mixture was quenched by slowly adding 10% HCl until acidic
pH, and the solvents were evaporated to dryness under
reduced pressure. The remaining solids were dissolved in 250
mL of hot ethanol, the insoluble inorganic salts were removed
by filtration, and the filtrate was concentrated under reduced
pressure, which yielded a mixture of 17 and 18 as a red-brown
foam (6.28 g, 73%): mp 160-180 °C dec; IR (KBr, cm-1) 2928,
1560; 1H NMR (D2O, 200 MHz, chemical shifts of major
3H), 4.04 (s, 2H), 4.41-4.56 (m, 1H), 4.69 (d, J ) 2.7, 1H),
6.79 (dd, J 1 ) 8.8, J 2 ) 2.9, 1H), 6.94 (d, J ) 2.9, 1H), 7.04 (d,
J ) 8.8, 1H), 7.33 (bd, 1H); 13C NMR δ 25.9, 42.3, 48.7, 55.2,
68.7, 115.4, 115.6, 122.1, 127.2, 134.5, 157.2, 166.5; HRMS
calcd (obsd) for C12H14NO3SCl 287.0383 (287.0409).
(()-tr a n s-3,4,4a ,10b-Tetr a h yd r o-9-m eth oxy-2H,5H-[1]-
ben zoth iop yr a n o[4,3-b]-1,4-oxa zin -3-on e (21). trans-Chlo-
roacetamide 19 (4.9 g, 17.1 mmol) was dissolved in 320 mL of
hot 2-propanol. After the solution had cooled to room temper-
ature, a solution of sodium hydroxide (1.4 g, 35.8 mmol) in
2.7 mL of water was added. The reaction was followed on TLC
and was complete after stirring for 2.5 h. The reaction mixture
was neutralized with 4 N HCl and concentrated under reduced
pressure. The remaining white solid, which contained both
product and sodium chloride, was transferred to a Soxhlet
thimble and extracted overnight in a Soxhlet apparatus with
500 mL of ethyl acetate. The ethyl acetate was concentrated
to 100 mL, and lactam 21 was filtered off as very fine white
crystals (2.9 g, 68% yield): mp 240 °C; EIMS m/e 251 (M+);
1H NMR ((CD3)2SO, 200 MHz) δ 2.99 (dd, J 1 ) 12.2, J 2 ) 3.9,
1H), 3.15 (t, J ) 12.0, 1H), 3.26-3.48 (m, 1H), 3.69 (s, 3H),
4.27 (d, J ) 3.2, 2H), 4.56 (d, J ) 9.8, 1H), 6.79 (dd, J 1 ) 8.6,
J 2 ) 2.7, 1H), 6.98-7.02 (m, 2H), 8.46 (s, 1H); 13C NMR δ 28.1,
51.7, 55.4, 67.8, 74.4, 111.1, 115.0, 122.0, 126.7, 133.5, 157.0,
167.7. Anal. (C12H13NO3S) C,H,N.
(()-cis-3,4,4a ,10b-Tetr a h ydr o-9-m eth oxy-2H,5H-[1]ben -
zoth iop yr a n o[4,3-b]-1,4-oxa zin -3-on e (22). cis-Chloroacet-
amide 20 (0.32 g, 1.11 mmol) was dissolved in 21 mL of
2-propanol. A solution of sodium hydroxide (0.09 g, 2.3 mmol)
in 0.18 mL of water was added, and the reaction was stirred
for 19 h. The reaction mixture was neutralized with 4 N HCl
and concentrated under reduced pressure. The remaining
solids were dissolved in water (30 mL), and the product was
extracted with ethyl acetate (2 × 30 mL). The combined
organic layers were extracted with brine, dried over Na2SO4,
and evaporated to dryness which yielded the cis-lactam 22 as
a light-yellow solid (0.24 g, 86%). An analytical sample was
obtained by recrystallization from ethyl acetate: mp 215-217
°C; EIMS m/e 251 (M+); 1H NMR (CDCl3, 500 MHz) δ 2.80
(dd, J 1 ) 12.7, J 2 ) 1.4, 1H), 3.37 (t, J ) 12.7, 1H), 3.80 (s,
3H), 3.90-3.93 (m, 1H), 4.37 (dd, J 1 ) 30.7, J 2 ) 16.7, 2H),
4.68 (d, J ) 3.0, 1H), 6.85 (dd, J 1 ) 8.6, J 2 ) 2.7, 1H), 6.92 (d,
J ) 2.7, 1H), 7.07-7.08 (m, 2H); 13C NMR δ 28.0, 51.2, 55.3,
67.6, 71.0, 116.6, 116.8, 123.3, 127.5, 131.0, 157.3, 168.5. Anal.
