N-substituted (2,3-dihydro-1,4-benzodioxin-2-yl)methylamine derivatives as 92 antagonists/5-HT(1A) partial agonists with potential as atypical antipsychotic agents

Article abstract of DOI:10.1021/jm9910122

A series of N-substituted 1-(2,3-dihydro-1,4-benzodioxin-2- yl)methylamine derivatives with D₂ antagonist/5-HT(1A) partial agonist activity has been prepared as potential atypical antipsychotic agents. Optimization of in vitro receptor binding activity and in vivo activity in rodent models of psychosis has led to compound 24, which showed good affinities for human D₂, D₃, and 5-HT(1A) receptors but significantly less affinity for human α₁ adrenoceptors and rat H₁ and muscarinic receptors. In rodents, 24 showed functional D₂-like antagonism and 5-HT(1A) partial agonism. After oral dosing, 24 showed good activity in rodent antipsychotic tests and very little potential to cause extrapyramidal side effects (EPS), as measured by its ability to induce catalepsy in rats only at very high doses. In the light of this promising profile of activity, 24 has been selected for clinical investigation as a novel antipsychotic agent with a predicted low propensity to cause EPS.

Full text of DOI:10.1021/jm9910122

3342
J . Med. Chem. 1999, 42, 3342-3355
N-Su bstitu ted (2,3-Dih yd r o-1,4-ben zod ioxin -2-yl)m eth yla m in e Der iva tives
a s D2 An ta gon ists/5-HT1A P a r tia l Agon ists w ith P oten tia l a s Atyp ica l
An tip sych otic Agen ts
Alan M. Birch,* Paul A. Bradley, J ulie C. Gill, Frank Kerrigan, and Pat L. Needham
Research and Development Department, Knoll Pharmaceuticals, Pennyfoot Street, Nottingham NG1 1GF, U.K.
Received J anuary 30, 1999
A series of N-substituted 1-(2,3-dihydro-1,4-benzodioxin-2-yl)methylamine derivatives with D2
antagonist/5-HT1A partial agonist activity has been prepared as potential atypical antipsychotic
agents. Optimization of in vitro receptor binding activity and in vivo activity in rodent models
of psychosis has led to compound 24, which showed good affinities for human D2, D3, and 5-HT1A
receptors but significantly less affinity for human R1 adrenoceptors and rat H1 and muscarinic
receptors. In rodents, 24 showed functional D2-like antagonism and 5-HT1A partial agonism.
After oral dosing, 24 showed good activity in rodent antipsychotic tests and very little potential
to cause extrapyramidal side effects (EPS), as measured by its ability to induce catalepsy in
rats only at very high doses. In the light of this promising profile of activity, 24 has been selected
for clinical investigation as a novel antipsychotic agent with a predicted low propensity to cause
EPS.
Ch a r t 1
In tr od u ction
Schizophrenia is a relatively common disorder, af-
fecting approximately 1% of the population, and one
where established therapies leave much room for im-
provement. Progress is still sought in terms of efficacy,
against both the positive symptoms of the disease, such
as delusions and hallucinations, and the negative
symptoms, such as social withdrawal and flattened
affect, and also in terms of lower side effect potential.
There is strong evidence1 that classical antipsychotic
agents, such as haloperidol (Chart 1), exert their
beneficial effects through blockade of central dopamine
D2 receptors. In addition, the acute extrapyramidal side
effects (EPS) induced by these agents, such as Parkin-
sonism, akathisia, and dystonia, are also thought to
result from their high degree of D2-receptor antagonism.
Clozapine is exceptional among established antipsy-
chotic agents, as it combines effectiveness against both
positive and negative symptoms of the disease with a
very low propensity to induce EPS. It has significant
affinity for a broad range of neurotransmitter receptors,2
which seems to account for its clinical effectiveness at
a relatively low occupancy of D2 receptors (40-60%),1
which in turn is probably responsible for the lack of EPS
seen with the drug. Clozapine was withdrawn from the
market over 20 years ago due to a relatively high
incidence of potentially fatal agranulocytosis, but was
reintroduced more recently, under carefully controlled
conditions, for the treatment of patients who failed to
respond to other drugs.
and D4, which would not be expected to be associated
with EPS.1 However, although a number of selective,
high-affinity compounds have been described, none of
these has yet been reported to be clinically effective.
More successful has been the combination of D2-receptor
antagonism with serotonergic activity. For example, in
the recently introduced drugs risperidone6 and sertin-
dole,7 5-HT2A-receptor antagonism is believed to play
an important role in their activity.
Typical D2-receptor antagonists, such as haloperidol
and raclopride, induce catalepsy in rats, an activity
which is predictive of the propensity of compounds to
cause EPS when administered to man. Following the
demonstration that the 5-HT1A-receptor full agonist
8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) re-
versed this catalepsy,8 we sought to find molecules
which combined antagonism at D2-like receptors with
agonism or partial agonism at 5-HT1A receptors. We
required these compounds to have much lower affinity
The search for alternatives to clozapine, which match
its effectiveness but lack its serious side effects, has led
to the structurally related compounds zotepine,3 olan-
zapine,4 and quetiapine,5 which are all newly approved
for the treatment of schizophrenia in major markets.
An alternative approach to the discovery of atypical
antipsychotics has been to target the other newly
identified dopamine receptor subtypes, e.g., D1/D5, D3,
10.1021/jm9910122 CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/11/1999

Preparation of Potential Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3343
to show agonist properties.17 Again the (S)-(-)-forms
showed the higher affinities.
Ch a r t 2
The chroman derivative BAY x 3702 is, in general
structural terms, similar to MDL 72832 and is also a
potent 5-HT1A ligand with some selectivity over R1 and
D2.18 A recent report describes aminotetralin deriva-
tives, which may be regarded as hybrid structures
between 8-OH-DPAT and the amides described above,
as having more balanced affinities for 5-HT1A and D2
receptors.19
We report here a new series of N-substituted 1-(2,3-
dihydro-1,4-benzodioxin-2-yl)methylamine derivatives,
the best of which have good affinity and selectivity for
the D2 and 5-HT1A receptors and show potential for
atypical antipsychotic activity in animal models.
Ch em istr y
The majority of the target compounds described in
this study (Table 1) were prepared either directly or
indirectly by reaction of the (2,3-dihydro-1,4-benzo-
dioxin-2-yl)methyl tosylates 25 with various amine
nucleophiles 26 as depicted in Scheme 1. The (2,3-
dihydro-1,4-benzodioxin-2-yl)methyl tosylates 25a -e
were synthesized by tosylation of the known (2,3-
dihydro-1,4-benzodioxin-2-yl)methanols 27a -e, which
were obtained from commercial sources or by prepara-
tion as described in the literature.20-23 Tosylates 25h ,i
were prepared from the methanols 27h ,i, which were
in turn obtained from the corresponding esters 28a ,b
by reduction with LiAlH4. The esters 28a ,b were
initially prepared as a mixture by literature methods.24
To separate the two regioisomers, the mixture was
converted into the carboxamides 29a ,b, which were
separated by fractional crystallization and then alco-
holized back to the individual esters (Scheme 2).
The remaining tosylates 25f,g were obtained by the
synthetic route outlined in Scheme 3. This involved
alkylation of the substituted salicylaldehydes 30a ,b
with (R)-glycidyl tosylate to give the ethers 31a ,b and
then subsequent Baeyer-Villiger oxidation to give the
formate esters 32a ,b. Alkylations of this type are known
to proceed predominantly with retention of configuration
at the glycidyl asymmetric center,25 thus leading to the
(S)-(2,3-dihydro-1,4-benzodioxin-2-yl)methanols. This ster-
eochemical course was confirmed for the final product
24 by X-ray crystallographic studies26 which showed it
to have the S-stereochemistry. All other levorotatory
final products are assumed, therefore, to share the
S-stereochemistry.
Treatment of 32a ,b with sodium methoxide in metha-
nol resulted in cleavage of the formate ester and
concomitant cyclization, yielding the (2,3-dihydro-1,4-
benzodioxin-2-yl)methanols 27f,g. Finally, reaction with
tosyl chloride in pyridine gave the tosylates 25f,g in
good yield.
The intermediate amines 26a -i were synthesized by
the route described in Scheme 4. Reaction of 1-chloro-
2,4-dinitrobenzene with isonicotinamide at 95 °C, in the
absence of solvent, led to the formation of the pyri-
dinium salt 33, which on amine exchange27 gave the
pyridinium salts 34a -g. Subsequent reduction of the
pyridine ring was accomplished using H2 on Pd/C,
affording piperidines 35a -g. Finally, reduction with
LiAlH4 furnished the key amines 26a -g.
for receptors associated with side effects, such as R1
adrenoceptors (sedation, postural hypotension), hista-
mine H1 (sedation), and muscarinic (dry mouth, blurred
vision).
Other research groups have also targeted D2-antago-
nist/5-HT1A-agonist compounds as potential antipsy-
chotics with low EPS. At least two such compounds,
RWJ -37796 (mazapertine, Chart 2)9 and 1192U9010
have reached early clinical development. These two
compounds are arylpiperazine derivatives which show
high and approximately equal affinities for D2, 5-HT1A
,
and R1 receptors. The clinical undesirability of high
affinity for R1 adrenoceptors was reinforced by reports
that mazapertine11,12 had encountered problems with
postural hypotension in clinical trials.
The arylpiperazine moiety found in the compounds
above would appear to be isosteric with the N-(2,3-
dihydro-1,4-benzodioxin-2-yl)methylamine substructure,
as evidenced by accounts of several derivatives of the
latter type which also have activity at various dopam-
inergic, serotoninergic, and noradrenergic receptor sub-
types. Often, compounds of this type show significant
affinity for D2, 5-HT1A, and R1 receptors, although the
relative proportion varies with the particular structural
class. For example, the spirocyclic imide derivatives
such as MDL 72832 and its aza-analogues13 show
selectivity for the 5-HT1A receptor, though with still
significant affinities for D2 and R1 receptors. Comparison
of the enantiomers of MDL 72832 with other standard
5-HT1A ligands, using molecular modeling techniques,14
allowed Hibert and co-workers to derive a pharmacoph-
ore for that receptor. The model correctly predicted that
the (S)-enantiomer would have the higher affinity. The
8-methyl-2,3-dihydro-1,4-benzodioxin derivative (-)HT-
90B is structurally related to the compound above and
shares a similar profile, with some selectivity for 5-HT1A
but still significant affinities for D2 and R1 receptors.15
MKC-242 is another benzodioxin derivative, and al-
though it lacks a polar amide group in its side chain,
its receptor binding profile remains similar.16
In contrast to these, a series of 7-hydroxy-2,3-dihy-
drobenzodioxin derivatives, together with their chroman
and tetralin analogues, were found to show some
preference for the D2 receptor (high-affinity form) and

