6-(4-Benzylpiperazin-1-yl)benzodioxanes as selective ligands at cloned primate dopamine D4 receptors

Article abstract of DOI:10.1016/S0968-0896(01)00226-7

A series of novel 6-(4-benzylpiperazin-1-yl)benzodioxanes were prepared and screened at selected dopamine receptor subtypes. 6-(4-[4-Chlorobenzyl]piperazin-1-yl)benzodioxane (2d) had high affinity and selectivity for the D₄ dopamine receptor su

Full text of DOI:10.1016/S0968-0896(01)00226-7

Bioorganic & Medicinal Chemistry 9 (2001) 3207–3213
6-(4-Benzylpiperazin-1-yl)benzodioxanes as Selective Ligands at
Cloned Primate Dopamine D4 Receptors
Kevin J. Hodgetts,* Andrzej Kieltyka, Robbin Brodbeck, Jennifer N. Tran,
Jan W. F. Wasley and Andrew Thurkauf
Neurogen Corporation, 35 Northeast Industrial Road, Branford, CT 06405, USA
Received 5 March 2001; accepted 8 June 2001
Abstract—A series of novel 6-(4-benzylpiperazin-1-yl)benzodioxanes were prepared and screened at selected dopamine receptor
subtypes. 6-(4-[4-Chlorobenzyl]piperazin-1-yl)benzodioxane (2d) had high affinity and selectivity for the D4 dopamine receptor
subtype and was identified as a D4 antagonist via its attenuation of dopamine-induced GTPg35S binding at the D4 receptor. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
dopamine D2 receptor family consists of D2, D3 and D4
subtypes.3 The distribution of the D4 receptor through-
out the brain differs from that of the D2 receptor. The
D4 receptor is localized primarily in areas other than the
striatum, such as the amygdala, thalamus, hypothala-
mus and cerebellum.4 Subsequently, the D4 receptor has
been shown to be overexpressed in the postmortem
brains of schizophrenics,5 although this finding is the
subject of some debate.3 The atypical antipsychotic clo-
zapine, which lacks the extrapyramidal side effects
associated with most other antipsychotics, has a higher
affinity for the D4 receptor over the D2 receptor.6 These
findings suggest that the D4 receptor is a good target for
the treatment of schizophrenia and a number of D4
selective antagonists have been reported.7 Several of
these, including NGD 94-1,8 L-745,8709 and U-
101387,10 have been advanced to the clinic.
Schizophrenia is
a
debilitating disease affecting
approximately 1% of the population. Affected indivi-
duals can experience delusions, hallucinations, dis-
organized thoughts and behavior resulting in an
impairment of normal social function. The dopamine
hypothesis postulates that schizophrenia is the result of
hyperactivity of the dopaminergic system and that inhi-
bition of dopaminergic processes by receptor blockade
can be an effective means of treatment.1 Classical anti-
psychotics such as haloperidol are effective in treating
schizophrenic patients, however, they can cause severe
side effects such as extrapyramidal symptoms and tard-
ive dyskinesia.2 These side effects have been attributed
to a blockade of dopamine receptors in the striatum.
Through cloning studies, it has been shown that the
*Corresponding author. Tel.: +1-203-488-8201; fax: +1-203-483-7027; e-mail: khodgetts@nrgn.com
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00226-7

3208
K. J. Hodgetts et al. / Bioorg. Med. Chem. 9 (2001) 3207–3213
Results and Discussion
representative electron-withdrawing group (chlorine)
and electron-donating group (methoxy) were introduced
as single substituents at each of the available positions
(2b–g, Table 1). Relative to the parent compound 2a,
both ortho-substituted compounds 2b and 2e were much
weaker at D4 and inactive at both D2 and D3. A sig-
nificant improvement in D4 affinity was achieved with
the meta-substituted analogues 2c and 2f and weak affi-
nity at both D2 and D3 receptors was observed. The
highest D4 potency was achieved with the para-sub-
stituted analogues 2d (Ki=2 nM) and 2g (Ki=5 nM).
Although there was an increase in both D2 and D3 affi-
nity, 2d displayed high selectivity over D2 (>250-fold)
and D3 (>500-fold). The finding that both electron-
withdrawing and electron-donating substituents at the
para-position gave compounds of similar potency indi-
cates that a steric rather than electronic interaction may
be responsible for these results. The para-fluoro 2h and
para-methyl 2i analogues had similar binding profiles to
2d and 2g. The introduction of a methyl group at the
benzylic position (2j) was detrimental to D4 binding.
