Fancy bioisosteres: Novel paracyclophane derivatives as super-affinity dopamine D3 receptor antagonists

Article abstract of DOI:10.1021/jm060138d

The exploration of the chemical diversity space depends on the discovery of novel bioisosteric elements. As a continuation of our project on bilayered arene surrogates, we herein report on [2.2]paracyclophane-derived dopamine D3 receptor antagonists of ty

Full text of DOI:10.1021/jm060138d

3628
J. Med. Chem. 2006, 49, 3628-3635
Fancy Bioisosteres: Novel Paracyclophane Derivatives As Super-Affinity Dopamine D3
Receptor Antagonists
Karin Schlotter, Frank Boeckler, Harald Hu¨bner, and Peter Gmeiner*
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander UniVersity, Schuhstrasse 19, D-91052 Erlangen, Germany
ReceiVed February 8, 2006
The exploration of the chemical diversity space depends on the discovery of novel bioisosteric elements.
As a continuation of our project on bilayered arene surrogates, we herein report on [2.2]paracyclophane-
derived dopamine D3 receptor antagonists of type 4 and 6. For the most promising test compound 6a,
bearing a 2-methoxyphenyl substituent, a stereocontrolled preparation was performed when the planar chirality
of enantiomers (R)-6a (FAUC 418) and (S)-6a caused a considerable differentiation of D3 binding, which
is indicated by Ki values of 0.19 and 3.0 nM, respectively. Functional experiments showed D3 antagonist
properties for the paracyclophane derivatives of type 6. To elucidate putative bioactive low-energy
conformations, DFT-based studies including the calculation of diagnostic magnetic shielding properties were
performed. An 89% increase in volume for the [2.2]paracyclophane moiety compared to that of the
monolayered benzofurane of lead compound 3b indicates higher plasticity of GPCR binding regions than
usually expected.
Introduction
electrostatic, and conformational properties of the novel bio-
isosteres, computational investigations were done involving the
comparison of DFT-based calculations with NMR-derived
experimental data.
To optimize potency and selectivity or to improve the overall
ADME profile of lead compounds, medicinal chemists have
widely pursued the strategy of bioisosteric replacement.1 The
concept of bioisosterism refers to compounds or substructures
of compounds that share similar shapes, volumes, electronic
distributions, and physicochemical properties, which together
effect similar biological activities. In addition to the successful
introduction of enynes and endiynes into dopaminergics,2 we
found out that hitherto unknown [2.2](4,7)indoloparacyclo-
phanes of type 2 can be used as double-layered aryl bioisosteres
of the highly selective D4 receptor ligand 1 (FAUC 213),
demonstrating that sterically demanding paracyclophane deriva-
tives are capable of approaching and recognizing transmembrane
receptor binding sites (Chart 1).3 Exploring the chemical
diversity space of bioactive compounds, we discovered metal-
locene-derived bioisosteres of type 5 (FAUC 378) as exception-
ally strong-binding G-protein receptor ligands when our studies
were built on the naphthalene-derived D3 receptor partial agonist
3a (BP 897) and its benzofuranyl congener 3b.4,5 Changing D3
selectivity of the monolayered lead compounds into a D4
preferential binding pattern, the ferrocenyl- and ruthenocenyl-
carboxamides investigated displayed not only subnanomolar
binding affinity but also D3 and D4 agonist properties.
Conceptional hybridization of the above-mentioned lead com-
pounds directed us to the double-layered target molecules of
type 4 and 6, when a [2.2]paracyclophane moiety was envisaged
to replace the benzofurane and metallocene units of dopamin-
ergics 3b and 5, respectively. We herein present chemical
synthesis of novel types of paracyclophanes and their biological
evaluation for GPCR ligand binding involving dopamine D1,
D2short, D2long, D3 and D4, serotonin 5-HT1A and 5-HT2, and
adrenergic R1-receptors. Because we were intrigued by the
question of whether the bilayered cyclophane systems will
behave as agonists or antagonists, functional experiments were
performed. To gain a better understanding of the steric,
Results and Discussion
For the synthesis of target compound 4 (Scheme 1), we started
from commercially available [2.2]paracyclophane (7). Following
a five step procedure,6a we obtained the double-layered salicylic
aldehyde 8, which was deprotonated with NaH and alkylated
with bromoacetic acid methyl ester to give carboxylate 9 in 87%
yield. Subsequent treatment with KH caused an intramolecular
attack and subsequent condensation. Saponification of furano-
paracyclophane 10 thus formed yielded carboxylic acid 11,
which could be activated by HATU in the presence of HOAt
and, subsequently, coupled to primary amine 12a to furnish
target compound 4.
To investigate double-layered analogues of carbocyclic D3
ligands,7 [2.2]paracyclophanyl carboxylic acid should be ex-
ploited as a further building block. In detail, HATU- or TBTU-
supported coupling of scaffold 156b with 2-methoxyphenylpip-
erazinyl-substituted butylamine 12a and its 2,3-dichlorophenyl
analogue 12b, being readily prepared following previously
described protocols,4 gave a 94% yield of paracyclophanyl
carboxamides 6a and 6b, respectively (Scheme 2). Structural
hybrid 12c and 2,3-difluoro analogue 12d were prepared
employing a Buchwald-Hartwig cross-coupling reaction,5,8,9
which was followed by N-alkylation with 4-bromobutyronitrile
and LiAlH4-promoted reduction. Subsequent coupling with the
planar-chiral building block 15, preactivated by TBTU, yielded
in bilayered carboxamides 6c and 6d, respectively.
Stereochemical information plays an important role in recep-
tor recognition processes. Thus, there is continuing interest in
the development of drugs as single enantiomers. Because a first
screening of the cyclophane derivatives identified the racemate
(R,S)-6a as a strong-binding test compound at the dopamine
D3 receptor, we decided to establish the chirospecific synthesis
of both enantiomers. Starting from racemic carboxylic acid 15,
pure enantiomers were obtained by resolution via the corre-
* To whom correspondence should be addressed. Tel: +49-(0)9131-
8529383.
Fax:
+49-(0)9131-8522585.
E-mail:
gmeiner@
pharmazie.uni-erlangen.de.
10.1021/jm060138d CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/20/2006

Fancy Bioisosteres
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3629
Chart 1
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) NaH, BrCH2CO2CH3, DMF, 0 °C to rt,
2 h (87%); (b) KH, NMP, 0 °C to rt, 1 h (59%); (c) 2n NaOH, MeOH,
THF, 0 °C to rt, 4 h (80%) (d) HATU, HOAt, DIPEA, DMF, 12a, CH2Cl2,
0 °C to rt, 1 h (81%).
sponding diastereomeric R-(p-nitrophenyl)ethylammonium salts.6b
Subsequent activation of the enantiopure building blocks (R)-
15 and (S)-15 by TBTU and coupling with primary amine 12a
gave access to the planar chiral enantiomers (R)-6a and (S)-6a
in 85 and 86% yield, respectively.
