Bioisosteric replacement leading to biologically active [2.2]Paracyclophanes with altered binding profiles for aminergic g-protein-coupled receptors

Article abstract of DOI:10.1021/jm100899z

Exploring the chemical diversity space of GPCR ligands, we recently discovered [2.2]paracyclophanes as valuable atypical bioisosteres for secondary affinity and selectivity generating moieties. To find out if such an exchange also works for structural moieties that simulate the endogenous neurotransmitter, π1 or π2 or both systems π1 and π2 of three representative privileged structures of types 1, 2, and 3 were replaced by a [2.2]paracyclophane unit. Contributions of the respective functionalities to the binding affinities of a panel of relevant monoaminergic GPCRs were systematically examined. The study led to the paracyclophanylpiperazine 3a displaying excellent D₃ affinity (K_i = 1.6 nM) and a strongly attenuated binding to D₄, 5-HT₁ and α₁. Whereas functional experiments showed neutral D₃ antagonist properties, mutagenesis studies indicated a binding mode that is similar to its lead compounds of type 3.

Full text of DOI:10.1021/jm100899z

J. Med. Chem. 2010, 53, 7219–7228 7219
DOI: 10.1021/jm100899z
Bioisosteric Replacement Leading to Biologically Active [2.2]Paracyclophanes with Altered Binding
Profiles for Aminergic G-Protein-Coupled Receptors
€
€
Marika Skultety, Harald Hubner, Stefan Lober, and Peter Gmeiner*
Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, 91052 Erlangen, Germany
Received July 19, 2010
Exploring the chemical diversity space of GPCR ligands, we recently discovered [2.2]paracyclophanes as
valuable atypical bioisosteres for secondary affinity and selectivity generating moieties. To find out if
such an exchange also works for structural moieties that simulate the endogenous neurotransmitter,
π1 or π2 or both systems π1 and π2 of three representative privileged structures of types 1, 2, and 3 were
replaced by a [2.2]paracyclophane unit. Contributions of the respective functionalities to the binding
affinities of a panel of relevant monoaminergic GPCRs were systematically examined. The study led to
the paracyclophanylpiperazine 3a displaying excellent D3 affinity (Ki = 1.6 nM) and a strongly atten-
uated binding to D4, 5-HT1 and R1. Whereas functional experiments showed neutral D3 antagonist
properties, mutagenesis studies indicated a binding mode that is similar to its lead compounds of type 3.
Introduction
Representative examples are the CNS active drugs haloper-
idol, iloperidone, paliperidone, fluanisone, fluspirilene, aripi-
prazole, buspirone,1-6 and the drug candidates F-13640
(bifiradol), RGH-188 (cariprazine), ABT-724, and FAUC
346.7-10 Interestingly, 1,4-DAPs have a common pattern of
ligand-receptor recognition that depends upon favorable
interactions with highly conserved amino acids of the receptor
binding site crevice.11 Containing a conjugated π-system (π1)
and a basic nitrogen, the phenylpiperidine/phenylpiperazine
scaffold represents the primary recognition element that
targets the binding site of the native biogenic amines. Addi-
tionally, in position 4 of the central piperidine/piperazine,
unsaturated carbocyclic or heterocyclic appendages (π2) are
attached via a spacer element. Interacting with a hydrophobic
microdomain provided by specific residues of the transmem-
brane domains (TMs) 2, 3, and 7 as well as parts of the extra-
cellular loop 2, this structural entity controls both binding
affinity and subtype selectivity.12
Exploring the chemical diversity space of bioactive com-
pounds, we recently discovered [2.2]paracyclophanes as valu-
able atypical bioisosteres for the secondary affinity and selec-
tivity generating system π2. Thus, we have discovered novel
paracyclophane derived dopamine D3 receptor antagonists
displaying particular binding profiles that might be a starting
point for the development of highly beneficial CNS active
drugs, especially for the treatment of schizophrenia and
addiction.13 Thus, we could show that the high steric demand
of [2.2]paracyclophanes is well tolerated by the hydrophobic
microdomain of the dopamine D3 receptor. Moreover, we
found that indoloparacyclophanes can be used as double
layered aryl bioisosteres of highly selective D4 receptor
ligands.14
G-Protein-coupled receptors (GPCRsa) represent the lar-
gest class of membrane proteins in the human genome. Within
the family of aminergic GPCR ligands, 1,4-disubstituted aro-
matic piperidines and piperazines (1,4-DAPs) are known as
privileged structural moieties simulating endogenous neuro-
transmitters, like dopamine, serotonin, and (nor)epinephrine.
*To whom correspondence should be addressed. Phone: þ49
9131 85-29383. Fax: þ49 9131 85-22585. E-mail: peter.gmeiner@
medchem.uni-erlangen.de.
a Abbreviations: GPCR, G-protein-coupled receptor; 1,4-DAPs, 1,4-
disubstituted aromatic piperidines and piperazines; CNS, central nervous
system; haloperidol, 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]-1-(4-
fluorophenyl)butan-1-one; iloperidone, 1-[4-[3-[4-(6-fluoro-1,2-benzisox-
azol-3-yl)-1-piperidinyl]propoxy]-3-methoxyphenyl]ethanone; paliperidone,
3-[2-[4-(6-fluorobenzo[d]isoxazol-3-yl)-1-piperidyl]ethyl]-7-hydroxy-4-
methyl-1,5-diazabicyclo[4.4.0]deca-3,5-dien-2-one; fluanisone, 1-(4-fluoro-
phenyl)-4-[4-(2-methoxyphenyl)piperazin-1-yl]butan-1-one; fluspirilene,
8-[4,4-bis(4-fluorophenyl)butyl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-
4-one; aripiprazole, 7-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butoxy]-
3,4-dihydroquinolin-2(1H)one; buspirone, 8-[4-(4-pyrimidin-2-ylpiper-
azin-1-yl)butyl]-8-azaspiro[4.5]decane-7,9-dione; F-13640, 3-chloro-4-
fluorophenyl-[4-fluoro-4-[[(5-methylpyridin-2-yl)methylamino]methyl]-
piperidin-1-yl]methanone; RGH-188, trans-N-[4-[2-[4-(2,3-dichloro-
phenyl)piperazin-1-yl]ethyl]cyclohexyl]-N0,N0-dimethylurea; ABT-724,
2-(4-pyridin-2-ylpiperazin-1-ylmethyl)-1H-benzimidazole; FAUC 346,
N-[4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl]benzo[b]thiophene-2-
carboxamide; TM, transmembrane helix; 5-HT, serotonin (5-hydroxy-
tryptamine); IBX, 2-iodoxybenzoic acid; TBTU, O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium tetrafluoroborate; SCH 23390, 7-chloro-
3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol; spiperone, 8-[4-
(4-fluorophenyl)-4-oxobutyl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one;
WAY100635, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)-
cyclohexanecarboxamide; ketanserin, 3-{2-[4-(4-fluorobenzoyl)piperidin-
1-yl]ethyl}quinazoline-2,4(1H,3H)-dione; prazosin, 2-[4-(2-furoyl)-
piperazin-1-yl]-6,7-dimethoxyquinazolin-4-amine; CHO, Chinese ham-
ster ovary; HEK, human embryonic kidney; SAR, structure-activity
relationship; NMR, nuclear magnetic resonance; MHz, megahertz; LC/
MS, liquid chromatography/mass spectrometry; APC, atmospheric
pressure chemical; TLC, thin layer chromatography; HRMS, high
resolution mass spectrometry; HPLC, high performance liquid chro-
matography; EI-MS, electron ionization mass spectrometry; DMF,
dimethylformamide.
To further validate the scope and limitations of the
[2.2]paracyclophane scaffold, π1, π2, or both systems π1 and
π2 of the three representative 1,4-DAPs of types 1, 2, and 3
were replaced by a [2.2]paracyclophane unit. To be able to
unambiguously allocate biological effects, 1,4-xylene derived
r
2010 American Chemical Society
Published on Web 09/14/2010
pubs.acs.org/jmc

7220 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Skultety et al.
Scheme 1a
Figure 1. Bioisosteric replacement leading to the target compounds
of types 1, 2, and 3.
analogues should be prepared and investigated for every
paracyclophane based test compound. Thus, the contribu-
tions of the respective functionalities to the binding affinities
of a panel of relevant monoaminergic GPCRs were system-
atically detected for the classical N-piperidinylbutyrophenones
1, the piperazinylbutyrophenones 2, and the homologous
(hetero)arene carboxamides 3 (Figure 1). To find out if such a
bioisosteric exchange also works for the structural moieties
that simulate the endogenous neurotransmitter, not only the
secondary unsaturated system π2 but also the primary recog-
nition element π1 was replaced. To learn if the increase of the
volumebyintroducingthe paracyclophane functionality leads
to a general modification of GPCR affinity and specificity
profiles, the binding properties toward eight structurally
a Reagents and conditions: (a) 1,4-dioxa-8-azaspirodecane, TEA or
Na2CO3/KI, DMF or toluene (29-98%); (b) 4-(4-chlorophenyl)-4-
hydroxypiperidine, Et3N, DMF (62-65%); (c) HCl (2 N) (63-68%);
(d) Br-R0, n-BuLi, Et2O (24-47%).
Scheme 2a
related GPCRs including D1, D2long, D2short, D3, D4, 5-HT1A
,
5-HT2, and R1 receptors were investigated. To examine ligand
efficacy and binding modes, highly promising test compounds
were further characterized by functional experiments and
mutagenesis studies, respectively.
a Reagents and conditions. (a) For 2a-e and 2g: N-arylphenylpipera-
zine, NaI, NaHCO3, CH3CN(27-96%). (b)For 2f:(1) NaHCO3, DMSO
(36%), (2) IBX, DMSO (41%), (3) 4-[2.2]paracyclophanylpiperazine,
Na(OAc3)BH, CH2Cl2 (55%).
Results and Discussion
Chemistry. All paracyclophane based target compounds
were synthesized in racemic form. Our initial investigations
were directed to the chemical synthesis of hydroxypiperidine
derivatives of type 1. The construction of the 4-aminobutyr-
ophenone moiety started from the commercially available
haloperidol intermediate 4a and the bromobutyrophenones
4b and 4c, which could be prepared by Friedel-Crafts
acylation of [2.2]paracyclophane and p-xylene, respectively
(Scheme 1). Introduction of an acetal-protected piperidone
unit was done by treatment of 4a-c with 1,4-dioxa-8-azas-
pirodecane. Ketal cleavage of the thus formed tertiary
amines 5a-c in the presence of aqueous hydrochloric acid
gave access to the piperidones 6a-c. Finally, introduction
of the second π-system was performed by halogen metal
exchange of bromofluorobenzene, bromoparacyclophane,
and bromoxylene and subsequent treatment with the piper-
idones 6a-c. Thus, the test compounds 1a,b,d,f could be
efficiently prepared. An alternative root was elaborated for
the 4-(p-chlorophenyl)piperidines 1c and 1e when 4b and 4c,
respectively, were reacted with commercially available
chlorophenyl-substituted piperidine-4-ol.