As part of a general program within the area of dopa-
minergic ligands, we have previously identified 1-
phenyl-4-benzylpiperazines as selective agents for the
D4 receptor.11 The 4-methoxyphenyl-piperazine 1 dis-
played modest potency at the D4 receptor (Ki=93 nM)
but was inactive (Ki >5000 nM) at the D2 and D3
receptors. The simplicity of the basic structure inferred
that selective modifications could lead to higher affinity
D4 selective compounds. In the course of these studies,
it was found that the alkoxy group could be incorpo-
rated into a benzodioxane ring system as shown for the
parent compound 2a (D4 Ki=47 nM). Compound 2a
displayed high selectivity for D4 over the related D2
(>100-fold) and D3 (>100-fold) receptor subtypes and
an exploration of the structure–activity relationship of
this compound was carried out in order to improve the
overall D4 affinity. The structural variations of com-
pound 2a examined fell into three classes: (i) Substitu-
tions upon the benzyl moiety by electron donating or
electron withdrawing substituents. (ii) Incorporation of
an alkyl group at the benzylic position. (iii) Variations
in the size and oxygen content of the cyclic ether
component of the original benzodioxane.
At this point, a further examination of the SAR was
carried out by selected variations of the 1,4-benzodiox-
ane portion of the molecule. The variations of ring size
and oxygen content included the chromane 7, the
As the number of dependent variables was high the
benzylic portion of the molecule was optimized first.
The required common intermediate, 1-(1,4-benzodiox-
ane-6-yl)piperazine (4), was prepared via a two-step lit-
erature procedure from commercially available 6-
bromo-1,4-benzodioxane (3).12 Condensation with the
appropriate benzyl halide furnished the target
compounds 2a–2j (Scheme 1).
1,3-dioxolane 8 and the benzofuran 9. The high affinity
4-chlorobenzylpiperazine unit of compound 2d was used
with these examples to provide a point of reference.
Each compound was prepared from the corresponding
arylpiperazine in a manner similar to that shown for the
preparation of compounds 2a–2j (Scheme 1). 6-Piper-
azinylchromane (6) was prepared via the three-step
procedure outlined in Scheme 2. 5-Piperazinyl-2H-ben-
zo[d]1,3-dioxolane13 and 5-piperazinyl-2,3-dihydro-
benzo[b]furan14 were prepared according to literature
procedures. All three compounds (7, 8, and 9) were
To determine how the D2, D3 and D4 affinities were
affected by substituents on the benzyl group, a
Scheme 1. Reaction conditions: (i) Pd2(dba)3, P(o-tolyl)3, tert-butyl 1-piperazinecarboxylate, NaOtBu, PhMe, 100 ꢀC, 55%; (ii) TFA, CH2Cl2, 20 ꢀC,
82%; (iii) benzyl halide, K2CO3, CH3CN, reflux, 48–89%.

K. J. Hodgetts et al. / Bioorg. Med. Chem. 9 (2001) 3207–3213
Table 1. Dopamine receptor subtype binding
3209
Ki (nM)a
Compound
R1
R2
R3
R4
n
X
D2
D3
D4
1
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
7
8
>5000
>5000
>5000
2076Æ645
526Æ96
>5000
3297Æ1414
1424Æ650
>5000
>5000
>5000
>5000
3770Æ1459
1396Æ558
>5000
93Æ15
47Æ8
434Æ43
10Æ3
2Æ1
H
Cl
H
H
OMe
H
H
H
H
H
H
H
Cl
H
H
OMe
H
H
H
H
H
H
H
H
Cl
H
H
OMe
F
Me
H
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
Me
H
H
H
2
2
2
2
2
2
2
O
O
O
O
O
O
O
O
O
O
160Æ23
19Æ3
5Æ2
>5000
1293Æ910
2
>5000
4Æ1
2
2
2
1
1
1212Æ148
2388Æ1318
>5000
>5000
>5000
>5000
Not determined
14Æ2
2Æ1
>5000
1174Æ315
4Æ1
H
H
H
CH2
O
CH2
2152Æ1189
2657Æ233
>5000
H
H
9Æ5
9
10Æ4
17Æ3
5Æ1
Clozapine
Haloperidol
113Æ9
2Æ1
aBinding data are the means of at least three independent experiments using standard displacement assays with [3H]YM 09151 as the competitive
ligand and primate dopamine receptor subtypes expressed in CHO cells.
