A series of 6- and 7-piperazinyl- and -piperidinylmethylbenzoxazinones with dopamine D4 antagonist activity: Discovery of a potential atypical antipsychotic agent

Article abstract of DOI:10.1021/jm990277d

As part of a program to develop dopamine D4 antagonists for the treatment of schizophrenia, we discovered a series of 6- and 7- (phenylpiperazinyl)- and -(phenylpiperidinyl)methylbenzoxazinones through mass screening of our compound library. A structure-a

Full text of DOI:10.1021/jm990277d

J . Med. Chem. 1999, 42, 5181-5187
5181
A Ser ies of 6- a n d 7-P ip er a zin yl- a n d -P ip er id in ylm eth ylben zoxa zin on es w ith
Dop a m in e D4 An ta gon ist Activity: Discover y of a P oten tia l Atyp ica l
An tip sych otic Agen t
Thomas R. Belliotti,* David J . Wustrow, Wouter A. Brink, Kim T. Zoski, Yu-Hsin Shih, Steven Z. Whetzel,
Lynn M. Georgic, Ann E. Corbin, Hyacinth C. Akunne, Thomas G. Heffner, Thomas A. Pugsley, and
Lawrence D. Wise
Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road,
Ann Arbor, Michigan 48105
Received J une 3, 1999
As part of a program to develop dopamine D4 antagonists for the treatment of schizophrenia,
we discovered a series of 6- and 7-(phenylpiperazinyl)- and -(phenylpiperidinyl)methylben-
zoxazinones through mass screening of our compound library. A structure-activity relationship
SAR study was carried out involving substituents on the phenyl ring, and several selective D4
antagonists were identified. The 7-substituted benzoxazinones showed more activity in
neurochemical and behavioral tests than the 6-substituted series. One of the most potent and
selective compounds (26) was found to have potent activity in animal tests predictive of
antipsychotic activity in humans after oral administration. This paper describes the SAR of
the benzoxazinone series and the preclinical characterization of 26.
In tr od u ction
a potential target in mediating the drug’s antipsychotic
effects.12 Clinical use of clozapine requires careful
monitoring due to a 1-2% occurrence of the potentially
fatal blood disorder agranulocytosis13 and it is limited
primarily to patients refractory to other treatments.
Therefore, improved agents are still needed. The pref-
erential binding of clozapine at the DA D4 receptor
along with the localization of the receptor suggests that
antagonists selective for the DA D4 receptor might offer
advantages over existing antipsychotic agents.
Schizophrenia is a complex psychiatric disorder af-
fecting up to 50 million people worldwide.1 The primary
therapy for the disease is administration of dopamine
(DA) antagonists such as olanzapine (1), which is an
antagonist at DA D2 receptors. The blockade of postsyn-
aptic DA D2 receptors in the mesolimbic and prefrontal
cortex regions of the brain is thought to be responsible
for decreasing the positive symptoms of schizophrenia
such as hallucinations and delusions.2 Unfortunately,
blockade of DA D2 receptors in the striatum causes
extrapyramidal side effects (EPS) and tardive dyskine-
sia.3 In addition, DA D2 receptor blockade can lead to
increased prolactin levels resulting in galactorrhea.4
Cloning studies showed that the DA D2 receptor family
consists of the DA D2, D3,5 and D46 receptor subtypes.
The discrete localization of DA D4 receptor mRNA in
cortical and mesolimbic regions of the brain (regions
believed to be involved in thought and emotion) and its
relative absence from nigral-striatal regions controlling
motor function suggest the DA D4 receptor is a good
target for the drug treatment of schizophrenia.6-8 Most
of the typical antipsychotic agents were found to have
similar affinities for DA D2 and DA D4 receptors, but
the atypical antipsychotic agent clozapine (2) was found
to have a 7-14-fold greater affinity for DA D4 receptors
compared to DA D2 receptors.6-9
Clozapine has been found to be effective in relieving
the positive symptoms of schizophrenia in a portion of
the patients resistant to treatment by other DA antago-
nists. Since it also has activity against negative symp-
toms (i.e., withdrawal, loss of drive, flattened effect) and
it has a decreased incidence of EPS,10 clozapine is an
atypical antipsychotic agent.11 Correlation of DA D4 (but
not D2 or D3) receptor binding affinity with concentra-
tions of clozapine in plasma of patients after clinically
efficacious doses also suggests that DA D4 receptors are
Several laboratories have reported success in generat-
ing DA D4 receptor antagonists with greater than 100-
fold selectivity in affinity for the DA D4 receptor subtype
compared to the DA D2 receptor subtype as exemplified
by compounds 3,14 4,15 5,16 6,17 and others.18-26 Clinical
trials with the DA D4 antagonist L-745,870 (5) have
been disappointing.27 This result is not surprising in
retrospect given the lack of activity of the compounds
in animal behavioral tests predictive of antipsychotic
efficacy.16 A recent report28 suggests that 5 acts as a
10.1021/jm990277d CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/23/1999

5182 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Ta ble 1. Receptor Binding of Target Compoundsa
Belliotti et al.
and 11 with a variety of phenylpiperazines or phenylpi-
peridines in the presence of sodium cyanoborohydride
(method A, Scheme 2). Alternatively, aldehydes 10 and
Sch em e 2. Preparation of Target Compounds by
Method Aa
6/7
D4
D3
D2
compd
R
X
subs Ki (nM) Ki (nM)
Ki (nM)
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
3,4-di Me CH
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
1.8
2.8
3.8
4.3
5.1
12.3
18.3
62.4
2.6
2.6
4.3
5
>3030
>3030
>3030
2490
864
572
52.5
698
4-Me
4-OMe
4-Me
3,4-di Me
4-OMe
H
CH
CH
N
N
N
493
2340
5590
2200
>5880
287
a
Reagents and conditions: (a) substituted phenylpiperazine or
>3030
phenylpiperidine, NaCNBH3, 1,2-dichloroethane.
