Conjugated enynes as nonaromatic catechol bioisosteres: Synthesis, binding experiments, and computational studies of novel dopamine receptor agonists recognizing preferentially the D3 subtype

Article abstract of DOI:10.1021/jm991098z

To evaluate nonaromatic catechol bioisosteres, the conformationally restrained enynes I and enediynes 2 were synthesized via palladium-catalyzed coupling as the key reaction step. Subsequent receptor binding studies at the dopamine receptor subtypes D

Full text of DOI:10.1021/jm991098z

756
J . Med. Chem. 2000, 43, 756-762
Brief Articles
Con ju ga ted En yn es a s Non a r om a tic Ca tech ol Bioisoster es: Syn th esis, Bin d in g
Exp er im en ts, a n d Com p u ta tion a l Stu d ies of Novel Dop a m in e Recep tor Agon ists
Recogn izin g P r efer en tia lly th e D3 Su btyp e
Harald Hu¨bner, Christian Haubmann, Wolfgang Utz, and Peter Gmeiner*
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander University, Schuhstrasse 19,
D-91052 Erlangen, Germany
Received J une 14, 1999
To evaluate nonaromatic catechol bioisosteres, the conformationally restrained enynes 1 and
enediynes 2 were synthesized via palladium-catalyzed coupling as the key reaction step.
Subsequent receptor binding studies at the dopamine receptor subtypes D1, D2 long, D2 short, D3,
and D4 showed highly interesting binding profiles for the enynes 1a and 1b when compared to
dopamine. At the guanine nucleotide-sensitive high-affinity binding site of the D3 receptor,
the target compound 1b (Ki ) 5.2 nM) was 10-fold more potent than dopamine but less potent
at the D2 and D4 subtypes. In contrast to dopamine the agonists 1a and 1b showed strong
selectivity for the receptors of the D2 family (D2-D4). As far as we know, this study represents
the first report on nonaromatic dopamine agonists. Comparison of molecular electrostatic
potentials, derived from semiempirical molecular orbital calculations, and lipophilicity maps
was performed.
Ch a r t 1
In tr od u ction
Employing genuine substrates, hormones, or neu-
rotransmitters as lead structures, the combination of
conformational restrictions and bioisosteric replace-
ments has become a valuable tool for the design of
highly selective enzyme inhibitors, antimetabolites, or
receptor ligands.1,2 Starting from the respective biogenic
amines, this strategy led to agonists or antagonists
selectively recognizing dopamine (DA), norepinephrine
(NA), serotonin (5-HT), or GABA receptors. As an
example, incorporation of the inhibitory neurotransmit-
ter GABA into a bicyclic ring system as well as bioisos-
teric exchange of the carboxylate function by an imidate
substructure resulted in the GABAA receptor agonist
THIP (Chart 1).3 In the field of DA receptor agonists,
which play an important role in the treatment of
Parkinson’s disease and are of potential interest as
atypical antipsychotics,4 the aminothiazole derivative
pramipexole can be regarded as a result of the above-
mentioned approach.5 In this case, an aromatic hetero-
cyclic system has been used as a bioisosteric surrogate
for the catechol nucleus. The dopaminergic effects of
ergoline derivatives including SAR studies with ergoline
partial structures show that indole or pyrrole rings as
well as aza analogues thereof can also mimic the
catechol moiety of DA.6-9 Heterocyclic bioisosteres play
also an important role in the field of 5-HT receptor and
adrenoreceptor agonists.10,11 However, in all cases aro-
matic substructures are involved. As far as we know,
nonaromatic agonists of DA receptors, 5-HT receptors,
or adrenoreceptors have not been described, yet.
In continuation of our SAR studies on selective DA
receptor ligands,12-15 we were intrigued by the question
whether nonaromatic, conjugated π-systems can mimic
the catechol nucleus of DA. In general, this seemed
unlikely since not only the aromatic system but also the
polar hydroxy functionalities are expected to be involved
in the receptor binding of DA.16-18 However, there is
more and more evidence in the literature that optimized
hydrophobic effects can compensate for the attractive
forces resulting from hydrogen bonds.19 In this paper,
we describe the synthesis, DA receptor binding, and
calculated molecular properties of the conformationally
restrained enynes of type 1 and enediynes of type 2
including aza and diaza analogues, respectively.
* To whom correspondence should be addressed. Tel: 09131/85-
29383. Fax: 09131/85-22585. E-mail: gmeiner@pharmazie.uni-erlan-
gen.de.
10.1021/jm991098z CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/29/2000

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 757
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (a) CBr4, PPh3, benzene, reflux, 4 h
(84%); (b) RCCH (R ) SiMe3, Ph), (Ph3P)2PdCl2, CuI, piperidine,
THF, rt, 20 h, 24 h (78%, 75%); (c) CH2(CN)2, piperidine, MeOH,
0 °C, 2 h (47% of 2d ), CH3CN, KOH, reflux, 3 h (48% of 6); (d)
Bu4NF, THF, -15 °C, 30 min (70%).
a
Reagents and conditions: (a) Tf2O, 2,6-di-tert-butyl-4-meth-
ylpyridine, 1,2-dichloroethane, reflux, 4.5 h (43%); (b) RCCH (R
) SiMe3, Ph), Pd(PPh3)4, CuI, EtMe2N, THF, rt, 15 min (77%,
96%); (c) KCN, Pd(PPh3)4, 18-crown-6, benzene, reflux, 2.5 h (70%);
(d) Bu4NF, THF, -15 °C, 30 min (90%).
6. All target compounds were synthesized in racemic
form. An asymmetric synthesis is in progress.
The dopaminergic binding profiles of the test com-
pounds 1a -d , 2a -d , and 6 to human DA D2 long, D2
Resu lts a n d Discu ssion
,
25 D3, 26 and D427 receptors heterologously expressed
short
For the preparation of the target compounds we
started from the aminocyclohexanone 3 that was readily
prepared from 1,4-cyclohexanedione monoethylene ac-
etal, according to the literature.20 Transformation of the
ketone 3 into an enol triflate and palladium-catalyzed
coupling reactions should give access to the conjugated
enynes of type 1 (Scheme 1). In practice, the central
synthetic intermediate 4 was obtained by treatment of
3 with trifluoromethanesulfonic anhydride when 2,6-
di-tert-butyl-4-methylpyridine was used as a nonnucleo-
philic acid scavenger.21 Transition-metal-catalyzed re-
action of the enol triflate 4 afforded the enynes 1a and
1c. Applying Cacchi’s conditions (Pd(PPh3)4, CuI,
EtMe2N, THF)22 the coupling products 1a and 1c were
produced in 77% and 96% yield when trimethylsilyl-
acetylene and phenylacetylene were used, respectively.