Scheme 2. Reaction conditions: (i) HNO3, 0 ꢀC, 79%; (ii) Raney nickel, H2, 1 atm, 91%; (iii) bis-(2-chloroethyl)amine hydrochloride, K2CO3,
chlorobenzene, reflux, 53%; (iv) 4-chlorobenzyl bromide, K2CO3, CH3CN, reflux, 79%.
potent (Ki <10 nM) at the D4 receptor with high selec-
tivity over D2 (>500 fold) and D3 (>500 fold).
Compound 2d was also tested for affinity at the ser-
otonergic and adrenergic receptors at which other D4
antagonists have shown activity. Compound 2d was
inactive at 5HT-2 (>10 mM) and displayed weak activity
at both alpha-1 (42 mM) and alpha-2 (5 mM).
It next became important to determine the functional
activity of these compounds at the D4 receptor. Ago-
nist-stimulated GTPg35S binding has been widely used
for many G protein coupled receptors to distinguish
agonists from antagonists and to determine their
potency and efficacy for a given receptor. The GTPg35S
binding functional assay can be used to demonstrate a
dose-dependent agonist stimulation by the full D4
receptor agonist, dopamine, in CHO cells producing
stable expression of primate D4.2 receptors. A typical
assay exhibited a 3-fold stimulation over baseline, with
an EC50 of 65 nM. When used alone compound 2d gave
no stimulation over baseline, suggesting that 2d does
not possess agonist properties at the human D4 recep-
tor. In combination with an EC74 level of dopamine
(333 nM), 2d reversed the agonist effect in a dose
dependent manner (IC50=20 nM) leading to complete
reversal. Thus, 2d exerts functional antagonism within
the D4 receptor system.
Conclusion
A series of 6-(4-benzyl]piperazin-1-yl)benzodioxanes
and related analogues were prepared and found to be
high affinity, high selectivity D4 receptor antagonists.
Several compounds were more potent (Ki <10 nM) at
D4 and several orders of magnitude weaker at D2 than
clozapine. Reports on the antipsychotic effects in animal
models and in clinical trials of selective D4 receptor
antagonists have so far proved disappointing. It appears
that D4 selective antagonists are no longer to be expec-
ted to reverse the symptoms of schizophrenia.15 It is
reasonable to assume, however, that compounds that
include a component of D4 antagonism in combination
with other activities may lead to the desired therapeutic

3210
K. J. Hodgetts et al. / Bioorg. Med. Chem. 9 (2001) 3207–3213
effect with reduced side effects. Genomic studies have
revealed that D4 receptor gene polymorphism may be
associated with other disorders and these may prove to
be better targets for D4 receptor antagonists. Indeed it
was recently reported that the selective D4 receptor
antagonist, U-101387, prevents stress-induced cognitive
deficits in monkeys.16 The ever widening variety of
structural classes with high affinity for the dopamine D4
receptor holds promise for elaboration of the function
of this receptor system in vivo.
with ether/hexane (1:1) gave tert-butyl 4-(2H,3H-ben-
zo[e]1,4-dioxin-6-yl)piperazinecarboxylate (4.37 g, 57%)
as a pale yellow solid: mp 53–55 ꢀC; 1H NMR
(400 MHz, CDCl3) d 1.47 (s, 9H), 2.97–2.99 (m, 4H),
3.53–3.56 (m, 4H), 4.19–4.24 (m, 4H), 6.45–6.48 (m,
2H), 6.78 (d, J=9.0 Hz, 1H). Trifluoroacetic acid (10
mL) was added slowly at 0 ꢀC to a solution of tert-butyl
4-(2H,3H-benzo[e]1,4-dioxin-6-yl)piperazinecarboxylate
(3.2 g, 10 mmol) in dichloromethane (10 mL). The mix-
ture was stirred for 1 h then poured onto ice water (50
mL). The aqueous solution was made basic by the
addition of aqueous ammonia solution and extracted
with dichloromethane (2Â100 mL). The combined
extracts were washed with brine (150 mL), dried
(MgSO4) and concentrated. The residue was purified by
flash chromatography on silica gel eluting with di-
chloromethane/methanol (4:1) and gave the title com-
pound (1.65 g, 75%) as a white solid: mp 134–137 ꢀC;
1H NMR (400 MHz, CDCl3) d 3.02 (brs, 8H), 4.17–4.23
(m, 4H), 6.44–6.47 (m, 2H), 6.76 (d, J=9.5 Hz, 1H).