(C12H13NO3S) C,H,N.
diastereomer (trans, 17) are given) δ 3.25 (dd, J 1 ) 13.7, J 2
)
6.8, 1H), 3.50 (dd, J 1 ) 13.7, J 2 ) 3.4, 1H), 3.87 (s, 3H), 3.89-
3.98 (m, 1H), 4.87 (d, J ) 6.4, 1H), 6.97 (dd, J 1 ) 8.6, J 2 ) 3.0,
1H) 7.14 (d, J ) 3.0, 1H), 7.19 (d, J ) 8.6, 1H); 13C NMR δ
41.8, 66.6, 72.0, 83.8, 131.6, 132.3, 138.5, 144.2, 149.3, 173.4;
HRMS calcd (obsd) for C10H13NO2S 211.0667 (211.0687).
tr a n s- a n d cis-3-Ch lor oa ceta m id o-6-m eth oxy-3,4-d i-
h yd r o-2H-[1]ben zoth iop yr a n -4-ol (19 a n d 20). A mixture
of amino alcohols 17‚HCl and 18‚HCl (6.0 g, 24.3 mmol) was
dissolved in a well-stirred two-layer system of NaHCO3 (5.7
g, 67 mmol) in 70 mL of water and 170 mL of ethyl acetate.
The mixture was cooled to 0 °C and a solution of chloroacetyl
chloride (2 mL, 25.4 mmol) in 50 mL of ethyl acetate was added
dropwise. After the addition was completed the mixture was
stirred for 30 min on ice and was allowed to warm to room
temperature. The layers were separated and the aqueous layer
was extracted once with ethyl acetate (100 mL). The combined
organic layers were washed once with brine, dried over MgSO4
and concentrated until crystallization started. The solution
was put aside overnight, and the light-green crystalline solid
was obtained by suction filtration, which yielded most of the
trans-chloroacetamide 19 (4.78 g). The mother liquor contained
a mixture of both diastereomers 19 and 20, which were
separated by MPLC on silica (initial eluent 100% hexane, final
eluent hexane:ethyl acetate 1:1) yielding cis-chloroacetamide
20 as a light-brown oil (0.39 g, 6% yield, Rf ) 0.26 in hexane:
ethyl acetate 1:1), and the rest of the trans-chloroacetamide
19 (0.30 g, 78% total yield, Rf ) 0.21 in hexane:ethyl acetate
1:1).
(()-tr a n s-3,4,4a ,10b-Tetr a h yd r o-9-m eth oxy-2H,5H-[1]-
ben zoth iop yr a n o[4,3-b]-1,4-oxa zin e (23). LiAlH4 (1.1 g,
28.9 mmol) was suspended in 22 mL of dry ether. A suspension
of the trans-lactam 21 (2.8 g, 11.3 mmol) in 220 mL of dry
ether was added in 30 mL portions to the LiAlH4 suspension.