3344 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Birch et al.
Ta ble 1. Structures and Comparative in Vitro and in Vivo Activities for Target and Standard Compounds

Preparation of Potential Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3345
Ta ble 1 (Continued)
a
Ki values (nM). Values are from single experiments where dose-response curves were determined with at least six concentrations of
b
compound, and each displacement was measured in triplicate. Ki values (nM) ( SEM. Percentages are for displacement of radioligand
at 10-6 M. c ED50 values (mg/kg po) for antagonism of apomorphine-induced climbing in the mouse. Percentages are for reduction in
climbing at dose given. Duration is defined by the time at which level of climbing has returned to 50% of control following a dose of 2 ×
d
ED50
.
ED50 values (mg/kg ip) for induction of catalepsy in the rat. IA, inactivescausing <50% reduction of climbing at highest dose; NT,
not tested.
Sch em e 1
Sch em e 3
benzodioxin, giving the ester 37, which on base hydrol-
ysis gave the carboxylic acid 38. Subsequent mixed
anhydride formation with ethyl chloroformate and reac-
tion with 2-methoxyaniline gave the amide 39, which
was reduced with borane-dimethyl sulfide complex to
furnish the target compound 22 (Scheme 6).
Sch em e 2
The open-chain analogue 10 was synthesized by the
route described in Scheme 7. Reduction of 2,3-dihydro-
1,4-benzodioxin-2-carbonitrile with LiAlH4 afforded the
methylamine 40 which, when reacted with N-(3-bro-
mopropyl)phthalimide, gave intermediate 41. Subse-
quent cleavage of the phthalimide group with hydrazine
produced the primary amine 42, which upon reaction
with 2-chloropyrimidine gave a mixture of the desired
target compound 10 and the bis(2-pyrimidinyl) deriva-
tive 43.
Compound 11 was prepared in 46% yield by direct
methylation of 1 using a mixture of sodium formate,
formaldehyde, and formic acid. The 8-hydroxy derivative
5 was prepared by hydrolysis of the corresponding
8-tosyloxy compound 49 (itself obtained via ditosylate
48) using potassium hydroxide in ethanol (Scheme 8).
The amine 26h was prepared by direct reaction of
2-chloropyridine with 4-(aminomethyl)piperidine 36a ,
whereas amine 26i was prepared from 2-chloropyrimi-
dine and the imine 36b followed by acid hydrolysis, as
depicted in Scheme 4.
In the pyrimidine series, analogues in which the
bridging aminomethylpiperidine group was reversed
were prepared by reaction of tosylate 25a with the
protected amine 45, followed by hydrolysis to the
primary amine and reaction with 2-chloropyrimidine
(Scheme 5). Preparative chiral HPLC then gave enan-
tiomers 8 and 9. The corresponding 2-methoxyphenyl
analogue was prepared by alkylation of ethyl 4-piperidi-
necarboxylate with 2-(chloromethyl)-2,3-dihydro-1,4-
Biologica l Resu lts
Compound library screening gave the lead N-substi-
tuted (2,3-dihydro-1,4-benzodioxin-2-yl)methylamine de-
rivative 1 and its enantiomer 2, originally prepared for
another CNS project, which showed moderate to good

3346 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Birch et al.
Sch em e 4
Sch em e 6
Sch em e 7
Sch em e 5
Sch em e 8
affinities for rat D2-like and 5-HT1A receptors, with the
(-)-isomer 1 being the more active (Table 1). Compound
1 also showed moderate activity in the mouse apomor-
phine climbing test (AC), a model of antipsychotic
activity, having an ED50 of 9 mg/kg after oral admin-
istration. The (+)-isomer was less active, giving a 77%
reduction in climbing at 50 mg/kg.
does not result from selective attenuation of postsyn-
aptic dopamine receptor function in the limbic area of
the brain.
Compound 1 caused a relatively weak induction of
catalepsy3 in rats, the ED50 value (Table 1) being 21
times greater than that for antagonism of the limbic
amphetamine-induced locomotion effects. At 30 mg/kg
Further in vivo profiling of 1, in the two-component
amphetamine stereotypy test,3 showed the compound
to be approximately equipotent in antagonizing the
effects of amphetamine-induced dopamine release in the
limbic and striatal regions of rat brain (ED50 values of
2 mg/kg and 1.5 mg/kg, respectively). This indicates that
the relative lack of EPS potential shown by 1 (see below)

Preparation of Potential Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3347
1 caused a significant 20% reduction in mouse brain
levels of the 5-HT precursor 5-HTP, indicating that the
compound has agonist activity at 5-HT1A receptors.
As the classical antipsychotic haloperidol, a D2-like
antagonist with little or no 5-HT1A receptor affinity,
induces catalepsy in rats with an ED50 of only 4 times
greater than that in the limbic amphetamine-induced
locomotion test, the results for 1 strongly suggested that
combining D2-like antagonism with 5-HT1A (partial)
agonism in a single molecule would give effective
antipsychotic compounds with a reduced propensity to
induce extrapyramidal side effects.
The relatively high R1 affinity of 1 was seen as a
shortcoming, as this would be likely to lead to unwanted
cardiovascular effects, including postural hypotension.
In addition to reduced R1 affinity, greater potency in vivo
and a further reduction in cataleptic potential were also
considered desirable. A further requirement for a de-
velopment compound was that it should have the
potential for once or twice daily dosing in man. As a
predictor of this, greater than 6 h duration (see Table 1
for definition) would be required in the AC test. There-
fore, further analogues of compound 1 were prepared
in order to address these concerns.
Substitution in the aromatic ring of the dihydroben-
zodioxin system was not explored extensively in this
pyrimidine-containing subseries, although 5- and 8-meth-
oxy substituents were found to have opposite effects on
receptor affinity. The racemic 8-methoxy compound 3
had significantly higher affinity for D2-like, 5-HT1A, and
R1 receptors than 1, and it was correspondingly more
active in AC. The 5-methoxy compound 4, in contrast,
had much reduced affinity for these three receptors. The
8-hydroxy compound 5 also had good affinity for D2-like
and 5-HT1A receptors, but was only weakly active in AC.
Introduction of a methyl substituent into the 2- or
3-position of the dihydrobenzodioxin system, as in
compounds 6 and 7, led to significant loss of receptor
affinities.
Reversal of the bridging 4-(aminomethyl)piperidine
grouping found in 1 gave the enantiomers 8 and 9,
which showed receptor binding profiles similar to 1 and
2, respectively. Incorporation of an acyclic bridge, as in
the racemate 10, also led to activity similar to that of
1, both in receptor affinities and in AC activity.
N-Methylation of 1, to give the tertiary amine 11, led
to significant loss of receptor affinities.
binding profiles, with the 4- and 2-isomers showing
significant in vivo activity. The (-)-enantiomer 18 of the
2-methoxy compound showed acceptable potency and
duration of action in the AC test. This compound also
showed good activity in antagonizing limbic amphet-
amine-induced stereotypy (ED50 4.4 mg/kg), combined
with only a moderate ability to induce catalepsy. The
corresponding (+)-isomer 19 was again less active both
in vitro and in vivo.
A concern with the methoxy substituted compounds
was their propensity for metabolic demethylation, which
could have adverse effects on the compound’s pharma-
cokinetics. Investigation of heterocyclic alternatives to
2-methoxyphenyl, which may be more stable metaboli-
cally, led to the 2,3-dihydrobenzofuran 20 and the 2,3-
dihydro-1,4-benzodioxin 21. Disappointingly, both had
inferior activity in AC.
Reversal of the 4-(aminomethyl)piperidyl bridging
group gave racemate 22, which had significantly re-
duced activity compared to compound 18, consistent
with the pyrimidine series.
At the time of this work, the only suggestions in the
literature of how to obtain selectivity over R1 activity
in this class of compounds was through 7,8-benzo-
fusion.14 Therefore, substitution in these two positions
was explored in order to determine whether selectivity
over R1 could be improved. As reported above, incorpo-
ration of an 8-methoxy substituent into the pyrimidine
subseries actually increased affinity for R1 receptors. A
similar effect was seen in the 2-methoxyphenyl contain-
ing subseries, where introduction of an 8-fluoro sub-
stituent (compound 23) led to good activity in vitro and
in vivo. The relatively high R1 affinity of this compound,
however, precluded its further development, despite its
low cataleptic activity. Introduction of a 7-chloro sub-
stituent (compound 24) was found to give a more useful
improvement, as affinity for rat R1 receptors was not
too high. Indeed, when human receptors were examined,
selectivity for D2, D3, and 5-HT1A receptors over R1
receptors was much improved (see later). In addition,
the duration of action of 24 in AC was now greater than
6 h, and the ED50 for catalepsy was increased to 188
mg/kg, 100-fold greater than its ED50 for antagonism
of limbic amphetamine-induced locomotor activity, 1.8
mg/kg. The corresponding ratios for haloperidol, cloza-
pine, olanzapine, and risperidone are 4, 9, 10, and 22,
respectively (Figure 1). Because compound 24 had a
desirable receptor binding profile and showed good in
vivo activity, it was selected for further evaluation.
It seemed at this point that neither substituent
variation in the benzodioxin ring nor alternative con-
figurations of the bridging portion of the side chain
would give us the potency and selectivity we required.
Alternatives to the 2-pyrimidinyl grouping were there-
fore explored, in the form of the 2-pyridyl containing
enantiomers 12 and 13, and the phenyl and 2-chlo-
rophenyl containing racemates 14 and 15. Although
receptor affinities were maintained, activity in AC was
moderate at best. It was not clear which of many
potential factors, including degree of functional activity
at D2 and 5-HT1A receptors and bioavailability, were
adversely affecting the in vivo activity of these com-
pounds, but it seemed worthwhile to try to mimic the
H-bond acceptor properties of the pyrimidines with
alkoxyphenyl groups. Thus, the 4-, 3-, and 2-methox-
yphenyl analogues 16-19 were found to show similar
Against a panel of other neurotransmitter receptors,
including 5-HT1A and R1 receptors from postmortem
human brain (Table 2), 24 was found to have high
affinity only for hD2L, hD3, and h5-HT1A subtypes, with
moderate affinity being shown for hD4.2, 5-HT7, and
5-HT2C. The encouraging separation between affinities
shown by 24 for human D2-like and 5-HT1A versus R1
receptors suggests postural hypotension is unlikely to
be significant in clinical studies with this compound.
Compound 24 increased levels of the dopamine me-
tabolites DOPAC and HVA in the rat brain with ED200
of 2 mg/kg for DOPAC in both limbic and striatal tissue,
thereby demonstrating functional dopamine antago-
nism. Also in rat brain, 24 reduced levels of the 5-HT