Experimental
Chemistry
Melting points were determined using a Thomas–
Hoover capillary melting apparatus and are uncor-
rected. Elemental analyses were obtained for all com-
pounds tested for receptor binding. Elemental analysis
were performed at Robertson Microlabs, Madison, NJ,
USA and are within 0.4% of theoretical C, H, and N
with the exception of 2c where carbon analysis was
0.54% above the theoretical value. 1H NMR spectra
were recorded in CDCl3 (unless otherwise noted) with
tetramethylsilane (TMS) as the internal standard on a
Varian Unity 400 MHz spectrometer. Electron ioniza-
tion mass spectra (MS) were recorded on a Hewlett-
Packard 5890 mass spectrometer. Reagents and solvents
were used as obtained from commercial suppliers with-
out further purification. Column chromatography was
performed using silica gel and the flash technique.
Representative procedures and physical properties for
selected compounds are described.
General procedure for the preparation of compounds 2a–2j
A solution of 1-(1,4-benzodioxane-6-yl)piperazine (4)
(110 mg, 0.5 mmol) in acetonitrile (5 mL) was treated
with potassium carbonate (0.4 g) and the appropriate
benzyl halide (0.55 mmol). The mixture was heated to
reflux for 4 h, or until complete by TLC, cooled to room
temperature and diluted with dichloromethane (15 mL).
The mixture was washed with saturated NH4Cl solution
(15 mL), brine (20 mL), dried (MgSO4) and con-
centrated. The residue was purified by flash chromato-
graphy on silica gel eluting with the appropriate hexane/
ether mixture. The free base was finally converted to the
salt form indicated in the text.
1-(4-Methoxyphenyl)-4-benzylpiperazine (1). A solution
of 1-(4-methoxyphenyl)piperazine (96 mg, 0.5 mmol) in
acetonitrile (5 mL) was treated with potassium carbon-
ate (0.4 g) and benzyl bromide (94 mg, 0.55 mmol). The
mixture was heated to reflux for 4 h, cooled to room
temperature and diluted with ethyl acetate (15 mL). The
mixture was washed with water (20 mL), saturated
NH4Cl solution (15 mL), brine (20 mL), dried (MgSO4)
and concentrated. The residue was purified by flash
chromatography on silica gel eluting with hexane/ether
(1:1) and gave the title compound as a white solid (130
6-(4-Benzylpiperazin-1-yl)benzodioxane dihydrochloride
(2a). 89% yield from 1-(1,4-benzodioxane-6-yl)piper-
azine (4) and benzyl bromide: mp 144–145 ꢀC; 1H NMR
(free base, 400 MHz, CDCl3) d 2.58–2.61 (m, 4H), 3.07–
3.08 (m, 4H), 3.56 (s, 2H), 4.19–4.24 (m, 4H), 6.45–6.47
(m, 2H), 6.76 (d, J=8.5 Hz, 1H), 7.26–7.34 (m, 5H); MS
(LC-MS) m/e 311 (MH+). Anal. (C19H22N2O.22HCl) C,
H, N.
6-(4-[2-Chlorobenzyl]piperazin-1-yl)benzodioxane oxa-
late (2b). 71% from 1-(1,4-benzodioxane-6-yl)piper-
azine (4) and 2-chlorobenzyl chloride: mp 175–176 ꢀC;
1H NMR (free base, 400 MHz, CDCl3) d 2.66–2.68 (m,
4H), 3.09–3.11 (m, 4H), 3.68 (s, 2H), 4.19–4.25 (m, 4H),
6.46–6.48 (m, 2H), 6.77 (d, J=8.5 Hz, 1H), 7.19–7.27
(m, 2H), 7.36 (d, J=8 Hz, 1H), 7.50 (d, J=8 Hz, 1H);
mg, 92%): mp 78 ꢀC; H NMR (free base, 400 MHz,
1
CDCl3) d 2.60–2.63 (m, 4H), 3.08–3.11 (m, 4H), 3.57 (s,
2H), 3.76 (s, 3H), 6.83 (d, J=9.0 Hz, 2H), 6.89 (d,
J=9.0 Hz, 1H), 7.26–7.36 (m, 5H); MS (LC-MS) m/e
283 (MH+). Anal. (C18H22N2O) C, H, N.