Starting from 4a-c and 4-substituted piperazines, nucleo-
philic displacement reaction gave the fluanisone-like test
compounds 2a-e and 2g (Scheme 2). As the bis-paracyclo-
phane 2f could not be prepared following this method, the
synthesis had to be modified for this particular target com-
pound. The final product 2f could be accessed by hydrolysis
of the bromide 4b and subsequent IBX-promoted oxidation
of the resulting alcohol to a corresponding carbaldehyde.
Reductive amination with paracyclophanylpiperazine14b in
the presence of sodium triacetoxyborohydride afforded the
test compound 2f.
For the synthesis of the carboxamides of type 3, the
o-methoxyphenyl-, paracyclophanyl-, and piperazines were sub-
jected to N-alkylation with 4-bromobutyronitrile (Scheme 3).
Subsequent reduction of the intermediates 7a-c by lithium
aluminum hydride yielded the primary amines 8a-c. Finally,
TBTU-promoted coupling reaction of 8a-c with benzothio-
phene, p-xylene, and paracyclophanecarboxylic acids resulted in
formation of the carboxamides 3a-e.
Biological Investigations. Receptor binding experiments
were established to evaluate the binding properties of the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7221
Scheme 3a
when compared to the classical antipsychotic haloperidol. It
is interesting to note that the same structural modification on
the piperazine analogues leading to the test compound 2b
caused a significant reduction of D4 affinity (Ki = 120 nM).
In contrast, 2b showed strongly improved D3 binding (Ki =
9.9 nM) when compared to the lead compound 2a. The
analogous π1-exchange within the compounds of type 3 led
to an almost unchanged binding to D1, D2, and D3 and
5-HT2. Interestingly, the paracyclophane 3a displayed ex-
cellent D3 affinity (Ki = 1.6 nM) and a strongly attenuated
binding to D4, 5-HT1, and R1. As a consequence, 3a revealed
a 50- to 500-fold target selectivity. Compared to the lead
compound FAUC 346, the 300-fold preference over the
antitarget R1 indicates a more than 10-fold enhancement,
which is worthy of note because R1 antagonism is known to
induce cardiovascular side effects limiting the therapeutic
value of many CNS-active drug candidates.19 Comparison of
the above-mentioned effects with the binding profiles of the
p-xylene derived reference compounds 1b, 2c, and 3b indi-
cated that none of the above-mentioned improvements could
be performed by the xylene moiety. Thus, the major SAR
effects are obviously due to the huge steric demand of the
double-layered system.
a Reagents and conditions: (a) 4-bromobutyronitrile, Na2CO3,
CH3CN (84-98%); (b) LiAlH4, Et2O (93-99%); (c) RCOOH, TBTU,
DIPEA, CH2Cl2, DMF (65-83%).
Displacement of π2 by [2.2]paracyclophane was less effec-
tive. Thus, reduced GPCR affinities were observed for 1c and
2d. Interestingly, however, the bioisosteric exchange that was
leading to the recently described paracyclophanyl carboxa-
mide 3f13 resulted in a binding pattern that was competitive
to the lead compound FAUC 346. The binding properties of
the xylene analogues 1e, 2e, and 3c were comparable, leading
to the observation that the structural modifications on π2 are
in fact less crucial than those of the primary recognition
element π1.
Analysis of the bis-paracyclophanes 1d, 2f, and 3d in
comparison to the bis-xylene analogues 1f, 2g, and 3e
indicated preferential D2, D3, and D4 binding for all com-
pounds when R1 affinity was substantially attenuated for the
paracyclophanes. Because R1 is regarded to be an antitarget
leading to cardiovascular problems, this observation might
be highly beneficial for further efforts in the drug discovery
of selective dopamine receptor agonists and antagonists.
As a measure of functional D3 activity, ligand efficacy of
the [2.2]paracyclophanylpiperazine 3a was determined by
a mitogenesis assay measuring the rate of [3H]thymidine
incorporation into a CHO dhfr- cell line stably expressing
the human D3 receptor when the full agonist quinpirole and
the partial agonist FAUC 346 were used as reference
agents.17,20,21 Interestingly, the test compound 3a did not show
any ligand efficacy at the D3 subtype, indicating neutral D3
antagonist properties.
Site-directed mutagenesis was employed to identify crucial
interactions between the binding pocket of the D3 receptor and
the paracyclophanylpiperazine 3a compared to the xylene
analogue 3b as a representative test compound. The binding
site crevice of dopamine receptors is thought to be determined
by several highly conserved amino acids incuding F6.52 as a part
of the aromatic microdomain binding the catechol substructure
of the endogenous ligand dopamine (Figure 2).22-24 As a crucial
residue for aromatic carboxamide moiety of compounds of type
3, V2.61 was identified.12 To inspect the binding mode of 3a, we
mutated V2.61 and F6.52 into the phenylalanine and trypto-
phan derivatives D3 V2.61F and D3 F6.52W,25 respectively.
In accordance with previously recorded mutagenesis data
for D3 ligands of type 3,12 the single mutant V2.61F induced
paracyclophanes 1a,c,d, 2b,d,f, 3a,d, and 3f13 and the
p-xylene derived analogues 1b,e,f, 2c,e,g, 3b,c,e. Haloperidol,
the fluanisone analogue 2a, and FAUC 346 were used as
reference agents (Table 1). D1 receptor affinities were deter-
mined utilizing porcine striatal membranes and the D1
selective radioligand [3H]SCH 23390.15 D2long, D2short, D3,
and D4 receptor affinities were investigated employing the
16
cloned human dopamine receptor subtypes D2long, D2short
,
D3,17 and D4.418 stably expressed in Chinese hamster ovary
cells (CHO) and the radioligand [3H]spiperone.15 The com-
petition data were analyzed according to a sigmoid model by
nonlinear regression. Because of the observation that our
lead compounds are known for serotonergic and adrenergic
activity as well, the test compounds were also investigated for
their potency to displace [3H]WAY 100635, [3H]ketanserin,
and [3H]prazosin when employing porcine 5-HT1A, 5-HT2,
and R1 receptors, respectively. The data of the radioligand
binding studies unambiguously proved that the double-
layered paracyclophane derived moiety of the target com-
pounds is specifically recognized by the binding sites of the
GPCRs investigated (Table 1). Whereas only weak to mod-
erate binding affinities were determined for D1, 5-HT2, and
5-HT1A, respectively, D2, D4, and R1 receptor recognition
resulted in Ki values reaching even the single-digit nanomolar
range.
Initially, we analyzed the impact of the replacement of the
π1-system of haloperidol, the fluanisone derivative 2a, and
FAUC 346 by the [2.2]paracyclophane scaffold. Interest-
ingly, the paracyclophane 1a and its xylene analogue 1b dis-
played a binding pattern that was very similar to that of its
lead compound haloperidol with comparable or slightly lower
affinities. However, the D4 receptor binding of 1a turnedout to
be significantly stronger resulting in a Ki value of 0.72 nM.
Thus, the introduction of a [2.2]paracyclophane scaffold led
to a significant change of the overall affinity profile. Besides
significant binding affinity to adrenergic, serotonergic, choli-
nergic, and histaminergic receptors, the atypical antipsychotic
clozapine displays D4 preference within the dopaminergic
receptor family. Thus, the above-mentioned bioisosteric
replacement could exert substantial therapeutic advantages

7222 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Skultety et al.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7223
and 13C NMR (90 or 150 MHz) spectra were determined on a
Bruker AM 360 or a Bruker AVANCE 600 spectrometer in
solution. LC/MS analyses were conducted in an Agilent binary
gradient system (MeOH/0.1% aqueous HCO2H, 10/90 to 90/
10) in combination with ChemStation software and UV detec-
tion at 254 nm using a Zorbax SB-C8 (4.6 mm ꢀ 150 mm, 5 μm)
with a flow rate of 0.5 mL/min. Mass detection was done with a
Bruker Esquire 2000 ion-trap mass spectrometer using an APC
ionization source. HRMS spectra were recorded on JEOL
GCmatell instrument. Flash chromatography was done using
silica gel (40-63 μm) as stationary phase. TLC analyses were
done on Merck 60 F254 glass plates and analyzed by UV light
(254 nm) or by iodine vapor. Purity was determined by elemen-
tary analysis or by HPLC using an Agilent 1100 HPLC systems
in combination with UV detection. As column, a Zorbax Eclipse
XDB-C8 (4.6 mm ꢀ 150 mm, 5 μm) was used. HPLC was run
with MeOH (eluent I) and 0.1% aqueous formic acid (eluent II)
and the following gradient: MeOH 10% for 3 min, ascending to
100% in 15 min, 100% for 6 min. The flow rate was 0.5 mL/min,
and the wavelength λ was 254 nm. All SAR compounds were
determined to be >95% pure.
Figure 2. Conceptional binding model for D3 and the paracyclo-
phane 3a as well as mutagenesis data for 3a compared to the xylene
analogue 3b.
a significant loss of binding affinity. Comparison of wild-type
D3 and D3 V2.61F, which were both expressed in transiently
transfected HEK cells, led to a 24-fold and 40-fold reduction
of affinity for the benzothiophene carboxamides 3a and 3b,
respectively, indicating the same molecular environment of
π2. On the other hand, the D3 F6.52W resulted in a significant
reduction of binding for the xylene 3b (38-fold) but only in a
slight impairment (2.4-fold) for the paracyclophane 3a. Thus,
the structural modification of the catechol-simulating moiety
π1 leads to a differential susceptibility of the ligand-receptor
contact in position 6.52 of the aromatic microdomain.