N
839
4-Cl
N
>3030
232
859
679
1790
11 could be reduced to give the benzylic alcohol, which
was activated with tosyl chloride and displaced with
various phenylpiperazines or phenylpiperidines (method
B, Scheme 3).
4-Me
CH
3,4-di Me CH
610
413
4-Cl
N
4-OMe
H
N
N
>5880
6.1
6.7
6.9
10.7
10.4
428
78.5
129
4-OMe
3,4-di Me
4-Me
CH
N
N
1130
346
2980
908
Sch em e 3. Synthesis of 26 by Method Ba
208
L-754870
0.91
>3030
a
Measured as ability of test compound to displace radioligand
in CHO cells transfected with receptor cDNA. Tests were run in
triplicate and results did not vary by more than 25%.
a
Reagents and conditions: (a) MeOH, NaBH4. (b) TsCl, pyri-
partial agonist at the DA D4 receptor and may explain
its lack of effect in animal tests predictive of antipsy-
chotic activity. Our research efforts have focused on
finding compounds that not only are potent and selective
antagonists at the DA D4 receptor, but also have
neurochemical and behavioral effects in vivo that are
predictive of antipsychotic efficacy. Desirable com-
pounds would also be inactive in tests predictive of side
effects associated with the standard neuroleptics. In this
article we outline the discovery of a series of piperazi-
nylmethylbenzoxazinones of the general structure I
having this desired profile.
dine, 0 °C. (c) 4-chlorophenylpiperazine, K2CO3, CH3CN, reflux.
Resu lts a n d Discu ssion
Table 1 shows the binding data for the target com-
pounds at the DA D2, D3, and D4 receptors. Earlier
work in our laboratories and others showed that sub-
stituents in the 3 or 4 position of the phenyl ring
attached to the piperazine or piperidine led to com-
pounds with increased selectivity and affinity at the DA
D4 receptor.29 In the series with 6-substituted benzox-
azinones (16-23), the affinity for the DA D4 receptor
ranges from 2 to 62 nM. In general, phenylpiperidines
(16-18) have slightly higher DA D4 affinity than the
corresponding phenylpiperazines (19-21), although
they are less selective for DA D4 vs D2 receptors.
In the 7-substituted benzoxazinones (24-31) there is
not such a wide range of binding affinities. This series,
with the exception of compound 28, all have about 100-
fold selectivity for the DA D4 receptor over the DA D2
receptor. The most noticeable difference between the 6-
and 7-benzoxazinones is that the binding affinity of the
4-chloro compound improves from 62 nM in the 6-sub-
stituted series (23) to 4.6 nM in the 7-substituted series
(26). Because of its potency and selectivity at the DA
D4 receptor, 26 was tested in a [3H]thymidine functional
assay.30 It blocked the quinpirole-stimulated uptake of
thymidine with an IC50 of 1.5 nM, while having no effect
on [3H]thymidine uptake on its own (intrinsic activity
) 0). These results indicate that 26 is an antagonist at
the DA D4 receptor. At the DA D2 receptor, 26 is a weak
partial agonist (intrinsic activity ) 41%) with an IC50
of 932 nM. In this functional assay 26 shows greater
than 600-fold selectivity for the DA D4 vs the DA D2
receptor.
Ch em istr y
A general route for the preparation of 3-oxobenzox-
azine carboxaldehydes was developed as outlined in
Scheme 1. The 4-carboxaldehyde derivative of 2-nitro-
Sch em e 1. Synthesis of Benzoxazinone Aldehydesa
a
Reagents and conditions: (a) 1,3-propanediol, pTsOH, toluene,
reflux. (b) (1) RaNi, THF, (2) chloroacetyl chloride, NaHCO3, THF,
(3) HCl, MeOH, (4) K2CO3, CH3CN.
phenol 8 was reacted with propanediol forming the
corresponding propylidine acetal 9. In a multistep
procedure the nitro group of 9 was reduced, the result-
ing 2-aminophenol was treated directly with chloro-
acetyl chloride, and the acetal was hydrolyzed. Treat-
ment of the resulting product with potassium carbonate
in acetonitrile gave the 3-oxobenzoxazine-6-carboxalde-
hyde 10. The 3-oxobenzoxazine-7-carboxaldehyde 11
was prepared in a similar manner from the 3-carbox-
aldehyde derivative of 2-nitrophenol. The target com-
pounds in Table 1 were made by reacting aldehydes 10
To test for in vivo functional effects, those compounds
that showed the highest affinity and selectivity for the
DA D4 receptor were tested for their ability to increase

Benzoxazinones with Dopamine D4 Antagonist Activity
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5183
Ta ble 2. Effects of Compounds on DA Synthesis in Rat
Hippocampus (HI) and Striatum (ST) Measured as
Accumulation of DOPAa
% change in
DA synthesis
6/7
dose
compd
R
X
subs (mg/kg) route
HI
ST
16
18
19
20
21
25
26
27
29
31
3,4-diMe CH
6
6
6
6
6
7
7
7
7
7
10
30
20
10
30
10
10
10
20
10
po 128 ( 10 104 ( 4
4-OMe
4-Me
CH
N
po
po
ip
93 ( 5
96 ( 8
89 ( 5 111 ( 9
122 ( 8 126 ( 7
91 ( 8 110 ( 9
3,4-diMe
4-OMe
N
N
po
3,4-diMe CH
po 103 ( 7
99 ( 7
4-Cl
N
po 146 ( 5 110 ( 7
po 122 ( 9 104 ( 9
po 132 ( 4 126 ( 2
po 133 ( 7 140 ( 5
4-OMe
4-OMe
4-Me
N
CH
N
F igu r e 1. Effects of 26 on dopamine synthesis in wild type
a
Compounds and NSD1015 (100 mg/kg, ip) were administered
60 and 30 min, respectively, before animal termination. Striatum
and mesolimbic brain regions were removed and homogenized. The
supernatant was analyzed by HPLC with electrochemical detection
for the presence of DOPA. Each value is the mean of 4-8 animals
and is expressed as percent of control values (ng/g ( SEM) that
were 119 ( 8 and 1378 ( 96 for hippocampus and striatum,
respectively. Data were analyzed by one-way analysis of variance
and a Newman-Keuls test was used to determine significantly
different group means, with P < 0.05 being considered significant.