1-(1,4-Benzodioxane-6-yl)piperazine (4). Tri(o-tolyl)pho-
sphine (0.3 g, 1.0 mmol) was added to a suspension of
tris(dibenzylideneacetone)dipalladium(0) (0.3 g, 1.0
mmol) in toluene (150 mL) followed by the addition of
sodium tert-butoxide (3.3 g, 34 mmol), tert-butyl 1-
piperazinecarboxylate (5.2 g, 28.2 mmol) and 1,4-ben-
zodioxan-6-bromide (5.16 g, 24 mmol). The mixture was
heated to reflux for 18 h and then cooled. The reaction
mixture was washed with water (200 mL), brine (200
mL), dried (MgSO4) and concentrated. Purification of
the residue by flash chromatography on silica gel eluting
MS
(C19H21ClN2O2.HO2CCO2H) C, H, N.
(LC-MS)
m/e
345
(MH+).
Anal.
6-(4-[3-Chlorobenzyl]piperazin-1-yl)benzodioxane dihydro-
chloride (2c). 67% from 1-(1,4-benzodioxane-6-yl)piper-
azine (4) and 3-chlorobenzyl chloride: mp 217–218 ꢀC;
1H NMR (free base, 400 MHz, CDCl3) d 2.57–2.60 (m,
4H), 3.07–3.09 (m, 4H), 3.52 (s, 2H), 4.19–4.25 (m, 4H),
6.45–6.47 (m, 2H), 6.76 (d, J=8 Hz, 1H), 7.23–7.26 (m,
3H), 7.36 (s, 1H); MS (LC-MS) m/e 345 (MH+). Anal.
(C19H21ClN2O.22HCl) C, H, N.

K. J. Hodgetts et al. / Bioorg. Med. Chem. 9 (2001) 3207–3213
3211
6-(4-[4-Chlorobenzyl]piperazin-1-yl)benzodioxane dihydro-
chloride (2d). 75% from 1-(1,4-benzodioxane-6-yl)pi-
perazine (4) and 4-chlorobenzyl chloride: mp 215–
(d, J=6.5 Hz, 3H), 2.49–2.56 (m, 2H), 2.61–2.68 (m,
2H), 3.04–3.07 (m, 4H), 3.40 (q, J=6.5 Hz, 1H), 4.18–
4.23 (m, 4H), 6.43–6.45 (m, 2H), 6.77 (d, J=9.5 Hz,
1H), 7.25–7.34 (m, 5H); MS (LC-MS) m/e 325 (MH+).
Anal. (C20H24N2O.22HCl) C, H, N.
216 ꢀC; H NMR (free base, 400 MHz, CDCl3) d 2.57–
1
2.59 (m, 4H), 3.09–3.11 (m, 4H), 3.49 (s, 2H), 4.18–4.25
(m, 4H), 6.42–6.44 (m, 2H), 6.78 (d, J=8.5 Hz, 1H),
7.22–7.25 (m, 4H); MS (LC-MS) m/e 345 (MH+). Anal.
(C19H21ClN2O2.2HCl) C, H, N.
Chromane (5). A solution of 4-chromanone (2.00 g,
13.50 mmol) in 20 mL of acetic acid was added to a
suspension of zinc dust (20 g) in acetic acid (40 mL).
The mixture was heated at 100 ꢀC for 4 h. After cooling
to room temperature, the mixture was filtered through
Celite and the filtrate evaporated. The residue was par-
titioned between ethyl acetate (50 mL) and 1 N NaOH
(50 mL) and extracted with further ethyl acetate (2Â50
mL). The organic layer was dried (MgSO4) and con-
centrated. The residue was purified by flash chromato-
graphy on silica gel eluting with hexane/ethyl acetate
(9:1) and gave the title compound 1.63 g (91%) as a
yellow oil: 1H NMR (400 MHz, CDCl3) d 1.99–2.06 (m,
2H), 2.80 (t, J=6.5 Hz, 2H), 4.19 (t, J=5.0 Hz, 2H),
6.79–6.86 (m, 2H), 7.03–7.11 (m, 2H).
6-(4-[4-Methoxybenzyl]piperazin-1-yl)benzodioxane dihy-
drochloride (2e). 48% from 1-(1,4-benzodioxane-6-yl)pi-
perazine (4) and 4-methoxybenzyl chloride: mp 195–
ꢀ
1
196 C; H NMR (free base, 400 MHz, CDCl3) d 2.56–
2.59 (m, 4H), 3.06–3.08 (m, 4H), 3.49 (s, 2H), 3.81 (s,
3H), 4.20–4.22 (m, 4H), 6.43–6.46 (m, 2H), 6.76 (d,
J=8.5 Hz, 1H), 6.87 (d, J=8.5 Hz, 2H), 7.25 (d, J=8.5
Hz, 2H) ; MS (LC-MS) m/e 341 (MH+). Anal.