When the addition was completed, the reaction mixture was
heated to reflux for 2 h. After cooling to room temperature
and further cooling on ice, the reaction was quenched by the
cautious addition of 1.1 mL of water, 1.1 mL of 4 N NaOH
and 3.3 mL of water (in that order). The mixture was heated
to reflux until all precipitates had turned white (15 min),
cooled to room temperature, and filtered over Celite. The
yellow filtrate was dried over Na2SO4 and concentrated under
reduced pressure, which yielded 23 as a yellow oil which
solidified upon cooling (2.6 g, 97%): mp 74-76 °C; EIMS m/e
1
237 (M+); H NMR (CDCl3, 200 MHz) δ 1.95 (br s, 1H), 2.68
(d, J ) 2.7, 1H), 2.88-3.12 (m, 4H), 3.70-3.84 (m, 1H), 3.75
(s, 3H), 4.08 (dd, J 1 ) 11.0, J 2 ) 3.7, 1H), 4.20 (d, J ) 8.5,
1H), 6.72 (dd, J 1 ) 8.5, J 2 ) 2.9, 1H), 6.97 (d, J ) 8.5, 1H),
7.13 (d, J ) 2.7, 1H); 13C NMR δ 29.6, 45.8, 55.18, 55.21, 67.8,
tr a n s-3-Ch lor oa cet a m id o-6-m et h oxy-3,4-d ih yd r o-2H -
[1]ben zoth iop yr a n -4-ol (19): mp 178-180 °C; EIMS m/e 287
1
(M+); H NMR (CDCl3, 200 MHz) δ 2.83 (dd, J 1 ) 12.9, J 2
)
5.1, 1H), 3.65 (dd, J 1 ) 13.3, J 2 ) 2.3, 1H), 3.79 (s, 3H), 4.01
(s, 2H), 4.48-4.60 (m, 1H), 4.62 (d, J ) 3.7, 1H), 6.84 (dd, J 1
) 8.7, J 2 ) 2.8, 1H), 6.90 (d, J ) 2.9, 1H), 7.10 (d, J ) 8.6,
1H), 7.24 (bd, 1H); 13C NMR (CD3OD, 125.7 MHz) δ 27.0, 42.5,
49.4, 55.2, 69.0, 115.7, 116.8, 123.4, 127.7, 135.2, 158.2, 168.3.
Anal. (C12H14NO3SCl) C,H,N.
78.1, 110.9, 114.6, 122.0, 126.6, 134.7, 157.3. Anal. (C12H15-
NO2S‚1/4H2O) C,H,N.
(()-cis-3,4,4a ,10b-Tetr a h ydr o-9-m eth oxy-2H,5H-[1]ben -
zoth iop yr a n o[4,3-b]-1,4-oxa zin e (24). The procedure de-
scribed above for the preparation of 23 was used for the
reduction of the cis-lactam 22 (0.2 g, 0.80 mmol) to 24, which
was obtained as a light-yellow oil (0.18 g, 95%): 1H NMR
(CDCl3, 200 MHz) δ 2.98-3.08 (m, 3H), 3.25 (dd, J 1 ) 12.9, J 2
) 7.3, 1H), 3.45-3.48 (m, 1H), 3.59-3.64 (m, 2H), 3.78 (s, 3H),
cis-3-Ch lor oa ceta m id o-6-m eth oxy-3,4-d ih yd r o-2H-[1]-
ben zoth iop yr a n -4-ol (20): 1H NMR (CDCl3, 200 MHz) δ 3.00
(dd, J 1 ) 10.9, J 2 ) 3.4, 1H), 3.13 (t, J ) 10.9, 1H), 3.76 (s,

Thiopyran Analogues of PD 128907
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2879
MHz) δ 11.7, 18.6, 27.3, 51.4, 55.2, 55.4, 60.5, 66.8, 77.3, 110.8,
114.5, 121.7, 126.5, 135.6, 157.3; HRMS calcd (obsd) for C15H21
NO2S 279.1293 (279.1286).
4.63 (d, J ) 3.7, 1H), 6.76 (m, 1H), 7.00-7.07 (m, 2H); 13C
NMR δ 29.2, 43.0, 49.8, 55.2, 63.4, 72.0, 114.6, 115.3, 124.0,
127.1, 133.5, 157.6; HRMS calcd (obsd) for C12H15NO2S 237.0823
(237.0829).
-
(-)-(4aS,10bR)-3,4,4a,10b-Tetr ah ydr o-9-m eth oxy-4-pr o-
p yl-2H ,5H -[1]b en zot h iop yr a n o[4,3-b]-1,4-oxa zin e ((-)-
27). Compound (+)-23‚HCl (0.20 g, 0.73 mmol) was converted
to (-)-27 (0.20 g, 98%), as described above for 27. A sample
was converted to hydrochloride and recrystallized from 100%
ethanol, yielding white crystals: mp 205-210 °C; [R] ) -3.1°
(c ) 2.61, MeOH). All spectral data for (-)-27 were identical
with those described for 27. Anal. (C15H21NO2S‚HCl) C,H,N.