3348 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Birch et al.
F igu r e 1. Comparison of the effects on catalepsy and amphetamine hyperlocomotion for BTS 79 018 (24) and reference compounds.
Key: percent catalepsy (2-2), percent inhibition of amphetamine hyperlocomotion (9‚‚‚9), therapeutic index (extrapolated for
24) (T). Compound 24, like the comparator antipsychotics, potently inhibits amphetamine-induced hyperlocomotion in rats after
ip administration; but unlike the other drugs, it shows a very low propensity to induce catalepsy. The ratio of the ED50 of a drug
to attenuate amphetamine-induced hyperlocomotion (highly predictive of antipsychotic efficacy) to its ED50 to induce catalepsy
(highly predictive of movement disorder liability) provides a therapeutic index for the drug. The ratios were calculated from the
following ED50 values (mg/kg with 90% confidence limits). Compound 24: hyperlocomotion 1.8 (0.78-3.9); catalepsy 190 (79-
450). Clozapine: hyperlocomotion 3.9 (1.7-8.8); catalepsy 34 (19-160). Haloperidol: hyperlocomotion 0.08 (0.03-0.22); catalepsy
0.44 (0.15-1.4). Olanzapine: hyperlocomotion 0.63 (0.28-1.4); catalepsy 6.1 (3.1-12). Risperidone: hyperlocomotion 0.078 (0.029-
0.21); catalepsy 1.8 (0.82-3.8). Compound 24 clearly has the best therapeutic index of any drug tested; as expected haloperidol
has the worst (24 . risperidone > olanzapine > clozapine > haloperidol).
Ta ble 2. Further Receptor Binding Affinities for 24 (BTS 79 018)a
a
Ki values (nM) ( SEM.
precursor 5-HTP by 25% at a dose of 30 mg/kg, consis-
tent with an agonist effect at 5-HT1A receptors. Com-
pound 24 showed no effect in the Porsolt test at 100
mg/kg, but reversed the effect of 8-OH-DPAT in this
test, with an ED50 of 11.9 mg/kg. Taken together these
data demonstrate in vivo 5-HT1A partial agonist activity.
Compound 24, BTS 79 018, demonstrated the requi-
site activity in rodent models of antipsychotic activity,
together with a predicted low propensity to cause
extrapyramidal and cardiovascular side effects. It has,
therefore, been selected for clinical studies to determine
its potential as a novel atypical antipsychotic therapeu-
tic agent.
Con clu sion
Synthesis of analogues of the lead N-substituted (2,3-
dihydro-1,4-benzodioxin-2-yl)methylamine derivative 1
resulted in the optimization of in vitro receptor binding
activity and in vivo activity in rodent models of psycho-
sis, leading to compound 24, which had the required

Preparation of Potential Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3349
(dd, J ) 11.1, 2.7 Hz, 1H), 4.06 (m, 1H), 3.41 (m, 1H), 2.96 (t,
J ) 4.6 Hz, 1H), and 2.78 (m, 1H).
good affinities for human D2/D3 and 5-HT1A receptors,
with significantly lower affinity for human R1 adreno-
ceptors and rat H1 and muscarinic receptors. In rodents,
24 showed functional D2-like antagonism and 5-HT1A
partial agonism. After oral dosing most analogues
showed good activity in rodent antipsychotic tests. This
was combined in compound 24 with a long duration of
action and very little potential to cause EPS, as mea-
sured by its ability to induce catalepsy in rats only at
very high doses compared to doses effective in antipsy-
chotic models. In light of this promising profile of
activity, 24 has been selected for clinical investigation
as a novel antipsychotic agent with a predicted low
propensity to cause EPS.
3-Chloroperoxybenzoic acid (86%; 9.20 g, 45.87 mmol) was
added to a stirred solution of 31a (8.10 g, 38.11 mmol) in
dichloromethane (100 mL), and the mixture was heated under
reflux for 20 h and then cooled to room temperature. The
resulting solid was filtered off and washed with dichlo-
romethane. The filtrate was washed with saturated aqueous
sodium metabisulfite solution (100 mL), saturated aqueous
sodium bicarbonate solution (2 × 100 mL), and brine (100 mL),
dried, and concentrated under reduced pressure to give (R)-
5-chloro-2-(2,3-epoxypropoxy)phenyl formate 32a (7.98 g, 92%)
as an orange oil: IR (film) 1745 cm-1; 1H NMR (CDCl3) δ 8.25
(s, 1H, OCHO), 7.19 (dd, J ) 8.8, 2.6 Hz, 1H, aromatic H),
7.12 (d, J ) 2.6 Hz, 1H, aromatic H), 6.96 (d, J ) 8.8 Hz, 1H,
aromatic H), 4.26 (dd, J ) 8.0, 2.9 Hz, 1H), 3.95 (m, 1H), 3.30
(m, 1H), 2.88 (t, J ) 4.7 Hz, 1H), and 2.72 (m, 1H).
Exp er im en ta l Section
A solution of the epoxide 32a (5.67 g, 24.81 mmol) in
methanol (80 mL) was added slowly to a stirred solution of
sodium methoxide, prepared from sodium (0.65 g, 28.26 mmol)
and methanol (50 mL) under nitrogen. The resulting solution
was stirred overnight at room temperature and concentrated
under reduced pressure, and the residue was partitioned
between ether and water. The ether layer was then washed
with brine, dried, and concentrated under reduced pressure
to give (S)-(7-chloro-2,3-dihydro-1,4-benzodioxin-2-yl)methanol
27f (4.40 g, 88%) as a pale-yellow solid, mp 61-62 °C: 1H NMR
(CDCl3) δ 6.90 (d, J ) 1.5 Hz, 1H, aromatic H), 6.80 (s, 2H,
aromatic H), 4.24 (m, 2H), 4.11 (m, 1H), 3.86 (m, 2H), and 1.96
(t, J ) 6.3 Hz, 1H, OH).
(S)-(8-F lu or o-2,3-d ih yd r o-1,4-ben zod ioxin -2-yl)m eth a -
n ol (27g). Alcohol 27g was prepared following the same
procedure as that for 27f starting from 6-fluorosalicylaldehyde
30b, which was synthesized by boron tribromide demethyla-
tion of 6-fluoro-2-methoxybenzaldehyde, which in turn was
prepared by lithiation of 3-fluoroanisole and subsequent
reaction with dimethylformamide following the procedure of
Bridges et al.28
(R,S)-(5-Met h oxy-2,3-d ih yd r o-1,4-b en zod ioxin -2-yl)-
m eth a n ol (27h ) a n d (R,S)-(8-Meth oxy-2,3-d ih yd r o-1,4-
ben zod ioxin -2-yl)m eth a n ol (27i). Reaction of 3-methoxy-
catechol (60.2 g, 0.42 mol) with ethyl 2,3-dibromopropionate
(90.0 g, 0.50 mol) and potassium carbonate (129.0 g, 0.94 mol)
in acetone, using the literature conditions,24 gave a mixture
of 5-methoxy and 8-methoxy-2,3-dihydro-1,4-benzodioxin-2-
carboxylic acid ethyl esters 28a and 28b (83.8 g, 84%) which,
by GC analysis, were in the approximate ratio of 2:1. A portion
of the mixture (65.0 g, 0.27 mol) in absolute ethanol (160 mL)
was treated with aqueous ammonia solution (SG 0.880; 500
mL) and the mixture stirred at room temperature for 90 min.
The resulting white solid was collected by filtration, washed
with a little water, and dried to give a mixture of the two
carboxamides 29a and 29b (43.45 g, 77%). The mixture was
then heated in boiling absolute ethanol (400 mL), and the
insoluble material was collected by filtration and then once
again heated in boiling absolute ethanol (25 mL). The resulting
white solid was collected by filtration, washed with ethanol
(20 mL), and dried to give 29a (18.55 g, 33%) as a white solid,
mp 196-198 °C; 97.3% pure by HPLC (S5 C8, MeCN/TEAF
buffer 20:80, λ ) 210 nm).
Ch em istr y. Proton magnetic resonance spectra were re-
corded on Bruker AM 360 or AC 250 spectrometers and are
reported in ppm on the δ scale from internal tetramethylsilane.
Infrared spectra were obtained using a Mattson-Unicam 3000
FTIR spectrometer. Elemental analyses were determined by
the Physical Chemistry Department (Knoll Pharmaceuticals,
Nottingham). Where analyses are indicated by the symbols of
the elements, analytical results were within (0.4% of the
theoretical values. Melting points were determined on a
Gallenkamp melting point apparatus and are uncorrected.
Thin-layer chromatography was performed using Merck silica
gel 60 F254 plates. Flash column chromatography was carried
out on silica 60 A. The term “dried” refers to use of anhydrous
magnesium or sodium sulfate. A general description of the
synthetic procedure is given where applicable.
P r ep a r a t ion of (2,3-Dih yd r o-1,4-b en zod ioxin -2-yl)-
m eth yl 4-Tolu en esu lfon a tes 25a -i, 48. Gen er a l P r oce-
d u r e for th e 4-Tolu en esu lfon yla tion of (2,3-Dih yd r o-1,4-
ben zod ioxin -2-yl)m eth a n ols 27a -i, 47, e.g., (R)-(8-F lu or o-
2,3-dihydro-1,4-benzodioxin-2-yl)methyl4-Toluenesulfonate
25. A stirred solution of (S)-(8-fluoro-2,3-dihydro-1,4-benzo-
dioxin-2-yl)methanol 27g (3.85 g, 20.9 mmol) in pyridine (50
mL) was treated with 4-toluenesulfonyl chloride (4.10 g, 21.6
mmol), and the resulting solution was stirred overnight at
room temperature. The reaction mixture was poured into
excess hydrochloric acid (5 M) and extracted with ethyl acetate.
The combined extracts were washed with brine, dried, and
concentrated under reduced pressure to give 25g (4.10 g,
58%): 1H NMR (DMSO-d6) δ 7.77 (d, J ) 8.3 Hz, 2H,
4-toluenesulfonyl m-H), 7.42 (d, J ) 8.3 Hz, 2H, 4-toluene-
sulfonyl o-H), 6.75 (m, 2H, aromatic H), 6.58 (m, 1H, aromatic
H), 4.48 (m, 1H), 4.25 (m, 3H), 4.04 (m, 1H), and 2.38 (s, 3H,
Me).
P r ep a r a t ion of (2,3-Dih yd r o-1,4-b en zod ioxin -2-yl)-
m eth a n ols 27b-i, 47. 2,3-Dihydro-1,4-benzodioxin-2-ylmeth-
anols 27b,20 27c,20 27d ,21 27e,22 and 4723 were synthesized
according to literature procedures. Compound 27a was pur-
chased from a commercial supplier.
(S)-(7-Ch lor o-2,3-d ih yd r o-1,4-ben zod ioxin -2-yl)m eth a -
n ol (27f). A stirred solution of 5-chlorosalicylaldehyde 30a
(8.92 g, 57.0 mmol) in dry dimethylformamide (200 mL) was
treated with potassium carbonate (7.87 g, 57.0 mmol) and (R)-
glycidyl 4-toluenesulfonylate (13.0 g, 57.0 mmol) and the
mixture heated with stirring at 60 °C for 5 h. The mixture
was then cooled and water (200 mL) added. The resulting
mixture was extracted with ether (3 × 150 mL), and the
combined extracts were washed with brine, dried, and con-
centrated under reduced pressure to give an oil. The residue
was purified by flash column chromatography on silica eluting
with a 25:1 mixture of petroleum ether (bp 40-60 °C) and ethyl
acetate and then a 10:1 mixture of petroleum ether (bp 40-
60 °C) and ethyl acetate to give (R)-5-chloro-2-(2,3-epoxypro-
poxy)benzaldehyde 31a (8.10 g, 67%) as a colorless oil which
solidified on standing: 1H NMR (CDCl3) δ 10.45 (s, 1H, CHO),
7.80 (d, J ) 2.7 Hz, 1H, aromatic H), 7.48 (dd, J ) 8.9, 2.7 Hz,
1H, aromatic H), 6.97 (d, J ) 8.9 Hz, 1H, aromatic H), 4.40
The filtrates from all of the ethanol digestions were com-
bined and concentrated under reduced pressure. The residue
was then purified by preparative HPLC (HP 1040M, sherisorb
5 µm C8, MeCN/TEAF buffer 20:80, λ ) 205 nm) to give 29b
(8.10 g, 14%) as a white solid, mp 184-187 °C.
A stirred solution of the amide 29a (18.40 g, 0.088 mol) in
absolute ethanol (330 mL) was saturated with gaseous hydro-
gen chloride, and the mixture was heated under reflux for 16
h. The cooled reaction mixture was filtered, and the filtrate
was concentrated under reduced pressure. The residue was
redissolved in ether, washed successively with water, 1 M
aqueous sodium hydroxide solution, and water, dried, and
concentrated under reduced pressure to give 28a (18.55 g, 88%)
as an oil, 97.5% pure by GC (3 ft, 10% OV-7).