(C20H24N2O3.2HCl) C, H, N.
6-(4-[3-Methoxybenzyl]piperazin-1-yl)benzodioxane dihy-
drochloride (2f). 48% from 1-(1,4-benzodioxane-6-yl)pi-
perazine (4) and 3-methoxybenzyl chloride: mp 215–
ꢀ
1
216 C; H NMR (free base, 400 MHz, CDCl3) d 2.58–
2.62 (m, 4H), 3.04–3.08 (m, 4H), 3.58 (s, 2H), 3.82 (s,
3H), 4.18–4.24 (m, 4H), 6.42–6.44 (m, 2H), 6.78–6.84
(M, 2H), 6.98 (M, 21H), 7.22 (t, J=7.5 Hz, 1H); MS
(LC-MS) m/e 341 (MH+). Anal. (C20H24N2O3. 2HCl)
C, H, N.
6-Piperazinylchromane (6). Chromane (5) (1.34 g, 10
mmol) was added portionwise to 70% HNO3 (6 mL) at
0 ꢀC. The mixture was stirred at room temperature for a
further 1 h. The solution was poured into ice water (15
mL) and extracted with ethyl acetate (3Â25 mL), dried
and concentrated. The residue was triturated with etha-
nol and gave 6-nitrochromane 1.41 g (79%) as pale yel-
low crystals: mp 99–100 ꢀC; 1H NMR (400 MHz,
CDCl3) d 2.01–2.09 (m, 2H), 2.85 (t, J=6.0 Hz, 2H),
4.28 (t, J=5.5 Hz, 2H), 6.83 (d, J=9.5 Hz, 1H), 7.96–
7.98 (m, 2H). 6-Nitrochromane (1.25 g, 7 mmol) was
dissolved in ethanol (50 mL), treated with Raney nickel
(1.5 g) and hydrogenated for 3 h at atmospheric pres-
sure. The resulting mixture was filtered and con-
centrated to afford chromane-6-ylamine 950 mg (91%)
as a yellow oil: 1H NMR (400 MHz, CDCl3) d 1.93–1.97
(m, 2H), 2.69 (t, J=5.5 Hz, 2H), 3.25 (brs, 2H), 4.10 (t,
J=5.0 Hz, 2H), 6.39 (s, 1H), 6.45 (d, J=8.5 Hz, 1H),
6.61 (d, J=8.5 Hz, 2H). A solution of chromane-6-yla-
6-(4-[2-Methoxybenzyl]piperazin-1-yl)benzodioxane oxa-
late (2g). 66% from 1-(1,4-benzodioxane-6-yl)piper-
azine (4) and 2-methoxybenzyl chloride: mp 143–144 ꢀC;
1H NMR (free base, 400 MHz, CDCl3) d 2.62–2.64 (m,
4H), 3.07–3.10 (m, 4H), 3.61 (s, 2H), 3.82 (s, 3H), 4.19–
4.25 (m, 4H), 6.42–6.44 (m, 2H), 6.78 (d, J=8.5 Hz,
1H), 6.88 (d, J=7 Hz, 1H), 6.93 (t, J=7.5 Hz, 1H), 7.23
(t, J=7.5 Hz, 1H), 7.39 (d, J=7 Hz, 1H); MS (LC-
MS) m/e 341 (MH+). Anal. (C20H24N2O.3HO2CCO2H)
C, H, N.
6-(4-[4-Fluorobenzyl]piperazin-1-yl)benzodioxane dihydro-
chloride (2h). 80% from 1-(1,4-benzodioxane-6-yl)-
piperazine (4) and 4-fluorobenzyl chloride: mp 178–
mine (0.745 g,
5 mmol), bis-(2-chloroethyl)amine
hydrochloride (1.07 g, 6 mmol) and potassium carbon-
ate (1.66 g, 12 mmol) in 25 mL chlorobenzene was
heated to reflux for 24 h. The dark brown reaction
mixture was partitioned between 3N NaOH (25 mL)
and dichloromethane (50 mL). The organic layer was
separated, dried and concentrated. The residue was
purified by flash chromatography on silica gel eluting
with dichloromethane/methanol (19:1) and gave the title
compound 0.58 g (53%) as a yellow oil: 1H NMR
(400 MHz, CDCl3) d 1.97–2.00 (m, 2H), 2.75 (t, J=6.0
Hz, 2H), 3.18 (brs, 8H), 4.13 (t, J=5.0 Hz, 2H), 6.36 (s,
1H), 6.72–6.73 (m, 2H).
