Chirospecific and subtype selective dopamine receptor binding of heterocyclic methoxynaphthamide analogs

Article abstract of DOI:10.1002/ardp.200400997

Employing the D₃ and D₄ selective methoxynaphthalines nafadotride and FAUC 182, respectively, as lead compounds, the pyrazolo[1,5-a]pyridine-3-carboxamides of type 1a and 2a as well as their 2-substituted regioisomers 1b and 2b were synthesized when following an ex-chiral pool approach. Dopamine receptor binding studies involving the target compounds (1a,b, 2a,b) and the respective optical antipodes ent-1a,b and ent-2a,b revealed the heterocyclic carboxamide 2a as a strong and selective D₄ ligand (K_i = 8.6 nM). According to a mitogenesis assay, 2a shows D₄ partial agonist effects (29%, EC₅₀ = 6.7 nM) and, thus, might be of interest for the treatment of sexual dysfunction.

Full text of DOI:10.1002/ardp.200400997

276
DOI 10.1002/ardp.200400997
Arch. Pharm. Chem. Life Sci. 2005, 338, 276−280
Chirospecific and Subtype Selective Dopamine
Receptor Binding of Heterocyclic Methoxy-
naphthamide Analogs
Laura Bettinetti, Harald Hübner, and Peter Gmeiner
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University, Erlangen,
Germany
Employing the D3 and D4 selective methoxynaphthalines nafadotride and FAUC 182, respectively, as
lead compounds, the pyrazolo[1,5-a]pyridine-3-carboxamides of type 1a and 2a as well as their
2-substituted regioisomers 1b and 2b were synthesized when following an ex-chiral pool approach.
Dopamine receptor binding studies involving the target compounds (1a,b, 2a,b) and the respective
optical antipodes ent-1a,b and ent-2a,b revealed the heterocyclic carboxamide 2a as a strong and
selective D4 ligand (Ki ϭ 8.6 nM). According to a mitogenesis assay, 2a shows D4 partial agonist
effects (29%, EC50 ϭ 6.7 nM) and, thus, might be of interest for the treatment of sexual dysfunction.
Keywords: Dopamine; receptor; medicinal chemistry
Received: November 25, 2004; Accepted: February 22, 2005 [FP997]
Introduction
2-Methoxybenzamides including the aminomethylpyrrolid-
ine derivative sulpiride are known as highly active agents
for the treatment of schizophrenia [1]. Due to the affinity
of this family of compounds to dopamine D2 receptors in
striatal regions of the brain, their application is associated
with extrapyramidal side effects [2]. Structural modifi-
cations led to the 2-methoxynaphthalenes nafadotride and
FAUC 182 revealing binding preference for the D3 and D4
subtype, respectively [3, 4]. Due to their distribution in the
brain and a series of functional studies, dopamine D3 and
D4 receptors seem to be especially involved in the symptoms
of schizophrenia [5, 6].
Very recently, we reported that the bioisosteric replacement
of the naphthalene subunit of the dopamine D3 receptor
partial agonist BP 897 by a pyrazolopyridine moiety led to
the neuroprotective D3 receptor ligand FAUC 329 [7, 8]. As
an extension of these studies, we herein describe the syn-
thesis of the enantiomerically pure pyrazolo[1,5-a]pyridine
carboxamides 1 and 2 (Figure 1) as well as their optical
antipodes ent-1 and ent-2. To investigate the effect of the
spatial orientation of the 7a-azaindole moiety onto the
dopamine receptor binding profiles, both 2- and the 3-sub-
stituted regioisomeres should be prepared.
Figure 1. Ratio for structural modifications.
Correspondence: Department of Medicinal Chemistry, Emil Fischer
Center, Friedrich Alexander University, Schuhstraße 19, DϪ91052
Erlangen, Germany. Phone: ϩ49 9131 8529383, Fax: ϩ49 9131
8522585, e-mail: gmeiner@pharmazie.uni-erlangen.de
Results and discussion
Chemical synthesis of the building block 3 was done by
1,3-dipolar cycloaddition of N-aminopyridinium iodide and
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 276−280
Heterocyclic methoxynapththamide analogs for dopamine receptor
277
compounds 1a, 1b, 2a, 2b, and their enantiomers ent-1a,
ent-1b, ent-2a and ent-2b were obtained in 83Ϫ99% yield.
