WB 4101-related compounds. 2. Role of the ethylene chain separating amine and phenoxy units on the affinity for α1-adrenoreceptor subtypes and 5-HT(1A) receptors

Article abstract of DOI:10.1021/jm991065j

WB 4101 (1)-related benzodioxanes were synthesized by replacing the ethylene chain separating the amine and the phenoxy units of 1 with a cyclopentanol moiety, a feature of 6,7-dihydro-5-[[(cis-2-hydroxy-trans-3- phenoxycyclopentyl)amino]methyl]-2-methylb

Full text of DOI:10.1021/jm991065j

4214
J . Med. Chem. 1999, 42, 4214-4224
WB 4101-Rela ted Com p ou n d s. 2.1 Role of th e Eth ylen e Ch a in Sep a r a tin g Am in e
a n d P h en oxy Un its on th e Affin ity for r1-Ad r en or ecep tor Su btyp es a n d 5-HT1A
Recep tor s
Maria L. Bolognesi,† Roberta Budriesi,† Andrea Cavalli,† Alberto Chiarini,† Roberto Gotti,† Amedeo Leonardi,‡
Anna Minarini,† Elena Poggesi,‡ Maurizio Recanatini,† Michela Rosini,† Vincenzo Tumiatti,† and
Carlo Melchiorre*,†
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, and Research and
Development Division, Recordati S.p.A., Via Civitali 1, 20148 Milano, Italy
Received April 23, 1999
WB 4101 (1)-related benzodioxanes were synthesized by replacing the ethylene chain separating
the amine and the phenoxy units of 1 with a cyclopentanol moiety, a feature of 6,7-dihydro-
5-[[(cis-2-hydroxy-trans-3-phenoxycyclopentyl)amino]methyl]-2-methylbenzo[b]thiophen-4(5H)-
one that was reported to display an intriguing selectivity profile at R1-adrenoreceptors. This
synthesis strategy led to 4 out of 16 possible stereoisomers, which were isolated in the case of
(-)-3, (+)-3, (-)-4, and (+)-4 and whose absolute configuration was assigned using a chiral
building block for the synthesis of (-)-3 starting from (+)-(2R)-2,3-dihydro-1,4-benzodioxine-
2-carboxylic acid ((+)-9) and (1S,2S,5S)-2-amino-5-phenoxycyclopentan-1-ol ((+)-10). The aim
of this project was to further investigate whether it is possible to differentiate between these
compounds with respect to their affinity for R1-adrenoreceptor subtypes and the affinity for
5-HT1A receptors, as 1 binds with high affinity at both receptor systems. The biological profiles
of reported compounds at R1-adrenoreceptor subtypes were assessed by functional experiments
in isolated rat vas deferens (R1A), spleen (R1B), and aorta (R1D) and by binding assays in CHO
and HeLa cells membranes expressing the human cloned R1-adrenoreceptor subtypes and
5-HT1A receptors, respectively. Furthermore, the functional activity of (-)-3, (+)-3, (-)-4, and
(+)-4 toward 5-HT1A receptors was evaluated by determining the induced stimulation of [35S]-
GTPγS binding in cell membranes from HeLa cells transfected with human cloned 5-HT1A
receptors. The configuration of the cyclopentane unit determined the affinity profile: a 1R
configuration, as in (+)-3 and (-)-4, conferred higher affinity at R1-adrenoreceptors, whereas
a 1S configuration, as in (-)-3 and (+)-4, produced higher affinity for 5-HT1A receptors. For
the enantiomers (+)-4 and (-)-4 also a remarkable selectivity was achieved. Functionally, the
stereoisomers displayed a similar R1-selectivity profile, that is R1D > R1B > R1A, which is different
from that exhibited by the reference compound 1. The epimers (-)-3 and (+)-4 proved to be
agonists at the 5-HT1A receptors, with a potency comparable to that of 5-hydroxytryptamine.
In tr od u ction
ity for R1-adrenoreceptor subtypes in comparison to the
prototype WB 4101 (1). It was also our aim to investi-
gate further the possibility to differentiate between the
affinity for R1-adrenoreceptors on the one hand and the
affinity for 5-HT1A receptors on the other, as it is known
that 1 binds with high affinity at both receptors.9 In a
previous project we observed that the structural modi-
fications performed on the benzodioxane moiety as well
as on the N-substituent of 1 resulted in a parallel effect
on the affinity for both R1-adrenoreceptors and 5-HT1A
receptors.10
The finding that receptor systems are comprised of
multiple subtypes has stimulated an intense research
to clarify the structural elements that allow a ligand to
selectively bind to one receptor subtype rather than to
another.2 In the field of R1-adrenoreceptors, it is now
accepted that they can be divided into at least three
subtypes, named R1a (R1A), R1b (R1B), and R1d (R1D), with
upper and lower case subscripts being used to designate
native or recombinant receptors, respectively.3-5 It
should be noted, however, that an additional R1-adreno-
receptor subtype, named R1L, may exist although efforts
to clone it have been unsuccessful so far.6-8 Whereas it
has been claimed that R1A-adrenoreceptor antagonists
can be useful in the treatment of benign prostatic
The starting point for the search described in this
paper was the observation that 6,7-dihydro-5-[[(cis-2-
hydroxy-trans-3-phenoxycyclopentyl)amino]methyl]-2-
methylbenzo[b]thiophen-4(5H)-one (2) displayed an in-
triguing selectivity profile.11 It turned out to be a rather
potent antagonist (pA2 ) 8.13) in blocking the vasocon-
strictor effects of phenylephrine in rabbit aorta, while
not showing any antagonism at R2-adrenoreceptors in
the rat vas deferens. Since it is now clear that rabbit
aorta7 and rat vas deferens12 contain different R1-
adrenoreceptor subtypes, we thought that 2 might
hyperplasia,2 a potential therapeutic use of either R1B
or R1D-subtype antagonists has not been defined yet.
The goal of this project was to discover WB 4101-
related compounds, which display an improved selectiv-
-
† University of Bologna.
‡ Recordati S.p.A.
10.1021/jm991065j CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/23/1999