3350 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Birch et al.
A solution of the ester 28a (18.54 g, 0.077 mol) in dry ether
(100 mL) was added dropwise to a stirred suspension of lithium
aluminum hydride (2.0 g, 0.053 mol) in dry ether (150 mL)
while the temperature of the reaction was maintained between
10 and 20 °C. The mixture was stirred at room temperature
for 2 h and cooled to 5 °C, and then water (5 mL) was added
cautiously. Aqueous sodium hydroxide solution (1 M; 12 mL)
and water (12 mL) were then added, and the mixture was
filtered through Celite. The filtrate was extracted with ether,
and the combined extracts were washed with water, dried, and
concentrated under reduced pressure to give (R,S)-(5-methoxy-
2,3-dihydro-1,4-benzodioxin-2-yl)methanol 27h (12.50 g, 79%).
The above procedure was repeated for 29b (8.0 g, 0.038 mol)
giving 27i (3.25 g, 36%), 95.69% pure by GC (OV-7).
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Am in es
26a-g. 1-(1-P h en yl-4-piper idyl)m eth ylam in e (26a). A mix-
ture of 1-chloro-2,4-dinitrobenzene (400 g, 1.98 mol) and
isonicotinamide (200 g, 1.64 mol) was heated at 95 °C for 1 h.
The resulting solid material was cooled, collected by filtration,
and washed with a 10:1 solution of ether and methanol. The
solid was then triturated with hot methanol (1000 mL),
collected, and dried to give 4-carbamoyl-1-(2,4-dinitrophenyl)-
pyridinium chloride 33 (449.1 g, 84%) as a cream solid, mp
233-36 °C: IR 1687 cm-1; 1H NMR (DMSO-d6) δ 9.58 (d, J )
6.8 Hz, 2H), 9.13 (d, J ) 2.5 Hz, 1H, aromatic H), 9.05 (br s,
1H, NH), 9.01 (dd, J ) 8.7, 2.5 Hz, 1H, aromatic H), 8.75 (d,
J ) 6.8 Hz, 2H, aromatic H), and 8.44 (d, J ) 8.7 Hz, 2H,
aromatic H).
A mixture of 33 (50.0 g, 0.15 mol) and aniline (35.60 g, 0.38
mol) in methanol (1000 mL) was stirred at room temperature
for 48 h. The resulting suspension was heated at 50 °C for 1
h, cooled, and concentrated under reduced pressure. The solid
residue was triturated with acetone (2 × 1000 mL), filtered,
and dried to give 4-carbamoyl-1-phenylpyridinium chloride 34a
(33.24 g, 92%) as an off-white solid, mp 290-292 °C: IR 1681
cm -1; 1H NMR (DMSO-d6) δ 9.56 (d, J ) 6.9 Hz, 2H, aromatic
H), 9.12 (br s, 1H, NH), 8.67 (d, J ) 6.9 Hz, 2H, aromatic H),
8.41 (br s, 1H, NH), 7.92 (m, 2H, aromatic H), and 7.76 (m,
3H, aromatic H). Anal. (C12H11ClN2O) C, H, Cl, N.
A solution of the pyridinium salt 34a (10.0 g, 42.64 mmol)
in ethanol (250 mL) was hydrogenated at atmospheric pressure
using hydrogen over a 10% palladium upon carbon catalyst
(1.0 g). The catalyst was removed by filtration through Celite,
and the filtrate was concentrated under reduced pressure to
resulting solution, and the mixture was heated at 95 °C with
stirring for 18 h. The cooled reaction mixture was extracted
with hydrochloric acid (5 M; 2 × 200 mL), and the combined
extracts were heated at 95 °C for 6 h. The resulting cooled
acidic solution was washed with ether and then basified with
5 M aqueous sodium hydroxide solution. The basic solution
was extracted with ether, and the extracts were dried and
concentrated under reduced pressure to leave a brown oil. The
basic aqueous solution was also concentrated to dryness, and
the resulting solid mass was extracted with ether. The
combined extracts were dried and concentrated under reduced
pressure to afford a brown oil. The combined brown oils were
dissolved in ether and saturated with dry hydrogen chloride
gas. The solid obtained was recrystallized from industrial
methylated spirit to yield 4-(aminomethyl)-1-(2-pyrimidinyl)-
piperidine 1.6 hydrochloride 26i (16.50 g, 66%) as a yellow
solid, mp 245-248 °C: 1H NMR (DMSO-d6) δ 8.43 (d, J ) 4.9
Hz, 2H, pyrimidine 4,6-H), 8.24 (br s, 2H, NH2), 6.72 (t, J )
4.9 Hz, 1H, pyrimidine 5-H), 4.66 (br d, J ) 13.3 Hz, 2H), 2.98
(br t, J ) 12.2 Hz, 2H), 2.69 (m, 2H), 1.92 (m, 1H), 1.83 (br d,
J ) 13.3 Hz, 2H), and 1.17 (dq, J ) 12.2, 4.0 Hz, 2H). Anal.
(C10H16N4‚1.6HCl) C, H, N, Cl.
The amine 26h was prepared in a similar manner by direct
reaction of 4-(aminomethyl)piperidine with 2-chloropyridine
and sodium carbonate in iso-amyl alcohol.
Gen er a l P r oced u r e for Alk yla tion of Am in es 26a -i by
th e (2,3-Dih yd r o-1,4-ben zod ioxin -2-ylm eth yl 4-Tolu en e-
su lfon a tes 25a -i, 48, e.g., P r ep a r a tion of N-(2,3-Dih y-
d r o-1,4-b en zod ioxin -2-ylm et h yl)-1-(1-p h en ylp ip er id -4-
yl)m eth yla m in e Dih yd r och lor id e (14). A stirred mixture
of the 4-toluenesulfonylate 25a (10.10 g, 0.032 mol), the amine
26a (6.0 g, 0.032 mol), and potassium carbonate (15.0 g, 0.11
mol) in acetonitrile (200 mL) was heated under reflux for 72
h and then cooled to room temperature. The resulting mixture
was then filtered, and the filtrate was concentrated under
reduced pressure. The residue was dissolved in ethyl acetate
and purified by elution through a silica pad, using ethyl acetate
as eluant, to give an oil after evaporation of solvent. The oil
was dissolved in ether and saturated with hydrogen chloride.
The resulting solid was collected by filtration and triturated
with a 25:1 solution of ethyl acetate in methanol to give N-(2,3-
dihydro-1,4-benzodioxin-2-yl)-1-(phenylpiperid-4-yl)methy-
lamine dihydrochloride (14) (5.64 g, 43%), mp 278-280 °C:
1H NMR (DMSO-d6) δ 9.75 (br s, 1H, HCl), 9.55 (br s, 1H, HCl),
7.87 (br s, 2H aromatic H), 7.50 (m, 3H aromatic H), 6.89 (m,
4H aromatic H), 4.81 (m, 1H), 4.44 (dd, J ) 11.7, 2.3 Hz, 1H),
4.08 (dd, J ) 11.7, 6.6 Hz, 1H), 3.30 (m, 8H) and 2.09 (m, 5
H). Anal. (C21H26N2O2‚2HCl) C, H, Cl, N.
give 1-phenylpiperidine-4-carboxamide hydrochloride (35a )
-1
(8.79 g, 86%) as a solid: IR 1679 cm
.
The above amide hydrochloride 35a (1.50 g, 6.24 mmol) was
added portionwise to a stirred suspension of lithium aluminum
hydride (0.50 g, 13.15 mmol) in dry tetrahydrofuran (100 mL),
and the mixture was stirred at room temperature for 2 h and
then at reflux for 2 h. The mixture was cooled, and water (0.5
mL) was carefully added. Concentrated aqueous sodium
hydroxide solution (12.5 M; 0.5 mL) was then added, and the
resulting precipitate was filtered through Celite. The filtrate
was then dried and concentrated under reduced pressure to
give 1-(1-phenyl-4-piperidyl)methylamine 26a (1.15 g, 97%) as
an oil: 1H NMR (CDCl3) δ 7.23 (m, 2H, aromatic H), 6.96 (d, J
) 7.0 Hz, 2H, aromatic H), 6.82 (br t, J ) 7.0 Hz, 1H, aromatic
H), 3.70 (br d, J ) 12.6 Hz, 2H), 2.63 (m, 4H), 1.81 (br d, J )
9.8 Hz, 2H), and 1.39 (m, 5H).
The remaining amines (26b-g) were synthesized in the
same manner. 5-Amino-2,3-dihydro-1,4-benzodioxin (precursor
to 26e) was prepared according to the literature method,29 and
7-amino-2,3-dihydrobenzo[b]furan (precursor to 26d ) was pre-
pared by Raney nickel reduction of the corresponding known
7-nitro-2,3-dihydrobenzo[b]furan.30
Compounds 1-4, 6-7, 12-13, 15-21, 23-24, and 49 were
prepared by similar procedures.
For chiral compounds 1 and 18, the (R)-4-toluenesulfonylate
25b, prepared from the (S)-alcohol 27b, was used in the
reaction with the appropriate amine. Similarly, for 2 and 19
the (S)-4-toluenesulfonylate 25c, prepared from the (R)-alcohol
27b, was employed. The enantiomers 12 and 13 were sepa-
rated by chiral HPLC (Chiralcel OC column eluting with
hexane/ethanol 1:1 at 5 then 10 mL/min). Specific rotations
and, where determined, HPLC derived enantiomeric excesses
for all chiral target compounds are given below:
1 [R]D -51.2° (c ) 1.0, MeOH) 85.4% e.e. (Chiralcel OC,
hexane/ethanol 1:1); 2 [R]D +49.5° (c ) 1.0, MeOH) 84.8% e.e.
(Chiralcel OC, hexane/ethanol 1:1); 8 [R]D -63.0° (c ) 1.0,
MeOH) 100% e.e.; 9 [R]D +63.0° (c ) 1.0, MeOH) 100% e.e.;
12 [R]D -63.0° (c ) 1.0, MeOH) 100% e.e. (Chiralcel OC,
hexane/ethanol 1:1); 13 [R]D +63.0° (c ) 1.0, MeOH) 100% e.e.
(Chiralcel OC, hexane/ethanol 1:1); 18 [R]D -27.0° (c ) 1.0,
CH2Cl2); 19 [R]D +25.3° (c ) 1.0, CH2Cl2); 23 [R]D -49.2° (c )
1.0, MeOH); 24 [R]D -42.6° (c ) 0.5, EtOH) 99.8% e.e.
(Chiralcel OD, isohexane/2-propanol 1:1 containing 0.1% di-
ethylamine).
P r ep a r a tion of th e Am in es (26h -i). 4-(Am in om eth yl)-
1-(2-p yr im id in yl)p ip er id in e (26i). Acetophenone (24.0 g, 0.2
mol) and 4-toluenesulfonic acid (0.40 g, 2.32 mmol) were added
to a stirred solution of 4-(aminomethyl)piperidine (22.8 g, 0.2
mol) in dry toluene (200 mL), and the mixture was heated
under reflux for 5 h under a Dean and Stark apparatus to give
intermediate 36b, which was not isolated. 2-Chloropyrimidine
(22.9 g, 0.2 mol) and triethylamine (14 mL) were added to the
N-(8-Hydr oxy-2,3-dih ydr o-1,4-ben zodioxin -2-ylm eth yl)-
1-[1-(2-m eth oxyp h en yl)p ip er id -4-yl]m eth yla m in e (5). A
solution of potassium hydroxide (1.0 g, 17.8 mmol) in a mixture
of water (19.7 mL) and ethanol (19.7 mL) was added portion-