4-Bromo-1-([2.2]paracyclophan-4-yl)butan-1-one (4b). To a
solution of [2.2]paracyclophane (1.2 g, 5.7 mmol) in dichloro-
methane (6.0 mL) was added a mixture of AlCl3 (1.4 g, 10.3
mmol) and 4-bromobutyric acid chloride (1.4 mL, 12 mmol) in
dichloromethane (3.3 mL) at -50 °C. The mixture was stirred
for 20 min when it was allowed to warm to -20 °C. Then the
suspension was filtered and added to a mixture of 6 N HCl
(10 mL) and ice-water (20 mL). After extraction with diethyl
ether, the organic layer was subsequently extracted with satu-
rated NaHCO3 solution and saturated NaCl solution. The
organic layer was dried (Na2SO4) and evaporated and the
residue was purified by flash chromatography (hexane/EtOAc
4:1) to give pure 4b (1.6 g, 79%) as a pale greenish solid (mp
Conclusion
1
77 °C). H NMR (CDCl3, 360 MHz) δ (ppm): 2.12-2.36 (m,
In conclusion, bioisosteric exchange of π1, π2, or both
systems π1 and π2 of the three representative privileged
structures of types 1, 2, and 3 by a [2.2]paracyclophane unit
led to GPCR ligands with novel selectivity profiles. As an
example, the paracyclophane 1a and its xylene analogue 1b
displayed a binding pattern that was very similar to that of its
lead compound haloperidol with comparable or slightly lower
affinities. However, the D4 receptor binding of 1a (Ki = 0.72
nM) turned out to be significantly stronger and the selectivity
higher (5- to 70-fold over D2long, D2short, and D3). The
contribution of the respective functionalities to the binding
affinities of a panel of relevant monoaminergic GPCRs was
systematically detected. The study led to the paracyclopha-
nylpiperazine 3a with excellent D3 affinity (Ki = 1.6 nM) and
a strongly attenuated binding to D4, 5-HT1A, and R1 resulting
in selectivity ratios of 550, 5300, and 300, respectively. The π1
exchange within the compounds of type 3 led to an almost
unchanged binding to D1, D2, and D3 and 5-HT2. As a
consequence, 3a revealed a 50- to 500-fold target selectivity.
Compared to the lead compound FAUC 346, the 300-fold
preference over the antitarget R1 indicates a more than 10-fold
enhancement, which is worthy of note because R1 antagonism
is known to induce cardiovascular side effects limiting
the therapeutic value of many CNS-active drug candidates.
Because of the chirality of the paracyclophane-derived final
products, aymmetric synthesis or separation of the enantio-
mers and pharmacological investigations of a configurational
impact will be investigated.
2H), 2.77-2.87 (m, 2H), 2.98-3.08 (m, 3H), 3.12-3.22 (m, 5H),
3.50-3.59 (m, 2H), 6.37 (dd, J = 7.8, 1.7 Hz, 1H), 6.45-6.56 (m,
4H), 6.66 (dd, J = 7.8, 1.7 Hz, 1H), 6.93 (d, J = 1.7 Hz, 1H). 13
C
NMR (CDCl3, 90 MHz) δ (ppm): 27.2, 33.7, 35.0, 35.2, 36.0,
38.4, 131.2, 132.1, 132.9, 133.0, 133.4, 136.4, 137.5, 139.2, 139.9,
140.2, 141.5, 201.1. IR (NaCl) ν (cm-1): 3008, 2927, 1674, 1551,
1500, 1230, 721. HPLC (254 nm) tR = 22.6 min, purity 100%.
HRMS (m/z): [M]þ calcd for C20H21BrO, 356.0776; found,
356.0776.
4-Bromo-1-(2,5-dimethylphenyl)butan-1-one (4c). To a mix-
ture of 4-bromobutyric acid chloride (1.1 mL, 9.8 mmol) and
p-xylene (2.8 mL, 22.8 mmol) was added AlCl3 (1.4 g, 10.5 mmol)
at 0 °C. After 30 min, stirring was continued at room tempera-
ture for another 30 min. Then the mixture was added to aqueous
HCl (1.5%) at 0 °C. After extraction with hexane, the organic
layer was dried (MgSO4) and evaporated and the residue was
purified by flash chromatography (hexane/EtOAc 10:1) to give
1
pure 4c (70%) as a colorless oil. H NMR (CDCl3, 600 MHz)
δ (ppm): 2.26-2.30 (m, 2H), 2.37 (s, 3H), 2.45 (s, 3H), 3.09 (t,
J = 7.0 Hz, 2H), 3.54 (t, J = 6.3 Hz, 2H), 7.13 (d, J = 7.8 Hz,
1H), 7.19 (dd, J = 7.8, 1.7 Hz, 1H), 7.47 (d, J = 1.7 Hz, 1H). 13
C
NMR (CDCl3, 90 MHz) δ (ppm): 20.9, 21.0, 27.1, 33.6, 39.4,
129.1, 131.9, 132.2, 134.9, 135.3, 137.6, 203.0. IR (NaCl) ν
(cm-1): 3021, 2964, 1685, 1567, 1496, 1241, 818. HPLC (254
nm) tR = 19.1 min, purity 100%. HRMS (m/z): [M]þ calcd for
C12H15BrO, 245.0306; found, 245.0305.
4-(1,4-Dioxa-8-azaspiro[4.5]dec-8-yl)-1-(4-fluorophenyl)butan-
1-one (5a). A mixture of 8-aza-1,4-dioxaspiro[4.5]decane (1.8 mL,
14.0 mmol), 4-chloro-40-fluorobutyrophenone (2.0 mL, 12.0 mmol),
KI (30 mg, 0.2 mmol), Na2CO3 (25 mg, 0.24 mmol), and toluene
(75 mL) was heated to reflux for 77 h. The mixture was filtered at
room temperature, and the filtrate was extracted with saturated
NaHCO3 solution. The organic layer was dried (Na2SO4), evapo-
rated and the residue was purified by flash chromatography
(CH2Cl2/MeOH 9:1) to give pure 5a (48%) as a pale yellow oil.
Experimental Section
Chemistry. IR spectra were registered on a JASCO model
FTIR 410 instrument via KBr pellet. 1H NMR (360 or 600 MHz)

7224 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Skultety et al.
1H NMR (CDCl3, 360 MHz) δ (ppm): 1.68-1.71 (m, 4H),
1.90-1.98 (m, 2H), 2.45 (t, J = 7.2 Hz, 2H), 2.51-2.54 (m, 4H),
2.97 (t, J = 7.0 Hz, 2H), 3.94 (s, 4H), 7.10-7.15 (m, 2H), 7.98-8.02
(m, 2H). IR (NaCl) ν (cm-1): 2955, 2812, 1685, 1598, 1506, 1228,
1096, 836. EIMS (m/z): [M]þ 307.
6.7 Hz, 2H), 7.13 (d, J = 7.8 Hz, 1H), 7.18 (dd, J = 7.8, 1.6 Hz,
1H), 7.46 (d, J = 1.6 Hz, 1H). 13C NMR (CDCl3, 90 MHz) δ
(ppm): 20.8, 21.0, 22.1, 39.2, 41.2, 53.1, 56.6, 129.0, 131.8, 131.9,
134.7, 135.1, 138.3, 204.3, 209.2. IR (KBr) ν (cm-1): 2960, 2808,
1718, 1682, 1568, 1221, 1093, 816. HPLC (254 nm) tR = 16.1
min, purity 100%. HRMS (m/z): [M]þ calcd for C17H23NO2,
273.1729; found, 273.1728.
1-([2.2]Paracyclophan-4-yl)-4-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-
butan-1-one (5b). To a solution of 4b (520 mg, 1.5 mmol) in DMF
(5.9 mL) were added 8-aza-1,4-dioxaspiro[4.5]decane (0.23 mL,
1.8 mmol) and triethylamine (0.26 mL). After the mixture was
stirred at room temperature for 3 h, H2O (40 mL) was added.
After extraction with diethyl ether, the organic layer was dried
(Na2SO4) and evaporated and the residue was purified by flash
chromatography (CH2Cl2/MeOH 93:7) to give 5b (99%) as a
colorless solid (mp 69 °C). 1H NMR (CDCl3, 360 MHz) δ (ppm):
1.74-1.78 (m, 4H), 1.85-1.96 (m, 2H), 2.45 (dt, J = 7.3, 3.0 Hz,
2H), 2.55-2.58 (m, 4H), 2.70 (ddd, J = 16.8, 7.7, 6.4 Hz, 1H),
2.79-2.93 (m, 2H), 2.96-3.05 (m, 2H), 3.11-3.22 (m, 4H), 3.86
(ddd, J = 12.4, 9.5, 2.5 Hz, 1H), 3.95 (s, 4H), 6.34 (dd, J = 7.7,
1.8 Hz, 1H), 6.48 (dd, J = 7.7, 1.8 Hz, 1H), 6.50 (d, J = 7.7 Hz,
1H), 6.51-6.56 (m, 2H), 6.63 (dd, J = 7.7, 1.8 Hz, 1H), 6.91 (d,
J = 1.8 Hz, 1H). 13C NMR (CDCl3, 90 MHz) δ (ppm): 22.0,
34.8, 35.1, 35.2, 35.9, 38.3, 51.3, 57.2, 64.2, 107.3, 131.2, 132.2,
132.8, 132.9, 133.3, 136.1, 136.3, 138.0, 139.2, 139.7, 140.2, 141.2,
202.6. IR (NaCl) ν (cm-1): 2927, 2810, 1674, 1593, 1231, 1095.
HPLC (254 nm) tR = 17.3 min, purity 97%. HRMS (m/z): [M]þ
calcd for C27H33NO3, 419.2460; found, 419.2460.
1-(2,5-Dimethylphenyl)-4-(1,4-dioxa-8-azaspiro[4.5]dec-8-yl)-
butan-1-one (5c). Compound 4c was reacted and worked up as
described for the synthesis of 5b to give 5c (52%) as a colorless
oil. 1H NMR (CDCl3, 360 MHz) δ (ppm): 1.75-1.78 (m, 4H),
1.89-1.97 (m, 2H), 2.35 (s, 3H), 2.43 (s, 3H), 2.49 (t, J = 7.4 Hz,
2H), 2.58-2.61 (m, 4H), 2.92 (t, J = 6.9 Hz, 2H), 3.95 (s, 4H),
7.11 (d, J = 7.7 Hz, 1H), 7.16 (dd, J = 7.7, 1.7 Hz, 1H), 7.43 (d,
J = 1.7 Hz, 1H). 13C NMR (CDCl3, 90 MHz) δ (ppm): 20.7, 20.9,
21.7, 34.6, 39.3, 51.2, 57.1, 64.3, 107.1, 129.0, 131.8, 134.6, 135.1,
138.2, 204.4. IR (NaCl) ν (cm-1): 2956, 2812, 1684, 1576, 1207,
1095, 814. HPLC (254 nm) tR = 14.5 min, purity 100%. HRMS
(m/z): [M]þ calcd for C19H27NO3, 317.1991; found, 317.1991.