The novel 7a-azaindole carboxamides were evaluated in vi-
tro for their ability to displace [3H]spiperone from the
cloned human dopamine receptors D2long, D2short [14], D3
[15] and D4 [16] being stably expressed in CHO cells [17].
D1 affinity was determined by employing porcine striatal
membrane preparations and the D1 selective antagonist
[3H]SCH 23390. For comparison of the binding data, the
antipsychotic drug sulpiride was investigated under the
same conditions (Table 1).
The dopamine receptor binding profiles of the test com-
pounds clearly indicate poor affinities for the D1, D2 and
D3 subtypes. Only the (S)-aminomethylpyrolidine derivative
1b displayed a Ki value in the nanomolar range (400 nM for
D3). Comparison of the D4 binding data shows significantly
higher affinities for the 2-aminopyrolidine derivatives (type
2). For both regioisomers, (R)-configuration proved su-
perior to the (S)-enantiomers. Interestingly, the pyrazolo-
pyridine-3-carboxamide 2a showed high and selective D4
binding leading to a Ki value of 8.6 nM when the selectivity
over the subtypes D1, D2long, D2short and D3 was higher than
300 fold for any subtype. To investigate the intrinsic effect
of the test compound 2a, an in vitro functional assay meas-
uring the [3H]thymidine uptake in growing CHO cells stab-
ily expressing the dopamine D4 receptor was performed
when a 29% stimulation of mitogenesis (compared to the
full agonist effect of quinpirole and the D4 antagonist cloza-
pine) was determined (EC50 ϭ 6.7 nM compared to 13 nM
for quinpirole) indicating that the test compound behaves
as a partial agonist (Figure 2) [18].
In conclusion, SAR investigations on the heterocyclic car-
boxamide regio- and stereoisomers of type 1 and 2 indicated
that the ortho-methoxy substituent of the lead compounds
might be of special importance for D2 and D3 but not for
D4 receptor binding. Thus, the D2 and D3 receptor recog-
nition was significantly reduced when compared to sulpir-
ide. However, for the D4 subtype, the (R)-configured re-
gioisomers of type 2 displayed substantial receptor binding
resulting in a 250 fold lower Ki value than the reference
agent. However, it is not clear whether they adopt the same
binding modes as the structural family of 2-methoxybenza-
mides and 2-methoxynaphthamides. Due to recent obser-
vations on the dopamine D4 receptor partial agonist ABT-
724 inducing penile erection in rats [19], the pyrazolo-
pyridine carboxamide 2a is of potential interest for further
investigations.
Scheme 1. Synthesis routes for 2a and 2b.
ethyl propiolate under oxidative conditions and subsequent
saponification employing a modified protocol of R. Huis-
gen’s pioneering work [9, 10]. For the preparation of the 2-
substituted regioisomer 4, acetylene dicarboxylate was util-
ized as a dipolarophile taking advantage of a selective de-
carboxylation in position 3 [11, 12]. Whereas the benzyl pro-
tected 2-aminomethylpyrrolidenes could be derived enanti-
omerically pure from (S)- or (R)-N-benzylproline ethyl ester
by aminolysis and lithium aluminum hydride promoted re-
duction, chiral N-benzyl protected 3-aminopyrrolidine was
synthesized in both configurations starting from (S)- or (R)-
aspartic acid following a protocol that we have recently
elaborated [4, 13].
Acknowledgments
Chemical coupling of the two amine building blocks and
their enantiomers to the heteroarene carboxylic acids 3 and
4 was accomplished by oxalyl chloride promoted activation
and subsequent aminolysis (Scheme 1). Thus, the target
The authors wish to 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 Dr. J. Shine (The
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

278
Gmeiner et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 276−280
Table 1. Receptor binding data for the test compounds 1, 2 and ent-1,2 and the neuroleptic drug sulpiride to porcine dopamine
D1 and human D2long, D2short, D3 and D4 receptors†.