Preparation of Potential Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3351
wise to 2-({1-[1-(2-methoxyphenyl)piperid-4-yl]methylamino}-
methyl)-2,3-dihydro-1,4-benzodioxin-8-yl 4-toluenesulfonate 49
(0.59 g, 0.93 mmol), and the mixture was heated under reflux
with stirring for 2.5 h. The cooled mixture was neutralized
with glacial acetic acid and extracted with ether (3 × 100 mL).
The combined extracts were left to stand overnight, and the
resulting white solid was collected by filtration and dried to
give N-(8-hydroxy-2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-1-
[1-(2-methoxyphenyl)piperid-4-yl]methylamine 5 (0.20 g, 64%),
0.1EtOAc, 0.7H2O) C, H, Cl, N. The second product obtained
was N-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-N-(2-pyrim-
idinyl)-3-(2-pyrimidinylamino)propylamine dihydrochloride 43
(0.46 g, 17%), mp 50-55 °C: 1H NMR (DMSO-d6) δ 8.99 (br s,
1H, HCl), 8.61 (d, J ) 5.3 Hz, 2H, pyrimidine 4,6-H), 8.43 (d,
J ) 5.3 Hz, 2H, pyrimidine 4,6-H), 6.95 (t, J ) 5.3 Hz, 1H,
pyrimidine 5-H), 6.82 (m, 4 H, aromatic H), 6.73 (t, J ) 5.3
Hz, 1H, pyrimidine 5-H), 6.30 (br s, 1H, NH), 4.55 (m, 1H),
4.35 (dd, J ) 11.6, 2.2 Hz, 1H), 4.04 (dd, J ) 11.6, 6.8 Hz,
1H), 3.70 (m, 6 H), and 1.95 (quintet, J ) 6.4 Hz, 2H). Anal.
(C20H22N6O2‚2HCl, 0.2EtOAc, 1.4H2O) C, H, Cl, N.
1
mp 116-119 °C: IR 3552 cm -1; H NMR (DMSO-d6) δ 9.00
(br s, 1H, OH), 6.88 (m, 4H, aromatic H), 6.58 (t, J ) 8.1 Hz,
1H, aromatic H), 6.36 (dd, J ) 8.0, 1.5 Hz, 1H, aromatic H),
6.31 (dd, J ) 8.0, 1.5 Hz, 1H, aromatic H), 4.30 (dd, J ) 11.2,
2.1 Hz, 1H), 4.15 (m, 1H), 3.91 (dd, J ) 11.2, 7.6 Hz, 1H), 3.76
(s, 3H, OMe), 2.77 (m, 2H), 1.78 (br d, J ) 12.1 Hz, 2H), 1.51
(m, 1H), and 1.31 (m, 2H)sremaining signals are obscured by
water and DMSO peaks. Anal. (C22H28N2O4‚3.5H2O) C, H, N.
N-(2-P yr im id in yl)-3-[1-(2,3-d ih yd r o-1,4-ben zod ioxin -2-
yl]m eth yla m in o)p r op yla m in e (10). A suspension of 2,3-
dihydro-1,4-benzodioxin-2-carbonitrile31 (15.0 g, 0.093 mol) in
dry ether (50 mL) was added portionwise to a stirred suspen-
sion of lithium aluminum hydride (5.25 g, 0.14 mol) in dry
ether (100 mL). The mixture was then stirred and heated
under reflux for 1 h. The cooled mixture was carefully treated
with water (7 mL), aqueous sodium hydroxide solution (2 M;
7 mL), and then water (30 mL). The resulting mixture was
filtered, and the solid was washed with ethyl acetate and
water. The filtrate was separated, and the aqueous phase was
extracted with ethyl acetate. The extracts and the original
organic phase were combined, dried, and concentrated under
reduced pressure to give 1-(2,3-dihydro-1,4-benzodioxan-2-yl)-
methylamine 40 (12.20 g, 80%) as an orange oil, 98% pure by
GC (OV-7).
A mixture of the product 40 from the previous reaction (6.0
g, 0.036 mol), N-(3-bromopropyl)phthalimide (9.75 g, 0.036
mol), and potassium carbonate (9.94 g, 0.072 mol) in dry
acetonitrile (100 mL) was heated under reflux with stirring
for 24 h. The mixture was cooled and filtered, and the filtrate
was concentrated under reduced pressure to give an orange
gum. The gum was then stirred in hydrochloric acid (5 M; 100
mL) to give a brown solid which was recrystallized from
methanol to give the phthalimide hydrochloride 41 (7.10 g,
56%) as a cream solid, mp 225-300 °C. Anal. (C20H21ClN2O4)
H, N; C: calcd, 61.8; found, 61.2.
Hydrazine hydrate (0.46 mL, 9.30 mmol) was added to a
stirred suspension of the phthalimide 41 (3.00 g, 7.7 mmol) in
methanol (50 mL), and the mixture was heated under reflux
for 1 h. Concentrated hydrochloric acid (4 drops) was then
added, and the mixture was stirred at room temperature for
1 h. A colorless solid which separated was removed by
filtration, and the filtrate was concentrated under reduced
pressure. The resulting residue was triturated with ether to
give the propylamine dihydrochloride 42 (2.00 g, 88%) as a
cream solid, mp 235-240 °C; 97.3% pure by HPLC (Asahipak
ODP-50, MeCN/TEAF buffer 5:95, λ ) 249 nm).
A stirred mixture of the propylamine 42 (1.61 g, 7.25 mmol),
potassium carbonate (2.00 g, 14.5 mmol), 2-chloropyrimidine
(0.87 g, 7.25 mmol), and sodium iodide (0.1 g) in dry acetoni-
trile (60 mL) was heated at reflux under nitrogen for 5 days.
The cooled mixture was filtered, and the filtrate was concen-
trated under reduced pressure to give a viscous oil which was
purified by flash chromatography on silica using a 10:1 solution
of ethyl acetate and triethylamine as eluant to give two
products, both as colorless oils. The dihydrochloride salts of
these two products were prepared by dissolution in ether and
then saturation with gaseous hydrogen chloride. This gave
3-[1-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)amino]-N-(2-py-
rimidinyl)propylamine dihydrochloride 10 (0.80 g, 37%), mp
50-55 °C: 1H NMR (DMSO-d6) δ 9.91 (br s, 1H, HCl), 9.56
(br s, 1H, HCl), 8.96 (br s, 1H, NH), 8.61 (d, J ) 5.2 Hz, 2H,
pyrimidine 4,6-H), 6.87 (m, 5H, 4 aromatic H and pyrimidine
5-H), 4.74 (m, 1H), 4.42 (dd, J ) 11.7, 2.3 Hz, 1H), 4.08 (dd, J
) 11.7, 6.8 Hz, 1H), 3.57 (t, J ) 6.5 Hz, 2H), 3.35 (m, 1H),
3.10 (m, 4H), and 2.04 (m, 2H). Anal. (C16H20N4O2‚2HCl‚
(S)-N-(2,3-Dih ydr o-1,4-ben zodioxin -2-ylm eth yl)-N-m eth -
yl-1-[1-(2-p yr im id in yl)-4-p ip er id yl]m et h yla m in e d ih y-
d r och lor id e (11). A mixture of (S)-N-(2,3-dihydro-1,4-benzo-
d i o x a n - 2 - y l m e t h y l ) - 1 - [ 1 - ( 2 - p y r i m i d i n y l ) 4 -
piperidyl]methylamine 1 (0.23 g, 0.56 mmol), sodium formate
(0.13 g, 1.9 mmol), and formaldehyde (38-40% aqueous
solution; 7 mL) was treated with formic acid (3 mL) while the
temperature of the reaction was maintained between 5 and
10 °C. The mixture was then stirred at room temperature for
104 h, poured into water, and basified with 5 M aqueous
sodium hydroxide solution. The cooled solution was then
extracted with ether, washed with water, dried, and concen-
trated under reduced pressure to leave an oil (0.16 g). The oil
was dissolved in ether and treated with hydrogen chloride gas
giving (S)-N-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-N-meth-
yl-1-[1-(2-pyrimidinyl)-4-piperidyl]methylamine dihydrochlo-
ride (11) (0.11 g, 46%) as a white solid, mp 235-240 °C: IR
1
3417 and 2800-2400 cm -1; H NMR (CDCl3) δ 10.68 (br s,
1H), 8.41 (d, J ) 4.8 Hz, 2H, pyrimidine 4,6-H), 6.90 (m, 4H,
aromatic H), 6.68 (t, J ) 4.8 Hz, 1H, pyrimidine 5-H), 4.95
(m,1H), 4.65 (br d, J ) 13.0 Hz, 2H), 4.36 (d, J ) 11.4 Hz,
1H), 2.80-3.60 (m, 10H), 2.19 (m, 1H), 1.92 (m, 2H), and 1.16
(m, 2H), some peaks obscured by broad water peak δ 4.1. Anal.
(C20H28Cl2N4O2‚1.2H2O) C, H, N.
(-)-1-[1-(2,3-Dih yd r o-1,4-ben zod ioxin -2-ylm eth yl)p ip -
er id -4-yl]-N-(p yr im id in -2-yl)m eth yla m in e (8) a n d (+)-1-
[1-(2,3-Dih yd r o-1,4-ben zod ioxin -2-ylm eth yl)p ip er id -4-yl]-
N-(p yr im id in -2-yl)m eth yla m in e (9). A solution of 4-(amino-
methyl)piperidine (11.63 g, 0.1 mol) and 5-chlorosalicylalde-
hyde (16.0 g, 0.1 mol) in a 1:1 mixture of ethanol and methanol
(400 mL) was stirred at room temperature for 4.5 h. The
solution was then concentrated under reduced pressure to give
N-(5-ch lor o-2-h ydr oxyben zyliden e)-1-piper id-4-ylm et h y-
lamine (45) (25.7 g, 100%) as a yellow solid which was used
without purification.
A mixture of 45 (25.5 g, 0.1 mol) and the 4-toluenesulfony-
late 25a (16.97 g, 0.053 mol) in dry dimethyl sulfoxide (200
mL) was heated with stirring at 100 °C for 3 h. The cooled
mixture was poured into water (400 mL) and extracted with
ethyl acetate. The combined extracts were dried and evapo-
rated under reduced pressure to afford a brown oil which
solidified on standing. Recrystallization from ethanol furnished
1-[1-(1,4-benzodioxan-2-ylmethyl)piperid-4-yl]-N-(5-chloro-2-
hydroxybenzylidene)methylamine (46) (14.02 g, 66%) as a
yellow solid, mp 94-96 °C: 1H NMR (DMSO-d6) δ 13.71 (br s,
1H, OH), 8.52 (br s, 1H, NdCH), 7.55 (d, J ) 2 Hz, 1H,
aromatic H), 7.35 (dd, J ) 8 Hz, 2 Hz, 1H, aromatic H), 6.85
(m, 5H, aromatic H), 4.30 (m, 2H) 3.93 (m, 1H), 3.50 (d, J )
5.5 Hz, 2H), 2.93 (m, 2H), 2.55 (m, 2H), 2.03 (br q, J ) 12 Hz,
2H), 1.61 (m, 3H), and 1.26 (m, 2H).
A solution of the product from the previous reaction (14.0
g, 0.036 mol) in a 4:1 mixture of methanol and water (300 mL)
and hydrochloric acid (5 M; 45 mL) was stirred at room
temperature for 1.5 h. The mixture was then concentrated
under reduced pressure, and the resulting mixture was washed
with diethyl ether. The aqueous solution was then basified
with 5 M sodium hydroxide and extracted with ethyl acetate.
The combined extracts were dried and evaporated under
reduced pressure to give 1-[1-(1,4-benzodioxan-2-ylmethyl)-
piperid-4-yl]methylamine (8.00 g, 87%) as a brown oil: GC
96.6% (10% OV-7).
The amine product above (9.25 g, 0.035 mol) and 2-chloro-
pyrimidine (4.01 g, 0.035 mol) were added to a mixture of
toluene (300 mL) and triethylamine (14 mL), and the resulting