(WT) versus D4 knockout (KO) mice.
binding to other major central nervous system (CNS)
receptors and ion channels (Table 3). It had only modest
affinity (Ki ) 200-800 nM) for adrenergic, 5-HT-1A, and
σ receptors. Only micromolar activity at muscarinic M5
and 5-HT2 receptors was observed, and 26 was found
to be inactive at a large number of other ion channels
and receptors.
dopamine synthesis (measured as increased levels of
DOPAC) in the rat hippocampus (HI) and striatum (ST)
(Table 2). Desirable compounds would selectively in-
crease DA synthesis in the hippocampus, a brain area
rich in DA D4 receptors, and not in the striatum, a brain
area with low DA D4 receptor concentration.6-8 We also
looked for oral activity in this assay because this is the
preferred route of administration for antipsychotic
agents. As can be seen from Table 2, the 6-substituted
benzoxazinones had weak activity in this assay. The
most active 6-benzoxazinone (16) increases DA synthe-
sis in the HI by only 28% after oral administration. The
7-substituted series had more oral activity. The most
active compound (26) increases DA synthesis by 46%
in the HI while having no significant effect in the ST
after oral administration. This is consistent with earlier
findings that the DA D4 receptor is localized in the
mesolimbic regions of the brain.7 Because of this com-
pound’s selectivity and potency it was carried on to
further in vitro and in vivo studies.
Another indication that the observed effects are D4-
mediated is the fact that 26 increased DOPAC levels in
the hippocampus of wild-type mice while it had no
effects on DOPAC levels in D4 knockout mice in our
assay (Figure 1). It is also interesting to note that there
was no effect on the DA synthesis in the striatum of
either type of mice. This is to be expected since there
are few D4 receptors in the striatum.7
The oral activity of 26 in tests predictive of antipsy-
chotic activity suggests that it may be efficacious in drug
treatment of the symptoms of schizophrenia. Figure 2
shows that the compound blocked amphetamine-stimu-
lated locomotor activity after oral administration in the
rat. As can be seen in the figure, administration of
amphetamine (0.5 mg/kg ip) increased locomotor activity
by a factor of 3-4-fold. As the oral dose of 26 was
increased, the locomotor activity dose dependently
returned to near control levels. The ED50 for 26 in this
test is 2.2 mg/kg (po). Activity in this paradigm suggests
that 26 will have antipsychotic activity in humans.31
Another test predictive of antipsychotic activity is the
restoration of prepulse inhibition of the acoustic startle
To see if the observed effects are selectively mediated
by the DA D4 receptor, compound 26 was tested for
Ta ble 3. In Vitro Profile of 26: Estimated Binding Affinities (Panlabs) of 26 for Various Receptor Binding Sites Where Affinity Was
Less Than 50% at 10 µMa
receptor
ligand
tissue
∼IC50 (nM)
∼Ki (nM)
ref cmpd. Ki(nM)
adrenergic R1A
adrenergic R1B
adrenergic-R2A
adrenergic-R2B
5-HT-1A
[3H]prazosin
[3H]prazosin
[3H]MK-912
rat submaxillary gland
rat liver
human recombinant
rat kidney cortex
human recombinant
rat brain
guinea pig brain
rat brain
human recombinant
500
1425
1100
630
1400
2700
285
203
787
411
368
792
1678
232
231
964
prazosin, 0.07
prazosin, 0.15
yohimbine, 3.1
yohimbine, 2.0
metergoline, 13
ketanserin, 1.3
haloperidol, 1.8
ifenprodil, 4.9
4-DAMP, 0.86
[3H]yohimbine
[3H]-8-OH-DPAT
[3H]ketanserin
[3H]pentazocine
[3H]Ifenprodil
[3H]NMS
5-HT-2
σ-1
σ-2
375
1340
muscarinic M5
a
The Panlabs screen indicated that 26 had no significant affinity for a large number of other receptors or ion channels (IC50 values >
10 µM) including the following: adenosine A1, A2A, and A3, adrenergic â-1, â-2, and â-3, angiotensin ATI and AT2, bombesin, bradykinin
B1 and B2, calcium L channels (three different sites), calcium N channels, cannabinoid CB1 and CB2, cholecystokinin CCKA and CCKB,
dopamine D1 and D5, endothelin ETA and ETB, estrogen, GABA-A (agonist site, benzodiazepine and chloride channel), glucocorticoid,
glutamate, kainate and NMDA, glycine, strychnine-sensitive, histamine H1, H2, and H3, interleukin-1R, leukotriene D4, muscarinic M1,
M2, M3, and M4, neurokinin NK1 and NK2, neuropeptide Y1 and Y2, nicotinic acetylcholine (central), opiate δ, κ and µ, K+ channel
[KATP], [KV], [SKCa], serotonin 5-HT3, serotonin 5-HT4, testosterone, thromboxane A2, TNF-R, vasoactive intestinal peptide-VIP1, and
vasopressin V1.