Fluoride-induced desilylation of 1a gave the terminal
alkyne 1b. Palladium-catalyzed coupling of the triflate
4 with KCN resulted in formation of the aza analogue
1d . For this reaction, the application of 18-crown-6 in
benzene was advantageous.23
in Chinese hamster ovary cells (CHO) and to bovine D1
receptors were investigated and compared with the
reference agonist DA (Table 1). In our initial series of
experiments, we evaluated the ability of the ligands to
displace the radioligands [3H]spiperone (for the recep-
tors of the D2 family) and [3H]SCH 23390 (a selective
D1 antagonist). The binding properties of the geminal
enediynes of type 2 and the aza enynes 1d and 6 were
disappointing. The data indicated Ki values in the
micromolar range and only a one-site competition or the
affinities were too low to be identified. Analogous results
turned out for the phenylacetylene derivative 1c. How-
ever, careful analysis of the D2, D3, and D4 competition
experiments for the enynes 1a and 1b employing a large
number of test concentrations clearly showed biphasic
curves. The calculated Hill coefficients (nH) between
-0.76 and -0.50 and a better fit of equations indicated
a two-site competition rather a one-site model resulting
only in a K0.5 value. Whereas the Ki values for the low-
affinity state were found to be guanine nucleotide-
insensitive, Gpp(NH)p induced rightward shift and
steepening of the curves indicating that the high-affinity
binding sites are representing the G protein-coupled
ternary complex. Analogous results were obtained when
employing bovine striatal membranes and the selective
D2 antagonist [3H]spiperone. Furthermore, high-affinity
bovine D2 receptors were exclusively labeled with [3H]-
pramipexole,28,29 known as a selective autoreceptor
agonist. Under this condition, monophasic curves with
Ki values at 30, 15, and 1.8 nM were observed for 1a ,
1b, and DA, respectively. In contrast to the reference
agonist DA, which shows strong binding to the high-
affinity binding sites of all the DA receptor subtypes
investigated, the enynes 1a and 1b show high-affinity
binding only for subtypes of the D2 family. For the
terminal enyne 1b Ki values of 5.2, 22, 270, and 250
nM were determined for the high-affinity states of
human subtypes D3, D4, D2 long, and D2 short, respectively.
Within this group of receptors, the affinities of the more
The aminocyclohexanone 3 was also chosen as a
suitable starting material for the synthesis of the
geminal enediynes of type 2 (Scheme 2). Thus, synthesis
of the dibromoalkene 5, which should serve as a key
intermediate, was accomplished by treatment of the
ketone 3 with CBr4 in the presence of PPh3. Subsequent
palladium-catalyzed coupling with trimethylsilylacety-
lene or phenylacetylene resulted in formation of the
enediynes 2a and 2c, respectively. It is worthy to note
that the reaction gave only poor product yields when
Pd(PPh3)4 was employed as a catalyst. Substantial
improvement could be achieved when we changed to
(Ph3P)2PdCl2.24 Transformation of the silylalkyne 2a
into the terminal alkyne 2b was performed in the
presence of Bu4NF. The diaza analogue 2d was prepared
from the amino ketone 3 and malononitrile under
classical Knoevenagel conditions. Using KOH as a base,
we also prepared the acetonitrile condensation product

758 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Brief Articles
Ta ble 1. Binding Data of the Conjugated Enynes 1a -d , 2a -d , and 6 and DA to Human and Bovine DA Receptorsa
Ki (nM) ( SEM
[3H]spiperone
[3H]SCH 23390 [3H]spiperone [3H]pramipexole
compd
human D2 long human D2 short human D3
human D4.4
bovine D1
bovine D2
bovine D2
DA Ki high
Ki low
20 ( 2.7
1900 ( 140
42%
17 ( 1.7
1100 ( 76
34%
50 ( 8.1
1600 ( 180
52%
1.2 ( 0.2
62 ( 7.5
57%
7 ( 3.6
650 ( 200
36%
11 ( 2.0
1600 ( 550
51%
1.8 ( 0.3
RH
Ki GppNHp 15000 ( 3000 15000 ( 2500
290 ( 31
94 ( 17
160 ( 35
3800 ( 1400
54%
nd
640 ( 0
-0.76
22 ( 0.5
380 ( 120
38%
1600 ( 300
12000 ( 2800
150 ( 53
7500 ( 2500
29%
12000 ( 2000
1600 ( 340
-0.65
57 ( 9.7
4100 ( 470
40%
1a Ki high
Ki low
160 ( 20.0
>20 µM
16%
970 ( 140
>20 µM
17%
47 ( 9.2
>20 µM
30.00 ( 2.9
15 ( 2.2
1600 ( 85
23%
1900 ( 150
820 ( 90
-0.74
5.20 ( 1.6
590 ( 120
23%
RH
Ki GppNHp >20 µM
>20 µM
>20 µM
-0.63
K0.5
nH
>20 µM
-0.46
1b Ki high
Ki low
270 ( 33
14000 ( 910
38%
250 ( 51
12000 ( 1600
34%
>20 µM
RH
Ki GppNHp 19000 ( 3000 17000 ( 2000
720 ( 130
350 ( 100
-0.69
3500 ( 450
13000 ( 500
2800 ( 50.0
4400 ( 1080
1800 ( 250
1600 ( 50.0
nd
170 ( 30
-0.69
16000 ( 4400 >20 µM
>20 µM
1900 ( 150
3900 ( 650
1400 ( 50.0
>20 µM
17000 ( 1500
750 ( 100
-0.55
K0.5
nH
2800 ( 500
-0.51
2900 ( 750
-0.55
1c
1d
2a
2b
2c
2d
6
15000 ( 1000 15000 ( 2000
nd
nd
nd
nd
nd
nd
nd
nd
>20 µM
>20 µM
>20 µM
nd
nd
nd
nd
nd
nd
11000 ( 2100 13000 ( 2500
12000 ( 1500 >20 µM
3900 ( 150
16000 ( 500
2200 ( 500
>20 µM
5200 ( 400
>20 µM
>20 µM
7300 ( 950
15000 ( 1000
>20 µM
13000 ( 2000 >20 µM
>20 µM
a
Ki values are the means of two to five independent experiments ( SEM; Ki high, Ki low, and RH (%) represent the inhibition constants
at the high- and low-affinity sites and the relative proportion of high-affinity sites, respectively; Ki GppNHp values were determined in the
presence of 100 µM Gpp(NH)p for decoupling of the ternary complex; K0.5 represents the dissociation constant using a one-site model
when nH indicates the existence of two binding sites; nd, not determined.