(+)-(4aR,10bS)-3,4,4a,10b-Tetr ah ydr o-9-m eth oxy-4-pr o-
p yl-2H ,5H -[1]b en zot h iop yr a n o[4,3-b]-1,4-oxa zin e ((+)-
27). Compound (-)-23‚HCl (0.24 g, 0.87 mmol) was converted
to (+)-27 (0.23 g, 95%), as described for 27. A sample was
converted to hydrochloride and recrystallized from 2-propanol,
yielding pink crystals: mp 207-212 °C; [R] ) +3.3° (c ) 2.73,
MeOH). All spectral data for (+)-27 were identical with those
described for 27. Anal. (C15H21NO2S‚HCl) C,H,N.
(()-cis-3,4,4a ,10b -Te t r a h yd r o-9-m e t h oxy-4-p r op yl-
2H,5H-[1]ben zoth iop yr a n o[4,3-b]-1,4-oxa zin e (28). Amine
24‚HCl (0.16 g, 0.59 mmol) was converted to 28 as described
above for 27 and was obtained as a light-yellow oil (0.08 g,
49%): 1H NMR (CDCl3, 200 MHz) δ 0.94 (t, J ) 7.3, 3H), 1.44-
1.62 (m, 2H), 2.47-2.72 (m, 5H), 3.28 (dd, J 1 ) 11.5, J 2 ) 2.0,
1H), 3.49 (t, J ) 11.7, 1H), 3.77 (s, 3H), 3.91-3.97 (m, 2H),
4.45 (s, 1H), 6.76 (dd, J 1 ) 8.5, J 2 ) 2.7, 1H), 6.85 (d, J ) 2.4,
1H), 7.01 (d, J ) 8.5, 1H); 13C NMR δ 11.5, 18.6, 19.9, 46.4,
55.2, 55.9, 57.3, 67.5, 75.0, 115.9, 116.8, 123.9, 126.9, 133.8,
156.9; HRMS calcd (obsd) for C12H15NO2S 279.1293 (279.1282).
(()-tr a n s-4-n -P r op yl-3,4,4a ,10b-tetr a h yd r o-2H,5H-[1]-
ben zoth iop yr a n o[4,3-b]-1,4-oxa zin -9-ol (9). Amine 27 (0.35
g, 1.25 mmol) was dissolved in 3.5 mL of ethanethiol, and the
solution was cooled on ice. Aluminum chloride (0.50 g, 3.8
mmol) was added as 3 portions of 0.17 g, with an interval of
30 min. After all the aluminum chloride was added, the
reaction mixture was stirred on ice for 1 h. The reaction was
quenched with water, alkalinized (10% NaHCO3) and extracted
with ethyl acetate, adding sodium chloride to enhance layer
separation. The combined organic layers were washed once
with brine, dried over Na2SO4 and concentrated to give 9 as a
light-yellow solid (0.26 g, 78%). A sample was converted to
hydrochloride and recrystallized from ethanol 100%, yielding
off-white crystals: mp 263-267 °C dec; EIMS m/e 265 (M+);
IR (KBr, cm-1) 3099, 2553, 1474, 1324, 1105; 1H NMR (CDCl3,
500 MHz) δ 0.91 (t, J ) 7.3, 3H), 1.49-1.57 (m, 2H), 2.33-
2.39 (m, 1H), 2.48-2.53 (m, 1H), 2.56-2.60 (m, 1H), 2.76-
2.82 (m, 1H), 2.88-2.96 (m, 2H), 3.12 (dd, J 1 ) 12.5, J 2 ) 4.2,
1H), 3.86-3.91 (m, 1H), 4.05 (dd, J 1 ) 11.2, J 2 ) 2.4, 1H),
4.33 (d, J ) 9.3, 1H), 6.64 (dd, J 1 ) 8.3, J 2 ) 2.4, 1H), 6.93 (d,
J ) 8.3, 1H), 7.06 (d, J ) 2.4, 1H); 13C NMR (CDCl3, 125.7
MHz) δ 11.8, 18.6, 27.3, 51.5, 55.5, 60.8, 66.8, 77.2, 113.4,
115.3, 121.5, 126.8, 135.6, 153.4. Anal. (C14H19NO2S‚HCl)
C,H,N.