5184 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Belliotti et al.
F igu r e 4. Catalepsy profile of 26. At 22 mg/kg, it has no
significant effects.
F igu r e 2. Effects of 26 on the amphetamine-stimulated
locomotor activity in rats. Control rats were given only vehicle;
the rest were given 0.5 mg/kg amphetamine sc before testing.
**p < 0.01 from vehicle. #p < 0.05 from amphetamine. ##p <
0.01 from amphetamine.
mice after ip administration of 30 mg/kg (10 times
ED50), indicating that it does not act at DA D2 receptors.
Con clu sion
Unlike recent DA D4 antagonists reported in the
literature such as 516 and 3,14 26 is orally active in
animal models predictive of antipsychotic activity. It
restores prepulse inhibition of acoustic startle in apo-
morphine-treated rats and it reverses amphetamine-
stimulated locomotor activity after oral administration
in the rat. Both of these results suggest that 26
(identified as CI-1030) will be an efficacious antipsy-
chotic., It is not likely to have the side effects associated
with typical antipsychotics because it does not increase
prolactin secretion or cause catalepsy in the rat.
Exp er im en ta l Section
F igu r e 3. Compound 26 reverses the apomorphine disruption
of prepulse inhibition of acoustic startle. Control rats were
given only vehicle while the rest were given 0.25 mg/kg
apomorphine sc. **p < 0.01 from vehicle, #p < 0.05 from
apomorphine.
Ch em istr y. Phenylpiperazines were purchased from Ald-
rich Chemical Co. or J anssen. Phenylpiperidines were pre-
pared as described in the literature.35 All reactions were
monitored by thin-layer chromatography (TLC) on Merck glass
plates precoated with 0.25 mm of silica gel. Chromatography
for purification was done with Merck silica gel (230-400 mesh)
or Elution Solution Flashelute cartridges packed with 32-63
µM silica gel. Solvents are reported as v/v solutions. Melting
points were recorded on a Thomas-Hoover capillary melting
point apparatus and are uncorrected. Mass spectra were
recorded on a Finnigan 4500 mass spectrometer or on a
Micromass Platform LC mass spectrometer and are chemical
ionization (CI) spectra. The spectra are reported as M+ peaks
and their intensity relative to the base peak (100%). Proton
NMR spectra were recorded on either a Varian 300 or a Varian
400 spectrometer with tetramethylsilane (TMS) as an internal
standard. Multiplicity is indicated as follows: s ) singlet, d
) doublet, dd ) doublet of doublets, t ) triplet, and m )
multiplet. Combustion analyses were performed by either
Robertson Microlit Laboratories or QTI Laboratories, and the
results were within 0.4% of the theoretical values for the
elements indicated.
3-Oxo-3,4-d ih yd r o-2H-ben z[1,4]oxa zin e-6-ca r boxa ld e-
h yd e (10). 4-Hydroxy-3-nitrobenzaldehyde (25 g, 149.6 mmol)
was added to a solution of 1,3-propanediol (17.1 g, 224.4 mmol)
in 250 mL of toluene. A catalytic amount of p-toluenesulfonic
acid was added, and the mixture was warmed to reflux with
removal of water by a Dean-Stark trap. After 12 h, the mixture
was cooled to room temperature and extracted with saturated
NaHCO3 (3 × 150 mL) followed by 100 mL of brine. Drying
over Na2SO4 followed by evaporation of the solvent gave 31.5
g of an oil, which was hydrogenated over Ra Ni in THF. When
the theoretical amount of H2 was taken up, the catalyst was
removed by filtration, and the solution was immediately
treated with chloroacetyl chloride (16.6 g, 146.8 mmol) and
reflex in rats. It is known that schizophrenic patients
exhibit diminished prepulse inhibition of acoustic startle
(PPI), and this phenomenon can be mimicked in rats
by treatment with apomorphine, a known dopamine
agonist.32,33 The antipsychotics currently on the market
will restore PPI in these apomorphine-treated rats. In
this test, rats were placed in a chamber and exposed to
a startle stimulus. The rats exhibited a startle response
and the magnitude of the response was measured. If
the same rat was exposed to a “prepulse” immediately
before the startle stimulus, the startle response was
diminished. This is prepulse inhibition of acoustic
startle. Figure 3 shows that rats treated with only
vehicle exhibited about 75% prepulse inhibition (PPI)
while those rats treated with apomorphine exhibited no
significant PPI. Compound 26 dose-dependently re-
stored PPI to near control levels with an oral minimum
effective dose (MED) of 3 mg/kg, indicating that it may
be efficacious as an antipsychotic agent.
In tests predictive of side effects associated with the
standard neuroleptics, 26 is inactive. Figure 4 shows
that 26 did not cause catalepsy34 in rats at 22 mg/kg po
(7 times ED50), indicating little or no potential for caus-
ing the EPS associated with other neuroleptics. Figure
5 shows that 26 did not increase the prolactin levels of

Benzoxazinones with Dopamine D4 Antagonist Activity
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5185
F igu r e 5. Effects of 26 on prolactin levels in mice. At 30 mg/kg, 26 does not increase prolactin levels, indicating that it does not
act at D2 receptor.