Receptor binding experiments were established to evaluate
the binding properties of target compounds 4, (R)-6a, (S)-6a,
and 6b-d in comparison to reference agent 3a,10a a D3 partial
agonist known for highly interesting anti-dyskinetic properties
and benefitial effects on cocaine-seeking behavior10b (Table 1).
D1 receptor affinities were determined by utilizing porcine
striatal membranes, and the D1 selective radioligand [3H]SCH
23390.2a D2long, D2short, D3, and D4 receptor affinities were
a Reagents and conditions: (a) Pd2(dba)3, BINAP, NaOt-Bu, toluene,
piperazine, 13, 115 °C, 18 h (55%); (b) 1. Br(CH2)3CN, K2CO3, MeCN,
reflux, 15 h (77%); 2. LiAlH4, Et2O, 0 °C to rt, 1 h (88%); (c) for 6a and
b: HATU, DIPEA, NMP, 12a and b 0 °C to rt, 1-2 h (94%); for 6c and
d: TBTU, DIPEA, DMF, CH2Cl2, 12c and d, CH2Cl2, 0 °C to rt, 1-2h
(64-91%).
investigated by employing the cloned human dopamine receptor
11
subtypes D2long, D2short
,
D3,12 and D4.4,13 stably expressed
in Chinese hamster ovary cells (CHO) and radioligand [3H]-
spiperone.2a The competition data were analyzed according to
a sigmoid model by nonlinear regression. Because of the
observation that lead compound 3a reveals serotonergic and

3630 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Schlotter et al.
Table 1. Receptor Binding Data of 4 and 6a-d Compared to Those of Reference Compound 3a Utilizing Human D2long, D2short, D3, and D4.4
Receptors and Porcine D1, 5-HT1A, 5-HT2, and R1 Receptors
Ki (nM)a
[3H]8-OH-
DPAT
[3H]ketan-
serin
compd
R
[3H]SCH 23390
D1
[3H]spiperone
[3H]prazosin
R′
D2long
D2short
D3
D4.4
5-HT1A
5-HT2
R1
4
780 ( 5.0
660 ( 30
490 ( 35
2800 ( 300
970 ( 140
570 ( 25
760 ( 60
170 ( 30
15 ( 0.50
11 ( 2.1
220 ( 45
42 ( 5.8
120 ( 15
220 ( 19
84 ( 2.5
13 ( 1.5
5.6 ( 0.15
140 ( 35
28 ( 3.4
35 ( 3.5
200 ( 5.0
16 ( 1.6
3.0 ( 0.69
0.19 ( 0.009
3.6 ( 2.3
0.46 ( 0.089
1.9 ( 0.050
1.3 ( 0.096
140 ( 15
9.50 ( 1.5
5.40 ( 0.15
120 ( 24
33 ( 9.5
190 ( 38
25 ( 5.5
58 ( 5.5
660 ( 90
22 ( 5.0
290 ( 30
81 ( 8.0
2700 ( 300
2300 ( 400
1600 ( 250
3500 ( 500
470 ( 95
1200 ( 380
840 ( 120
8.4 ( 2.6
3.9 ( 0.20
2.5 ( 0.0
480 ( 80
80 ( 2.0
(S)-6a
(R)-6a
6b
6c
6d
OMe
OMe
Cl
OMe
F
H
H
Cl
Cl
F
18 ( 0.0
45 ( 2.0
3a
44 ( 7.6
5.0 ( 0.40
a Ki values in nM ( SEM are based on the means of 2-10 experiments, each done in triplicate.
adrenergic activities as well,10a the test compounds were also
investigated for their potency to displace [3H]8-OH-DPAT, [3H]-
ketanserin, and [3H]prazosin, when employing porcine 5-HT1A,
5-HT2, and R1 receptors, respectively.
The data from the radioligand binding studies unambiguously
proved that the double-layered cyclophane-derived moiety of
the target compounds is specifically recognized by the binding
sites of the investigated GPCRs (Table 1). Whereas only weak
to moderate binding affinities were determined for D1, 5-HT2,
and 5-HT1A, D2, D4, and R1 receptor recognition resulted in Ki
values reaching even the single-digit nanomolar range. Except
for furanocyclophane 4 displaying preferential R1 binding (Ki
) 8.4 nM), all test compounds preferentially associated to the
dopamine D3 receptor subtype.
Because our initial screening data for paracyclophane 6a in
racemic form indicated extraordinarily high D3 binding com-
pared to that of heterocyclic analogue 4 (Ki ) 16 nM), we
synthesized the pure enantiomers and subjected (R)-6a and (S)-
6a to heterologous displacement experiments. In fact, we
determined a Ki value of 0.19 nM for (R)-6a (FAUC 418),
indicating almost 100-fold improvement compared to that of 4,
bearing the bilayered unit that is more remote from the
carboxamide function. Interestingly, the structural difference
being caused by the planar chirality led to a eudysmic ratio of
more than 15 (Ki ) 3.0 for (S)-6a). However, the 5-HT1A
component of (S)-6a (Ki ) 25 nM) was superior compared to
that of (R)-6a (Ki ) 58 nM). To investigate the sensitivity of
the binding profiles toward the substitution pattern of the phenyl
substituent, the 2,3-dichloro, 3-chloro-2-methoxy, and 2,3-
difluoro analogues 6b, 6c, and 6d, respectively, were tested. In
contrast to our recent observations with lead compounds of type
3 and 5, D3 receptor binding considerably decreased for all
GPCRs in this study when the 2-methoxy group was displaced
(Ki ) 3.6 and 1.9 nM for 6b and 6d, respectively). An interesting
binding pattern was observed for structural hybrid 6c with an
ortho positioned methoxy group and a chloro atom in meta
position displaying both high D3 affinity (Ki ) 0.46 nM) and
considerable selectivity over the potential anti-target R1 (Ki )
80 nM).
Table 2. Intrinsic Activities Derived from the Stimulating Effect on
Mitogenesis of D3 Receptor Expressing Cells and Calculated
Physicochemical Properties of 4, 6a-d and Reference Compounds
Quinpirole, 3a, and 5
[3H]thymidine uptake
(mitogenesis)a
calcd physicochemical
propertiesb
agonist
effectc
EC50
kB
compd
(nM)d
(nM)e
MW
637.7
clogP
4
n.df
0%
0%
0%
0%
0%
0%
56%
38%
100%
n.d.
n.d.
8.5
75
4.7
110
18
6.21
5.65
5.65
5.65
5.65
7.17
6.28
6.08
n.d.