Ki values in nM SEM
[3H]SCH 23390
pD1
[3H]spiperone
hD3
Compound
hD2long
hD2short
hD4
1a
ent1a
1b
ent1b
2a
ent2a
2b
26000 1500
20000 1000
17000 2000
23000 1000
22000 2000
24000 1000
77000 23000
16000 2000
26000 1500
11000 2000
16000 3000
35000 3500
35000 7000
34000 9500
140 22
60000 17000
14000 3000
19000 4000
7700 150
15000 3000
36000 5000
24000 1000
25000 3500
51 11
7000 950
3800 750
400 5.0
3800 450
2900 300
5100 900
4300 350
5200 3300
89 32
4600 800
4000 150
3800 250
410 35
8.6 1.2
100 8.0
55 5.0
16000
21000
0
0
ent2b
sulpiride
160 15
2300 260
50000 13000
†
Ki values in nM SEM are based on the means of 2Ϫ3 experiments each done in triplicate.
Chemistry
[(S)-1-Benzylpyrrolidin-2-yl]-methylamine [20, 21]
(S)-N-Benzylproline ethyl ester (432 mg, 1.85 mmol) in a NH3
MeOH solution (20 mL) was stirred at RT for 10 days. After evapo-
ration of NH3 at RT, the solvent was removed in vacuum to give
375 mg of a yellow pasty solid which was used without purification.
A suspension of the crude intermediate (153 mg, 0.75 mmol) in abs
THF (4 mL) was treated with a 1.0 M LiAlH4 solution in THF
(1.26 mL) and stirred under nitrogen atmosphere at RT for 6 h.
EtOAc (0.1 mL) and a 2 N NaOH solution (0.2 mL) were added,
the obtained solid was removed by filtration and the solution dried
(Na2SO4). Evaporation of the solvent gave an oil purified by flash
chromatography (CHCl3 : MeOH ϭ 8:2). Yield: 144 mg (76%).
[α]2D0: Ϫ71.3° (c 1.0, CHCl3), ([α]D25 (CHCl3): Ϫ55°) [21]. IR (NaCl)
ν: 3367, 3027, 2962, 2792, 1643, 1573, 1118, 740, 698 cmϪ1 1H
.
NMR (CDCl3) δ: 1.64Ϫ1.73 (m, 3H); 1.86Ϫ1.94 (m, 1H),
2.17Ϫ2.24 (m, 1H), 2.54Ϫ2.59 (m, 1H), 2.70 (dd, J ϭ 13.0 Hz, 3.4
Hz, 1H), 2.76 (dd, J ϭ 12.8 Hz, 5.3 Hz, 1H), 2.91Ϫ2.96 (m, 1H),
3.30 (d, J ϭ 12.8 Hz, 1H), 3.96 (d, J ϭ 13.1 Hz, 1H), 7.23Ϫ7.31
(m, 5H). 13C NMR (CDCl3) δ: 22.9, 27.9, 44.0, 54.5, 59.0; 65.4,
126.8, 128.2 (2 C, isochrones), 128.7 (2 C, isochrones), 139.8. MS
(EI): m/z 190 (Mϩ).
Figure 2. Determination of intrinsic activity of 2a (29%,
EC50 = 6.7 nM) compared to the full agonist quinpirole by
measuring the incorporation of [3H]thymidine into growing
CHO cell expressing the rat D4.2 receptor.
Garvan Institute of Medical Research, Sydney) for provid-
ing dopamine D4, D3 and D2 receptor expressing cell lines,
respectively. This work was supported by the Fonds der
Chemischen Industrie.
[(R)-1-Benzylpyrrolidin-2-yl]-methylamine
[(R)-1-Benzylpyrrolidin-2-yl]-methylamine was synthesized under
the same reaction conditions starting from (R)-N-benzylproline
ethyl ester; yield: 78.5 mg (55%). [α]2D0: ϩ68.8° (c 1.0, CHCl3).
Experimental
General procedure for the preparation of carboxamides 1 and 2
General
Three equivalents of oxalyl chloride were added to a suspension
of the corresponding acid in abs. toluene (2 mL) under nitrogen
atmosphere. The mixture was heated slowly to 40°C until gas devel-
opment was observed, then stirred for 1 h at RT and finally 4 h at
60°C. Evaporation of the liquid gave a solid, which was dissolved
in abs. CH2Cl2 (2 mL) and subsequently added to a solution of 1.1
equivalents of amine in abs. CH2Cl2 at Ϫ60°C. The reaction mix-
ture was allowed to warm to RT and stirred for 45 min. A saturated
NaHCO3 solution (5 mL) was added, the mixture extracted with
CH2Cl2, the solvent dried (Na2SO4) and evaporated giving a solid,
which was purified by flash chromatography.