NaHCO3 (23.5 g, 279.7 mmol) at room temperature. After 1.5
h, the THF was evaporated and the residue was taken up in
a 50/50 mixture of MeOH/H2O and acidified to pH ) 1 with
concentrated HCl. The resulting solution was stirred at room
temperature overnight. The MeOH was evaporated and the
solid that formed was collected by filtration. It was treated
with K2CO3 (57.9 g, 419.6 mmol) in 200 mL of CH3CN at room-
temperature overnight. The solvent was evaporated, and the
solid remaining was triturated with 200 mL of H2O. The
remaining solid was dried at room temperature under vacuum
to give 19.7 g (79% yield) of 10. An aliquot was recrystallized
from EtOAc. Mp ) 219-221 °C. MS 178 (M+, 10%) 219 (M +
CH3CN, 100%). 1H NMR (DMSO-d6) δ 4.67 (s, 2Η), 7.10 (d,
1Η, J ) 8 Hz), 7.33 (d, 1H, J ) 2 Hz), 7.49 (dd, 1H, J ) 8 and
2 Hz), 9.80 (s, 1H), 10.96 (s, 1H). Analysis for C9H7NO3: C, H,
N.
3-Oxo-3,4-d ih yd r o-2H-ben z[1,4]oxa zin e-7-ca r boxa ld e-
h yd e (11) was prepared by the same method as 10 starting
from 3-hydroxy-4-nitrobenzaldehyde. Mp ) 220-222 °C. MS
178 (M+, 100%). 1H NMR (DMSO-d6) δ 4.65 (s, 2H), 7.08 (d,
1H, J ) 8.0 Hz), 7.32 (d, 1H, J ) 1.9 Hz), 7.47 (dd, 1H, J )
8.3 and 2.0 Hz), 9.78 (s, 1H), 10.9 (s, 1H). Analysis for C9H7-
NO3: C, H, N.
4-toluenesulfonyl chloride (0.74 g, 3.9 mmol) in 10 mL of
pyridine at 0 °C. The mixture was stirred at 0 °C for 5 h and
warmed to room temperature. The pyridine was evaporated
and the solid remaining was dried under vacuum overnight
to give 1.2 g of 7-[[[(4-methylphenyl)sulfonyl]oxy]methyl]-4H-
benz[1,4]oxazin-3-one (15), which was used without further
purification.
7-[[4-(4-Ch lor op h en yl)p ip er a zin -1-yl]m eth yl]-4H-ben z-
[1,4]oxa zin -3-on e (26). A suspension of 7-[[[(4-methylphenyl)-
sulfonyl]oxy]methyl]-4H-benz[1,4]oxazin-3-one (15) (12.7 g,
38.1 mmol), 1-(4-chlorophenyl)piperazine (7.5 g, 38.1 mmol),
and K2CO3 (26.4 g, 190.5 mmol) in 300 mL of acetonitrile was
warmed to reflux under Ar overnight. The mixture was cooled
to room temperature, and the solvent was evaporated. The
solid remaining was taken up in 200 mL of H2O and stirred
at room temperature. The solid was collected and air-dried.
The dry solid was recrystallized twice from EtOAc to give 6.8
g (50% yield) of 7-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-
4H-benz[1,4]oxazin-3-one (26). Mp ) 234-236 °C MS 358 (M+,
100%). 1H NMR (DMSO-d6) δ 2.45 (m, 4H), 3.10 (m, 4H), 3.40
(s, 2H), 6.85 (m, 3H), 6.89 (d, 2H, J ) 9.2 Hz), 7.10 (d, 2H, J
) 9.2 Hz). Analysis for C19H20ClN3O2: C, H, N, Cl.
6-[[4-(3,4-Dim eth ylp h en yl)p ip er id in -1-yl]m eth yl]-4H-
ben z[1,4]oxa zin -3-on e (16) was prepared by method A. Mp
6-[[4-(4-Meth ylp h en yl)p ip er a zin -1-yl]m eth yl]-4H-ben z-
[1,4]oxa zin -3-on e (19). Acetic acid (0.34 g, 5.6 mmol) was
added to a solution of 3-oxo-3,4-dihydro-2H-benz[1,4]oxazine-
6-carboxaldehyde (10) (1.0 g, 5.6 mmol) and (4-methylphenyl)-
piperazine (0.98 g, 5.6 mmol) in 50 mL of dichloroethane.
NaBH(OAc)3 (2.5 g, 11.9 mmol) was added, and the reaction
mixture was stirred at room temperature overnight. The
reaction was quenched by addition of 50 mL of H2O, and the
layers were separated. The aqueous layer was extracted with
CH2Cl2 (2 × 50 mL) and the combined organic layers were
dried over Na2SO4. Evaporation of the solvent followed by
recrystallization of the solid from EtOAc gave 0.81 g (43%
1
) 175-177 °C. MS 351 (M+, 100%). H NMR (CDCl3) δ 1.75
(m, 4H), 2.05 (m, 2H), 2.20 (s, 3H), 2.21 (s, 3H), 2.40 (m, 1H),
2.93 (d, 2H, J ) 12 Hz), 3.40 (s, 2H), 4.59 (s, 2H), 6.79 (s, 1H),
6.90 (s, 2H), 6.99 (d, 1H, J ) 8 Hz), 6.97 (s, 1H), 7.02 (d, 1H,
J ) 8 Hz). Analysis for C22H26N2O2: C, H, N.
6-[[4-(p-Tolyl)piper idin -1-yl]m eth yl]-4H-ben z[1,4]oxazin -
3-on e (17) was prepared by method A. Mp ) 178-180 °C. MS
337 (M+, 100%). 1H NMR (CDCl3) δ 1.75 (m, 4H), 2.05 (m, 2H),
2.30 (s, 3H), 2.45 (m, 1H), 2.93 (d, 2H, J ) 12 Hz), 3.40 (s,
2H), 4.60 (s, 2H), 6.83 (s, 1H), 6.95 (s, 2h), 7.10 (s, 4H). Analysis
for C21H24N2O2: C, H, N.