6a
(S)-6a
(R)-6a
6b
6c
6d
3a
497.7
497.7
497.7
497.7
536.5
532.1
503.6
n.d.
10
1.8
2.0
2.6
n.d.
n.d.
n.d.
5
quinpirole
n.d.
n.d.
a The values have been determined with CHO dhfr- mutant cells stably
expressing the human D3 receptor in 4-10 independent experiments carried
out in hexaduplicates. b The clogP and MW values were calculated with
ChemDraw Ultra 6. c The rate of incorporation of [3H]thymidine as evidence
for mitogenesis activity relative to the maximal effect of the full agonist
quinpirole ()100%) is used as a derived reference. d The EC50 values are
derived from a mean curve of three or four independent experiments. e The
dose-dependent inhibition of the intrinsic activity of 5 nM quinpirole
(inducing a 65% max. effect) derived from six to eight independent
experiments, each carried out in hexaduplicates. f n.d. means not determined.
human D3 receptor.12,14,15 Although the metallocenes of type 5
(38%, EC50 ) 2.0 nM) could mimic the partial agonist activities
of 3a (56%, EC50 ) 1.8 nM),5 paracyclophane derivatives 6a-d
revealed an approximately neutral antagonism at the D3 receptor.
The data listed in Table 2 displays Kb values between 4.7 and
110 nM indicating good correlation to the D3 binding data when
the (R)-enantiomer of planar-chiral paracyclophane 6a again
displayed higher biological activity than the (S)-isomer. This
finding is surprising and complementary to our previous work,
where we observed partial agonist properties for all metallocenes
of type 5, whereas the intrinsic activity of the monolayered
dopaminergics of type 3 strongly depended on the nature of
both the heterocyclic system and the phenyl substituents giving
rise to both neutral antagonists and partial agonists.
As a measure of functional activity, the intrinsic activities of
the paracyclophanyl carboxamides 6a-d were determined by
a mitogenesis assay measuring the rate of [3H]thymidine incor-
poration into growing CHO dhfr- cells stably expressing the
Because of the finding that not only metallocenes 5 but also
sterically demanding paracyclophane derivatives can provide
efficient binding to the D3 dopamine receptor subtype, we tried

Fancy Bioisosteres
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3631
successfully to confirm a dominant conformer in similar
studies.18,19 These calculations were found to give a highly
consistent ranking of the conformers with almost identical
relative energy differences. The energy gaps between m1 and
m2 as well as between m1 and m3 only marginally decreased
to 1.25 and 2.51 kcal/mol, respectively, at the highest calculation
level (B3PW91/6-311+G(2df,2p)). Thus, the order of the energy
content of all three conformations is retained during all
calculations. According to the Boltzmann equation, the ratio of
structures in the m3 versus m1 conformation at room temper-
ature (298.15 K) is only about 1.44%, whereas the m2/m1 ratio
of 12.11% indicates a putative relevance of both conformations
for its biological activity. An evaluation of the energy of the
transition state (ts) confirms that at room temperature a moderate
probability exists for the transition between m1 and m2.
To evaluate the relevance of the preferential structures
resulting from DFT calculations and to gain further evidence
for the putative bioactive conformations, we envisioned a
comparison of calculated magnetic shielding with experimental
NMR data. Therefore, we calculated the magnetic shielding
tensor using gauge invariant atomic orbitals20 (GIAO) within
B3PW91/6-311+G(2d,p) or B3PW91/6-311+G(2df,2p) single-
point calculations. The different orientations of the carboxamide
in the three investigated conformational states and, thus, their
changes of the local magnetic field by state-specific proximities
to certain carbon atoms were used as a criterion to compare
experimental with calculated 13C NMR data. As described in
detail in the Supporting Information, this comparison suggests
a preference for the m1 conformer, although it is possible that
the m2 conformation also plays a role at room or body
temperatures.
Figure 1. DFT grid-calculation of the rotational barriers and minima
of the N-methylcarboxamide fragment of paracyclophanes 6a-d with
B3LYP/6-31G(d). For better readability, the relative energies in kcal/
mol applied to the lowest energy calculated (-827.314748089 hartree)
are denoted. Structures shown with the identified minima, m1-m3,
and the transition state ts between m1 and m2.
to characterize the conformational properties by employing DFT-
based quantum chemical calculations on the methyl-paracyclo-
phanylcarboxamide fragment of 6. We further evaluated the
resulting preferential structures for the conformity of their
calculated magnetic shielding versus experimental NMR data.
Initially, we performed a semiempirical AM1 optimization with
VAMP16 on the fragment, followed by two steps of DFT
calculations in Gaussian98.17 We used a B3LYP density
functional with a 3-21G basis set to produce a reasonable
geometry in an appropriate time. Then, we increased the basis
set to the double-valence level 6-31G(d) to enhance the quality
of the structure. At the same level, we accomplished a grid
calculation varying the dihedral angle Φ3-4-17-19 in steps of
10°. With this dihedral being frozen, the rest of the system was
allowed to freely adapt to the new geometry by the minimization
of energy. As depicted in Figure 1, we obtained three local
minima (called m1, m2, and m3), one with the carbonyl pointing
toward the proximate ethano-bridge (m1, Φ3-4-17-18 ≈ 143.6°),
the second directing the carbonyl to the interior of the bilayer
(m2, Φ3-4-17-18 ≈ 43.6°), and the third orienting its N-H bond
toward the interior of the bilayer (m3, Φ3-4-17-18 ≈ 213.6°).
On this level of calculation, the lowest relative energy was found
for m1, whereas m2 and m3 exceeded the energy of m1 by
1.64 and 2.95 kcal/mol, respectively. The minima m2 and m3
are separated by a large rotational barrier of 4.70 and 6.01 kcal/
mol for m3 and m2, respectively. The height of this barrier can
be explained not only by the fact that the orientation of the
carboxamide is approximately perpendicular to the plane of the
aromatic system, thus, minimizing the overlap of the π-systems,
but also by the fact that the hydrogen of the amide closely
approaches a hydrogen in the second aromatic layer (∼1.9 Å).
(For an extension of the discussion of rotational barriers and
the characterization of the transition state ts, please see the
Supporting Information.)