(()-tr a n s-3,4,4a ,10b-Tetr a h yd r o-4-[(S)-m eth oxyp h en -
yla cet yl]-9-m et h oxy-2H ,5H -[1]b en zot h iop yr a n o[4,3-b]-
1,4-oxa zin e, Dia ster eom er ic Am id es 25 a n d 26.30 (S)-(+)-
R-Methoxyphenylacetic acid (0.91 g, 5.5 mmol) and thionyl
chloride (7.6 mL, 104 mmol) were dissolved in 26 mL of
methylene chloride. The mixture was stirred and heated to
reflux for 1 h, then the excess thionyl chloride and the solvent
were evaporated. The resulting acid chloride (a clear oil) was
redissolved in 25 mL of methylene chloride and added dropwise
to a vigorously stirred solution of amine 23 (1.2 g, 5.0 mmol)
in 25 mL of methylene chloride, layered with 50 mL of 5%
NaOH (aq). After 20 min all starting amine was consumed,
and the layers were separated. The organic layer was washed
once with water, dried over Na2SO4 and concentrated under
reduced pressure which yielded a yellow oil (1.9 g, 99%). The
two diastereomeric amides were separated by careful and
repeated MPLC on silica with diisopropyl ether. The combined
fractions of the diastereomeric amide that eluted first (25) (Rf
) 0.30, 100% diisopropyl ether) yielded a light-yellow oil (0.70
g, 36%) of 96% purity according to GLC and crystallized
overnight: mp 128 °C. Anal. (C21H23NO4S) C,H,N.
The diastereomeric amide that eluted second (26) (Rf ) 0.21,
100% diisopropyl ether) was obtained as a light-yellow oil (0.62
g, 32%) of 96% purity according to GLC: HRMS calcd (obsd)
for C21H23NO4S 385.1348 (385.1349).
(+)-(4aS,10bR)-3,4,4a,10b-Tetr ah ydr o-9-m eth oxy-2H,5H-
[1]ben zot h iop yr a n o-[4,3-b]-1,4-oxa zin e ((+)-23). The di-
astereomeric amide that eluted first (25) (0.60 g, 1.56 mmol)
was dissolved in dry THF (53 mL). Potassium tert-butoxide
(1.12 g, 9.75 mmol) and water (0.083 mL, 4.5 mmol) were
added, and the mixture was stirred at room temperature for
1.5 h. The reaction mixture was partitioned between ether and
water, and the water layer was extracted once more with ether.
The combined organic layers were extracted with 5% HCl (3
× 50 mL), the water phase was alkalinized (10% Na2CO3) and
extracted with ether (3 × 50 mL). The combined organic layers
were washed once with brine, dried over Na2SO4 and concen-
trated under reduced pressure, which yielded (+)-(23) as a
light-yellow solid (0.34 g, 92%). A sample was converted to the
hydrochloride and recrystallized from 100% ethanol, yielding
off-white crystals: mp 238-242 °C; [R] ) +51.6° (c ) 1.36,
MeOH). All spectral data for (+)-23 were identical with those
described for 23. Anal. (C12H15NO2S‚HCl) C,H,N.
(-)-(4aR,10bS)-3,4,4a,10b-Tetr ah ydr o-9-m eth oxy-2H,5H-
[1]b en zot h iop yr a n o[4,3-b]-1,4-oxa zin e ((-)-23). The di-
astereomeric amide that eluted second (26) (0.60 g, 1.56 mmol)
was converted to (-)-23 (0.29 g, 78%) as described above for
(+)-23. A sample was converted to hydrochloride, yielding off-
white crystals: mp 234-236 °C; [R] ) -48.3° (c ) 4.62,
MeOH). All spectral data for (-)-23 were identical with those
described for 23. Anal. (C12H15NO2S‚HCl) C,H,N.