3352 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Birch et al.
solution was stirred and heated at 95 °C for 72 h. The cooled
mixture was evaporated, and the residue was dissolved in
water (100 mL). The solution was extracted with ethyl acetate,
and the combined extracts were dried and evaporated to leave
a brown oil. Trituration with diethyl ether afforded a brown
solid (1.55 g), mp 88-94 °C. The aqueous phase was re-
extracted with ethyl acetate, the combined extracts were dried
and evaporated, and the resulting brown oil was triturated
with isopropyl alcohol to give additional brown solid (0.93 g),
mp 94-96 °C. On standing, a solid precipitated from the
filtrate and was collected by filtration. The solid was washed
with diethyl ether to give a tan colored solid (1.01 g). The three
solids isolated above were combined and washed with diethyl
ether to give racemic 1-[1-(2,3-dihydro-1,4-benzodioxin-2-yl-
methyl)piperid-4-yl]-N-(pyrimidin-2-yl)methylamine (1.23 g,
10%) as a tan solid. The enantiomers were then separated by
preparative chiral HPLC (column Chiralcel OD, mobile phase
20% ethanol in cyclohexane) to give (-)-1-[1-(2,3-dihydro-1,4-
benzodioxin-2-ylmethyl)piperid-4-yl]-N-(pyrimidin-2-yl)methy-
lamine (0.44 g) as a solid, mp 88-90 °C: [R]D ) - 24.6° (c )
1.0, CH2Cl2); 1H NMR (DMSO-d6) δ 8.23 (d, J ) 5 Hz, 2H,
pyrimidine 4,6-H), 7.17 (t, J ) 6 Hz, 1H, HNCH2), 6.81 (m,
4H, aromatic H), 6.51 (t, J ) 5 Hz, 1H, pyrimidine 5-H), 4.29
(m, 2H), 3.93 (m, 1H), 3.15 (t, J ) 6 Hz, 2H), 2.91 (m, 2H),
2.54 (m, 2H), 1.95 (br q, J ) 12 Hz, 2H), 1.61 (m, 3H), and
1.20 (m, 2H), and (+)-1-[1-(2,3-dihydro-1,4-benzodioxin-2-
ylmethyl)piperid-4-yl]-N-(pyrimidin-2-yl)methylamine (0.52 g)
as a solid, mp 84-87 °C; which was less enantiomerically pure
as shown by its optical rotation [R]D ) +16° (c ) 1.0, CH2Cl2):
1H NMR (DMSO-d6) δ 8.23 (d, J ) 5 Hz, 2H, pyrimidine 4,6-
H), 7.17 (t, J ) 5 Hz, 1H, HNCH2), 6.82 (m, 4H, aromatic H),
6.51 (t, J ) 5 Hz, 1H, pyrimidine 5-H), 4.29 (m, 2H), 3.93 (m,
1H), 3.15 (t, J ) 6 Hz, 2H), 2.88 (m, 2H), 2.52 (m, 2H), 1.97
(br q, J ) 12 Hz, 2H), 1.62 (m, 3H), and 1.18 (m, 2H).
s, 1H, NH), 6.90 (m, 7H), 4.32 (m, 2H), 4.02 (m, 1H), 3.89 (s,
3H, OMe), 3.06 (m, 2H), 2.63 (m, 2H), 2.24 (m, 3H), and 1.90
(m, 4H).
A stirred solution of the amide 39 (1.69 g, 4.4 mmol) in dry
tetrahydrofuran (100 mL) was treated with borane-dimethyl
sulfide complex (1.0 M solution in tetrahydrofuran; 1.9 mL,
19 mmol), and the mixture was heated at reflux for 2 h. The
mixture was cooled, and the solvent was removed under
reduced pressure. Hydrochloric acid (2 M) was added to the
residue, and the mixture was heated at 95 °C for 90 min. The
mixture was cooled, basified with 5 M aqueous sodium
hydroxide solution, and extracted with dichloromethane. The
organic solution was washed with water, dried, and concen-
trated under reduced pressure. The residue was dissolved in
ethyl acetate, and a warm solution of oxalic acid (0.20 g, 16
mmol) in ethyl acetate was added. The resulting precipitated
solid was collected by filtration and dried to give 1-[1-(2,3-
dihydro-1,4-benzodioxin-2-ylmethyl)-4-piperidyl]-N-(2-methox-
yphenyl)methylamine oxalate 22 (0.78 g, 38%), mp 215-218
°C: IR 3424 cm-1; 1H NMR (DMSO-d6) δ 6.80 (m, 6H, aromatic
H), 6.51 (m, 2H, aromatic H), 4.63 (m, 1H), 4.29 (dd, J ) 11.5,
2.3 Hz, 1H), 4.00 (m, 1H), 3.77 (s, 3H, OMe), 3.35 (m, 2H),
3.10 (m, 2H), 2.99 (d, J ) 6.1 Hz, 2H), 2.73 (m, 2H), 1.83 (m,
3H), and 1.41 (m, 2H). Anal. (C22H28N2O3(COOH)2‚0.1H2O) C,
H, N.
Bin d in g Assa ys. Assay methods and radioligands for the
cloned human dopamine D1-5 receptors and animal derived
D2, 5-HT2A, R1, R2D, muscarinic, and H1 receptors were as
previously described.3 For percent displacement of radioligand
at 10-6 M the specific binding in the absence and presence of
test compound was determined. The percentage displacement
of specific binding was then calculated manually. For Ki
determinations, the IC50 values were calculated using the
iterative curve-fitting program EBDA. Ki values were then
calculated using the Cheng and Prusoff equation.
1-[1-(2,3-Dih yd r o-1,4-b en zod ioxin -2-ylm et h yl)-4-p ip -
er id yl]-N-(2-m eth oxyp h en yl)m eth yla m in e Oxa la te (22).
A stirred mixture of 2-(chloromethyl)-2,3-dihydro-1,4-benzo-
dioxin32 (20.0 g, 0.108 mol) and ethyl piperidine-4-carboxylate
(34.0 g, 0.217 mol) was heated at 130 °C for 3 h. The mixture
was then cooled, diluted with ether, and filtered. The filtrate
was concentrated under reduced pressure, and volatile byprod-
ucts were removed by vacuum distillation. The residual oil was
then purified by flash column chromatography on silica using
a 30:1 solution of dichloromethane and industrial methylated
spirit as eluant to give ethyl 1-(2,3-dihydro-1,4-benzodioxin-
2-ylmethyl)piperidine-4-carboxylate 37 (23.3 g, 71%) as a
brown oil. Anal. (C17H23NO4) C, H, N.
5-HT1A Recep tor Bin d in g Assa y in Ra t Br a in . 1. Mem -
br a n e P r ep a r a tion . Adult male CD rats were killed by
cervical dislocation, their brains removed, and hippocampii
(100-120 mg) immediately dissected. Tissue was homogenized
in ice-cold 50 mM Tris-HCl, pH 7.7 (at 25 °C, 1:40 w/v), using
a
motor-driven Teflon pestle (12 strokes, 800 rpm) and
centrifuged at 40000g for 10 min. To remove endogenous 5-HT,
the pellet was rehomogenized in 50 mM Tris-HCl, pH 7.7 (1:
40 w/v), incubated at 37 °C for 10 min, and then recentrifuged
at 40000g for 10 min. The final pellet was resuspended in 50
mM Tris-HCl, pH 7.7, containing 4 mM calcium chloride, 0.1%
L-ascorbic acid, and 10 µM pargyline hydrochloride (equivalent
to 6.25 mg wet weight of tissue/mL) and used immediately in
the binding assay. All centrifugations were carried out at 4
°C.
2. Bin d in g Assa y. Membranes (400 µL; equivalent to 2.5
mg wet weight of tissue/tube) were incubated with 50 µL of
[3H]8-OH-DPAT at a single concentration of 2 nM and 50 µL
of distilled water (total binding) or 50 µL of test compound
(10-6 M) or 50 µL of 5-HT (10 µM; nonspecific binding) for 30
min at 25 °C.
Membrane-bound radioactivity was recovered by filtration
under vacuum through Skatron 11734 filters using a Skatron
cell harvester. Filters were rapidly washed with ice-cold 50
mM Tris-HCl, pH 7.7 (wash setting 9,9,0), and radioactivity
determined by liquid scintillation counting (1 mL Packard MV
Gold scintillator).
5-HT1B Recep tor Bin d in g Assa y in P ig Br a in . 1. Mem -
br a n e P r ep a r a tion . Pig caudate was homogenized in ice-cold
50 mM Tris-HCl, pH 7.7 (at 25 °C, 1:100 w/v), using a motor-
driven Teflon pestle (8 strokes, 800 rpm) and centrifuged at
40000g for 10 min. The resulting pellet was resuspended in
50 mM Tris-HCl, pH 7.7 (1:100 w/v), incubated at 37 °C for
10 min to remove endogenous 5-HT, and then recentrifuged
at 40000g for 10 min. The final pellet was resuspended in 50
mM Tris-HCl, pH 7.7 (equivalent to 33.3 mg wet weight of
tissue/mL), and used immediately in the binding assay. All
centrifugations were carried out at 4 °C.
A solution of the ester 37 (5.0 g, 0.164 mol) in industrial
methylated spirit (100 mL) was added to a stirred solution of
potassium hydroxide (4.0 g, 0.071 mol) in water (50 mL), and
the mixture was stirred at room temperature for 1 h and then
at reflux for 6 h. The cooled solution was concentrated under
reduced pressure, and the residue was diluted with water and
neutralized with hydrochloric acid (5 M). The aqueous mixture
was then concentrated under reduced pressure, and the
residue was triturated with a 25:1 solution of dichloromethane
and methanol. The inorganic material was removed by filtra-
tion, and the filtrate was concentrated under reduced pressure
to give crude 1-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)pip-
eridine-4-carboxylic acid 38 (5.03 g).
Ethyl chloroformate (0.90 g, 8.30 mmol) was added dropwise
to a stirred solution of the carboxylic acid 38 (2.52 g, 8.97
mmol) and triethylamine (1.08 g, 10.70 mmol) in chloroform
(90 mL) at 0 °C. After 30 min a solution of 2-methoxyaniline
(1.08 g, 8.80 mmol) in chloroform (45 mL) was added, and the
mixture was stirred at room temperature overnight and then
at 50 °C for 1 h. The cooled mixture was then concentrated
under reduced pressure, and the residue was purified by flash
column chromatography on silica using a 1:1 mixture of ethyl
acetate and petroleum ether (bp 40-60 °C) as eluant to give
1-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-N-(2-methoxyphe-
nyl)piperidine-4-carboxamide 39 (1.05 g, 31%): 1H NMR
(CDCl3) δ 8.39 (dd, J ) 7.8, 1.7 Hz, 1H, aromatic H), 7.85 (br
2. Bin d in g Assa y. Membranes (300 µL; equivalent to 10