To understand the molecular similarity of the monolayered
benzofurane-2-carboxamide moiety (A), the ferrocene-derived
(B), and the respective cyclophane-derived bioisosteric element
(C), we investigated both the steric demand and the charge
distribution by mapping the electrostatic potentials onto the van
der Waals surfaces.21 In detail, the structure of the ferrocene
was derived from X-ray data22 of a suitable precursor, whereas
for the cyclophane, we chose the preferential m1 conformer from
our previous DFT calculations. ESP charges were obtained
consistently for all three structures using the PM3(tm) Hamil-
tonian within the Spartan program package.23 The distribution
of charge on the molecular surfaces was visualized with
MOLCAD, implemented in Sybyl 6.9.24 Figure 2 clearly shows
a high degree of similarity when the molecular electrostatic
properties are compared. In addition to our findings for the
metallocenes, this might be the structural origin for the
remarkable high target binding of the double-layered bioiso-
steres. Nevertheless, the ability of double-layered cyclophanes
6 to specifically bind to the GPCRs investigated is surprising
because the height of the cyclophane-derived π-systems is
approximately doubled (6.0 Å) compared to that of the benzo-
furancarboxamide substructure (3.0 Å). Moreover, the volume
of the aryl fragment is increased from benzofurane (86.1 Å3)
to paracyclophane (162.5 Å3) by 89%, whereas the exchange
to ferrocene (108.0 Å3) extends the volume only by 25%. Thus,
we were able to reconfirm that the plasticity of the binding
region is substantially higher than usually expected. One main
structural difference between the cyclophanylcarboxamide and
the ferrocenylcarboxamide or benzofurane-2-carboxamide moi-
eties is that one side of the carbonyl function in the cyclophane-
carboxamides is prohibited for hydrogen-bond interactions with
the receptor by the preferred orientation of the bulky bilayer
system. This difference might have relevance for the ligand’s
Subsequently, we subjected all three preferential conformers
(m1, m2, and m3) to a series of higher-level calculations (Table
S1, Supporting Information), a method we have already applied

3632 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Schlotter et al.
Figure 2. Electrostatic potentials mapped onto the van der Waals surfaces of N-methylcarboxamide fragments A, B, and C of monolayered benzofurane
3 and the ferrocene (5)- and paracyclophane (6)-derived bilayered bioisosteres, respectively.
2H), 2.96-3.03 (m, 4H), 3.13-3.15 (m, 4H), 3.31-3.36 (m, 1H),
3.50-3.56 (m, 2H), 3.60-3.65 (m, 1H), 3.85 (s, 3H), 6.03 (dd, J
)1.9 Hz, J ) 7.9 Hz, 1H), 6.13 (dd, J ) 1.9 Hz, J ) 7.9 Hz), 6.42
(dd, J ) 1.8 Hz, J )7.8 Hz), 6.47 (dd, J ) 1.8 Hz, J ) 7.8 Hz),
6.55 (d, J ) 7.6 Hz, 1H), 6.65 (d, J ) 7.6 Hz, 1H), 6.81 (br t, J )
5.5 Hz, 1H), 6.85 (dd, J ) 1.2 Hz, J ) 8.2 Hz, 1H), 6.83-6.95
(m, 2H), 6.99 (dd, 1.2 Hz, J ) 8.2 Hz, 1H), 7.27 (s, 1H). 13C NMR
(CDCl3, 90 MHz): δ 24.2, 27.7, 29.9, 32.4, 34.2, 34.7, 39.3, 50.5,
53.5, 55.3, 58.1, 111.2, 111.4, 118.2, 120.9, 122.8, 123.3, 125.3,
128.1, 128.9, 131.1, 131.9, 132.6, 136.1, 137.9, 138.4, 141.3, 147.4,
152.3, 154.5, 159.4. EI-MS m/z: 537 (M+). Anal. (C34H39N3O3):
C, H, N.
inability to activate the D3 receptor observed by the absence
of any intrinsic activity.
In conclusion, we have discovered novel paracyclophane-
derived D3 antagonists displaying interesting binding profiles,
which might be a starting point for the development of highly
beneficial CNS active drugs, especially for the treatment of
schizophrenia. Because of the planar chirality of the cyclophane
skeleton, stereochemical differentiation was observed when the
(R)-enantiomer (R)-6a showed significantly higher D3 affinity.
Moreover, we could show that the high steric demand of the
paracyclophanes of type 6 is well tolerated by the binding site
of the dopamine D3 receptor, indicating substantial plasticity
of the receptor-excluded volumes. Thus, the paracyclophane-
derived D3 antagonists will serve as valuable molecular probes
for the investigation of GPCR-ligand interactions. In contrast
to our findings on the metallocenes of type 5 displaying partial
agonist properties, the paracyclophanes of type 6 revealed
neutral D3 antagonism.
N-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl][2.2]-
paracyclophane-4-carboxamide (6a). To a solution of racemic
1525 (synth. according to literature6b) (30.0 mg, 0.12 mmol) and
DIPEA (0.05 mL, 0.24 mmol) in CH2Cl2 (4 mL) was added HATU
(47.0 mg, 0.12 mmol) in NMP (2 mL) at 0 °C. After the addition
of 12a (34.0 mg, 0.13 mmol), the mixture was stirred at room
temperature for 0.5 h. Then, CH2Cl2 and aqueous saturated NaHCO3
were added. The organic layer was dried (MgSO4) and evaporated,
and the residue was purified by flash chromatography (CH2Cl2/
Experimental Section
1
MeOH 98:2) to give pure 6a (56 mg, 94%) as a colorless oil. H
Materials and Methods. The chemicals and solvents were
purchased in the highest purity available. All reactions were carried
out under a nitrogen atmosphere except for ester hydrolysis. Column
chromatography was performed using 60 µm silica gel. For TLC,
silica gel 60 F254 plates were used (UV, I2 or ninhydrin detection).
Melting temperatures were uncorrected. NMR data were noted in
ppm relative to TMS. IR spectroscopy was carried out on a FT/IR
410 spectrometer. MS were run on a MAT TSQ 70 spectrometer
by EI (70 eV) with solid inlet. HPLC-MS were performed on an
analytic HPLC system with a VWL detector, coupled to a mass
spectrometer with electron spray ionization. CHN elementary
analyses were performed at the Department of Organic Chemistry
of the Friedrich-Alexander University.
NMR (CDCl3, 600 MHz): δ 1.69-1.87 (m, 4H), 2.82-3.23 (m,
17H), 3.41-3.50 (m, 2H), 3.60-3.69 (m, 1H), 3.84 (s, 3H), 6.32
(br t, J ) 5.9 Hz, 1H), 6.44-6.50 (m, 3H), 6.54 (s, 1H), 6.57 (dd,
J ) 7.9 Hz, J ) 1.8 Hz, 1H), 6.68-6.73 (m, 2H), 6.81-6.93 (m,
3H), 6.97-7.01 (m, 1H). 13C NMR (CDCl3, 90 MHz): δ 24.4,
27.5, 34.7, 35.1, 35.3, 35.4, 39.6, 50.3, 53.3, 55.3, 57.9, 111.2,
118.2, 120.9, 122.9, 131.7, 132.0, 132.4, 132.5, 132.6, 134.8, 135.2,
135.8, 138.9, 139.1, 139.8, 140.1, 141.2, 152.2, 169.4. EI-MS m/z:
498 (M+). Anal. (C32H39N3O2 ‚0.3 H2O): C, H, N.