(()-tr a n s-3,4,4a ,10b -Tet r a h yd r o-9-m et h oxy-4-p r op yl-
2H,5H-[1]ben zoth iop yr a n o[4,3-b]-1,4-oxa zin e (27). Amine
23 (0.72 g, 3.04 mmol) and cesium carbonate (2.95 g, 9.1 mmol)
were suspended in dry acetonitrile (19 mL). 1-Iodopropane
(0.83 mL, 8.3 mmol) was added and the mixture was heated
to reflux overnight. The reaction mixture was concentrated
under reduced pressure, water was added to the residue and
the product was extracted with ether (2 × 100 mL). The
combined organic layers were washed with brine, dried over
Na2SO4 and concentrated under reduced pressure, which
yielded a yellow oil. The product was purified with MPLC on
silica (initial eluent 100% hexane, final eluent hexane:ethyl
acetate ) 4:1) yielding 27 as a light-yellow oil (0.65 g, 77%):
1H NMR (CDCl3, 500 MHz) δ 0.92 (t, J ) 7.3, 3H), 1.49-1.57
(m, 2H), 2.32-2.37 (m, 1H), 2.48-2.54 (m, 1H), 2.55-2.59 (m,
1H), 2.75-2.81 (m, 1H), 2.88-2.95 (m, 2H), 3.15 (dd, J 1 ) 12.4,
J 2 ) 4.0, 1H), 3.79 (s, 3H), 3.86-3.91 (m, 1H), 4.08-4.11 (m,
1H), 4.33 (d, J ) 9.2, 1H), 6.72-6.75 (m, 1H), 6.99 (d, J ) 8.6,
1H), 7.16 (dd, J 1 ) 2.8, J 2 ) 0.9, 1H); 13C NMR (CDCl3, 50.3
(-)-tr a n s-(4a S,10bR)-3,4,4a ,10b-Tetr a h yd r o-4-p r op yl-
2H,5H-[1]ben zoth iop yr a n o[4,3-b]-1,4-oxa zin -9-ol ((-)-9).
Amine (-)-27 (0.20 g, 0.72 mmol) was converted to (-)-9 (0.18
g, 94%) as described above for 9. The product was converted
to hydrochloride and recrystallized from 100% ethanol yielding
pink crystals: mp 250-260 °C dec; [R] ) -8.8° (c ) 2.49,
MeOH). All spectral data for (-)-9 were identical with those
described for 9. Anal. (C14H19NO2S‚HCl) C,H,N.
(+)-tr a n s-(4a R,10bS)-3,4,4a ,10b-Tetr a h yd r o-4-p r op yl-
2H,5H-[1]ben zoth iop yr a n o[4,3-b]-1,4-oxa zin -9-ol ((+)-9).
Amine (+)-27 (0.21 g, 0.72 mmol) was converted to (+)-9 (0.19
g, 96%) as described above for 9. The product was converted
to hydrochloride and recrystallized from 100% ethanol yielding
pink crystals: mp 251-255 °C dec; [R] ) +10.1° (c ) 2.76,
MeOH). All spectral data for (+)-9 were identical with those
described for 9. Anal. (C14H19NO2S‚HCl) C,H,N.
(()-cis-3,4,4a ,10b -Tet r a h yd r o-4-p r op yl-2H ,5H -[1]b en -
zoth iop yr a n o[4,3-b]-1,4-oxa zin -9-ol (10). Amine 28 (0.09 g,
0.32 mmol) was converted to 10 (0.080 g, 94%) as described
above for 9. The product was converted to hydrochloride and

2880 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
van Vliet et al.
recrystallized from 100% ethanol yielding a pink solid: mp
189-192 °C dec; EIMS m/e 265 (M+); IR (KBr, cm-1) 2579,
2498, 2411, 1480, 1276; 1H NMR (CDCl3, 500 MHz) δ 0.95 (t,
J ) 7.2, 3H), 1.52-1.60 (m, 2H), 2.52-2.64 (m, 3H), 2.67-
2.76 (m, 2H), 3.29-3.33 (m, 1H), 3.48 (t, J ) 11.9, 1H), 3.91-
3.98 (m, 2H), 4.44 (s, 1H), 6.68 (dd, J 1 ) 8.6, J 2 ) 2.6, 1H),
6.76 (d, J ) 2.6, 1H), 6.96 (d, J ) 8.6, 1H); 13C NMR (CDCl3,
125.7 MHz) δ 12.4, 19.5, 20.7, 47.3, 56.8, 58.1, 68.1, 75.5, 117.6,
119.8, 124.4, 127.8, 134.5, 153.7. Anal. (C14H19NO2S‚HCl‚1/
4H2O) C,H,N.
arrival prior to use in the experiments. Experimental drugs
were first dissolved in a few drops of ethanol 96% and slightly
warmed, then saline was added. The administered volume was
1 mL/kg.