potent enyne 1b were compared to those of DA. It
turned out that 1b is 5-20-fold less potent at the
subtypes D2 and D4 but 10-fold more potent than DA
at the D3 receptor. Since the D3 subtype is expressed
predominantly in the limbic brain areas, the receptor
is an important target for antipsychotic drugs.
receptor affinity. The lipophilic potentials mapped onto
the Connolly surfaces, which are presented in Figure
2, indicate higher lipophilicity for 1b and 1d when
compared to DA and pramipexole. This can be observed
not only for the differently substituted amino groups but
also for the π-regions.
From the physicochemical point of view, the enyne
substructure of 1b and the catechol fragment of DA look
quite different. Whereas the catechol group of DA
contains two acidic HO functions capable of taking part
in Coulomb or H-bonding interactions, the enyne sub-
structure of 1b was expected less polar. Here, binding
energy could result from hydrophobic forces. To gain
more insight into the structural requirements that are
necessary for agonist binding at the receptors of the D2
family, we compared the molecular electrostatic poten-
tial (MEP) and lipophilicity maps of the test compounds
1b and 1d with those of the active analogues prami-
pexole and DA. To facilitate a suitable alignment, the
R-rotamer of DA, representing an extended conforma-
tion with the m-OH group projected over the ethylamine
side chain, was selected, which is in agreement with
previous studies on the ligand-based design of rigid DA
surrogates.30 Figure 1 shows isopotential surfaces at -1
kcal/mol for DA, pramipexole, the active agonist 1b, and
the inactive aza analogue 1d representing the interac-
tion between a positive probe and the calculated charge
distribution. Similar shapes and locations were observed
for the MEP maps of the respective amino functions and
π-systems of DA, pramipexole, and the enyne 1b.
Nevertheless, the negative electrostatic potential de-
rived from the enyne 1b turned out to be smaller than
those generated by the aromatic moieties of DA and
pramipexole. Due to the polarization of the CtN triple
bond, the negative isopotential region of the nitrile 1d
was significantly more extended away from the core of
the π-system. This might be the reason for its poor DA
Although the nonaromatic conjugated system of the
novel DA receptor agonist 1b revealed a more lipophilic
and less polar character than the catechol ring of DA
and the aminothiazole portion of pramipexole, the
computational study corroborated our assumption that
the conjugated enyne functionality of 1b shows molec-
ular properties that are similar to those of the catechol
subunit of DA. Especially, at the D3 receptor the ability
of the more lipophilic enyne system to facilitate hydro-
phobic interactions seems to compensate for slightly
reduced attractive forces resulting from electrostatic
interactions.
Exp er im en ta l Section
Solvents were purified and dried by standard procedures.
If not otherwise stated reactions were performed under dry
N2. MS and HRMS were run on Finnigan MAT TSQ 70 and
8200 spectrometers, respectively, by EI (70 eV) with solid inlet.
1H NMR spectra were obtained on a Bruker AM 360 (360 MHz)
spectrometer, if not otherwise stated in CDCl3 relative to TMS
(J values in Hz); 13C NMR spectra were run on a Bruker AC
250 (63 MHz) in CDCl3 relative to the solvent resonance (δ )
77.0). Chromatographic purification was performed using silica
gel 60 (Merck).
Dip r op yl(4-t r im e t h ylsilyle t h yn ylcycloh e x-3-e n yl)-
a m in e (1a ). To a solution of 4 (69 mg, 0.21 mmol) in THF (6
mL) were added EtMe2N (225 µL, 2.08 mmol), trimethylsilyl-
acetylene (60 µL, 0.42 mmol), CuI (6 mg, 0.03 mmol), and
(Ph3P)4Pd0 (12 mg, 0.01 mmol) in a nitrogen-purged flask. After
being stirred at room temperature for 15 min, aqueous
NaHCO3 (5%) and Et2O were added. The organic layer was
dried (MgSO4) and evaporated and the residue was purified
by flash chromatography (petroleum ether-EtOAc-EtMe2N

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 759
F igu r e 1. Molecular electrostatic isopotential maps (from left to right) contoured at -1 kcal/mol for DA, pramipexole, the
nonaromatic DA receptor ligand 1b, and the inactive aza analogue 1d .
F igu r e 2. Lipophilic potentials (from left to right) mapped onto the calculated Connolly surfaces of DA, pramipexole, the
nonaromatic DA receptor ligand 1b, and the inactive aza analogue 1d .
95:5:1) to give 1a (45 mg, 77%) as a colorless oil: IR 3030,
δ 11.8, 22.1, 25.1, 28.5, 30.2, 52.6, 55.4, 74.6, 85.1, 119.5, 135.9;
EIMS 205 (M+). Anal. (C14H23N) C,H,N.
1
2960, 2870, 2810, 2140, 1630, 1250 cm-1; H NMR δ 0.17 (s,
9H), 0.85 (t, 6H, J ) 7.4), 1.35-1.49 (m, 5H), 1.79-1.88 (m,
1H), 1.99-2.11 (m, 1H), 2.14-2.28 (m, 3H), 2.34-2.41 (m, 4H),
2.74 (dddd, 1H, J ) 12.0, 10.5, 5.1, 2.7), 6.11-6.16 (m, 1H);
13C NMR δ 0.0, 11.8, 22.1, 25.1, 28.5, 30.3, 52.6, 55.5, 91.3,
106.6, 120.5, 135.6; EIMS 277 (M+). Anal. (C17H31NSi) C,H,N.