179 ꢀC; H NMR (free base, 400 MHz, CDCl3) d 2.58–
1
2.59 (m, 4H), 3.08–3.10 (m, 4H), 3.51 (s, 2H), 4.19–4.23
(m, 4H), 6.41–6.44 (m, 2H), 6.79 (d, J=8.5 Hz, 1H),
6.99–7.03 (m, 2H), 7.25–7.31 (m, 2H); MS (LC-MS) m/e
329 (MH+). Anal. (C19H21FN2O2.2HCl) C, H, N.
6-(4-[4-Methylbenzyl]piperazin-1-yl)benzodioxane dihydro-
chloride (2i). 56% from 1-(1,4-benzodioxane-6-yl)piper-
azine (4) and 4-methylbenzyl chloride: mp 199–201 ꢀC;
1H NMR (free base, 400 MHz, CDCl3) d 2.35 (s, 3H),
2.58–2.60 (m, 4H), 3.03–3.08 (m, 4H), 3.52 (s, 2H),
4.19–4.26 (m, 4H), 6.42–6.45 (m, 2H), 6.77 (d, J=8.5
Hz, 1H), 7.16 (d, J=8.5 Hz, 2H), 7.22 (d, J=8.5 Hz,
2H); MS (LC-MS) m/e 325 (MH+). Anal.
(C20H24N2O.22HCl) C, H, N.
6-[4-(4-Chlorobenzyl)piperazinyl]chromane (7). A solu-
tion of 6-piperazinylchromane (6) (0.11 g, 0.5 mmol) in
acetonitrile (5 mL) was treated with potassium carbon-
ate (0.4 g) and 4-chlorobenzyl chloride (89 mg, 0.55
mmol). The mixture was heated to reflux for 4 h, cooled
to room temperature and diluted with dichloromethane
(15 mL). The mixture was washed with saturated
NH4Cl solution (15 mL), brine (20 mL), dried (MgSO4)
6-[4-(Phenylethyl)piperazinyl]-2H,3H-benzo[e]1,4-dioxin
dihydrochloride (2j). 51% from 1-(1,4-benzodioxane-6-
yl)piperazine (4) and (1-bromoethyl)benzene: mp 218–
220 ꢀC; H NMR (free base, 400 MHz, CDCl3) d 1.40
1

3212
K. J. Hodgetts et al. / Bioorg. Med. Chem. 9 (2001) 3207–3213
and concentrated. The residue was purified by flash
chromatography on silica gel eluting with hexane/ether
(1:1) and gave the title compound 135 mg (79%) as a
white solid: mp 94–95 ꢀC; 1H NMR (free base,
400 MHz, CDCl3) d 1.95–1.99 (m, 2H), 2.58 (brs, 4H),
2.75 (t, J=6.5 Hz, 2H), 3.06 (brs, 4H), 3.52 (s, 2H), 4.12
(t, J=5.0 Hz, 2H), 6.62 (s, 1H), 6.72 (s, 2H), 7.28–7.29
(m, 4H); MS (LC-MS) m/e 343 (MH+). Anal.
(C20H23ClN2O.C2H2O4) C, H, N.
Binding assay. For D2, D3, and D4 binding, each sample
was tested in triplicate in a final volume of 0.25 mL in
Polypropylene microtube trips containing 0.1 nM
[3H]YM 09151 (81.4 Ci/mmol, NEN DuPont), and
CHO cell homogenate (40 mg protein) in 50 mM Tris
buffer (pH 7.4 at 25 ꢀC) containing 120 mM NaCl. After
a 2 h incubation at room temperature, the samples were
rapidly filtered through 1% PEI treated GF/C filters
using a Tomtec Harvester 96. The filters were rinsed
with two washes of assay buffer. After air drying, bound
radioactivity was then quantitated via the BetaPlate
Scintillation Counter at an efficiency of 65%. Non-
specific binding was defined with 10 mM spiperone for
D2 and D4 and with 1 mM haloperidol for D3.