1-[4-(4-Fluorophenyl)-4-oxobutyl]piperidin-4-one (6a). A mix-
ture of 5a (1.5 g, 5.0 mmol), 2 N HCl (12.4 mL), and acetone
(24.5 mL) was refluxed for 29 h. The mixture was neutralized
with NaHCO3 at room temperature, and acetone was removed
under vacuum. The residue was extracted with ethyl acetate
after addition of water. The organic layer was dried (Na2SO4)
and evaporated and the residue was purified by flash chroma-
tography (Ca2Cl2/MeOH 95:5) to give pure 6a (72%) as a
colorless solid (mp 63 °C). 1H NMR (CDCl3, 360 MHz) δ
(ppm): 1.98-2.05 (m, 2H), 2.37-2.42 (m, 4H), 2.56 (t, J = 6.4
Hz, 2H), 2.74-2.78 (m, 4H), 3.03 (t, J = 6.7 Hz, 2H), 7.11-7.16
(m, 2H), 7.99-8.03 (m, 2H). IR (NaCl) ν (cm-1): 2924, 2809,
1715, 1683, 1598, 1506, 1226, 1096, 837. EIMS (m/z): [M]þ 263.
1-[4-([2.2]Paracyclophan-4-yl)-4-oxobutyl]piperidin-4-one (6b).
Compound 5b was reacted and worked up as described for the
preparation of 6a to give pure 6b (68%) as a colorless solid (mp
127 °C). 1H NMR (CDCl3, 360 MHz) δ (ppm): 1.90-1.98 (m,
2H), 2.41-2.44 (m, 4H), 2.52 (t, J = 6.8 Hz, 2H), 2.73-2.76 (m,
4H), 2.80-3.06 (m, 5H), 3.12-3.20 (m, 4H), 3.88 (ddd, J = 12.2,
8.9, 3.2 Hz, 1H), 6.35 (dd, J = 7.7, 1.7 Hz, 1H), 6.48 (dd, J = 7.7,
1.7 Hz, 1H), 6.50-6.53 (m, 2H), 6.56 (dd, J = 7.7, 1.7 Hz, 1H),
6.65 (dd, J = 7.7, 1.7 Hz, 1H), 6.92 (d, J = 1.7 Hz, 1H). 13C
NMR (CDCl3, 150 MHz) δ (ppm): 22.2, 35.1, 35.2, 36.0, 38.1,
41.2, 51.0, 56.6, 131.2, 132.1, 132.9, 133.0, 133.3, 136.2, 136.4,
137.9, 139.2, 139.8, 140.3, 141.2, 202.4, 209.3. IR (NaCl) ν
(cm-1): 2925, 2810, 1718, 1674, 1592, 1227, 1093. EIMS (m/z):
[M]þ 375.
1-(4-Fluorophenyl)-4-[4-([2.2]paracyclophan-4-yl)-4-hydroxy-
piperidin-1-yl]butan-1-one (1a). Compound 6a and 4-bromo-
[2.2]paracyclophane were reacted and worked up as described
for 1b to give 1a (47%) as a pale orange oil. 1H NMR (CDCl3,
360 MHz) δ (ppm): 1.78-1.83 (m, 2H), 1.95-1.99 (m, 2H), 2.08
(t, J = 7.1 Hz, 2H), 2.30-2.41 (m, 2H), 2.60-2.75 (m, 4H),
2.90-3.19 (m, 9H), 3.75-3.81 (m, 1H), 6.30 (dd, J = 7.8, 1.5 Hz,
1H), 6.34-6.39 (m, 2H), 6.43 (dd, J = 7.8, 1.5 Hz, 1H), 6.50 (d,
J = 1.5 Hz, 1H), 6.59-6.63 (m, 2H), 7.11-7.15 (m, 2H),
7.99-8.03 (m, 2H). 13C NMR (CDCl3, 90 MHz) δ (ppm):
23.2, 35.2, 35.6, 36.1, 36.2, 37.1, 49.1, 49.3, 57.4, 71.3, 115.6,
115.8, 128.4, 130.7, 130.8, 132.0, 132.1, 132.3, 132.5, 132.7,
137.3, 137.7, 139.5, 139.6, 139.7, 167.2, 198.0. IR (NaCl) ν
(cm-1): 3364, 2928, 2852, 1685, 1598, 1230, 1157, 836, 731.
HPLC (254 nm) tR = 18.3 min, purity 100%. HRMS (m/z):
[M]þ calcd for C31H34FNO2, 471.2574; found, 471.2572.
4-[4-(2,5-Dimethylphenyl)-4-hydroxypiperidin-1-yl]-1-(4-fluoro-
phenyl)butan-1-one (1b). To a mixture of 2-bromo-p-xylene (0.05
mL, 0.35 mmol) in diethyl ether (20 mL) were added n-butyl-
lithium (0.28 mL, 2.5 M in hexane) at -78 °C. The mixture was
allowed to warm to room temperature within 90 min. After the
mixture was cooled to -30 °C, a precooled solution of 6a (210
mg, 0.80 mmol) in diethyl ether (6.4 mL) was added. After the
mixture was stirred at -30 °C for 5 min, stirring was continued
for 5.5 h at room temperature. Then H2O was added, and the
mixture was extracted by CH2Cl2. The organic layer was dried
(Na2SO4) and evaporated and the residue was purified by flash
chromatography (CH2Cl2/MeOH 95:5) to give pure 1b (61 mg,
47%) as a pale yellow solid (mp 99 °C). 1H NMR (CDCl3, 360
MHz) δ (ppm): 1.88-1.92 (m, 2H), 1.94-2.02 (m, 2H),
2.06-2.10 (m, 2H), 2.30 (s, 3H), 2.45-2.52 (m, 4H), 2.54 (s,
3H), 2.75-2.80 (m, 2H), 2.98 (t, J = 7.1 Hz, 2H), 6.97 (dd, J =
7.7, 1.7 Hz, 1H), 7.04 (d, J = 7.7 Hz, 1H), 7.09-7.14 (m, 2H),
7.15 (d, J = 1.7 Hz, 1H), 7.99-8.03 (m, 2H). 13C NMR (CDCl3,
90 MHz) δ (ppm): 21.2, 21.8, 22.0, 36.3, 36.9, 49.4, 57.8, 72.4,
115.5, 115.7, 126.1, 127.7, 130.6, 130.8, 133.0, 133.4, 133.7,
135.0, 144.7, 167.0, 198.5. IR (NaCl) ν (cm-1): 3466, 2921,
2850, 1686, 1598, 1230, 1121, 773. EIMS (m/z): [M]þ 369.
4-[4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl]-1-([2.2]paracy-
clophan-4-yl)butan-1-one (1c). To a solution of 4b (24 mg, 0.067
mmol) and 4-(4-chlorophenyl)-4-hydroxypiperidine (14 mg,
0.067 mmol) in DMF (0.27 mL) was added triethylamine (0.01
mL, 0.073 mmol) at room temperature. After the mixture was
stirred for 6 h, H2O (4 mL) and diethyl ether were added. The
organic layer was dried (Na2SO4) and evaporated and the
residue was purified by flash chromatography (CH2Cl2/
MeOH 9:1) to give pure 1c (20 mg, 62%) as a colorless solid
(mp 75 °C). 1H NMR (CDCl3, 360 MHz) δ (ppm): 1.77-1.81
(m, 2H), 2.02-2.10 (m, 2H), 2.32-2.42 (m, 2H), 2.66-2.90 (m,
6H), 2.94-3.06 (m, 5H), 3.14-3.20 (m, 4H), 3.85-3.92 (m,
1H), 6.37 (dd, J = 7.7, 1.7 Hz, 1H), 6.46 (dd, J = 7.7, 1.7 Hz,
1H), 6.50-6.57 (m, 3H), 6.66 (dd, J = 7.7, 1.7 Hz, 1H), 6.93 (d,
J = 1.7 Hz, 1H), 7.31-7.33 (m, 2H), 7.43-7.46 (m, 2H). 13
C
NMR (CDCl3, 90 MHz) δ (ppm): 20.9, 35.1, 35.2, 36.0, 38.0,
49.1, 49.3, 57.5, 70.6, 126.1, 128.6, 131.2, 132.2, 132.9, 133.0,
133.1, 133.4, 136.4, 137.7, 139.3, 139.9, 140.2, 141.4, 146.0,
201.9. IR (NaCl) ν (cm-1): 3366, 2926, 2853, 1673, 1490, 1242,
1093, 826, 753. HRMS (m/z): [M]þ calcd for C31H34NO2Cl,
487.2278; found, 487.2279.
1-[4-(2,5-Dimethylphenyl)-4-oxobutyl]piperidin-4-one (6c).
Compound 5c was reacted and worked up as described for 6a to
give 6c (78%) as a colorless oil. 1H NMR (CDCl3, 600 MHz) δ
(ppm): 1.93-1.98 (m, 2H), 2.36 (s, 3H), 2.41-2.43 (m, 4H), 2.45
(s, 3H), 2.53 (t, J = 7.1 Hz, 2H), 2.74-2.76 (m, 4H), 2.96 (t, J =
4-[4-([2.2]Paracyclophan-4-yl)-4-hydroxypiperidin-1-yl]-1-([2.2]-
paracyclophan-4-yl)butan-1-one (1d). Compound 6b and 4-bromo-
[2.2]paracyclophane were reacted and worked up as described
for 1b to give pure 1d (24%) as a colorless oil. 1H NMR (CDCl3,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7225
360 MHz) δ (ppm): 1.88-1.99 (m, 4H), 2.22-2.29 (m, 2H),
2.45-2.54 (m, 4H), 2.67-2.79 (m, 2H), 2.82-2.93 (m, 4H),
2.96-3.06 (m, 4H), 3.08-3.19 (m, 8H), 3.78-3.91 (m, 2H), 6.29
(dd, J = 7.7, 3.0 Hz, 1H), 6.34-6.37 (m, 3H), 6.46 (d, J = 8.7 Hz,
1H), 6.49-6.56 (m, 5H), 6.61-6.65 (m, 3H), 6.92 (d, J = 1.7 Hz,
1H). 13C NMR (CDCl3, 90 MHz) δ (ppm): 21.8, 35.1, 35.2, 35.3,
35.7, 36.0, 36.2, 37.9, 38.4, 39.1, 49.2, 49.4, 49.6, 49.7, 57.8, 71.8,
128.4, 131.3, 132.1, 132.2, 132.3, 132.6, 132.8, 132.9, 133.3, 136.1,
136.3, 137.2, 137.8, 138.0, 139.2, 139.5, 139.6, 139.7, 140.3, 141.3,
202.6. IR (NaCl) ν (cm-1): 3332, 2927, 2852, 1672, 1591, 1219,
1120, 756. HPLC (254 nm) tR = 20.2 min, purity 95%; tR = 17.8
min, purity 100%. HRMS (m/z): [M]þ calcd for C41H45NO2,
583.3450; found, 583.3448.
NMR (CDCl3, 90 MHz) δ (ppm): 21.7, 34.1, 35.1, 35.2, 35.3,
36.2, 51.6, 53.8, 57.8, 115.5, 115.8, 121.4, 127.0, 128.8, 130.7,
130.8, 131.3, 132.3, 132.8, 133.2, 133.7, 136.2, 138.8, 139.9,
140.9, 150.5, 167.1, 198.5. IR (NaCl) ν (cm-1): 2924, 2812,
1686, 1597, 1232, 1155, 835, 773. APCI-MS (m/z): [M þ 1]þ 457.