(4-Eth yn ylcycloh ex-3-en yl)d ip r op yla m in e (1b). To a
solution of 1a (24 mg, 0.086 mmol) in THF (4 mL) was added
Bu4NF (100 µL, 1 M solution in THF) at -15 °C. After being
stirred at this temperature for 30 min, aqueous NaHCO3 (5%)
and Et2O were added. The organic layer was dried (MgSO4)
and evaporated and the residue was purified by flash chro-
matography (petroleum ether-EtOAc-EtMe2N 95:5:1) to give
1b (16 mg, 90%) as a colorless liquid: IR 3310, 3030, 2960,
2870, 2810, 2100, 1630 cm-1; 1H NMR δ 0.85 (t, 6H, J ) 7.4),
1.36-1.52 (m, 5H), 1.81-1.89 (m, 1H), 2.00-2.12 (m, 1H),
2.15-2.30 (m, 3H), 2.32-2.45 (m, 4H), 2.76 (dddd, 1H, J )
12.1, 10.5, 5.1, 2.7), 2.79 (s, 1H), 6.13-6.18 (m, 1H); 13C NMR
(4-P h en yleth yn ylcycloh ex-3-en yl)d ip r op yla m in e (1c).
A solution of 4 (46 mg, 0.14 mmol) in THF (5 mL), EtMe2N
(150 µL, 1.38 mmol), phenylacetylene (30 µL, 0.27 mmol), CuI
(3 mg, 0.01 mmol), and (Ph3P)4Pd0 (8 mg, 0.007 mmol) was
reacted and worked up as described for 1a to give 1c (38 mg,
96%) as a colorless oil: IR 3030, 2960, 2870, 2810, 2200, 1590
cm-1; 1H NMR δ 0.87 (t, 6H, J ) 7.4), 1.37-1.57 (m, 5H), 1.85-
1.93 (m, 1H), 2.05-2.18 (m, 1H), 2.20-2.47 (m, 7H), 2.80
(dddd, 1H, J ) 12.1, 10.6, 5.1, 2.7), 6.14-6.19 (m, 1H), 7.25-
7.32 (m, 3H), 7.37-7.44 (m, 2H); EIMS 281 (M+). Anal.
(C20H27N) C,H,N.
4-Dip r op yla m in ocycloh ex-1-en eca r bon itr ile (1d ). To a
solution of 4 (66 mg, 0.20 mmol) in benzene (5 mL) were added
KCN (53 mg, 0.81 mmol), 18-crown-6 (70 mg, 0.26 mmol), and
(Ph3P)4Pd0 (14 mg, 0.01 mmol). After being refluxed for 2.5 h
the mixture was cooled to room temperature; aqueous NaHCO3
(5%) and Et2O were added. The organic layer was dried

760 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Brief Articles
(MgSO4) and evaporated and the residue was purified by flash
2.28 (m, 2H), 2.32-2.54 (m, 6H), 2.81 (dddd, 1H, J ) 12.1,
10.0, 5.5, 2.8), 5.69-5.74 (m, 1H); EIMS 329 (M+), 196 (M -
133)+; HREIMS (M+) 329.1266 (329.1272 calcd for C13H22O3-
NF3S).
chromatography (CH2Cl2-MeOH 95:5) to give 1d (29 mg, 70%)
as a colorless oil: IR 3030, 2960, 2870, 2810, 2215, 1640 cm-1
;
1H NMR δ 0.86 (t, 6H, J ) 7.4), 1.36-1.54 (m, 5H), 1.88-1.96
(m, 1H), 2.08-2.20 (m, 1H), 2.25-2.43 (m, 7H), 2.78 (dddd,
1H, J ) 12.1, 10.5, 5.0, 2.7), 6.57-6.62 (m, 1H); EIMS 206
(M+). Anal. (C13H22N2) C,H,N.
(4-Dibr om om eth ylen ecycloh exyl)d ip r op yla m in e (5).
To a solution of PPh3 (1.31 g, 5 mmol) in benzene (20 mL) was
added CBr4 (837 mg, 2.5 mmol). The mixture was stirred at
room temperature for 30 min when a solution of 3 (200 mg,
1.01 mmol) in benzene (5 mL) was added. After being refluxed
for 4 h, the mixture was cooled to room temperature and
filtrated and the solvent was evaporated. The residue was
dissolved in CH2Cl2 and washed with aqueous NaHCO3 (5%).
The organic layer was dried (MgSO4) and evaporated and the
residue was purified by flash chromatography (petroleum
ether-EtOAc 3:7) to give 5 (302 mg, 84%) as a colorless oil:
IR 2960, 2870, 2800, 1620, 800 cm-1; 1H NMR δ 0.85 (t, 6H, J
) 7.4), 1.28-1.47 (m, 6H), 1.79-1.96 (m, 4H), 2.34-2.40 (m,
4H), 2.65 (tt, 1H, J ) 11.4, 3.4), 2.93-3.01 (m, 2H); EIMS 355
(M+), 353 (M+). Anal. (C13H23NBr2) C,H,N.
Dip r op yl[4-(3-t r im et h ylsilyl-1-t r im et h ylsilylet h yn yl-
p r op -2-yn ylid en e)cycloh exyl]a m in e (2a ). To a suspension
of (Ph3P)2PdCl2 (26 mg, 0.03 mmol) and CuI (10 mg, 0.05
mmol) in THF (10 mL) were added a solution of 5 (134 mg,
0.38 mmol) in THF (5 mL), trimethylsilylacetylene (370 µL,
2.61 mmol), and piperidine (375 µL, 3.79 mmol). After being
stirred at room temperature for 20 h, aqueous NaHCO3 (5%)
was added and the mixture was extracted with Et2O. The
organic layer was dried (MgSO4) and evaporated and the
residue was purified by flash chromatography (petroleum
ether-EtOAc-EtMe2N 90:10:1) to give 2a (115 mg, 78%) as a
colorless solid: mp 70 °C; IR 2960, 2870, 2810, 2150, 1590,
1250 cm-1; 1H NMR δ 0.19 (s, 18H), 0.85 (t, 6H, J ) 7.2), 1.29-
1.47 (m, 6H), 1.83-1.92 (m, 2H), 1.98 (dt, 2H, J ) 13.3, 4.4),
2.33-2.39 (m, 4H), 2.68 (tt, 1H, J ) 11.4, 3.3), 3.03-3.13 (m,
2H); 13C NMR δ 0.0, 11.8, 22.3, 28.8, 31.5, 52.9, 59.2, 96.1,
98.8, 101.0, 162.2; EIMS 387 (M+). Anal. (C23H41NSi2) C,H,N.