Preparation of Potential Atypical Antipsychotic Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17 3353
mg wet weight of tissue/tube) were incubated with 50 µL of
[3H]sumatriptan at a single concentration of 4 nM; 50 µL of
50 mM Tris-HCl, pH 7.7; 50 µL of 50 mM Tris-HCl, pH 7.7,
containing 50 mM calcium chloride, 100 µM pargyline hydro-
chloride, and 1% L-ascorbic acid; and 50 µL of distilled water
(total binding), or 50 µL of drug solution (10-6 M), or 50 µL of
5-HT (10 µM; nonspecific binding) for 45 min at 25 °C.
Membrane-bound radioactivity was recovered by filtration
under vacuum through Whatman GF/B filters, previously
soaked in 0.5% polyethylenimine using a Brandel cell har-
vester. Filters were rapidly washed with 16 mL of ice-cold 50
mM Tris-HCl, pH 7.7, and radioactivity was determined by
liquid scintillation counting (2 mL Packard MV Gold scintil-
lator).
5-HT2C Recep tor Bin d in g Assa y in P ig Br a in . 1. Mem -
br a n e P r ep a r a tion . Pig choroid plexus was homogenized in
ice-cold 0.32 M sucrose (1:30 w/v) with a Kinematic polytron
(speed setting 6 for 30 s) and centrifuged at 1000g for 10 min.
The supernatant was stored on ice, and the pellet was
rehomogenized in 0.32 M sucrose (1:15 w/v) and centrifuged
at 850g for 12 min. Combined supernatants were diluted to
1:80 w/v with ice-cold 50 mM Tris-HCl, pH 7.4 (at 25 °C), and
centrifuged at 40000g for 10 min. The resulting pellet was
resuspended in 50 mM Tris-HCl, pH 7.4 (1:80 w/v), preincu-
bated for 10 min at 37 °C to remove endogenous 5-HT, and
centrifuged at 40000g for 10 min. The final pellet was
resuspended in 50 mM Tris-HCl, pH 7.4, containing 4 mM
calcium chloride, 0.1% L-ascorbic acid, and 10 µM pargyline
hydrochloride (equivalent to 12.5 mg wet weight of tissue/mL).
All centrifugations were carried out at 4 °C.
2. Bin d in g Assa y. Membranes (800 µL; equivalent to 10
mg wet weight of tissue/tube) were incubated with 100 µL of
[3H]mesulergine at a single concentration of 1 nM and 100 µL
of distilled water (total binding) or 100 µL of drug solution
(10-6 M or at 10 concentrations ranging from 10-11-10-4 M)
or 100 µL of 5-HT (10 µM; nonspecific binding) for 30 min at
37 °C. Membrane-bound radioactivity was recovered by filtra-
tion under vacuum through Whatman GF/C filters presoaked
for 1 h in 0.5% polyethylenimine, using a Brandel cell
harvester. Filters were rapidly washed with 12 mL of ice-cold
50 mM Tris-HCl, pH 7.4, and radioactivity determined by
liquid scintillation counting (2 mL Packard MV Gold scintil-
lator).
5-HT3 Recep tor Bin d in g Assa y in Ra t Br a in . 1. Mem -
br a n e P r ep a r a tion . Rat entorhinal cortices were homog-
enized in ice-cold 50 mM HEPES (N-2-hydroxyethylpiperazine-
N′-2-ethanesulfonic acid) buffer (pH 7.4 at 4 °C; 1:10 w/v) using
a Soniprep 150 (setting 5-6 for 8 s) and centrifuged at 49000g
for 10 min. The resulting pellet was homogenized in 50 mM
HEPES buffer (1:10 w/v) and recentrifuged at 49000g for 10
min. The final pellet was resuspended in 50 mM HEPES
buffer, pH 7.4 (equivalent to 40 mg wet weight of tissue/mL),
and used immediately in the binding assay. All centrifugations
were carried out at 4 °C.
wet weight of tissue/tube) were incubated with 50 µL of [3H]-
GR 113808 at a single concentration of 0.1 nM and 50 µL of
50 mM HEPES buffer (total binding) or 50 µL of drug solution
(10-6 M) or 50 µL of 5-HT (30 µM; nonspecific binding) for 30
min at 37 °C. Membrane-bound radioactivity was recovered
by filtration under vacuum through Skatron 11734 filters,
presoaked for 1 h in 0.5% polyethylenimine, using a Skatron
cell harvester. Filters were rapidly washed with ice-cold 50
mM HEPES buffer, pH 7.4 (wash setting 9,9,0), and radioac-
tivity determined by liquid scintillation counting (1 mL Pack-
ard MV Gold scintillator).
Ra t Recom bin a n t 5-HT6 Recep tor Bin d in g Assa y. 1.
Mem b r a n e P r ep a r a t ion . Frozen membranes from SF9
insect cells, infected with baculovirus to express the rat
recombinant 5-HT6 receptor, were used. Membrane fragments
were thawed, resuspended in 50 mM Tris-HCl, pH 7.4 (at 25
°C), containing 10 mM magnesium sulfate and 0.5 mM
ethylenediaminetetraacetic acid (EDTA) (equivalent to 35 µg
of membrane protein/mL), and used immediately in the
binding assay.
2. Bin d in g Assa y. Membranes (400 µL; equivalent to 14
µg of membrane protein/tube) were incubated with 50 µL of
[3H]lysergic acid diethylamide ([3H]LSD) at a single concentra-
tion of 3 nM and 50 µL of distilled water (total binding) or 50
µL of drug solution (10-6 M) or 50 µL of methiothepin (10 µM;
nonspecific binding) for 90 min at 27 °C. Membrane-bound
radioactivity was recovered by filtration under vacuum through
Whatman GF/C filters, presoaked for 1 h in 0.3% polyethyl-
enimine, using a Skatron cell harvester. Filters were washed
with ice-cold 50 mM Tris-HCl, pH 7.4 (wash setting 9,9,0), and
radioactivity was determined by liquid scintillation counting
(1 mL Packard MV Gold scintillator).
Ra t Recom bin a n t 5-HT7 Recep tor Bin d in g Assa y. 1.
Mem b r a n e P r ep a r a t ion . Frozen membranes from SF9
insect cells, infected with baculovirus to express the rat
recombinant 5-HT7 receptor, were used. Membrane fragments
were thawed, resuspended in 50 mM Tris-HCl, pH 7.4 (at 25
°C), containing 10 mM magnesium sulfate and 0.5 mM EDTA
(equivalent to 5 µg of membrane protein/mL), and used
immediately in the binding assay.
2. Bin d in g Assa y. Membranes (400 µL; equivalent to 2 µg
of membrane protein/tube) were incubated with 50 µL of [3H]-
lysergic acid diethylamide ([3H]LSD) at a single concentration
of 3 nM and 50 µL of distilled water (total binding) or 50 µL of
drug solution (10-6M) or 50 µL of methiothepin (10 µM;
nonspecific binding) for 90 min at 27 °C. Membrane-bound
radioactivity was recovered by filtration under vacuum through
Whatman GF/C filters, presoaked for 1 h in 0.3% polyethyl-
enimine, using a Skatron cell harvester. Filters were washed
with ice-cold 50 mM Tris-HCl, pH 7.4 (wash setting 9,9,0), and
radioactivity determined by liquid scintillation counting (1 mL
Packard MV Gold scintillator).
r1 Adr en oceptor Bin din g in P ostm or tem Hu m an Br ain .
1. Mem br a n e P r ep a r a tion . Postmortem human cortex was
homogenized in ice-cold 0.25 M sucrose (1:30 w/v) using a
motor-driven Teflon pestle and centrifuged at 1000g for 10
min. The supernatant was stored on ice, and the pellet was
resuspended in 0.25 M sucrose (1:15 w/v) and centrifuged at
750g for 10 min. Combined supernatants were diluted to 1:80
w/v with ice-cold 50 mM Tris-HCl, pH 7.4 (at 25 °C), and
centrifuged at 28000g for 10 min. The resulting pellet was
stored at -80 °C until the day of assay. The pellet was then
resuspended in 50 mM Tris-HCl, pH 7.6 (1:40 w/v), and
centrifuged at 40000g for 10 min. The final pellet was
resuspended in 50 mM Tris-HCl, pH 7.6 (equivalent to 12.5
mg wet weight of tissue/mL), and used immediately in the
binding assay. All centrifugations were carried out at 4 °C.
2. Bin d in g Assa y. Membranes (200 µL; equivalent to 8 mg
wet weight of tissue/tube) were incubated with 200 µL of [3H]-
GR 65630 at a single concentration of 0.2 nM and 100 µL of
50 mM HEPES buffer (total binding) or 100 µL of drug solution
(10-6 M) or 100 µL of metoclopramide (30 µM; nonspecific
binding) for 30 min at 37 °C. Membrane-bound radioactivity
was recovered by filtration under vacuum through Skatron
11734 filters using a Skatron cell harvester. Filters were
rapidly washed with ice-cold 50 mM HEPES buffer, pH 7.4
(wash setting 9,9,0), and radioactivity determined by liquid
scintillation counting (1 mL Packard MV Gold scintillator).
5-HT4 Recep tor Bin d in g Assa y in P ig Br a in . 1. Mem -
br a n e P r ep a r a tion . Pig hippocampii were homogenized in
ice-cold 50 mM HEPES buffer (pH 7.4 at 4 °C; 1:15 w/v) using
a Soniprep 150 (setting 5-6 for 8 s) and centrifuged at 40000g
for 15 min. The resulting pellet was resuspended in 50 mM
HEPES buffer, pH 7.4 (equivalent to 20 mg wet weight of
tissue/mL), and used immediately in the binding assay. All
centrifugations were carried out at 4 °C.
2. Bin d in g Assa y. Membranes (400 µL; equivalent to 5 mg
wet weight of tissue/tube) were incubated with 50 µL of [3H]-
prazosin at a single concentration of 0.1 nM and 50 µL of
distilled water (total binding) or 50 µL of drug solution (10-6
M or a range of 10 concentrations) or 50 µL of phentolamine
(5 µM; nonspecific binding) for 30 min at 30 °C. Membrane-
bound radioactivity was recovered by filtration under vacuum
2. Bin d in g Assa y. Membranes (400 µL; equivalent to 8 mg