N-[4-(4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl][2.2]-
paracyclophane-4-carboxamide (6b). Compound 1325 (30.0 mg,
0.12 mmol), DIPEA (0.05 mL, 0.24 mmol), HATU (47.0 mg, 0.13
mmol), and 12b (0.04 g, 0.13 mmol) were reacted and worked up
as described for 6a to give 6b (60.0 mg, 94%) as a colorless oil.
1H NMR (CDCl3, 360 MHz): δ 1.60-1.69 (m, 4H), 2.44 (t, J )
6.6 Hz, 2H), 2.55-2.57 (m, 4H), 2.88-3.20 (m, 8H), 3.41-3.47
(m, 2H), 3.62-3.69 (m, 1H), 6.28 (br t, J ) 5.5 Hz, 1H), 6.42 (d,
J ) 7.8 Hz, 1H), 6.45 (d, J ) 7.8 Hz, 1H), 6.53-6.57 (m, 3H),
6.65 (d, J ) 2.0 Hz, 1H), 6.84-6.88 (m, 2H), 7.09-7.16 (m, 2H).
13C NMR (CDCl3, 90 MHz): δ 24.5, 27.6, 34.7, 35.1, 35.3, 35.4,
39.7, 51.0, 53.2, 57.9, 118.6, 124.5, 127.3, 127.5, 131.7, 132.0,
132.5, 132.6, 134.8, 133.9, 135.2, 135.8, 138.9, 139.1, 139.8, 140.1,
151.1, 169.4. EI-MS m/z: 535, 537 (M+). Anal. (C31H35Cl2N3O):
C, H, N.
N-4-(4-(2-Methoxyphenyl)piperazin-1-yl)butyl-1(4,7)-benzo-
furano-4-(1,4)-benzolhexaphanyl-12-carboxamide (4). To a solu-
tion of 11 (35.0 mg, 0.12 mmol) and DIPEA (0.05 mL, 0.24 mmol)
in CH2Cl2 (5 mL) was added HATU (47.0 mg, 0.13 mmol) and
HOAt (17.0 mg, 0.12 mmol) in DMF (2 mL) at 0 °C. After the
addition of 12a (34.0 mg, 0.13 mmol) in CH2Cl2 (5 mL), the mixture
was stirred at 0 °C for 1 h. Then, CH2Cl2 and aqueous saturated
NaHCO3 were added. The organic layer was dried (MgSO4) and
evaporated, and the residue was purified by flash chromatography
(CH2Cl2/MeOH 98:2) to give 4 (52.0 mg, 81%) as a white solid.
1
Mp 81-82 °C. H NMR (CDCl3, 360 MHz): δ 1.69-1.82 (m,
4H), 2.57 (t, J ) 7.4 Hz, 2H), 2.74-2.76 (m, 4H), 2.86-2.93 (m,

Fancy Bioisosteres
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12 3633
N-4-[4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl]butyl][2.2]-
paracyclophane-4-carboxamide (6c). To a solution of 1525 (30.0
mg, 0.12 mmol) and DIPEA (0.07 mL, 0.42 mmol) in CH2Cl2 (4
mL) was added TBTU (42.0 mg, 0.13 mmol) in DMF (0.30 mL)
at 0 °C. After the addition of 12c (39.0 mg, 0.13 mmol) in CH2Cl2
(5 mL), the mixture was stirred for 30 min at room temperature.
Then, CH2Cl2 and aqueous saturated NaHCO3 were added. The
organic layer was dried (MgSO4) and evaporated, and the residue
was purified by flash chromatography (CH2Cl2/MeOH 98:2) to give
2812, 1638 cm-1. APCI-MS m/z: 498 ((M + H)+). Anal.
(C32H39N3O2 0.3 H2O): C, H, N.
[2.2]Paracyclophane-4-carboxylic Acid (15) Compound 1525
was synthesized according to literature.6b To a stirred solution of
4-bromo[2.2]paracyclophane26 (2.08 g, 7.25 mmol) in dry Et2O (135
mL), n-butyllithium (4.50 mL of a 2.5 M solution in hexane, 11.20
mmol) was added dropwise under nitrogen. Then, the mixture was
stirred at room temperature for 2 h. Then, an excess of dried dry
ice was added. The reaction was allowed to warm to room
temperature, the solvent was evaporated to dryness, and the solid
residue was dissolved in H2O (200 mL). Insoluble [2.2]paracyclo-
phane was filtered off, and the aqueous phase was thoroughly
washed with ether and CH2Cl2 to remove traces of the added CH2-
Cl2, and the mixture was extracted with CH2Cl2. The aqueous layers
were combined and acidified with concentrated hydrochloric acid
to pH 3 and extracted with CHCl3.The combined organic layer was
dried (MgSO4) and evaporated to give [2.2]paracyclophanyl-4-
carboxylicacid (1.25 g, 61%) as a white solid. Mp 223-224 °C
(ref 25 mp 223.5-224.5 °C).
(S)-[2.2]Paracyclophane-4-carboxylic Acid (S)-15. The resolu-
tion of 1525 was accomplished according to ref 6b6b to give 0.30 g
(63%) of (S)-(+)-[2.2]paracyclophane-4-carboxylic acid. [R]D +172
(c 0.5 CHCl3), (ref 27 [R]D +164).
(R)-[2.2]Paracyclophane-4-carboxylic Acid (R)-15. The resolu-
tion of 1525 was accomplished according to ref 6b to give 5.00 mg
(20%) of (R)-(-)-[2.2]paracyclophane-4-carboxylic acid. [R]D
-164.5 (c 0.5 CHCl3), (ref 25 [R]D -157).
1
pure 6c (58.0 mg, 91%) as a colorless oil. H NMR (CDCl3, 360
MHz): δ 1.61-1.69 (m, 4H), 2.43 (t, J ) 6.7 Hz, 2H), 2.54-2.56
(m, 4H), 2.82-3.00 (m, 8H), 3.42-3.47 (m, 2H), 3.62-3.69 (m,
1H), 6.23 (br t, J ) 3.4 Hz, 1H), 6.41 (d, J ) 7.8 Hz, 1H), 6.45 (d,
J ) 7.8 Hz, 1H), 6.54-6.57 (m, 3H), 6.64 (d, J ) 2.1 Hz, 1H),
6.71 (d, J ) 1.6 Hz, d, J ) 8.1 Hz, 2H), 6.99 (d, J ) 1.6 Hz, d, J
) 8.1 Hz, 2H). 13C NMR (CDCl3, 90 MHz): δ 24.5, 27.5, 34.7,
35.1, 35.3, 35.4, 39.6, 49.9, 53.6, 57.9, 58.9, 117.1, 123.2, 124.5,
128.7, 131.7, 132.0, 132.4, 132.5, 132.6, 134.9, 135.2, 135.8, 138.9,
139.1, 139.8, 140.1, 146.5, 148.6, 169.4. EI-MS m/z: 531, 533
(M+). Anal. (C32H38ClN3O2 ‚0.33 H2O): C, H, N.