[4-(1-E t h yn ylp r op -2-yn ylid en e)cycloh exyl]d ip r op yl-
a m in e (2b). To a solution of 2a (79 mg, 0.2 mmol) in THF (8
mL) was added Bu4NF (430 µL, 1 M solution in THF) at -15
°C. After being stirred at -15 °C for 30 min, aqueous NaHCO3
(5%) and Et2O were added. The organic layer was dried
(MgSO4) and evaporated and the residue was purified by flash
chromatography (petroleum ether-EtOAc-EtMe2N 90:10:1)
to give 2b (35 mg, 70%) as a colorless liquid: IR 3310, 2960,
2870, 2810, 2100, 1590 cm-1; 1H NMR δ 0.85 (t, 6H, J ) 7.4),
1.31-1.47 (m, 6H), 1.85-1.94 (m, 2H), 2.03 (dt, 2H, J ) 13.5,
4.4), 2.34-2.40 (m, 4H), 2.70 (tt, 1H, J ) 11.5, 3.3), 3.07 (s,
2H), 3.08-3.15 (m, 2H); EIMS 243 (M+). Anal. (C17H25N)
C,H,N.
[4-(3-P h en yl-1-p h en yleth yn ylp r op -2-yn ylid en e)cyclo-
h exyl]d ip r op yla m in e (2c). A suspension of (Ph3P)2PdCl2 (10
mg, 0.01 mmol) and CuI (4 mg, 0.02 mmol) in THF (4 mL)
was reacted with a solution of 5 (50 mg, 0.14 mmol) in THF (2
mL), phenylacetylene (110 µL, 1.0 mmol), and piperidine (150
µL, 1.51 mmol). After being stirred at room temperature for
24 h the mixture was worked up as described for 2a to give
2c (42 mg, 75%) as a yellowish oil: IR 3050, 2950, 2870, 2810,
2210, 1590, 1490 cm-1; 1H NMR δ 0.87 (t, 6H, J ) 7.3), 1.38-
1.56 (m, 6H), 1.90-2.01 (m, 2H), 2.12 (dt, 2H, J ) 13.3, 4.2),
2.37-2.46 (m, 4H), 2.72-2.83 (m, 1H), 3.20-3.30 (m, 2H),
7.27-7.36 (m, 6H), 7.45-7.53 (m, 4H); EIMS 395 (M+). Anal.
(C29H33N) C,H,N.
(4-Dip r op yla m in ocycloh exylid en e)a ceton itr ile (6). A
suspension of KOH (28 mg, 0.5 mmol) in acetonitrile (6 mL)
was refluxed for 20 min. Then a solution of 3 (90 mg, 0.45
mmol) in acetonitrile (2 mL) was added dropwise to the boiling
mixture and refluxing was continued for 3 h. The mixture was
poured into ice water and extracted with Et2O. The organic
layer was dried (MgSO4) and evaporated, and the residue was
purified by flash chromatography (CH2Cl2-MeOH 95:5) to give
6 (48 mg, 48%) as a yellowish oil: IR 3050, 2960, 2870, 2810,
1
2220, 1630 cm-1; H NMR δ 0.86 (t, 6H, J ) 7.4), 1.34-1.48
(m, 6H), 1.88-2.01 (m, 2H), 2.07-2.24 (m, 2H), 2.34-2.47 (m,
5H), 2.72 (tt, 1H, J ) 11.2, 3.2), 2.96 (ddd, 1H, J ) 13.9, 5.8,
3.6), 5.05 (s, 1H); 13C NMR δ 11.7, 22.1, 28.8, 29.4, 31.8, 34.6,
52.7, 58.6, 92.5, 116.8, 167.5; EIMS 220 (M+). Anal. (C14H24N2)
C,H,N.
Bovin e Recep tor P r ep a r a tion . Fresh bovine brains were
obtained from the local slaughterhouse. The striata were
dissected and frozen at -80 °C. Membranes were prepared as
described previously.29 In brief, the striata were thawed, cut
up, and homogenized in an aqueous solution of sucrose (0.1
M). The suspension was washed by repeated centrifugation
at 1000g. Then, the resulting supernatant was pelleted by
centrifugation at 60000g for 1 h. The pellet was washed twice
by resuspension in Tris-EDTA buffer (50 mM TrisHCl, 1 mM
EDTA; pH 7.4) and subsequent centrifugation at 60000g for
15 min. Finally the membranes were suspended in Tris-EDTA
buffer, homogenized with a Potter-Elvehjam homogenizer, and
stored at -80 °C in small aliquots.
Bovin e Ra d ior ecep tor Assa y. For the D1 receptor binding
assay bovine striatal membranes were diluted with binding
buffer (50 mM TrisHCl, 1 mM EDTA, 5 mM MgCl2, 0.1 mM
dl-dithiothreitol, 100 µg/mL bacitracin, 5 µg/mL soybean
trypsin inhibitor; pH 7.4) to a final concentration of 25 µg
protein/assay tube. Tubes were prepared with the radioligand
[3H]SCH 23390 (0.3 nM) (specific activity 83.0 Ci/mmol;
Amersham Pharmacia Biotech, Freiburg, Germany) and vary-
ing concentrations of test compounds (from 0.01 to 100 000
nM). Total binding of [3H]SCH 23390 was measured in the
absence of any competing drug; nonspecific binding was
determined by coincubation with butaclamol (1 µM). Incuba-
tion was started by adding membranes to the assay tube with
a final volume of 200 µL. It was carried on for 60 min at 37 °C
and stopped by rapid filtration through GF/B filters precoated
with 0.3% polyethylenimine, using an automated cell harvester
(Inotech, Dottikon, CH). Filters were washed five times with
ice-cold wash buffer (50 mM TrisHCl, 120 mM NaCl; pH 7.4)
2-(4-Dipr opylam in ocycloh exyliden e)m alon on itr ile (2d).