Rats were reserpinized 18-24 h prior to the experiments
with 10 mg/kg sc reserpine (RBI). Reserpine was dissolved in
10% glacial acetic acid, 80% ultrapure water and 10% sucrose
(MERCK). On the day of the experiments the animals were
placed alone in PMMA boxes to acclimate. After the rats were
acclimated the compounds were administered subcutaneously
in the neck region, after which the locomotor activity counting
was started. Locomotor activity was measured using AU-
TOMEX II (Columbus Instruments, Columbus, OH) activity
monitors. Observations of behavior were made during the first
30 min of the locomotor activity measurements.
(()-tr a n s-4-n -P r op yl-3,4,4a ,10b-tetr a h yd r o-2H,5H-[1]-
ben zoth iopyr an o[4,3-b]-1,4-oxazin -9-ol S-Oxide (11). NaIO4
(0.083 g, 0.39 mmol) was dissolved in 2 mL of a 1/1 mixture of
water and methanol, and the solution was cooled to 0 °C on
ice. A suspension of sulfide 9 (0.073 g, 0.28 mmol) in 1 mL of
methanol, was added dropwise, and the reaction mixture was
stirred on ice for 2 h. The reaction was monitored on TLC. To
complete the oxidation extra NaIO4 (0.038 g, 0.18 mmol) was
added and the mixture was stirred for another 0.5 h. The
reaction mixture was extracted with chloroform (3 × 5 mL),
and the combined organic layers were dried over Na2SO4,
filtered and concentrated under reduced pressure. The re-
maining brown oil was purified with MPLC on silica (initial
eluent hexane:ethyl acetate ) 1:1, final eluent 100% ethyl
acetate) yielding 11 as an off-white oil (0.023 g, 29%). The
product was converted to hydrochloride and recrystallized from
2-propanol/isopropyl ether to yield white crystals: mp 236-
Micr od ia lysis in Ra t Str ia tu m . Microdialysis experi-
ments were performed at the Department of Medicinal Chem-
istry, University of Groningen, by N. Rodenhuis. Male Wistar
rats (from Harlan, Zeist, The Netherlands) weighing 280-320
g were used and housed as described for the locomotor activity
experiments.
On-line brain microdialysis in freely moving animals was
essentially performed as described previously by Westerink.55
Briefly, rats were anesthetized with chloral hydrate (400 mg/
kg ip) and 10% lidocaine was locally applied. The rats were
then mounted into a stereotaxic frame (Kopf). The incisor bar
was placed in position so that the skull was held in a horizontal
position. The skull was exposed and burr holes were drilled.
An Y-shaped cannula was used for the experiments, with an
exposed tip length of 3 mm. The dialysis tube (i.d.: 0.22 mm;
o.d.: 0.31 mm) was prepared from polyacrylonitrile methalys
sulfonate copolymer (AN 69, Hospal, Bologna, Italy). The
dialysis membrane was implanted in the striatum with
coordinates which were calculated relative to bregma: A1,
L(3, D-6, according to Paxinos and Watson (1982). The dura
was removed with a sharp needle. Two anchor screws were
positioned in different bone plates nearby. Before insertion into
the brain the dialysis probes were perfused with successively
ultrapure water, methanol, ultrapure water and Ringer solu-
tion (1.2 mM Ca). The dialysis probe was positioned in the
burr hole under stereotaxic guidance. The probe was cemented
in this position with phosphatine dental cement (Associated
Dental Products Ltd., Kemdent Works, Purdon, Swinden,
Wiltshire SN5 9HT, U.K.).
1
240 °C dec; IR (KBr, cm-1) 2972, 1596, 1475, 1115, 1023; H
NMR (CDCl3, 500 MHz, hydrochloride) δ 1.08 (t, J ) 7.4, 3H),
1.80-1.90 (m, 2H), 3.19-3.25 (m, 1H), 3.33-3.38 (m, 1H),
3.45-3.55 (m, 2H), 3.73 (d, J ) 12.7, 1H), 3.97-4.06 (m, 1H),
4.08-4.17 (m, 1H), 4.24 (t, J ) 12.5, 1H),4.37 (d, J ) 10.6,
1H), 4.90 (d, J ) 9.5, 1H), 6.98 (dd, J 1 ) 8.7, J 2 ) 2.4, 1H),
7.21 (dd, J 1 ) 2.7, J 2 ) 1.1, 1H), 7.62 (d, J ) 8.5, 1H); HRMS
calcd (obsd) for C14H19NO3S 281.1086 (281.1100).