1
yield) of 19. Mp ) 212-214 °C. MS 338 (M+, 100%). H NMR
6-[[4-(4-Meth oxyph en yl)piper idin -1-yl]m eth yl]-4H-ben z-
[1,4]oxa zin -3-on e (18) was prepared by method A. Mp )
(CDCl3) δ 2.22 (s, 3H), 2.56 (t, 4H, J ) 4.9 Hz), 3.09 (t, 4H, J
) 4.9 Hz), 3.44 (s, 2H), 4.57 (s, 2H), 6.79 (s, 2H), 6.81 (d, 2H,
J ) 8.5 Hz), 7.03 (d, 2H, J ) 8.5 Hz), 7.9 (s, 1H). Analysis for
1
177-178 °C. MS 353 (M+, 100%). H NMR (CDCl3) δ 1.75 (m,
4H), 2.05 (m, 2H), 2.40 (m, 1H), 2.93 (d, 2H, J ) 12 Hz), 3.40
(s, 2H), 3.75 (s, 3H), 4.60 (s, 2H), 6.80 (m, 3H), 6.90 (m, 2H),
7.14 (d, 2H, J ) 7 Hz). Analysis for C21H24N2O3: C, H, N.
6-[[4-(3,4-Dim eth ylp h en yl)p ip er a zin -1-yl]m eth yl]-4H-
ben z[1,4]oxa zin -3-on e (20) was prepared by method A. Mp
C
21H25N3O2: C, H, N.
7-(Hydr oxym eth yl)-4H-ben z[1,4]oxazin -3-on e (14).3-Oxo-
3,4-dihydro-2H-benzo[1,4]oxazine-7-carboxaldehyde (11) (1.0
g, 5.6 mmol) was added to a solution of NaBH4 (0.11 g, 2.8
mmol) in 25 mL of MeOH at 0 °C. After being stirred at 0 °C
for 1 h, the reaction was quenched with 10 mL of 1.0 N HCl,
and the MeOH was evaporated. The precipitate was collected
and air-dried overnight to give 0.7 g (76% yield) of 7-(hy-
droxymethyl)-4H-benz[1,4]oxazin-3-one (14). An analytical
sample was obtained by recrystallization from EtOAc. Mp )
1
) 148-153 °C. MS 352 (M+, 100%). H NMR (CDCl3) δ 2.19
(s, 3H), 2.20 (s, 3H), 2.58 (s, 4H), 3.10 (s, 4H), 3.24 (s, 2H),
4.60 (s, 2H), 6.62 (dd, 1H, J ) 8.3 and 2.5 Hz), 6.70 (d, 1H, J
) 2.9 Hz), 6.80 (s, 1H), 6.89 (s, 2H), 6.98 (d, 1H, J ) 8.5 Hz),
8.10 (s, 1H). Analysis for C21H25N3O2: C, H, N.
6-[[4-(4-Meth oxyph en yl)piper azin -1-yl]m eth yl]-4H-ben z-
[1,4]oxa zin -3-on e (21) was prepared by method A. Mp )
1
206-208 °C. MS 180 (M+, 100%). H NMR (DMSO-d6) δ 4.33
1
(d, 2H, J ) 5 Hz), 4.48 (s, 2H), 5.07 (t, 1H, J ) 5.6 Hz), 6.79
(d, 1H, J ) 14.7 Hz), 6.82-6.83 (m, 2H), 10.6 (s, 1H). Analysis
for C9H9NO3: C, H, N.
202-203 °C. MS 353 (M+, 95%). H NMR (CDCl3) δ 2.58 (m,
4H), 3.08 (m, 4H), 3.40 (s, 2H), 3.75 (s, 3H), 4.60 (s, 2H), 6.78
(m, 3H), 6.80-6.95 (m, 4H), 8.05 (s, 1H). Analysis for
7-[[[(4-Met h ylp h en yl)su lfon yl]oxy]m et h yl]-4H -b en z-
[1,4]oxa zin -3-on e (15). 7-(Hydroxymethyl)-4H-benz[1,4]oxa-
zin-3-one (14) (0.6 g, 3.7 mmol) was added to a solution of
C
20H23N3O3: C, H, N.
6-[4-P h en ylp ip er a zin -1-ylm eth yl]-4H-ben z[1,4]oxa zin -
3-on e (22) was prepared by method A. Mp ) 207-209 °C. MS

5186 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Belliotti et al.
1
323 (M+, 100%). H NMR (DMSO-d6) δ 2.48 (m, 4H), 3.15 (m,
Competition binding assays were conducted as follows:
CHO-K1 cell membranes (400 µL) in appropriate ice-cold buffer
(25 mM Tris-HCl, 5 mM MgCl2, 5 mM KCl, 1.5 mM CaCl2, 1
mM EDTA, and 120 mM NaCl, pH 7.4), 50 µL of either drug
(0.1-10 000 nM or 0.01-1000 nM for a total of six concentra-
tions) or buffer, and 50 µL of [3H]ligand (0.2 nM, final
concentration for D2 and D4, and 0.5 nM, final concentration
for D3) were added to polypropylene microtubes (Marsh
Biomedical Products, Inc., Rochester, NY) to give a total
volume of 500 µL. Incubation proceeded for 2 h at room
temperature and was terminated by rapid filtration followed
by four washes with 1 mL of buffer on Whatman GF/B filters
on a Brandel MLR96 cell harvester (Biomedical Research and
Development, Inc., Gaithersburg, MD). Following the addition
of liquid scintillation cocktail, the radioactivity remaining was
counted with a Wallac 1205 Betaplate liquid scintillation
counter (50% efficiency). Specific binding was defined as total
binding minus binding in the presence of 1 µM haloperidol and
this ranged from 90% to 95%. All assays were performed in
triplicate.