5-{4-[(4-Chlorophenyl)methyl]piperazinyl}-2H-benzo[d]1,3-
dioxolane dihydrochloride (8). 53% from 5-piperazinyl-
2H-benzo[d]1,3-dioxolane and 4-chlorobenzyl chloride
according to the procedure described for compound 7:
1H NMR (free base, 400 MHz, CDCl3) d 2.59–2.60 (m,
4H), 3.07–3.09 (m, 4H), 3.62 (s, 2H), 5.90 (s, 2H), 5.99–
6.04 (m, 2H), 6.49 (d, J=8 Hz, 1H), 7.01 (d, J=7 Hz,
2H), 7.15 (d, J=7 Hz, 2H); MS (LC-MS) m/e 331
(MH+). Anal. (C18H19ClN2O.22HCl) C, H, N.
Dopamine functional assays. CHO cells expressing pri-
mate D4.2 receptor were grown to confluency in Ham’s
media supplemented with 10% fetal calf serum, har-
vested, and then stored as pellets at À80 ꢀC. Thawed
cells were homogenized using a Polytron (30 s, setting 5)
in 50 mM Tris pH 7.4, 10 mM MgCl2 and 2 mM
EGTA. Membrane homogenates were centrifuged at
14,000g for 10 min and the pellet washed one time in
cold PBS. The final pellet was resuspended in homo-
ginization buffer and stored at À80 ꢀC. On the day of
the assay, thawed membrane homogenates were resus-
pended in assay buffer (50 mM Tris pH 7.4, 120 mM
NaCl, 10 mM MgCl2, 2mM EGTA, 0.1% BSA, 0.1 mM
bacitracin, 100 KIU/mmL aprotinin, 5 mM GDP) and
added to reaction tubes at a concentration of 25 mg/
0.200 mL. Reactions were initiated by the addition of
100 pM GTPg35S, dopamine (0.01–10 mM) and indivi-
dual compounds ranging in concentration from 0.1 nM
to 10 mM. Following a 45 min incubation at 22 ꢀC, the
reaction was terminated by vacuum filtration over GF/
C filters with ice-cold wash buffer (50 mM Tris pH 7.4,
120 mM NaCl). Bound GTPg35S was determined by
liquid scintillation spectrometry. Non-specific binding
was defined by 10 mM GTPgS and represented less than
10% of total binding.
5-{4-[(4-Chlorophenyl)methyl]piperazinyl}-2,3-dihydro-
benzo[b]furan dihydrochloride (9). 55% from 5-piper-
azinyl-2,3-dihydrobenzo[b]furan and 4-chlorobenzyl
chloride according to the procedure described for com-
pound 7: mp 218–219 ꢀC; 1H NMR (free base,
400 MHz, CDCl3) d 2.58–2.60 (m, 4H), 3.05–3.08 (m,
4H), 3.18 (t, J=7 Hz, 2H), 3.52 (s, 2H), 4.53 (t, J=7
Hz, 2H), 6.64–6.66 (m, 2H), 6.85 (s, 1H), 7.22–7.25 (m,
4H); MS (LC-MS) m/e 329 (MH+). Anal.
(C19H21ClN2O.2HCl) C, H, N.
Biological methods
Expression of recombinant dopamine receptors. The
recombinant primate D4.2 receptor was prepared from
the D4.2 minigene expression construct by replacement
of the NotI-KasI fragment containing two introns with
a synthetic DNA fragment encoding the intron-deleted
sequence (Genbank #HSD4DOP). Stable clones
expressing each receptor were isolated under G418
selection after calcium phosphate transfection of CHO-
K1 cells (the primate D4.2 plasmid was cotransfected
with pSV2Neo, Clontech). The membranes prepared
from cell pellets were stored at À80 ꢀC.
References and Notes
Membrane preparation. Pellets containing selected
cloned dopamine receptor membrane were thawed on
ice and resuspended in ice cooled 50 mM Tris buffer
(pH 7.4 at 25 ꢀC) containing 120 mM NaCl, 1 mM
EDTA and 5 mM MgCl2. All subsequent work was
performed on ice. The membranes were homogenized
on a Brinkmann Polytron (10 s, setting 5). The homo-
genate was centrifuged at 48,000g and 4 ꢀC for 10 min
(DuPont Sorvall RC5B). The pellet was resuspended in
fresh buffer and centrifugation was repeated. The pellet
was again resuspended in fresh buffer and centrifuged a
final time at 48,000 g and 4 ꢀC for 10 min. The pellet
was resuspended to a final concentration of 40 mg pro-
tein/mL with 50 mM Tris buffer (pH 7.4 at 25 ꢀC) con-
taining 120 mM NaCl, just prior to addition to the
assay tubes. The protein content was determined using
the Bio-Rad assay (Hercules, CA), with bovine plasma
gamma globulin as a standard.
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