4-[4-(2,5-Dimethylphenyl)piperazin-1-yl]-1-(4-fluorophenyl)-
butan-1-one (2c).26 Compound 4a and 2,5-dimethylphenypiper-
azine were reacted and worked up as described for 2a to give 2c
(96%) as a colorless oil. 1H NMR (CDCl3, 360 MHz) δ (ppm):
1.95-2.02 (m, 2H), 2.24, 2.30, 2.48 (t, J = 7.0 Hz, 2H), 2.56-
2.61 (m, 4H), 2.86-2.88 (m, 4H), 3.01 (t, J = 7.3 Hz, 2H), 6.77-
6.79 (m, 2H), 7.04 (d, J = 7.6 Hz, 1H), 7.11-7.15 (m, 2H),
8.00-8.04 (m, 2H). 13C NMR (CDCl3, 90 MHz) δ (ppm): 17.4,
21.2, 21.8, 36.2, 51.7, 53.7, 57.8, 115.5, 115.7, 119.7, 123.7, 129.3,
130.7, 130.8, 130.9, 133.7, 136.1, 151.4, 167.1, 198.6. IR (NaCl) ν
(cm-1): 2943, 2812, 1686, 1597, 1234, 1136, 834, 808. APCI-MS
(m/z): [M þ 1]þ 355.
4-[4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(2,5-dimethyl-
phenyl)butan-1-one (1e). Compound 4c and 4-(4-chlorophenyl)-
4-hydroxypiperidine were reacted and worked up as described for
1c to give pure 1e (1.1 g, 65%) as a colorless solid (mp 83 °C) when
1
4-[4-(4-Chlorophenyl)piperazin-1-yl]-1-([2.2]paracyclophan-4-
yl)butan-1-one (2d). Compound 4b and 4-chlorophenylpipera-
zine were reacted and worked up as described for 2a to give 2d
(27%) as a colorless oil. 1H NMR (CDCl3, 600 MHz) δ (ppm):
1.89-1.97 (m, 2H), 2.42-2.51 (m, 2H), 2.59-2.63 (m, 4H), 2.74
(ddd, J = 16.8, 7.4, 6.9 Hz, 1H), 2.82-2.86 (m, 1H), 2.91 (ddd,
J = 16.8, 7.4, 7.4 Hz, 1H), 2.99-3.04 (m, 2H), 3.12-3.20 (m,
8H), 3.87 (ddd, J = 12.4, 10.1, 2.2 Hz, 1H), 6.34 (dd, J = 7.6, 1.6
Hz, 1H), 6.49 (dd, J = 7.6, 1.6 Hz, 1H), 6.50-6.53 (m, 2H), 6.55
(dd, J = 7.6, 1.6 Hz, 1H), 6.65 (dd, J = 7.6, 1.6 Hz, 1H),
6.82-6.84 (m, 2H), 6.92 (d, J = 1.6 Hz, 1H), 7.19-7.20 (m, 2H).
13C NMR (CDCl3, 90 MHz) δ (ppm): 21.7, 35.1, 35.2, 36.0, 38.2,
49.2, 53.1, 57.7, 117.2, 124.5, 129.0, 131.2, 132.1, 132.8, 132.9,
133.9, 136.1, 136.4, 138.0, 139.2, 139.7, 140.3, 141.2, 150.0,
202.6. IR (NaCl) ν (cm-1): 2926, 2823, 1678, 1595, 1236, 1136,
806, 721. HPLC (254 nm) tR = 20.5 min, purity >99%. HRMS
(m/z): [M]þ calcd for C30H33ClN2O, 472.2281; found, 472.2284.
4-[4-(4-Chlorophenyl)piperazin-1-yl]-1-(2,5-dimethylphenyl)-
butan-1-one (2e). Compound 4c and 4-chlorophenylpiperazine
were reacted and worked up as described for 2a to give 2e (31%)
as a colorless oil. 1H NMR (CDCl3, 360 MHz) δ (ppm): 1.90-
1.98 (m, 2H), 2.35 (s, 3H), 2.44 (s, 3H), 2.46 (t, J = 7.1 Hz, 2H),
2.58-2.61 (m, 4H), 2.94 (t, J = 6.7 Hz, 2H), 3.13-3.16 (m, 4H),
6.81-6.84 (m, 2H), 7.12 (dd, J = 7.7, 1.0 Hz, 1H), 7.16-7.20 (m,
3H), 7.44 (d, J = 1.0 Hz, 1H). 13C NMR (CDCl3, 90 MHz) δ
(ppm): 20.7, 21.0, 21.5, 39.3, 49.2, 53.1, 57.6, 117.2, 124.5, 129.0,
131.8, 134.5, 135.1, 138.3, 150.0, 204.5. IR (NaCl) ν (cm-1):
CH2Cl2/MeOH (95:5) was used for flash chromatography. H
NMR (CDCl3, 360 MHz) δ (ppm): 1.69-1.75 (m, 2H), 1.95-2.02
(m, 2H), 2.10-2.18 (m, 2H), 2.36 (s, 3H), 2.44 (s, 3H), 2.47-2.55
(m, 4H), 2.84-2.90 (m, 2H), 2.94 (t, J = 7.1 Hz, 2H), 7.13 (d, J =
7.9 Hz, 1H), 7.18 (dd, J = 7.9, 1.7 Hz, 1H), 7.29-7.31 (m, 2H),
7.40-7.43 (m, 2H), 7.46 (d, J = 1.7 Hz, 1H). 13C NMR (CDCl3,
90 MHz) δ (ppm): 20.8, 21.0, 21.5, 38.3, 39.3, 49.4, 57.8, 71.0,
126.1, 128.4, 129.1, 131.8, 131.9, 132.9, 134.7, 135.1, 138.2, 146.7,
204.3. IR (NaCl) ν (cm-1): 3387, 2924, 2817, 1682, 1492, 1219,
1094, 825, 772. APCIMS (m/z): [M]þ 386, [M þ 2]þ 388
1-(2,5-Dimethylphenyl)-4-[4-(2,5-dimethylphenyl)-4-hydroxy-
piperidin-1-yl]butan-1-one (1f). Compound 6c and 2-bromo-p-
xylene were reacted and worked up as described for the pre-
paration of 1b to give 1f (45%) as a colorless solid (mp 108 °C).
1H NMR (CDCl3, 360 MHz) δ (ppm): 1.92-2.03 (m, 4H),
2.20-2.27 (m, 2H), 2.30 (s, 3H), 2.35 (s, 3H), 2.44 (s, 3H), 2.54
(s, 3H), 2.55-2.63 (m, 4H), 2.84-2.91 (m, 2H), 2.95 (t, J = 7.1
Hz, 2H), 6.97 (dd, J = 7.9, 1.7 Hz, 1H), 7.05 (d, J = 7.9 Hz, 1H),
7.11 (d, J = 7.8 Hz, 1H), 7.17 (dd, J = 7.8, 1.5 Hz, 1H), 7.20 (d,
J = 1.7 Hz, 1H), 7.45 (d, J = 1.5 Hz, 1H). 13C NMR (CDCl3, 90
MHz) δ (ppm): 20.7, 20.9, 21.2, 21.5, 21.7, 36.7, 39.3, 49.3, 57.7,
72.2, 126.1, 127.9, 129.0, 131.8, 133.0, 133.3, 134.7, 135.1, 138.2,
144.4, 204.4. IR (NaCl) ν (cm-1): 3533, 2924, 2816, 1682, 1500,
1248, 1124, 812. HRMS (m/z): [M]þ calcd for C25H33NO2,
379.2511; found, 379.2510.
4-[4-(4-Chlorophenyl)piperazin-1-yl]-1-(4-fluorophenyl)butan-
1-one (2a). To a solution of 4-chlorophenylpiperazine (200 mg,
1.0 mmol), NaI (4.4 mg, 0.3 mmol), and NaHCO3 (84 mg, 1.0
mmol) in acetonitrile (3.5 mL) was added 4a (0.08 mL, 0.5
mmol). The mixture was heated to reflux for 22 h. After
evaporation, saturated NaHCO3 solution and CH2Cl2 were
added. The organic layer was dried (Na2SO4) and evaporated
and the residue was purified by flash chromatography (hexane/
EtOAc 1:1) to give pure 2a (37 mg, 46%) as a pale yellow solid
2956, 2819, 1684, 1597, 1236, 1136, 816. HPLC (254 nm) tR
=
19.4 min, purity 100%. HRMS (m/z): [M]þ calcd for C22H27-
ClN2O, 370.1812; found, 370.1813.
4-[4-([2.2]Paracyclophan-4-yl)piperazin-1-yl]-1-([2.2]paracyc-
lophan-4-yl)butan-1-one (2f). A mixture of 4b (200 mg, 0.56 mmol)
and NaHCO3 (320 mg, 3.8 mmol) in DMSO (3.8 mL) was heated
to reflux for 1 h. Then saturated NaHCO3 solution and CH2Cl2
were added at room temperature. The organic layer was extracted
with saturated NaCl solution, dried (MgSO4), and evaporated
and the residue was purified by flash chromatography (hexane/
EtOAc 1:1) to give 4-hydroxy-1-([2.2]paracyclophan-4-yl)butan-
1
(mp 92 °C). H NMR (CDCl3, 360 MHz) δ (ppm): 1.94-2.02
(m, 2H), 2.46 (t, J = 7.4 Hz, 2H), 2.56-2.59 (m, 4H), 3.00 (t, J =
7.2 Hz, 2H), 3.09-3.11 (m, 4H), 6.80-6.82 (m, 2H), 7.10-7.14
(m, 2H), 7.17-7.20 (m, 2H), 7.98-8.02 (m, 2H). 13C NMR
(CDCl3, 90 MHz) δ (ppm): 21.6, 36.2, 49.1, 53.0, 57.6, 115.5,
115.7, 117.2, 124.4, 128.9, 130.6, 130.7, 133.7, 150.0, 167.1,
198.4. IR (NaCl) ν (cm-1): 2951, 2819, 1678, 1595, 1236, 1138,
810. APCI-MS (m/z): [M]þ 361, [M þ 2]þ 363.
1
1-one (97 mg, 59%) as a colorless solid (mp 93 °C). H NMR
(CDCl3, 360 MHz) δ (ppm): 1.89-2.07 (m, 2H), 2.76-2.89 (m,
2H), 2.96-3.06 (m, 3H), 3.11-3.24 (m, 4H), 3.72-3.76 (m, 2H),
3.87 (ddd, J = 12.5, 9.0, 2.5 Hz, 1H), 6.35 (dd, J = 7.8, 1.8 Hz,
1H), 6.47 (dd, J = 7.8, 1.8 Hz, 1H), 6.49-6.57 (m, 3H), 6.65 (dd,
J = 7.8, 1.8 Hz, 1H), 6.92 (d, J = 1.8 Hz, 1H). 13C NMR (CDCl3,
90 MHz) δ (ppm): 27.3, 35.1, 35.2, 36.0, 37.4, 62.5, 131.2, 132.2,
132.9, 133.0, 133.4, 136.3, 136.4, 137.7, 139.2, 139.8, 140.2, 141.4,
203.1. IR (NaCl) ν (cm-1): 3413, 3009, 2927, 2852, 1672, 1551,
721. HPLC (254 nm) tR = 20.9 min, purity >99%. HRMS (m/z):
[M]þ calcd for C20H22O2, 294.1620; found, 294.1619.