N-[4-[4-(2,3-Difluorophenyl)piperazin-1-yl]butyl][2.2]-
paracyclophane-4-carboxamide (6d). To a solution of 1525 (30.0
mg, 0.12 mmol) and DIPEA (0.07 mL, 0.42 mmol) in CH2Cl2 (4
mL) was added TBTU (42.0 mg, 0.13 mmol) in DMF (0.30 mL)
at 0 °C. After the addition of 12d (36.0 mg, 0.13 mmol) in CH2Cl2
(5 mL), the mixture was stirred at room temperature for 1 h. Then,
CH2Cl2 and aqueous saturated NaHCO3 were added. The organic
layer was dried (MgSO4) and evaporated, and the residue was
purified by flash chromatography (CH2Cl2/MeOH 98:2) to give 6d
(38.0 mg, 64%) as a colorless oil. 1H NMR (CDCl3, 360 MHz): δ
1.61-1.69 (m, 4H), 2.44 (t, J ) 6 Hz, 2H), 2.55-2.57 (m, 4H),
2.84-3.30 (m, 8H), 3.41-3.47 (m, 2H), 3.65 (ddd, J ) 2.5 Hz, J
) 10.2 Hz, J ) 12.7 Hz, 1H, 1H), 6.17 (br t, J ) 6.1 Hz, 1H),
6.41 (d, J ) 7.8 Hz, 1H), 6.45 (d, J ) 7.8 Hz, 1H), 6.53-6.60 (m,
4H), 6.65 (d, J ) 1.8 Hz, 1H), 6.71-6.79 (m, 1H), 6.84 (d, J )
7.3 Hz, 1H), 6.90-6.97 (m, 1H). 13C NMR (CDCl3, 90 MHz): δ
24.4, 27.6, 34.7, 35.1, 35.3, 35.4, 39.7, 50.2, 53.1, 57.9, 109.9 (d,
J ) 8.0 Hz), 113.6-113.7 (m,1C), 123.5 (dd, J ) 2.8 Hz, J ) 5.5
Hz, 1C), 131.6, 132.0, 132.4, 132.5, 132.6, 134.8 (C-7), 135.2,
135.8, 141.9 (dd, J ) 2.8 Hz, J ) 5.5 Hz), 143.5 (d, J ) 8.0 Hz,
J ) 247.3 Hz), 151.5 (dd, J ) 8.0 Hz, J ) 247.3 Hz), 169.4 (CO).
EI-MS m/z: 503 (M+) Anal. (C31H35F2N3O 0.25 H2O): C, H, N.
(S)-N-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl][2.2]-
paracyclophane-4-carboxamide (S)-(6a). Compound (S)-156b
(30.0 mg, 0.12 mmol), DIPEA (0.05 mL, 0.24 mmol), HATU (47.0
mg, 0.13 mmol), and 12a (34.0 mg, 0.13 mmol) were reacted and
worked up as described for 6a to give (S)-6a (51.0 mg, 85%) as a
colorless oil. [R]D + 65 °C (c 0.5, CHCl3). 1H NMR (CDCl3, 360
MHz): 1.64-1.70 (m, 4H), 2.49 (t, J ) 7.0 Hz, 2H), 2.64-2.66
(m, 4H), 2.87 (ddd, J ) 5.5 Hz, J ) 10.2 Hz, 12.7 Hz, 1H), 2.94-
2.97 (m, 1H), 2.97-3.03 (m, 4H), 3.07 (ddd, J ) 2.5 Hz, J )10.2
Hz, J ) 12.7 Hz, 1H), 3.11-3.17 (m, 2H), 3.25 (ddd, J ) 5.5 Hz,
J ) 10.2 Hz, J )12.7 Hz), 3.42-3.46 (m, 2H), 3.66 (ddd, J ) 2.5
Hz, J ) 10.2 Hz, J ) 12.7 Hz, 1H), 3.84 (s, 3H), 6.23 (br t, J )
5.5 Hz, 1H), 6.41-6.42 (m, 1H), 6.46 (d, J ) 8.0 Hz, 1H), 6.53
(m, 2H), 6.56 (dd, J ) 1.8 Hz, J ) 8.0 Hz), 6.66 (d, J ) 1.8 Hz,
1H), 6.81 (m, 1H), 6.845 (dd, J ) 1.2 Hz, J ) 8.5 Hz, 1H), 6.85
(dd, J ) 1.5 Hz, J ) 7.8 Hz, 1H), 6.90 (ddd, J ) 1.2 Hz, J ) 7.8
Hz, J ) 7.8 Hz), 6.99 (ddd, J ) 1.5 Hz, J ) 7.5 Hz, 7.5 Hz, 1H).
13C NMR (CDCl3, 90 MHz): δ (ppm): 24.3, 27.5, 34.8, 35.1, 35.3,
35.5, 39.6, 50.2, 53.4, 55.4, 57.9, 111.2, 118.3, 120.9, 123.0, 131.5,
132.1, 132.4, 132.5, 132.6, 134.9, 135.2, 135.9, 139.0, 139.2, 139.9,
140.1, 141.1, 152.3, 169.4. IR (NaCl): 3316, 2930, 2812, 1638
cm-1. APCI-MS m/z: 498 ((M + H)+). Anal. (C32H39N3O2 0.6
H2O): C, H, N.
(4-Formyl[2.2]paracyclophan-5-yloxy)acetic Acid Methyl Es-
ter (9). To a solution of 4-formyl-5-hydroxy[2.2]paracyclophane
8 (synthesized according to ref 6a) (70.0 mg, 0.28 mmol) in dry
DMF (5 mL) was added dropwise a suspension (3 mL) of NaH
(40.0 mg) in DMF (9 mL) at 0 °C. Then, the reaction mixture was
allowed to warm to room temperature and stirred for 1 h at room
temperature. The mixture was extracted with aqueous saturated
NaHCO3 and Et2O. After washing with citronic acid (5%), aqueous
saturated NaCl, and water, the combined organic layers were dried
(MgSO4) and evaporated, and the residue was purified by flash
chromatography (hexanes/EtOAc 9:1) to give (4-formyl[2.2]-
paracyclophan-5-yloxy)acetic acid methyl ester (9) (78.0 mg, 87%)
1
as a white solid. Mp 97-99 °C. H NMR (CDCl3, 360 MHz): δ
2.72-2.85 (m, 3H), 2.99-3.31 (m,9H), 3.75 (s, 3H), 4.03-4.11
(m, 1H), 4.43 (d, J ) 15.6 Hz, 1H), 4.72 (d, J ) 15.6 Hz, 1H),
6.40 (dd, J ) 1.8 Hz, J ) 7.8 Hz, 1H), 6.44 (d, J ) 7.8 Hz, 1H),
6.45 (d, J ) 7.8 Hz, 1H), 6.59 (dd, J ) 1.8 Hz, J ) 7.8 Hz, 1H),
6.67 (d, J ) 7.8 Hz, 1H), 6.84 (dd, J ) 1.8 Hz, J ) 7.8 Hz, 1H),
10.36 (s, 1H).13C NMR (CDCl3, 90 MHz): δ 31.5, 33.8, 34.1, 34.2,
52.1, 71.4, 129.4, 130.0, 130.9, 131.4, 131.7, 132.0, 132.4, 133.2,
133.4, 139.1, 140.8, 144.4, 161.6, 169.2, 192.8. EI-MS m/z: 324
(M+).