To a solution of 3 (50 mg, 0.25 mmol) in MeOH (3 mL) were
added malononitrile (18 mg, 0.27 mmol) and piperidine (30
µL) at 0 °C. After being stirred at 0 °C for 2 h, the solvent was
evaporated and the residue was purified by flash chromatog-
raphy (petroleum ether-EtOAc-EtMe2N 80:20:1) to give 2d
(29 mg, 47%) as a slightly yellowish oil: IR 2960, 2870, 2810,
1
2230, 1640 cm-1; H NMR δ 0.86 (t, 6H, J ) 7.4), 1.41 (sext,
4H, J ) 7.4), 1.58 (dddd, 2H, J ) 13.3, 12.1, 10.6, 4.0), 1.98-
2.07 (m, 2H), 2.33-2.44 (m, 6H), 2.80 (tt, 1H, J ) 10.6, 3.4),
3.01-3.09 (m, 2H); EIMS 245 (M+). Anal. (C15H23N3) C,H,N.
Tr iflu or om eth a n esu lfon ic Acid 4-Dip r op yla m in ocy-
cloh ex-1-en yl Ester (4). To a solution of 3 (300 mg, 1.52
mmol), which was synthesized according to ref 20, in 1,2-
dichloroethane (30 mL) were added 2,6-di-tert-butyl-4-meth-
ylpyridine (420 mg, 2.04 mmol) and Tf2O (500 µL, 2.97 mmol).
After being refluxed for 4.5 h, the mixture was cooled to room
temperature and aqueous NaHCO3 (5%) and CH2Cl2 were
added. The organic layer was dried (MgSO4) and evaporated,
and the residue was purified by flash chromatography (petro-
leum ether-EtOAc 9:1f6:4) to give 4 (219 mg, 43%) as a
yellow liquid: IR 2960, 2870, 2810, 1690, 1420, 1140 cm-1; 1H
NMR δ 0.86 (t, 6H, J ) 7.4), 1.42 (sext, 4H, J ) 7.4), 1.64
(dddd, 1H, J ) 12.5, 12.1, 11.2, 6.0), 1.90-1.98 (m, 1H), 2.08-
dried, and counted in
Freiburg, Germany).
a MicroBeta Trilux (Wallac ADL,
Protein concentration was established by the method of
Lowry using bovine serum albumin as standard.31
Competition binding analysis with the bovine D2 receptor
was done with the radioligand [3H]spiperone (0.5 nM) (specific
activity 99.0 Ci/mmol; Amersham Pharmacia Biotech, Freiburg,
Germany) as an antagonist and the agonist [3H]pramipexole
(0.5 nM) (specific activity 42.0 Ci/mmol; a generous gift from

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4 761
Boehringer Ingelheim, Ingelheim, Germany) for labeling se-
lectively the high-affinity state of the D2 receptor. The assay
was carried out in a final volume of 1500 µL by incubating
the radioligand, the test compounds (8 concentrations between
0.01 and 100 000 nM, and 16 concentrations between 0.1 and
100 000 nM for two-site determinations) or butaclamol for
investigating unspecific binding, and finally the membrane
suspension. Membranes were diluted in binding buffer with a
protein concentration of 200 µg/assay tube for [3H]spiperone
and 500 µg/assay tube for [3H]pramipexole, respectively.
Assays with [3H]spiperone also included ketanserin (50 nM)
to mask binding of the radioligand to serotonin sites. Incuba-
tion for 2 h at 23 °C was terminated by rapid filtration through
GF/C filters, using a Brandel cell harvester (Brandel, Gaith-
ersburg, MD). The filters were rinsed three times with ice-
cold Tris-EDTA buffer, and the radioactivity trapped on the
filters was counted using a Beckman LS 6500 scintillation
counter (Beckman).
calculated for a one-site model (nH - 1) or a two-site model
(nH < 1) depending on the slope factor. The bases of the
discrimination between monophasic and biphasic data analysis
were the nH values and the ability of the program PRISM to
fit the data; -0.8 was chosen as the cutoff value. If both
requirements were fulfilled (nH below 0.8 and PRISM calcu-
lated a better fit of equations for a two-site competition) a
biphasic curve was used. IC50 values were transformed to Ki
values according to the equation of Cheng and Prusoff.32
Molecu la r Mod elin g. Structures were built using the
SYBYL 6.5 software package.33 Geometry optimization as well
as calculation of ESP point charges of all the compounds
investigated were done with the AM1 Hamiltonian34 imple-
mented in MOPAC 6.0.35 All other graphical manipulations
were carried out within SYBYL 6.5, including visualization of
molecular electrostatic potentials (MEP; -1.0 kcal/mol) and
mapping of lipophilic potentials onto the calculated Connolly
surfaces (MOLCAD module). Generally, default parameters
were used. To force the lipophilic potential colors of each
molecule into common borders, the option global was selected.
Cell Cu ltu r e. All cell culture material was purchased from
LifeTechnologies, Karlsruhe, Germany. Chinese hamster ovary
cells (CHO-K1) stably expressing the human DA D2
and
long
D2
receptors25 were grown in DMEM-Ham’s F12 medium
short
Ack n ow led gm en t. The authors thank Dr. H. H. M.
Van Tol (Clarke Institute of Psychiatry, Toronto), Dr.
J .-C. Schwartz and Dr. P. Sokoloff (INSERM, Paris), and
Dr. J . Shine (The Garvan Institute of Medical Research,
Sydney) for providing dopamine D4, D3, and D2 receptor-
expressing cell lines, respectively. Thanks are also due
to Mrs. H. Ka¨ding, Mrs. B. Linke, and Mrs. P. Schmitt
for skillful technical assistance. This work was sup-
ported by the DFG and the Fonds der Chemischen
Industrie.
(1:1), supplemented with 0.05% sodium bicarbonate, 10% fetal
calf serum, and glutamine (2 mM). Stably expressed human
D3 receptors26 were obtained from dihydrofolate reductase
gene-deficient CHO cells. They were grown in DMEM medium
containing 4500 mg/L glucose, 10% heat-inactivated dialyzed
fetal calf serum, MEM amino acid supplement, and glutamine
(2 mM). CHO-K1 cells expressing the human DA receptor
27
subtype D4.4 were grown in MEM R-medium supplemented
with 2.5% fetal calf serum, 2.5% horse serum, and 400 µg/mL
G418. All cells were grown at 37 °C under a humidified
atmosphere of 5% CO2-95% air in the presence of 100 U/mL
penicillin G and 100 µg/mL streptomycin.