3354 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 17
Birch et al.
through Skatron 11734 filters using a Skatron cell harvester.
Filters were rapidly washed with ice-cold 50 mM Tris-HCl,
pH 7.6 (wash setting 9,9,0), and radioactivity determined by
liquid scintillation counting (1 mL Packard MV Gold scintil-
lator).
Ack n ow led gm en t. The authors gratefully acknowl-
edge the work of Mr. M. Andrew, Mr. S. A. Burkitt, Dr.
A. P. A. Crew, Dr. L. Mackenzie, Miss S. Orme, Mr. G.
J . Smith, and Dr. N. Wishart for synthesis of target
compounds, Dr. S. C. Cheetham for receptor binding
studies involving postmortem human brain tissue, Dr.
N. Walker (ex BASF, Ludwigshafen) for X-ray crystal-
lographic studies, and Dr. B. J . Sargent for useful
discussions.
5-HT1A Receptor Bin din g in P ostm or tem Hu m an Br ain .
1. Mem br a n e P r ep a r a tion . Postmortem human hippocampi
were homogenized in ice-cold 0.25 M sucrose (1:30 w/v) using
a motor-driven Teflon pestle and centrifuged at 1000g for 10
min. The supernatant was stored on ice and the pellet was
resuspended in 0.25 M sucrose (1:15 w/v) and centrifuged at
850g for 10 min. Combined supernatants were diluted to 1:80
w/v with ice-cold 50 mM Tris-HCl, pH 7.7 (at 25 °C), and
centrifuged at 40000g for 10 min. The pellet was then
resuspended in 50 mM Tris-HCl, pH 7.7 (1:40 w/v), and
preincubated at 37 °C for 10 min to remove endogenous 5-HT.
The membrane suspension was then centrifuged at 40000g for
10 min. The final pellet was resuspended in 50 mM Tris-HCl,
pH 7.7, containing 4 mM calcium chloride, 0.1% L-ascorbic
acid, and 10 µM pargyline hydrochloride (equivalent to 12.5
mg wet weight of tissue/mL) and used immediately in the
binding assay. All centrifugations were carried out at 4 °C.
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2. Bin d in g Assa y. Membranes (400 µL; equivalent to 5 mg
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OH-DPAT at a single concentration of 0.75 nM and 50 µL of
distilled water (total binding) or 50 µL of drug solution (10-6
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