Bioch em ica l Exp er im en ts. The effects of compounds on
DA synthesis were measured by monitoring DOPA (L-3,4-
dihydroxyphenylalanine) accumulation after inhibiting L-
aromatic amino acid decarboxylase with NSD-1015 in animals
treated with GBL to reduce DA neuronal firing.30,36 Test
compounds were administered 1 h and NSD-1015 was admin-
istered 30 min before animals were sacrificed by decapitation.
The brain was rapidly removed and placed on an ice-cooled
plate for dissection of striatum, nucleus accumbens plus
olfactory tubercle (mesolimbic region), and brain stem. Tissue
samples were frozen on dry ice and stored at -70 °C until
assayed.
On the day of assay samples were thawed, homogenized in
0.5-1.0 mL of 0.1 M phosphate-citrate buffer (pH 2.5)
containing 15% methanol, and centrifuged for 1 min in a
Beckman Microfuge B. Samples were analyzed on a C18
reverse-phase analytical column (3 µm spheres, 150 × 4.6 mm;
IB-SIL, Phenomenex, Torrance, CA) coupled to an electro-
chemical detector (Waters 464 pulsed electrochemical detector)
equipped with a glassy carbon electrode set at a potential of
+0.75 V relative to an Ag/AgCl reference. The HPLC mobile
phase consisted of 0.05 mM sodium phosphate, 0.03 M citrate
buffer (pH 2.7), 0.1 mM disodium EDTA, 0.035% sodium
octanesulfate, and 25% methanol. Data were analyzed by one-
way analysis of variance, and a Newman-Keuls test was used
to determine significantly different group means, with P < 0.05
being considered significant.
Mitogen esis Assa y. The effects of test compounds on [3H]-
thymidine uptake were measured as previously described.30
CHO p-5 cells transfected with human D2 or D4.2 cDNA were
seeded into 96-well plates at a density of about 5000 cells/
well and grown at 37 °C in a minimum essential medium.
(RMEM, Gibco) with 10% calf serum for 2 days. The wells were
rinsed with serum-free medium three times and fresh medium
was added along with either test compound in water or vehicle
alone. Eight wells of each plate received 100 µL of RMEM with
10% fetal calf serum. After culture for 16-17 h, [3H]thymidine
(1 µCi/well) was added for 4 h. The cells were trypsinized and
harvested onto filter mats with a 96-well Brandel cell har-
vester. The filters were counted in a Beta-Plate scintillation
counter.
4H), 3.45 (s, 2H), 4.30 (s, 2H), 6.70-6.98 (m, 6H), 7.14 (m, 2H),
10.80 (s, 1H). Analysis for C19H21N3O2‚0.5H2O: C, H, N.
6-[[4-(4-Ch lor op h en yl)p ip er a zin -1-yl]m eth yl]-4H-ben z-
[1,4]oxa zin -3-on e (23) was prepared by method A. Mp )
250-252 °C. MS 357 (M+, 80%), 162 (C7H8NO2, 100%). 1H
NMR (DMSO-d6) δ 2.48 (m, 4H), 3.15 (m, 4H), 3.19 (s, 2H),
4.30 (s, 2H), 6.80 (m, 3H), 6.90 (d, 2H, J ) 9.0 Hz), 7.15 (d,
2H, J ) 9.0 Hz), 10.60 s, 1H). Analysis for C19H20N3O2Cl‚
0.5H2O: C, H, N.
7-[[4-(p-Tolyl)piper idin -1-yl]m eth yl]-4H-ben z[1,4]oxazin -
3-on e (24) was prepared by method A. Mp ) 186-188 °C. MS
337 (M+, 100%). 1H NMR (CDCl3) δ 1.75 (m, 4H), 2.05 (m, 2H),
2.35 (s, 3H), 2.40 (m, 1H), 2.93 (d, 2H, J ) 12 Hz), 3.40 (s,
2H), 4.60 (s, 2H), 6.72 (d, 1H, J ) 8 Hz), 6.93 (d, 1H, J ) 7.4
Hz), 7.00 (s, 1H), 7.11 (s, 4H). Analysis for C21H24N2O2: C, H,
N.
7-[[4-(3,4-Dim eth ylp h en yl)p ip er id in -1-yl]m eth yl]-4H-
ben z[1,4]oxa zin -3-on e (25) was prepared by method A. Mp
) 192-194 °C. MS 351 (M+, 100%). 1H NMR (DMSO-d6) δ 1.60
(m, 4H), 1.95 (m, 2H), 2.09 (s, 3H), 2.11 (s, 3H), 2.35 (m, 1H),
2.79 (d, 2H, J ) 11.2 Hz), 3.35 (s, 2H), 4.70 (s, 2H), 6.75-6.90
(m, 4H), 6.92-6.98 (m, 3H). Analysis for C22H26N2O2: C, H, N.
7-[[4-(4-Meth oxyph en yl)piper azin -1-yl]m eth yl]-4H-ben z-
[1,4]oxa zin -3-on e (27) was prepared by method A. Mp ) 235
°C. MS 353 (M+, 100%). 1H NMR (CDCl3) δ 2.58 (m, 4H), 3.08
(m, 4H), 3.40 (s, 2H), 3.85 (s, 3H), 4.60 (s, 2H), 6.69 (d, 1H, J
) 7.8 Hz), 6.78-6.85 (m, 3H), 6.88-6.90 (m, 3H), 6.99 (s, 1H).
Analysis for C20H23N3O3: C, H, N.