Clon ed Recep tor P r ep a r a tion . Human D2 long, D2 short, D3,
and D4.4 receptor-expressing cell lines were grown in 145-cm2
culture dishes to 80% confluency. Cells were rinsed twice with
ice-cold PBS and scraped from the dishes using a sterile cell
scraper in the presence of harvest buffer (10 mM TrisHCl, 0.5
mM EDTA, 5.5 mM KCl, 140 mM NaCl; pH 7.4) supplemented
with protease inhibitors. After centrifugation (400g, 5 min) the
Refer en ces
(1) J ucker, E., Ed. Progress in Drug Research, Vol. 37; Birkha¨user
Verlag: Basel, Boston, Berlin, 1991.
(2) Casy, A. F. The Steric Factor in Medicinal Chemistry; Plenum
Press: New York, London, 1993.
(3) Krogsgaard-Larsen, P. Gamma-aminobutyric acid agonists,
antagonists and uptake inhibitors. J . Med. Chem. 1981, 24,
1377-1383.
(4) Seyfried, C. A.; Boettcher, H. Central D2-autoreceptor agonists,
with special reference to indolylbutylamines. Drugs Future 1990,
15, 819-832.
(5) Mierau, J .; Schingnitz, G. Biochemical and pharmacological
studies on pramipexole, a potent and selective dopamine D2
receptor agonist. Eur. J . Pharmacol. 1992, 215, 161-170.
(6) Bach, N. J .; Kornfeld, E. C.; J ones, N. D.; Chaney, M. O.;
Dorman, D. E.; Paschal, J . W.; Clemens, J . A.; Smalstig, E. B.
Bicyclic and Tricyclic Ergoline Partial Structures. Rigid 3-(2-
Aminoethyl)pyrroles and 3- and 4-(2-Aminoethyl)pyrazoles as
Dopamine Agonists. J . Med. Chem. 1980, 23, 481-491.
(7) Gmeiner, P.; Mierau, J .; Ho¨fner, G. Enantiomerically Pure
Aminoindolizines: Bicyclic Ergoline Analogues with Dopamine
Autoreceptor Activity. Arch. Pharm. (Weinheim) 1992, 325, 57-
60.
cells expressing D2
D2 short, and D4.4 receptors were resus-
long
pended in homogenate buffer (50 mM TrisHCl, 5 mM EDTA,
1.5 mM CaCl2, 5 mM MgCl2, 5 mM KCl, 120 mM NaCl; pH
7.4) and cells expressing D3 receptors in Tris-MgSO4 buffer
(10 mM TrisHCl, 5 mM MgSO4; pH 7.4), respectively, and
stored at -80 °C. After thawing, the cells were diluted in
homogenate buffer (D2 long, D2 short, and D4.4) or Tris-MgSO4
buffer (D3), homogenized using a Polytron (20 000 rpm, 5 times
for 5 s each in an ice bath), and spun at 50000g for 15 min.
The membrane pellet was resuspended in binding buffer,
homogenized with a Potter-Elvehjem homogenizer, and stored
in small aliquots at -80 °C with an estimated protein
concentration of ca. 500 µg/mL.
(8) Gmeiner, P.; Sommer, J .; Mierau, J .; Ho¨fner, G. Dopamine
Autoreceptor Agonists: Computational Studies, Synthesis and
Biological Investigations. Bioorg. Med. Chem. Lett. 1993, 3,
1477-1483.
(9) Doll, M. K.-H.; Nichols, D. E.; Kilts, J . D.; Prioleau, C.; Lawler,
C. P.; Lewis, M. M.; Mailman, R. B. Synthesis and Dopaminergic
Properties of Benzo-Fused Analogues of Quinpirole and Quinelo-
rane. J . Med. Chem. 1999, 42, 935-940.
(10) Blair, J . B.; Marona-Lewicka, D.; Kanthasamy, A.; Lucaites, V.
L.; Nelson, D. L.; Nichols, D. E. Thieno[3,2-b]- and Thieno[2,3-
b]pyrrole Bioisosteric Analogues of the Hallucinogen and Sero-
tonin Agonist N,N-Dimethyltryptamine. J . Med. Chem. 1999,
42, 1106-1111.
Bin d in g Assa y w ith Clon ed Hu m a n Recep tor s. Binding
analysis with human DA receptors was done in the same
manner as described for the bovine D1 receptors. In brief, cell
membranes containing the D2 long, D2 short, D3, and D4.4 receptors
were diluted in binding buffer to a final protein concentration
of 10-20 µg/assay tube. [3H]Spiperone (0.5 nM) was used as
the radioligand and haloperidol (10 µM) to determine unspe-
cific binding. Eight concentrations of the test compounds
between 0.01 and 100 000 nM and 16 concentrations between
0.1 and 100 000 nM for two-site determinations were inves-
tigated.
(11) Shih, T. L.; Candelore, M. R.; Cascieri, M. A.; Chiu, S.-H. L.;
Colwell J r., L. F.; Deng, L.; Feeney, W. P.; Forrest, M. J .; Hom,
G. J .; MacIntyre, D. E.; Miller, R. R.; Stearns, R. A.; Strader, C.
D.; Tota, L.; Wyvratt, M. J .; Fisher, M. H.; Weber, A. E. L-770,-
644: A potent and selective human â3 adrenergic receptor
agonist with improved oral bioavailability. Bioorg. Med. Chem.
Lett. 1999, 9, 1251-1254.
(12) Thomas, C.; Ohnmacht, U.; Niger, M.; Gmeiner, P. â-Analogues
of PLG (L-Prolyl-L-Leucyl-Glycinamide): Ex-Chiral Pool Syn-
theses and Dopamine D2 Receptor Modulating Effects. Bioorg.
Med. Chem. Lett. 1998, 8, 2885-2890.
To reveal the high-affinity sites of the receptor, competition
analysis was done in the absence and presence of the nonhy-
drolyzable GTP analogue Gpp(NH)p (100 µM).