4-[4-([2.2]Paracyclophan-4-yl)piperazin-1-yl]-1-(4-fluorophenyl)-
butan-1-one (2b). Compound 4a and ([2.2]paracyclophan-4-yl)-
piperazine14b were reacted and worked up as described for 2a to
give 2b (46%) as a pale yellow solid (mp 83 °C). 1H NMR
(CDCl3, 360 MHz) δ (ppm): 1.97-2.04 (m, 2H), 2.49-2.54 (m,
2H), 2.59-2.72 (m, 6H), 2.83-2.90 (m, 2H), 2.93-3.05 (m, 8H),
3.24 (ddd, J = 12.7, 7.8, 7.8 Hz, 1H), 3.36 (ddd, J = 12.7, 9.5, 2.3
Hz, 1H), 5.69 (d, J = 1.7 Hz, 1H), 6.26 (dd, J = 7.7, 1.7 Hz, 1H),
6.35 (dd, J = 7.7, 1.7 Hz, 1H), 6.40 (d, J = 7.7 Hz, 1H), 6.44 (dd,
J = 7.7, 1.7 Hz, 1H), 6.52 (dd, J = 7.7, 1.7 Hz, 1H), 6.68 (dd,
A solution of 4-hydroxy-1-([2.2]paracyclophan-4-yl)butan-1-
one (43 mg, 0.15 mmol) and IBX (73 mg, 0.30 mmol) in DMSO
(2.2 mL) was stirred at room temperature for 150 min. After
addition of saturated NaHCO3 solution and CH2Cl2, the organic
J = 7.7, 1.7 Hz, 1H), 7.10-7.15 (m, 2H), 7.99-8.03 (m, 2H). 13
C

7226 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Skultety et al.
layer was separated, dried (MgSO4), and evaporated and the
residue was purified by flash chromatography (hexane/EtOAc
4:1) to give 4-oxo-4-([2.2]paracyclophan-4-yl)butyraldehyde (33
mg, 77%) as a colorless solid (mp 85 °C). 1H NMR (CDCl3, 360
MHz) δ (ppm): 2.76-2.91 (m, 3H), 2.96-3.07 (m, 3H),
3.13-3.23 (m, 4H), 3.28-3.37 (m, 1H), 3.86-3.92 (m, 1H),
6.41 (dd, J = 7.7, 1.7 Hz, 1H), 6.49-6.56 (m, 4H), 6.67 (dd, J =
7.7, 1.7 Hz, 1H), 7.01 (d, J = 1.7 Hz, 1H), 9.95 (s, 1H). 13C NMR
(CDCl3, 90 MHz) δ (ppm): 32.8, 35.0, 35.2, 36.0, 38.0, 131.4,
132.2, 132.9, 133.0, 133.6, 136.4, 136.5, 137.2, 139.2, 139.9,
140.3, 141.7, 200.0, 200.8. IR (NaCl) ν (cm-1): 3012, 2927,
2852, 1720, 1672, 1551, 723. HPLC (254 nm) tR = 21.0 min,
purity 97%; tR = 23.6 min, purity 100%. HRMS (m/z): [M]þ
calcd for C20H20O2, 292.1463; found, 292.1463.
(m, 2H), 2.46 (t, J = 7.1 Hz, 2H), 2.55-2.59 (m, 2H),
2.62-2.73 (m, 5H), 2.88-3.06 (m, 9H), 3.24 (ddd, J = 12.8,
9.4, 6.4 Hz, 1H), 3.36 (ddd, J = 12.8, 9.4, 2.4 Hz, 1H), 5.71 (d,
J = 1.7 Hz, 1H), 6.27 (dd, J = 7.7, 1.7 Hz, 1H), 6.36 (dd, J =
7.7, 1.7 Hz, 1H), 6.40 (d, J = 7.7 Hz, 1H), 6.44 (dd, J = 7.7, 1.7
Hz, 1H), 6.53 (dd, J = 7.7, 1.7 Hz, 1H), 6.67 (dd, J = 7.7, 1.7 Hz,
1H). 13C NMR (CDCl3, 90 MHz) δ (ppm): 15.0, 22.7, 34.1, 35.0,
35.2, 35.3, 51.5, 53.8, 56.4, 119.7, 121.5, 127.2, 128.7, 131.3,
132.2, 132.8, 133.2, 136.3, 138.9, 139.9, 140.9, 150.3. IR (NaCl) ν
(cm-1): 2924, 2815, 2245, 1490, 1449, 1233, 1138, 772. HPLC
(254 nm) tR = 17.0 min, purity 100%. HRMS (m/z): [M]þ calcd
for C24H29N3, 359.2361; found, 359.2361.
4-[4-(2,5-Dimethylphenyl)piperazin-1-yl]butyronitrile (7c). 2,5-
Dimethylphenylpiperazine was reacted and worked up as
described for 7a to give 7c (99%) as a pale yellow solid (mp
45 °C). 1H NMR (CDCl3, 360 MHz) δ (ppm): 1.82-1.90 (m, 2H),
2.25 (s, 3H), 2.30 (s, 3H), 2.45 (t, J = 7.1 Hz, 2H), 2.53 (t, J = 6.7
Hz, 2H), 2.56-2.61 (m, 4H), 2.90-2.93 (m, 4H), 6.81 (dd, J = 7.5,
To a mixture of 4-oxo-4-([2.2]paracyclophan-4-yl)butyraldehyde
(6.1 mg, 0.02 mmol) and 4-[2.2]paracyclophanylpiperazine14
(8.6 mg, 0.03 mmol) in CH2Cl2 (0.27 mL) was added sodium
triacetoxyborohydride (5.8 mg, 0.03 mmol). After the mixture
was stirred for 16 h at room temperature, saturated NaHCO3
solution and CH2Cl2 were added. The organic layer was dried
(Na2SO4) and evaporated and the residue was purified by flash
chromatography (hexane/EtOAc 1:1) to give 2f (6.5 mg, 55%) as
2.0 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H). 13
C
NMR (CDCl3, 90 MHz) δ (ppm): 15.0, 17.5, 21.2, 22.9, 51.8, 53.7,
56.4, 119.8, 123.8, 129.3, 130.9, 136.1, 151.3. IR (NaCl) ν (cm-1):
2943, 2814, 2245, 1504, 1450, 1244, 1138, 771. EI-MS (m/z): [M]þ
257.
1
a colorless solid (mp 65 °C). H NMR (CDCl3, 360 MHz) δ
4-[4-(2-Methoxyphenyl)piperazin-1-yl]butylamine (8a).13 To a
solution of 7a (670 mg, 2.6 mmol) in diethyl ether (33 mL) was
added LiAlH4 (6.4 mL, 6.4 mmol, 1 M in diethyl ether) at 0 °C.
After being stirred for 1 h at room temperature, the solution was
cooled to 0 °C when saturated NaHCO3 solution was added.
After filtration through Celite, the filtrate was evaporated to
give pure 8a (670 mg, 99%) as a colorless solid. 1H NMR
(CDCl3, 600 MHz) δ (ppm): 1.49-1.54 (m, 2H), 1.56-1.62
(m, 2H), 2.43 (t, J = 7.0 Hz, 2H), 2.63-2.69 (m, 4H), 2.75 (t, J =
6.4 Hz, 2H), 3.07-3.14 (m, 4H), 3.86 (s, 3H), 6.86 (dd, J = 7.7,
1.3 Hz, 1H), 6.90-6.96 (m, 2H), 6.99 (ddd, J = 7.7, 7.0, 1.6 Hz,
1H). EI-MS (m/z): [M]þ 263.
(ppm): 1.89-2.02 (m, 2H), 2.52 (t, J = 7.0 Hz, 2H), 2.63-2.80
(m, 6H), 2.85-3.08 (m, 13H), 3.13-3.21 (m, 4H), 3.25 (ddd, J =
12.5, 8.2, 8.0 Hz, 1H), 3.38 (ddd, J = 12.5, 10.1, 1.8 Hz, 1H),
3.85-3.94 (m, 1H), 5.71 (d, J = 1.7 Hz, 1H), 6.27 (dd, J = 7.7,
1.7 Hz, 1H), 6.32-6.38 (m, 2H), 6.40 (d, J = 7.7 Hz, 1H), 6.44
(dd, J = 7.7, 1.7 Hz, 1H), 6.48-6.58 (m, 5H), 6.64 (dd, J = 7.6,
1.6 Hz, 1H), 6.70 (dd, J = 7.7, 1.7 Hz, 1H), 6.94 (d, J = 1.6 Hz,
1H). 13C NMR (CDCl3, 90 MHz) δ (ppm): 21.7, 34.1, 35.1, 35.2,
35.3, 36.0, 38.2, 51.7, 53.9, 57.8, 121.5, 127.1, 128.8, 131.2, 132.2,
132.3, 132.8, 133.0, 133.2, 133.4, 136.2, 136.3, 138.0, 138.8,
139.2, 139.8, 140.0, 140.3, 140.9, 141.3, 150.5, 202.7. IR
(NaCl) ν (cm-1): 3010, 2926, 2810, 1674, 1587, 1221, 771. HPLC
(254 nm) tR = 20.4 min, purity >99%. HRMS (m/z): [M]þ
calcd for C40H44N2O, 568.3454; found, 568.3453.
4-[4-([2.2]Paracyclophan-4-yl)piperazin-1-yl]butylamine (8b).