7-[[4-P h en ylpiper azin -1-yl]m eth yl]-4H-ben z[1,4]oxazin -
3-on e (28) was prepared by method A. Mp ) 191-193 °C. MS
324 (M+, 100%). 1H NMR (CDCl3) δ 2.55 (m, 4H), 3.10 (m, 4H),
3.45 (s, 2H), 4.58 (s, 2H), 6.64 (dd, 1H, J ) 8 and 2.6 Hz), 6.82
(m, 1H), 6.93 (m, 3H), 6.99 (s, 1H), 7.24 (d, 2H, J ) 12.4 Hz),
7.90 (s, 1H). Analysis for C19H21N3O2: C, H, N.
7-[[4-(4-Meth oxyph en yl)piper idin ]-1-ylm eth yl]-4H-ben z-
[1,4]oxa zin -3-on e (29) was prepared by method A. Mp )
198-199 °C. MS 353 (M+, 100%). H NMR (CDCl3) δ 1.75 (m,
1
4H), 2.05 (m, 2H), 2.45 (m, 1H), 2.93 (d, 2H, J ) 12 Hz), 3.40
(s, 2H), 3.79 (s, 3H), 4.60 (s, 2H), 6.64 (dd, 1H, J ) 8 and 2.6
Hz), 6.80 (d, 2H, J ) 8.2 Hz), 6.90 (dd, 1H, J ) 8.3 and 2.0
Hz), 6.98 (s, 1H), 7.10 (d, 2H, J ) 8.6 Hz), 8.0 (s, 1H). Analysis
for C21H24N2O3: C, H, N.
7-[[4-(3,4-Dim eth ylp h en yl)p ip er a zin -1-yl]m eth yl]-4H-
ben z[1,4]oxa zin -3-on e (30) was prepared by method A. Mp
) 195-197 °C. MS 352 (M+, 100%). H NMR (CDCl3) δ 2.18
1
(s, 3H), 2.20 (s, 3H), 2.55 (m, 4H), 3.10 (m, 4H), 3.45 (s, 2H),
4.58 (s, 2H), 6.64 (dd, 1H, J ) 8.2 and 2.4 Hz), 6.68 (d, 2H, J
) 7.8 Hz), 6.91 (dd, 1H, J ) 8.5 and 1.8 Hz), 6.97 (d, 2H, J )
6.4 Hz), 7.60 (s, 1H). Analysis for C21H25N3O2: C, H, N.
7-[[4-(p-Tolyl)p ip er a zin -1-yl]m eth yl]-4H-ben z[1,4]oxa -
zin -3-on e (31) was prepared by method A. Mp ) 208-210
°C. MS 338 (M+, 100%). H NMR (CDCl3) δ 2.24 (s, 3H), 2.55
1
(m, 4H), 3.10 (m, 4H), 3.45 (s, 2H), 4.58 (s, 2H), 6.70 (d, 1H, J
) 7.8 Hz), 6.80 (d, 2H, J ) 8.6 Hz), 6.91 (d, 1H, J ) 7.8 Hz),
6.97 (s, 1H), 7.02 (d, 2H, J ) 8.8 Hz), 7.72 (s, 1H). Analysis
for C20H23N3O2: C, H, N.
P h a r m a cology: Recep tor Bin d in g Assa ys for DA D2L,
D3, a n d D4.2 Recep tor s. CHO cells (provided by Dr. J ames
Granneman, Wayne State University, Detroit, MI) transfected
with human D2L, D3, and D4.2 cDNA were grown and
harvested as previously described.30 Confluent cultures were
harvested by replacing the media with cold phosphate-buffered
saline (PBS) containing 0.05% EDTA followed by centrifuga-
tion at 1000g for 2 min. The pellets obtained were suspended
in appropriate volume of ice-cold buffer (25 mM Tris-HCl and
1 mM EDTA, pH 7.4; TE buffer) and centrifuged at 20000g
for 15 min at 4 °C. The final pellets obtained were suspended
in TE buffer and homogenized with a Polytron (Brinkman
Instruments, Westbury, NY) at setting 6 for 5 s for use in
radioligand binding assays or stored at -80 °C. CHO-K1 cell
membranes for use in the assay were homogenized with a
Polytron at setting 6 for 5 s and stored at -80 °C in 1 mL
aliquots until used in the assay.
R a t Am p h et a m in e-St im u la t ed Locom ot or Act ivit y
Stu d ies. Rats were dosed po with water or test compound and
placed in Omnitech motion detection chambers for a 15 min
acclimation period. They were dosed ip with saline or D-
amphetamine and returned to the chamber for an additional
15 min. Locomotor activity was then recorded for 30 min, and
data were expressed as total distanced traveled in centimeters.
Statistical significance between groups was calculated by a
t-test, and amphetamine reversal ED50s and 95% confidence
limits were calculated by regression analysis.
P r ep u lse In h ibition of Acou stic Sta r tle in Ra ts. Rats
were dosed po with vehicle or with compound 26 30 min prior
to a sc injection of saline or apomorphine (0.25 mg/kg) and

Benzoxazinones with Dopamine D4 Antagonist Activity
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then placed in one of eight San Diego Instruments acoustic
startle chambers for a 30 min test. The test session consisted
of a total of 90 trials after a 5-min test acclimation period of
70-dB white noise. The fires and last 10 trials were 120 dB
pulse-alone trials (not included in the PPI calculation). The
middle 70 trials consisted of 20 120-dB pulse-alone trials and
10 trials of each of the following five trial types in pseudoran-
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to a 120-dB startle pulse, (3) 74-dB prepulse + 120-dB pulse,
(4) 78-dB prepulse + 120-dB pulse, and (5) 86-dB prepulse +
120-dB pulse. Intertrial interval was 7-23 s. The prepulses
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