1(4,7)Benzofurano-4(1,4)benzenehexaphanyl-12-carboxylic Acid
Methyl Ester (10). To a solution of (4-formyl[2.2]paracyclophan-
5-yloxy)acetic acid methyl ester (9) (0.13 g, 0.40 mmol) in dry
NMP (10 mL) was added dropwise a suspension (5 mL. 0.44 mmol)
of KH (36.0 mg) in DMF (10 mL) at 0 °C. Then, the reaction
mixture was allowed to warm to room temperature and stirred for
1 h at room temperature. The mixture was extracted with aqueous
saturated NaHCO3 and Et2O. After washing with aqueous saturated
NaCl, the combined organic layers were dried (MgSO4) and
evaporated, and the residue was purified by flash chromatography
(hexanes/EtOAc 9:1) to give 1(4,7)benzofurano-4(1,4)benzene-
hexaphanyl-12-carboxylic acid methyl ester (10) (72.0 mg, 59%)
1
as a white solid. Mp 215-217 °C. H NMR (CDCl3, 360 MHz):
δ 2.83-2.96 (m, 2H), 2.97-3.13 (m, 4H), 3.30-3.34 (m, 1H), 3.64
(m, 1H), 4.01 (s, 3H), 6.04 (d, J ) 1.8 Hz, J ) 7.8 Hz, 1H), 6.09
(d, J ) 1.8 Hz, J ) 7.8 Hz, 1H), 6.41 (dd, J ) 1.8 Hz, J ) 7.8 Hz,
1H), 6.47 (dd, J ) 1.8 Hz, J ) 7.8 Hz, 1H), 6.56 (d, J ) 7.5 Hz,
1H), 6.68 (d, J ) 7.5 Hz, 1H), 7.31 (s, 1H). 13C NMR (CDCl3, 90
MHz): δ 34.9, 35.1, 35.3, 36.2, 51.6, 112.8, 129.6, 131.8, 132.3,
132.8, 133.1, 136.2, 136.4, 137.4, 139.4, 140.0, 140.1, 143.8, 172.0.
EI-MS m/z: 306 (M+).
(R)-N-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl][2.2]-
paracyclophane-4-carboxamide (R)-(6a). Compound (R)-156b
(30.0 mg, 0.12 mmol), DIPEA (0.05 mL, 0.24 mmol), HATU (47.0
mg, 0.13 mmol), and 12a (34.0 mg, 0.13 mmol) were reacted and
worked up as described for 6a to give (R)-6a (53.0 mg, 88%) as a
colorless oil. [R]D -70 °C (c 0.5, CHCl3). IR (NaCl): 3316, 2930,

3634 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 12
Schlotter et al.
1-(4,7) Benzofurano-4-(1,4)benzene-hexaphanyl-12-carboxylic
Acid (11). To a solution of 1(4,7)benzofurano-4(1,4)benzene-
hexaphanyl-12-carboxylic acid methylester (10) (11.0 mg, 0.04
mmol) in THF (1 mL), MeOH (1 mL) and aqueous NaOH (2n,
0.50 mL) were added at 0 °C. The mixture was stirred at room
temperature for 4 h and left for 1 h under ultra sonic assistance.
After evaporation in vacuo (40 °C, 100 mbar), the residue was
diluted with water and washed with Et2O. After acidification with
HCl (2 N) to pH 2 and extraction with Et2O, the organic layer was
dried (MgSO4) and evaporated, and the residue was purified by
flash chromatography (CH2Cl2/MeOH 98:2) to give 11 (9.0 mg,
0.25-0.48 nM for D4.4. Unspecific binding was determined using
10 µM haloperidole.
The investigation of serotonin 5-HT1A and 5-HT2 and adrenergic
R1
binding was performed as described in literature.5 In brief,
porcine cortical membranes were subjected to the binding assay at
a concentration of 80-115 µg/assay tube for the determination of
5-HT1A
and 5-HT2 binding utilizing [3H]8-OH-DPAT and [3H]-
ketanserin, each at a final concentration of 0.5 nM with Kd values
of 0.92-2.6 nM (for 5-HT
1A) and 1.9-2.5 nM (for 5-HT2). Cortical
membranes at 55-80 µg/assay tube and the radioliogand [3
H]-
prazosin at a final concentration of 0.4 nM were applied to
1
80%) as a white solid. Mp 234-236 °C. H NMR (CDCl3, 360
determine adrenergic R1 binding with Kd values of 0.10 nM. For
the investigation of unspecific binding serotonin, methysergid and
prazosin were used, each at a concentration of 10 µM when
MHz): δ 2.87-2.96 (m, 2H), 2.98-3.16 (m, 4H), 3.34-3.71 (m,
2H), 4.77 (br s., 1H), 6.08 (d, J ) 1.8 Hz, J ) 7.8 Hz, 1H), 6.15
(d, J ) 1.8 Hz, J ) 7.8 Hz, 1H), 6.43 (dd, J ) 1.8 Hz, J ) 7.8
Hz), 6.49 (dd, J ) 1.8 Hz, J )7.8 Hz), 6.61 (d, J ) 7.5 Hz, 1H),
6.73 (d, J ) 7.5 Hz, 1H), 7.48 (s, 1H). 13C NMR (CDCl3, 90
MHz): δ 29.7, 31.7, 33.6, 34.2, 114.7, 123.7, 124.9, 127.6, 128.8,
130.6, 131.8, 132.4, 133.2, 136.5, 137.7, 138.6, 155.7, 161.8. EI-
MS m/z: 292 (M+). Anal. (C19H16O3. 0.11 H2O ): C, H, N.