Compound 7b was reacted and worked up as described for 8a
to give 8b (99%) as a colorless oil. 1H NMR (CDCl3, 360 MHz)
δ (ppm): 1.52-1.60 (m, 4H), 2.46 (t, J = 7.1 Hz, 2H), 2.61-2.77
(m, 7H), 2.89-3.05 (m, 9H), 3.20-3.28 (m, 1H), 3.37 (ddd, J =
12.8, 9.4, 2.4 Hz, 1H), 5.71 (d, J = 1.7 Hz, 1H), 6.27 (dd, J = 7.7,
1.7 Hz, 1H), 6.35 (dd, J = 7.7, 1.7 Hz, 1H), 6.40 (d, J = 7.7 Hz,
1H), 6.44 (dd, J = 7.7, 1.7 Hz, 1H), 6.53 (dd, J = 7.7, 1.7 Hz,
1H), 6.68 (dd, J = 7.7, 1.7 Hz, 1H). 13C NMR (CDCl3, 90 MHz)
δ (ppm): 24.4, 29.7, 30.9, 34.1, 35.0, 35.2, 35.3, 51.4, 53.8, 58.5,
121.5, 127.1, 128.7, 131.2, 132.2, 132.8, 133.2, 136.2, 138.9,
139.9, 140.9, 150.4. IR (NaCl) ν (cm-1): 3375, 3007, 2927,
2813, 1587, 1490, 1232, 1129, 753. HPLC (254 nm) tR = 14.4
min, purity >99%. APCI-MS (m/z): [M þ 1]þ 364.
1-(2,5-Dimethylphenyl)-4-[4-(2,5-dimethylphenyl)piperazin-1-
yl]butan-1-one (2g). Compound 4c and 2,5-dimethylphenylpi-
perazine were reacted and worked up as described for 2a to give
2g (31%) as a colorless oil. 1H NMR (CDCl3, 360 MHz) δ
(ppm): 1.91-1.99 (m, 2H), 2.25 (s, 3H), 2.29 (s, 3H), 2.36 (s, 3H),
2.44 (s, 3H), 2.48 (t, J = 7.2 Hz, 2H), 2.58-2.62 (m, 4H),
2.90-2.93 (m, 4H), 2.97 (t, J = 7.0 Hz, 2H), 6.79 (dd, J = 7.6,
1.3 Hz, 2H), 6.82 (d, J = 1.3 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H),
7.12 (d, J = 7.7 Hz, 1H), 7.17 (dd, J = 7.7, 1.5 Hz, 1H), 7.46 (d,
J = 1.5 Hz, 1H). 13C NMR (CDCl3, 90 MHz) δ (ppm): 17.5,
20.7, 21.0, 21.2, 21.6, 39.4, 51.8, 53.7, 57.8, 119.7, 123.7, 129.0,
129.3, 130.9, 131.8, 134.6, 135.1, 136.1, 138.4, 151.4, 204.7. IR
(NaCl) ν (cm-1): 2943, 2812, 1684, 1606, 1207, 1136, 808. HPLC
(254 nm) tR = 19.6 min, purity 96%. HRMS (m/z): [M]þ calcd
for C24H32N2O, 364.2515; found, 364.2512.
4-[4-(2,5-Dimethylphenyl)piperazin-1-yl]butylamine (8c). Com-
pound 7c was reacted and worked up as described for 8a to give 8c
(93%) as a colorless oil. 1H NMR (CDCl3, 360 MHz) δ (ppm):
1.46-1.62 (m, 4H), 2.24 (s, 3H), 2.29 (s, 3H), 2.42 (t, J = 7.0 Hz,
2H), 2.55-2.63 (m, 4H), 2.74 (t, J = 6.4 Hz, 2H), 2.92-2.93 (m,
4H), 6.78 (dd, J = 7.6, 1.3 Hz, 1H), 6.82 (d, J = 1.3 Hz, 1H), 7.04
(d, J = 7.6 Hz, 1H). 13C NMR (CDCl3, 90 MHz) δ (ppm): 17.5,
21.2, 24.4, 31.6, 42.1, 51.8, 53.8, 58.6, 119.8, 123.8, 129.3, 130.9,
136.1, 151.5. IR (NaCl) ν (cm-1): 3350, 2939, 2808, 1608, 1575,
1504, 1244, 1136, 995, 806. HPLC (254 nm) tR = 11.9 min, purity
>99%. HRMS (m/z): [M]þ calcd for C16H27N3, 261.2205; found,
261.2206.
N-[4-[4-([2.2]Paracyclophan-4-yl)piperazin-1-yl]butyl]benzo-
[b]thiophenyl-2-carboxamide (3a). Compound 8b and benzothio-
phene-2-carboxylic acid were reacted and worked up as
described for 3c to give pure 3a (75%) as a colorless solid (mp
114 °C). 1H NMR (CDCl3, 360 MHz) δ (ppm): 1.62-1.68 (m,
4H), 2.45 (t, J = 7.1 Hz, 2H), 2.58-2.66 (m, 5H), 2.81-2.88 (m,
4H), 2.91-2.98 (m, 5H), 3.16 (ddd, J = 12.7, 9.4, 6.3 Hz, 1H),
3.29 (ddd, J = 12.7, 9.4, 2.2 Hz, 1H), 3.45 (dt, J = 6.2, 5.9 Hz,
2H), 5.60 (d, J = 1.6 Hz, 1H), 6.20 (dd, J = 7.5, 1.6 Hz, 1H), 6.27
4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyronitrile (7a).13 To
a solution of 2-methoxyphenylpiperazine (600 mg, 3.1 mmol)
and NaHCO3 (800 mg, 7.5 mmol) in acetonitrile (16.4 mL) was
slowly added 4-bromobutyronitrile (0.25 mL, 2.5 mmol). After
heating to reflux for 16 h, the mixture was evaporated and
CH2Cl2 and water were added. The organic layer was dried
(MgSO4) and evaporated and the residue was purified by flash
chromatography (CHCl3/EtOAc 1:1) to give 150 (640 mg, 99%)
as a colorless solid (mp 77 °C). 1H NMR (CDCl3, 360 MHz) δ
(ppm): 1.82-1.90 (m, 2H), 2.45 (t, J = 7.1 Hz, 2H), 2.53 (t, J =
6.7 Hz, 2H), 2.62-2.65 (m, 4H), 3.07-3.10 (m, 4H), 3.86 (s, 3H),
6.86 (dd, J = 7.9, 1.0 Hz, 1H), 6.91-6.94 (m, 2H), 7.00 (ddd,
J = 7.9, 6.2, 2.7 Hz, 1H). IR (NaCl) ν (cm-1): 2941, 2816, 2245,
1500, 1450, 1240, 1140, 1024, 750. EI-MS (m/z): [M]þ 259.
4-[4-([2.2]Paracyclophan-4-yl)piperazin-1-yl]butyronitrile (7b).
[2.2]Paracyclophan-4-yl)piperazine was reacted and worked up
as described for 7a to give 7b (84%) as a pale yellow solid (mp
150 °C). 1H NMR (CDCl3, 360 MHz) δ (ppm): 1.85-1.93

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7227
(dd, J = 7.5, 1.6 Hz, 1H), 6.33 (d, J = 7.5 Hz, 1H), 6.37 (dd, J =
7.5, 1.6 Hz, 1H), 6.45 (dd, J = 7.5, 1.6 Hz, 1H), 6.52 (br t, J = 5.9
Hz, 1H), 6.60 (dd, J = 7.5, 1.6 Hz, 1H), 7.31 (ddd, J = 7.2, 7.2,
1.5 Hz, 1H), 7.35 (ddd, J = 7.2, 7.2, 1.6 Hz, 1H), 7.69 (br s, 1H),
7.72-7.79 (m, 2H). 13C NMR (CDCl3, 90 MHz) δ (ppm): 24.4,
27.4, 34.1, 35.0, 35.2, 35.3, 40.1, 51.5, 54.0, 58.1, 121.5, 122.7,
124.9, 125.0, 125.1, 126.3, 127.1, 128.8, 131.2, 132.3, 132.8,
133.2, 136.2, 138.7, 138.8, 139.1, 139.9, 140.7, 140.9, 150.4,
162.4. IR (NaCl) ν (cm-1): 3314, 2923, 2850, 1628, 1543, 1295,
1219, 772. APCI-MS (m/z): [M þ 1]þ 524.
2.39 (s, 3H), 2.49 (t, J = 6.7 Hz, 2H), 2.54-2.60 (m, 4H),
2.72-2.74 (m, 4H), 3.46 (dt, J = 6.2, 5.9 Hz, 2H), 6.67 (d, J =
1.3 Hz, 1H), 6.79 (dd, J = 7.7, 1.4 Hz, 1H), 7.02 (br t, J = 5.9 Hz,
1H), 7.03 (d, J = 7.5 Hz, 1H), 7.09-7.10 (m, 2H), 7.16 (d, J =
1.4 Hz, 1H). 13C NMR (CDCl3, 150 MHz) δ (ppm): 17.4, 19.2,
20.8, 21.2, 24.5, 27.7, 39.6, 51.2, 53.7, 58.0, 119.8, 123.9, 127.4,
129.3, 130.3, 130.8, 132.4, 135.2, 136.0, 137.2, 151.0, 170.4. IR
(NaCl) ν (cm-1): 3284, 2941, 2812, 1643, 1539, 1308, 1244, 810.
APCIMS (m/z): [M þ 1]þ 394.
Receptor Binding Studies. Receptor binding studies were
carried out as previously described.15 In brief, the dopamine
D1 receptor assay was done with porcine striatal membranes at a
final protein concentration of 40-60 μg/assay tube and the
N-[4-[4-(2,5-Dimethylphenyl)piperazin-1-yl]butyl]benzo[b]thi-
ophenyl-2-carboxamide (3b). Compound 8c and benzothio-
phene-2-carboxylic acid were reacted and worked up as
described for 3c to give pure 3b (73%) as a pale yellow solid (mp
radioligand [3H]SCH 23390 at 0.3 nM (KD = 0.53-0.95 nM).