P h a r m a cology, Gen er a l. All experimental drugs tested
were in the hydrochloride form.
Recep tor Bin d in g. The receptor binding studies were
performed at Parke Davis Pharmaceutical Research Division,
by T. A. Pugsley. Binding to cloned human DA D2, D3, and D4
receptors was carried out as described in ref 22. Binding to
rat hippocampal 5HT1A and cortical 5HT2 receptor binding was
carried out as described in ref 35.
In tr in sic Activity. The intrinsic activity determinations
were performed at Pharmacia & Upjohn, CNS Diseases
Research, by M. E. Lajiness, as described in refs 47, 48.
Con tr a la ter a l Tu r n in g in 6-OH-DA Lesion ed Ra ts.
Contralateral turning experiments were performed at Parke
Davis Pharmaceutical Research Division, by L. Meltzer, es-
sentially according to the original reference by Ungerstedt and
Arbuthnott.51 Briefly, rats were lesioned in the right medial
forebrain bundle (P4.8 mm, L1.1 mm, V-8.2 mm from bregma)
with 8 µg/4 µL 6-hydroxydopamine HBr in saline with ascorbic
acid 1 mg/mL added. After 3 weeks recovery, completeness of
lesion was assessed with apomorphine 50 µg/kg sc. Only
animals rotating more than 100 turns/h were used in subse-
quent experiments.
Rats were removed from home cages in morning, weighed,
dosed and placed into harnesses in rotorat apparatus. Rats
sat in stainless steel, flat-bottomed, hemispheric bowls and
were connected via the harness and a flexible spring tether to
an automated data collection system. Data are presented as
full rotations in contraversive directions. Rats were used once
weekly.
Locom ot or Act ivit y a n d Beh a vior in R eser p in ized
Ra ts. Locomotor activity experiments were performed at the
Department of Medicinal Chemistry, University of Groningen,
by L. A. van Vliet and N. Rodenhuis. Male Wistar rats (from
Harlan, Zeist, The Netherlands) weighing 180-200 g were
used. The rats were housed in PMMA cages, eight animals in
each cage, with free access to water and food. The cages were
placed in a room with controlled environmental conditions (21
°C; humidity 60-65%; 8 a.m. to 8 p.m., 8 p.m. to 8 a.m. light-
dark periods). The animals were housed at least 1 week after
The experiments were performed in conscious rats 17-56
h after implantation of the cannula. The striatum was perfused
with a Ringer solution (147 mM NaCl, 4 mM KCl, 1.2 mM
CaCl2, 1.1 mM MgCl2) at 2 µL/min (CMA/102 microdialysis
pump). After the experiments the rats were sacrificed and the
brains were removed. After removal the brains were kept in
4% paraformaldehyde solution until they were sectioned to
control the location of the dialysis probes.
Dopamine, DOPAC, and 5-HIAA were quantitated by HPLC
with electrochemical detection. An HPLC pump (LKB, Phar-
macia) was used in conjugation with an EC detector (Antec,
Leiden) working at 625 mV versus Ag/AgCl reference electrode.
The analytical column was a Supelco Supelcosil LC-18 column
(15 cm, 4.6 mm, 3 µm). The mobile phase consisted of a mixture
of 4.1 g/L sodium acetate (Merck), 85 mg/L octanesulfonic acid
(Aldrich), 50 mg/L EDTA (Merck), 8.5% methanol (Labscan)
and ultrapure water (pH ) 4.1 with glacial acetic acid).
Sta tistics. The microdialysis data were analyzed using
Friedman repeated measures analysis of variance on ranks
with as post hoc test Dunnett’s method.
Ack n ow led gm en t. Y.-H. Shih, K. Zoski, L. Georgic,
and H. Akunne are gratefully acknowledged for the in
vitro pharmacology testing.
Su p p or tin g In for m a tion Ava ila ble: X-ray analysis,
crystal data, and elemental analyses. This material is available
free of charge via the Internet at http://pubs.acs.org.

Thiopyran Analogues of PD 128907
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