4-[4-(2,3-Difluorphenyl)piperazin-1yl]butylamine (12d). To a
solution of piperazine (1.12 g, 13.0 mmol), NaOt-Bu (0.68 g, 7.0
mmol), Pd2(dba)3 (0.05 g, 0.50 mol %), BINAP (2,2′-bisdiphe-
nylphosphino)-1,1′-binaphthalin (0.09 g, 2.00 mol %) in toluene
(20 mL) was added 1-bromo-2,3-difluorobenzene (13) (0.56 mL,
5.00 mmol). The reaction mixture was heated for 18 h to 115 °C,
cooled to room temperature, filtered through Celite, and evaporated,
measuring 5-HT
1A, 5-HT2, and R1 affinities.
Protein concentration was established by the method of Lowry
using bovine serum albumin as the standard.28
Data analysis of the resulting competition curves was ac-
complished by nonlinear regression analysis using the algorithms
in PRISM (GraphPad Software, San Diego, CA). The Ki values
were derived from the corresponding EC50 data utilizing the
equation of Cheng and Prusoff.29
Mitogenesis Experiments. The determination of the stimulating
effect of the test compounds on mitogenesis as a functional assay
was done with a CHO dhfr- cell line stably transfected with the
human dopamine D3 receptor, according to literature.4a,12,14a In
detail, 10 000 cells/well were grown for 72 h in a 96-well plate in
the appropriate medium supplemented with serum. After washing
and adding a serum free medium, the cells were incubated for 20
h with the test compound at eight different concentrations in the
range of 0.01-10 000 nM as hexaduplicates. After the addition of
0.02 µCi/well of [3H]thymidine (specific activity 25 Ci/mmol,
Amersham Biosciences), incubation was continued for 4 h. Finally,
the cells were harvested, and the incorporated radioactivity was
measured with a microplate scintillation counter.
Experimental data resulting from the mitogenesis assay were
normalized and then combined to obtain a mean curve. Nonlinear
regression analysis of this curve provided the EC50 value as a
measure of potency. The top value of the curve represented intrinsic
activity, which was correlated to the effect of the full agonist
quinpirole (100%).
1
yielding 14 (0.55 g, 55%) as a yellow oil. H NMR (CDCl3, 360
MHz): δ 2.48-2.50 (m, 4H), 3.12-3.15 (m, 4H), 6.67-6.71 (m,
1H), 6.73-6.80 (m, 1H), 6.92-6.99 (m, 1H). IR (NaCl): 3421,
2925, 2852, 1620 cm-1. EI-MS m/z: 198 (M+).
To a solution of 14 (0.39 g, 1.95 mmol) and Na2CO3 (0.50 g,
4.70 mmol) in acetonitrile (10 mL) was added bromobutyronitrile
(0.15 mL, 1.55 mmol), and the mixture was refluxed for 15 h. After
cooling to room temperature, the mixture was dried (MgSO4) and
evaporated, and the residue was purified by flash chromatography
(CHCl3/EtOAc 1:1) to give 4-[4-(2,3-difluorophenyl)piperazin-1yl]-
1
butyronitrile (0.50 g, 77%) as a colorless oil. H NMR (CDCl3,
360 MHz): δ 1.83-1.90 (m, 2H), 2.46 (t, J ) 6.9 Hz, 2H), 2.53
(t, J ) 6.9 Hz, 2H), 2.44-2.48 (m, 4H), 2.60-2.63 (m, 4H), 6.66-
6.71 (m, 1H), 6.74-6.81 (m, 1H), 6.93-6.99 (m, 1H). EI-MS
m/z: 265 (M+).
Computational Investigations. Molecular modeling investiga-
tions were performed on Silicon Graphics Indigo2 and Octane2
workstations. Quantum chemical calculations with a total of
approximately 1700 processor hours were performed on a 4-node
dual Xeon mini-cluster as well as in part on a 150-node dual Xeon
cluster at the High Performance Computing division of the
Friedrich-Alexander University.
To a solution of 4-[4-(2,3-difluorophenyl)piperazin-1yl]buty-
ronitrile (0.15 g, 0.50 mmol) in Et2O (5 mL), LiAlH4 (1 M in Et2O,
1.00 mL) was added dropwise at 0 °C and stirred at room
temperature for 1 h. After the addition of aqueous saturated
NaHCO3 at 0 °C, the residue was filtered (Celite/MgSO4/Celite),
washed with CH2Cl2, and evaporated to give 12d (0.14 g, 95%) as
a yellow oil. 1H NMR (CDCl3, 360 MHz): δ 1.47-1.60 (m, 4H),
2.39-2.44 (m, 2H), 2.61-2.65 (m, 4H), 2.71-2.75 (m, 2H), 3.12-
3.15 (m, 4H), 6.67-6.71 (m, 1H), 6.73-6.80 (m, 1H), 6.92-6.99
(m, 1H). 13C NMR (CDCl3, 90 MHz): δ 24.2, 31.5, 41.9, 45.9,
51.5, 58.4, 109.9, 113.6-113.7), 123.5, 141.9, 144.0, 151.5. EI-
MS m/z: 269 (M+).
Determination of Enantiomeric Excess. Enantiomeric excess
was determined by HPLC analysis using an Agilent system in
combination with ChemStation software (hexane/2-propanol; iso-
gradic 60/40), a CHIRACEL OD chiral column (0.64 × 20 cm),
and a flow rate of 1 mL/min; 210 nm; Rt (S)-6a ) 16.8 min, Rt
(R)-6a ) 21.2 min.
Acknowledgment. We thank Dr. H. H. M. Van Tol (Clarke
Institute of Psychiatry, Toronto), Dr. J. -C. Schwartz, and Dr.
P. Sokoloff (INSERM, Paris) as well as J. Shine (The Garvan
Institute of Medical Research, Sydney) for providing dopamine
D4, D3, and D2 receptor expressing cell lines, respectively. We
also acknowledge Dr. G. Wellein and Dr. G. Hager (High
Performance Computing division of the “Regionales Rechen-
zentrum Erlangen”) for their collaboration during calculations
on a 150-node dual Xeon cluster. This work was supported by
the BMBF and Fonds der Chemischen Industrie.
Receptor Binding Experiments. Receptor binding studies were
carried out as described in the literature.2 In brief, the dopamine
D1 receptor assay was done with porcine striatal membranes at a
final protein concentration of 40 µg/assay tube and the radioligand
[3H]SCH 23390 at 0.3 nM (Kd ) 0.11-0.62 nM). Competition
experiments with the human D2long, D2short, D3, and D4.4 receptors
were run with preparations of membranes from CHO cells express-
ing the corresponding receptor and [3H]spiperone at a final
concentration of 0.1 nM. The assays were carried out with a protein
concentration of 3-20 µg/assay tube and Kd values of 0.12-0.15
nM for D2long, 0.10 nM for D2short, 0.18-0.35 nM for D3, and
Supporting Information Available: Extended discussion about
rotational barriers, calculation of magnetic shielding properties, and
elemental analysis data.
References
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