1
16
58 °C). H NMR (CDCl3, 360 MHz) δ (ppm): 1.72-1.76 (m,
Competition experiments with human D2long
,
D
,
16 D3,17
2short
4H), 2.23 (s, 3H), 2.27 (s, 3H), 2.66 (t, J = 7.2 Hz, 2H), 2.77-
2.81 (m, 4H), 2.97-3.00 (m, 4H), 3.52 (dt, J = 6.2, 5.9 Hz, 2H),
6.80 (dd, J = 7.7, 1.4 Hz, 1H), 6.77 (d, J = 1.4 Hz, 1H), 6.81 (dd,
J = 7.6, 1.4 Hz, 1H), 6.98 (br t, J = 5.9 Hz, 1H), 7.05 (d, J = 7.6
Hz, 1H), 7.37 (ddd, J = 7.2, 7.2, 1.6 Hz, 1H), 7.40 (ddd, J = 7.2,
7.2, 1.8 Hz, 1H), 7.81-7.85 (m, 3H). 13C NMR (CDCl3, 90 MHz)
δ (ppm): 17.4, 21.2, 23.6, 27.1, 39.7, 50.9, 53.7, 57.9, 119.9, 122.7,
124.2, 124.9, 125.1, 125.2, 126.2, 129.3, 130.9, 136.3, 138.9, 139.3,
140.8, 150.6, 162.6. IR (NaCl) ν (cm-1): 3305, 2941, 2812, 1630,
1545, 1294, 1246, 754. APCI-MS (m/z): [M þ 1]þ 422.
and D4.418 receptors were run with preparations of membranes
from CHO cells stably expressing the corresponding receptor
and [3H]spiperone at a final concentration of 0.1-0.3 nM. The
assays were carried out at a protein concentration of 1-8 μg/
assay tube and KD values of 0.05-0.11, 0.03-0.15, 0.06-0.28,
and 0.11-0.28 nM for the D2long, D2short, D3, and D4.4 recep-
tors, respectively. 5-HT and R1 receptor binding experiments
were performed with homogenates prepared from porcine cere-
bral cortex as described.27 Assays were run with membranes at a
protein concentration per assay tube of 80, 100, and 55 μg/mL
for 5-HT1A, 5-HT2, and R1 receptor, respectively, and radioli-
gand concentrations of 0.1 nM ([3H]WAY100635 and [3H]-
prazosin) and 0.5 nM ([3H]ketanserin) with KD values of 0.06
nM for 5-HT1A, 1.1 nM for 5-HT2, and 0.04-0.14 nM for the R1
receptor. Protein concentration was established by the method
of Lowry using bovine serum albumin as standard.28
Mitogenesis Experiments. Determination of the intrinsic acti-
vity of the representative compound was carried out by measur-
ing the incorporation of [3H]thymidine into growing cells after
stimulation with the test compound as described in the liter-
ature.10,21 For this assay D3 expressing CHO dhfr- cells have
been incubated with 0.02 μCi [3H]thymidine per well (specific
activity 25 μCi/mmol). Dose response curves of six experiments
have been normalized and pooled to get a mean curve from
which the EC50 value and the maximum intrinsic activity of each
compound could be compared to the effects of the full agonist
quinpirole.
Site Directed Mutagenesis. The pcDNA3.1(þ) of hDRD3
receptor was used as described previously.29 Oligonucleotidic
primers were purchased from Biomers.net or MWG Biotech
AG. Site directed mutagenesis was performed by polymerase
chain reaction (PCR) using oligonucleotides bearing the desired
mutation. Fidelity of PCR amplification and introduction of
mutations in the receptor cDNA were confirmed by sequencing
with the ABI sequencer system (ABI Systems, Weiterstadt,
Germany) at the laboratory of C.-M. Becker (Department of
Biochemistry, FAU Erlangen, Germany) using oligonucleotidic
primers.
Mutant Receptor Preparation. HEK-293 cells, transiently
transfected with the wild type and mutant receptor cDNA by
the CaHPO4 method or using TransIT-293 transfection reagent
(Mirus Bio Corp.), were cultured in 150 mm Petri plates contain-
ing 20 mL of MEM R medium supplemented with 10% (v/v)
fetal bovine serum, 100 U/mL penicillin G, 100 μg/mL strepto-
mycin, and 2 mM L-glutamine at 37 °C and 5% CO2.
N-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl]-2,5-dimethyl-
benzamide (3c). To a solution of 2,5-dimethylbenzoic acid (47
mg, 0.32 mmol) in CH2Cl2 (12 mL) was added diisopropylethy-
lamine (0.22 mL) at 0 °C. Then TBTU (120 mg, 0.38 mmol) in
DMF (0.9 mL) and subsequently 8a (100 mg, 0.38 mmol) in
CH2Cl2 were added. The mixture was stirred for 1 h at room
temperature when saturated NaHCO3 solution was added.
After extraction with CH2Cl2 the organic layer was dried
(MgSO4) and evaporated and the residue was purified by flash
chromatography (CH2Cl2/MeOH 95:5) to give pure 3c (88 mg,
1
71%) as a colorless solid (mp 126 °C). H NMR (CDCl3, 600
MHz) δ (ppm): 1.69-1.71 (m, 4H), 2.28 (s, 3H), 2.38 (s, 3H),
2.54 (t, J = 6.5 Hz, 2H), 2.65-2.71 (m, 4H), 2.90-2.97 (m, 4H),
3.46 (dt, J = 6.2, 5.9 Hz, 2H), 3.85 (s, 3H), 6.80 (dd, J = 7.7, 1.4
Hz, 1H), 6.85 (dd, J = 7.9, 1.0 Hz, 1H), 6.90 (ddd, J = 7.5, 7.0,
1.0 Hz, 1H), 6.93-6.95 (m, 1H), 7.00 (ddd, J = 7.9, 7.0, 1.7 Hz,
1H), 7.06-7.09 (m, 2H), 7.15 (d, J = 1.4 Hz, 1H). 13C NMR
(CDCl3, 90 MHz) δ (ppm): 19.3, 20.8, 24.2, 27.6, 39.5, 49.9, 53.3,
55.4, 58.0, 111.3, 118.3, 121.1, 123.1, 127.4, 130.3, 130.8, 132.4,
135.3, 137.1, 141.0, 152.3, 170.5. IR (NaCl) ν (cm-1): 3284, 2939,
2816, 1643, 1500, 1240, 1146, 748. EI-MS (m/z): [M]þ 395.
N-[4-[4-([2.2]Paracyclophan-4-yl)piperazin-1-yl]butyl][2.2]para-
cyclophanyl-4-carboxamide (3d). [2.2]Paracyclophane carboxy-
lic acid and 8b were reacted and worked up as described for 3c to
give 3d (65%) as a pale yellow oil. 1H NMR (CDCl3, 360 MHz) δ
(ppm): 1.65-1.70 (m, 4H), 2.48 (t, J = 5.7 Hz, 2H), 2.56-2.70
(m, 6H), 2.80-3.16 (m, 14H), 3.21-3.27 (m, 2H), 3.34 (ddd, J =
12.6, 9.5, 2.1 Hz, 1H), 3.43-3.47 (m, 2H), 3.61-3.70 (m, 1H),
5.64 (d, J = 1.6 Hz, 1H), 6.12 (br t, J = 5.6 Hz, 1H), 6.26 (dd,
J = 7.5, 1.6 Hz, 1H), 6.33 (dd, J = 7.5, 1.6 Hz, 1H), 6.39 (d, J =
7.5 Hz, 1H), 6.41-6.47 (m, 3H), 6.51-6.56 (m, 4H), 6.63-6.66
(m, 2H), 6.82 (dd, J = 7.9, 1.8 Hz, 1H). 13C NMR (CDCl3, 90
MHz) δ (ppm): 24.5, 27.6, 34.0, 34.8, 35.0, 35.1, 35.2, 35.3, 35.5,
39.7, 51.2, 53.9, 58.0, 121.5, 127.1, 128.7, 131.2, 131.6, 132.2,
132.0, 132.4, 132.5, 132.6, 132.8, 133.1, 134.8, 135.3, 135.9,
136.2, 138.8, 139.0, 139.1, 139.8, 139.9, 140.1, 140.8, 150.4,
169.4. IR (NaCl) ν (cm-1): 3305, 3007, 2926, 2850, 1641, 1512,
1290, 771. HPLC (254 nm) tR = 20.7 min, purity 96%; tR = 20.3
min, purity 100%. HRMS (m/z): [M]þ calcd for C41H47N3O,
597.3719; found, 597.3720.
HEK-293 cells were harvested 48 h after transfection. Cells
were harvested by removal of the medium, followed by a wash
with phosphate buffered saline, which was discarded, resuspen-
sion in 10 mL of harvest puffer (10 mM Tris-HCl, 0.5 mM
EDTA, 5.4 mM KCl, and 140 mM NaCl, pH 7.4), scraping of
the cells with a rubber spatula into a centrifuge tube, and
collection of the cells by centrifugation at 220g for 8 min. The
cellular pellet was resuspended in 5 mL of homogenate buffer for
D2 and D4 receptors (50 mM Tris-HCl, 5 mM EDTA, 1,5 mM
CaCl2, 5 mM MgCl2, 5 mM KCl, and 120 mM NaCl, pH 7,4)
N-[4-[4-(2,5-Dimethylphenyl)piperazin-1-yl]butyl]-2,5-dimet-
hylbenzamide (3e). 2,5-Dimethylbenzoic acid and 8c were
reacted and worked up as described for 3c to give 3e (83%) as
a pale yellow solid (mp 121 °C). 1H NMR (CDCl3, 360 MHz) δ
(ppm): 1.67-1.72 (m, 4H), 2.21 (s, 3H), 2.30 (s, 3H), 2.31 (s, 3H),

7228 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Skultety et al.
and for the D3 receptor (10 mM Tris-HCl and 5 mM MgSO4, pH
7,4). Cells were used as they were or stored at -80 °C.
After thawing or being used directly, the cells were diluted in
homogenate buffer, homogenized using a Polytron (20 000 rpm,
5 ꢀ 5 s each in an ice bath), and spun at 50000g for 18 min. The
membrane pellet was always resuspended in homogenate buffer
for D2 and D4 receptors, homogenized with a Potter-Elvehjem
homogenizer, and stored in small aliquots at -80 °C. Protein
concentration was estimated by the method of Lowry et al. using
bovine serum albumin as a standard.28
Data Analysis. The resulting competition curves of the
receptor binding experiments were analyzed by nonlinear re-
gression using the algorithms in PRISM 3.0 (GraphPad soft-
ware, San Diego, CA). The data were fit using a sigmoid model
to provide an IC50 value, representing the concentration corre-
sponding to 50% of maximal inhibition. IC50 values were trans-
formed to Ki values according to the equation of Cheng and
Prusoff.30
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tive dopamine D3 receptor antagonists and partial agonists.
J. Med. Chem. 2002, 45, 4594–4597.
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disubstituted aromatic piperidines and piperazines with extreme
selectivity for the dopamine D4 receptor interact with a common
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ized (basal effect = 0%; maximum effect of the full agonist
quinpirole = 100%) and then combined to get a mean curve.
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Acknowledgment. We thank Dr. J.-C. Schwartz and Dr. P.
Sokoloff (INSERM, Paris, France), Dr. H. H. M. Van Tol
(Clarke Institute of Psychiatry, Toronto, Canada), and Dr. J.
Shine (The Garvan Institute of Medical Research, Sydney,
Australia) for providing D3, D4.4, and D2 receptor expres-
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Forschungsgemeinschaft (DFG).
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phore models for biogenic amine binding GPCRs to avoid GPCR-
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Supporting Information Available: Elementary analysis re-
sults for 1b, 1c, 1e, 1f, 2b, 3a-c, 6b, and 7c. This material is
available free of charge via the Internet at http://pubs.acs.org.
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