MS spectra were recorded on FINNIGAN MAT TSQ 70 spec-
trometer. 1H NMR (360 MHz) and 13C NMR (90 MHz) spectra
were recorded in solution using a BRUCKER AM 360 instrument.
Infrared spectra were registered on a JASCO FT/IR 410 instrument,
using a film of substance on a NaCl pill. Melting points were deter-
mined on a BÜCHI apparatus. CHN elementary analyses were done
at the Department of Organic Chemistry (Friedrich Alexander Uni-
versity). Flash chromatography was performed using Silica Gel 60
(40Ϫ63 μm). For TLC, Merck 60 F254 aluminum plates were used
analyzed by UV light (254nm) or by iodine vapor.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 276−280
Heterocyclic methoxynapththamide analogs for dopamine receptor
279
N-[(S)-1-Benzylpyrrolidin-2-ylmethyl]-pyrazolo[1,5-a]pyridine-3-
m/z 320 (Mϩ). Anal. Calcd. for C19H20N4O ϫ 1/4H2O: C, 70.24;
carboxamide (1a)
H, 6.36; N 17.24; found C, 70.47; H, 6.33; N 17.15.
ent-2a could be synthesized under the reaction conditions described
for 2a; yield:106 mg (90%). [α]2D0: Ϫ12.1° (c 1.0, CHCl3).
The reaction was carried out using 3 (47.0 mg, 0.29 mmol) in tolu-
ene, oxalyl chloride (76 μL, 0.87 mmol) and [(S)-1-benzylpyrroli-
din-2-yl]-methylamine (60.6 mg, 0.32 mmol) in CH2Cl2. The prod-
uct was purified by flash chromatography (CH2Cl2 : MeOH ϭ
N-[(R)-1-Benzylpyrrolidin-3-yl]-pyrazolo[1,5-a]pyridine-2-carb-
oxamide (2b)
90:10) obtaining a white sticky solid. Yield: 86 mg (89%). [α]D20
:
Ϫ119.7° (c 0.4, CHCl3). IR (NaCl) ν: 3328, 2962, 2796, 1639, 1550,
1531, 1272, 748 cmϪ1 1H NMR (CDCl3) δ: 1.67Ϫ1.77 (m, 3H),
.
The reaction was carried out using 4 (53.0 mg, 0.33 mmol) in tolu-
ene, oxalyl chloride (86 μL, 0.99 mmol) and (3R)-(Ϫ)-1-benzyl-3-
aminopyrrolidine (63.5 mg, 0.36 mmol) in CH2Cl2. The product
was purified by flash chromatography (CH2Cl2 : MeOH ϭ 95:5)
1.94Ϫ2.02 (m, 1H), 2.29Ϫ2.37 (m, 1H), 2.86Ϫ2.93 (m, 1H),
3.05Ϫ3.10 (m, 1H), 3.36 (ddd, J ϭ 13.8 Hz, 4.3 Hz, 3.2 Hz, 1H),
3.43 (d, J ϭ 13.1 Hz, 1H), 3.76 (ddd, J ϭ 13.8 Hz, 7.4 Hz, 2.8 Hz,
1H), 4.00 (d, J ϭ 13.1 Hz, 1H), 6.64 (br s, 1H), 6.90 (br dd, J ϭ
7.1 Hz, 6.7 Hz, 1H), 7.23Ϫ7.27 (m, 1H), 7.29Ϫ7.35 (m, 5H), 8.13
(s, 1H), 8.28 (br d, J ϭ 8.9 Hz, 1H), 8.49 (br d, J ϭ 6.7 Hz, 1H).
13C NMR (CDCl3) δ: 22.9, 28.2, 39.9, 54.5, 58.5, 62.5, 106.8, 113.4,
119.5, 126.3, 127.1, 128.2, 128.5, 128.6, 128.8 (2 C, isochrones),
138.4, 140.4, 140.5, 162.6. MS (EI): m/z 334 (Mϩ). Anal. Calcd. for
C20H22N4O ϫ 3/4 H2O: C, 69.04; H, 6.81; N, 16.10; found C, 69.25;
H, 6.79; N, 15.54.