Da ta An a lysis. The resulting competition curves were
analyzed by nonlinear regression using the algorithms in
PRISM (GraphPad Software, San Diego, CA). The data were
initially fit using a sigmoid model to provide a slope coefficient
(nH) and an IC50 value, representing the concentration corre-
sponding to 50% of maximal inhibition. Data were then

762 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 4
Brief Articles
(13) Lo¨ber, S.; Hu¨bner, H.; Gmeiner, P. Azaindole Derivatives with
High Affinity for the Dopamine D4 Receptor: Synthesis, Ligand
Binding Studies and Comparison of Molecular Electrostatic
Potential Maps. Bioorg. Med. Chem. Lett. 1999, 9, 97-102.
(14) Thomas, C.; Hu¨bner, H.; Gmeiner, P. Enantio- and Diastereo-
controlled Dopamine D1, D2, D3 and D4 Receptor Binding of
N-(3-Pyrrolidinylmethyl)benzamides Synthesized from Aspartic
Acid. Bioorg. Med. Chem. Lett. 1999, 9, 841-846.
(15) Haubmann, C.; Hu¨bner, H.; Gmeiner, P. 2,2-Dicyanovinyl as a
Nonaromatic Aryl Bioisostere: Synthesis, Binding Experiments
and SAR Studies of Highly Selective Dopamine D4 Receptor
Ligands. Bioorg. Med. Chem. Lett. 1999, 9, 1969-1972.
(16) Chidester, C. G.; Lin, C.-H.; Lahti, R. A.; Haadsma-Svensson,
S. R.; Smith, M. W. Comparison of 5-HT1A and Dopamine D2
Pharmacophores. X-ray Structures and Affinities of Conforma-
tionally Constrained Ligands. J . Med. Chem. 1993, 36, 1301-
1315.
(17) Teeter, M. M.; Froimowitz, M.; Stec, B.; DuRand, C. J . Homology
Modeling of the Dopamine D2 Receptor and Its Testing by
Docking of Agonists and Tricyclic Antagonists. J . Med. Chem.
1994, 37, 2874-2888.
(18) Bikker, J . A.; Trumpp-Kallmeyer, S.; Humblet, C. G-Protein
Coupled Receptors: Models, Mutagenesis, and Drug Design. J .
Med. Chem. 1998, 41, 2911-2927.
(19) Davis, A. M.; Teague, S. J . Hydrogen Bonding, Hydrophobic
Interactions, and Failure of the Rigid Receptor Hypothesis.
Angew. Chem., Int. Ed. Engl. 1999, 38, 736-749.
(20) Demopoulos, V. J .; Gavalas, A.; Rekatas, G.; Tani, E. Synthesis
of 6,7,8,9-Tetrahydro-N,N-di-n-propyl-1H-benz[g]indol-7-amine,
a Potential Dopamine Receptor Agonist. J . Heterocycl. Chem.
1995, 32, 1145-1148.
(21) Ritter, K. Synthetic Transformations of Vinyl and Aryl Triflates.
Synthesis 1993, 735-762.
(22) Cacchi, S.; Morera, E.; Ortar, G. Palladium-Catalyzed Reaction
of Enol Triflates with 1-Alkynes. A New Route to Conjugated
Enynes. Synthesis 1986, 320-322.
(23) Quirante, J .; Escolano, C.; Massot, M.; Bonjoch, J . Synthesis of
2-Azabicyclo[3.3.1]nonanes by means of (Carbamoyl)dichloro-
methyl Radical Cyclization. Tetrahedron 1997, 53, 1391-1402.
(24) Neidlein, R.; Winter, M. Synthesis of Geminal Enediynes with
Saturated and Unsaturated Carbocyclic Backbones by Pal-
ladium-Catalyzed Alkynylation of Dibromoolefins. Synthesis
1998, 1362-1366.
(25) Hayes, G.; Biden, T. J .; Selbie, L. A.; Shine J . Structural
Subtypes of the Dopamine D2 Receptor Are Functionally Dis-
tinct: Expression of the Cloned D2A and D2B Subtypes in a
Heterologous Cell Line. Mol. Endocrinol. 1992, 6, 920-926.
(26) Sokoloff, P.; Andrieux, M.; Besanc¸on, R.; Pilon, C.; Martres, M.-
P.; Giros, B.; Schwartz, J .-C. Pharmacology of human dopamine
D3 receptor expressed in a mammalian cell line: comparison
with D2 receptor. Eur. J . Pharmacol. 1992, 225, 331-337.
(27) Asghari, V.; Sanyal, S.; Buchwaldt, S.; Paterson, A.; J ovanovic,
V.; Van Tol, H. H. M. Modulation of Intracellular Cyclic AMP
Levels by Different Human Dopamine D4 Receptor Variants.
J . Neurochem. 1995, 65, 1157-1165.
(28) Mierau, J .; Schneider, F. J .; Ensinger, H. A.; Chio, C. L.; Lajiness,
M. E.; Huff, R. M. Pramipexole binding and activation of cloned
and expressed dopamine D2, D3 and D4 receptors. Eur. J .
Pharmacol. 1995, 290, 29-36.
(29) Ohnmacht, U.; Tra¨nkle, C.; Mohr, K.; Gmeiner, P. [3H]Prami-
pexole: a selective radioligand for the high affinity dopamine
D2 receptor in bovine striatal membranes. Pharmazie 1999, 54,
294-297.
(30) Mailman, R. B.; Nichols, D. E.; Tropsha, A. Molecular Drug
Design and Dopamine Receptors. In The Dopamine Receptors;
Neve, K. A., Neve, R. L., Eds.; Humana Press: Totowa, NJ , 1997;
Chapter 5.
(31) Lowry, O. H.; Rosebrough, N. J .; Farr, A. L.; Randall, R. J .
Protein measurement with the folin phenol reagent. J . Biol.
Chem. 1951, 193, 265-275.
(32) Cheng, Y. C.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (IC50) of an enzymatic reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
(33) SYBYL 6.5, Tripos Inc., 1699 South Hanley Rd., St. Louis, MO
63144.
(34) Dewar, M. J . S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J . J . P.
AM1: A New General Purpose Quantum Mechanical Molecular
Model. J . Am. Chem. Soc. 1985, 107, 3902-3909.
(35) Stewart, J . J . P. QCPE #455.
J M991098Z