obtaining a white solid. Yield: 87 mg (83%). Mp 101°C. [α]D20
:
Ϫ30.4° (c 1.0, CHCl3). IR (NaCl) ν: 3401, 2919, 2796, 1662, 1546,
1515, 1257, 1025, 744. 1H NMR (CDCl3) δ: 1.74Ϫ1.84 (m, 1H),
2.33Ϫ2.43 (m, 2H), 2.69 (dd, J ϭ 9.6 Hz, 3.5 Hz, 1H,), 2.76 (dd,
J ϭ 9.7 Hz, 6.6 Hz, 1H), 2.83Ϫ2.90 (m, 1H), 3.61Ϫ3.69 (m, 2H),
4.65Ϫ4.75 (m, 1H), 6.84 (br dd, J ϭ 7.1 Hz, 6.7 Hz, 1H), 7.03 (s,
1H), 7.13 (ddd, J ϭ 8.9 Hz, 6.7 Hz, 1.1 Hz, 1H), 7.24Ϫ7.38 (m,
6H), 7.57 (br d, J ϭ 8.9 Hz, 1H), 8.38 (dd, J ϭ 7.1 Hz, 1.1 Hz,
1H). 13C NMR (CDCl3) δ: 32.5, 48.4, 52.7, 60.1, 60.7, 97.9, 113.5,
119.2, 123.6, 127.0, 128.1, 128.2, 128.4, 128.5, 128.7, 138.7, 141.3,
147.9, 161.4. MS (EI): m/z 320 (Mϩ). Anal. Calcd. for C19H20N4O:
C, 71.23; H, 6.29; N, 17.49; found: C, 71.49; H, 6.38; N, 17.49.
ent-1a could be synthesized under the reaction conditions described
for 1a; yield: 83 mg (98%). [α]2D0: ϩ121.0° (c 1, CHCl3).
N-[(S)-1-Benzylpyrrolidin-2-ylmethyl]-pyrazolo[1,5-a]pyridine-2-
carboxamide (1b)
ent-2b could be synthesized under the reaction conditions described
for 2b; yield:113 mg (97%). [α]2D0: ϩ32.8° (c 1.0, CHCl3).
The reaction was carried out using 4 (47.0 mg, 0.29 mmol) in tolu-
ene, oxalyl chloride (76 μL, 0.87 mmol) and [(S)-1-benzylpyrroli-
din-2-yl]-methylamine (60.6 mg, 0.32 mmol) in CH2Cl2. The prod-
uct was purified by flash chromatography (CH2Cl2 : MeOH ϭ 95:5)
Receptor binding experiments and data analysis
Receptor binding studies were carried out as described in literature
[17]. 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]SCH23390 at 0.3 nM
obtaining a white solid. Yield: 88 mg (91%). Mp 98°C. [α]D20
:
Ϫ127.0° (c 1.0, CHCl3). IR (NaCl) ν: 3401, 2962, 2796, 1666, 1546,
1515, 1257, 740 cm-1. 1H NMR (CDCl3) δ: 1.67Ϫ1.77 (m, 3H);
1.93Ϫ2.00 (m, 1H), 2.22Ϫ2.29 (m, 1H), 2.82Ϫ2.87 (m, 1H),
2.98Ϫ3.03 (m, 1H), 3.35Ϫ3.41 (m, 1H), 3.37 (d, J ϭ13.1 Hz, 1H),
3.78 (ddd, J ϭ 13.8 Hz, 7.5 Hz, 3.2 Hz, 1H), 6.85 (br dd, J ϭ 7.1
Hz, 6.7 Hz, 1H), 7.05 (s, 1H), 7.13 (ddd, J ϭ 8.9 Hz, 6.7 Hz, 1.1
Hz, 1H), 7.24Ϫ7.26 (m, 1H), 7.30Ϫ7.35 (m, 2H), 7.40Ϫ7.43 (m,
2H), 7.58 (br d, J ϭ 8.9 Hz, 1H), 7.64 (br s, 1H), 8.42 (br d, J ϭ
7.1 Hz, 1 H). 13C NMR (CDCl3) δ (ppm): 22.8, 28.4, 40.6, 54.3;
58.6, 62.3, 97.7, 113.3, 119.2, 123.5, 126.9, 128.2, 128.3, 128.5,
128.6, 128.8, 139.5, 141.3, 148.1, 162.5. MS (EI): m/z 334 (Mϩ).
Anal. Calcd. for C20H22N4O ϫ 1/5 H2O: C, 71.07; H, 6.68; N, 16.57;
found C, 71.19; H, 6.84; N 16.23.
(Kd ϭ 0.5 nM). Competition experiments with the human D2long
,
D2short, D3 and D4.4 receptors were run with preparations of mem-
branes from CHO cells expressing the corresponding receptor and
[3H]spiperone at a final concentration of 0.5 nM. The assays were
carried out at a protein concentration of 6Ϫ30 μg/assay tube and
K
d values of 0.10 nM for D2long, D2short and D3 and 0.10Ϫ0.13 nM
for D4.4
.
The resulting competition curves were analyzed by nonlinear re-
gression using the algorithms in PRISM (GraphPad Software, San
Diego, USA). The data were fit using a sigmoid model to provide
an IC50 value, representing the concentration corresponding to 50%
of maximal inhibition. IC50 values were transformed to Ki values
according to the equation of Cheng and Prusoff [22].
ent-1b could be synthesized under the reaction conditions described
for 1b; yield: 81 mg (97%). [α]2D0: ϩ127.3° (c 1.0, CHCl3).
Mitogenesis assay
N-[(R)-1-Benzylpyrrolidin-3-yl]-pyrazolo[1,5-a]pyridine-3-carb-
The mitogenesis experiments were done with a CHO10001A cell
line stably transfected with the rat dopamine D4.2 receptor accor-
ding to literature [18]. In brief, cells were grown in MEM
α-medium supplemented with fetal calf serum, L-glutamine, penicil-
lin G, streptomycin and hygromycin B at 37°C under a humidified
atmosphere of 5% CO2Ϫ95% air at a density of 10,000 cells/well.
After 72 h the growth medium was removed and the cells were
rinsed twice with serum free medium. Incubation was started by
adding seven different concentrations of the test compounds (with
a final concentration of 0.01Ϫ10,000 nM) diluted in 10 μL of sterile
water to each well containing 90 μL serum free medium. Eight wells
of every plate contained 100 μL serum free medium or medium
supplemented with 10% fetal calf serum to control stimulation of
growth. After incubation for 20 h, 0.02 μCi [3H]thymidine (specific
activity 25 Ci/mmol) in 10 μL serum free medium was added to
each well for 2 h at 37°C. Finally, cells were trypsinized and har-
oxamide (2a)
The reaction was carried out using 3 (60.6 mg, 0.37 mmol) in tolu-
ene, oxalyl chloride (98 μL, 1.12 mmol) and (3R)-(Ϫ)-1-benzyl-3-
aminopyrrolidine (72.5 mg, 0.41 mmol) in CH2Cl2. The product
was purified by flash chromatography (CH2Cl2 : MeOH ϭ 90:10)
obtaining a white sticky solid. Yield: 99 mg (83%). [α]2D0: ϩ11.8° (c
1.0, CHCl3). IR (NaCl) ν: 3320, 2958, 2796, 1635, 1550, 1531, 1272,
1068, 752 cmϪ1
.
1H NMR (CDCl3) δ: 1.73Ϫ1.81 (m, 1H),
2.29Ϫ2.44 (m, 2H), 2.65 (dd, J ϭ 9.9 Hz, 6.4 Hz, 1H), 2.75 (dd,
J ϭ 9.9 Hz, 2.5 Hz, 1H), 2.91Ϫ2.96 (m, 1H), 3.64 (s, 2H), 4.66Ϫ4.74
(m, 1H), 6.41 (br d, J ϭ 7.8 Hz, 1H), 6.89 (br dd, J ϭ 7.1 Hz, 6.7
Hz, 1H), 7.24Ϫ7.34 (m, 6H), 8.16 (s, 1H), 8.28 (br d, J ϭ 8.9 Hz,
1H), 8.47 (br d, J ϭ 7.1 Hz, 1H). 13C NMR (CDCl3) δ: 32.7, 48.4,
52.7, 60.0, 60.9, 106.8, 113.4, 119.5, 126.3, 127.1, 128.2, 128.5,
128.6, 128.8 (2 C, isochrones), 138.4, 140.5, 140.4, 162.6. MS (EI):
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Gmeiner et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 276−280
vested onto GF/C filters using an automated cell harvester. Filters
were washed four times with ice-cold PBS buffer and counted in a
microplate scintillation counter.
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