WB 4101-Related Compounds
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4215
Sch em e 1
F igu r e 1. Design strategy for the synthesis of compounds 3-8
(structure I) by replacing the oxyethylene chain of 1 with the
trisubstituted cyclopentane unit of 2.
represent a lead for the design of related compounds,
which, hopefully, are endowed with high affinity and
selectivity for R1-adrenoreceptor subtypes.
An analysis of Dreiding stereomodels of reference
compounds 1 and 2 reveals that they can be superim-
posed on to each other. It derives that the 6,7-dihydro-
2-methylbenzo[b]thiophen-4(5H)-one unit of 2 can be
replaced by the benzodioxane moiety of 1. To this end,
we have already demonstrated that the two oxygen
atoms at positions 1 and 4 of 1 are not essential for
affinity at R1-adrenoreceptors and can be replaced by a
carbonyl and a methylene, respectively, without affect-
ing the potency.13 Thus we designed structure I (Figure
1), which is a hybrid structure of both prototypes 1 and
2. Since it was demonstrated that the other stereoiso-
mers of 2, having a different relationship among the
substituents on the cyclopentane unit, were at the best
less potent at R1-adrenoreceptors,11 we decided to keep
constant in hybrid structure I and related compounds
the same spatial arrangement as in 2 to possibly achieve
the best fit with R1-adrenoreceptor subtypes. Consider-
ing the fact that the enantiomers of 1 have different
affinity for R1-adrenoreceptors,14,15 it is of interest to
investigate whether the 4 of 16 possible stereoisomers
of structure I derived from the combination of the chiral
moieties of 1 and 2 ((+)-3, (-)-3, (+)-4, and (-)-4) might
be able to better discriminate among R1-adrenoreceptor
subtypes and 5-HT1A receptors.
amine group. 2,3-Dihydro-1,4-benzodioxine-2-carboxylic
acid (9) was amidated with amines 10-12 to give a 1:1
mixture of the corresponding diastereomeric amides 17
and 18, 19 and 20, and 21 and 22, which were separated
by flash chromatography and reduced with borane
methyl sulfide complex to give racemic 3-8 (Scheme 1).
Attempts to separate the enantiomers of racemates
3 and 4 through the formation of diastereomeric mixture
by reaction with N-tosyl-S-proline chloride or (+)-di-
acetyl-L-tartaric anhydride were unsuccessful. Thus we
turned our attention to a different synthetic scheme in
which enantiomers (+)-9 and (-)-9 were used instead
of the racemic (()-9. This different approach gave us
the possibility to obtain, through an amidation reaction,
a mixture of diastereomeric amides (+)-17 and (+)-18
or (-)-17 and (-)-18, which could be easily separated
by chromatography. Although enantiomers (+)-9 and
(-)-9 have been already described,17 we preferred to
obtain them by using an adapted procedure described
for 7-methoxy-2,3-dihydro-1,4-benzodioxine-2-carboxylic
acid18 (Scheme 2). Thus, racemic 9 was treated with
(-)-(R)-dihydro-3-hydroxy-4,4-dimethyl-2(3H)-fura-
none ((-)-(R)-pantolactone) to afford a mixture of dia-
stereomeric esters 23 and 24, which were isolated by
flash chromatography followed by their hydrolysis with
6 N HCl in THF to afford (+)-9 and (-)-9, respectively.
Their optical purity was assessed by capillary electro-
phoresis using a citric acid/Tris base (pH 8.0) buffer con-
taining heptakis(2,6-di-O-methyl)-â-cyclodextrin (DMCD)
as chiral selector; baseline enantioresolution was achieved
with short analysis time (migration time of the first
eluting peak: 11 min). The enantiomericexcess amounted
to 98.2% for (-)-9 and to 98.0% for (+)-9.
Finally, we included in this work compounds 5-8 to
investigate whether the insertion of methoxy groups on
the phenoxy moiety of structure I might affect differ-
ently the affinity for R1-adrenoreceptors and 5-HT1A
receptors.
Ch em istr y
The compounds used in the present investigation were
synthesized by standard procedures and characterized
1
by H NMR and elemental analysis.
The key compounds for the synthesis of stereoisomers
3-8 were 2,3-dihydro-1,4-benzodioxine-2-carboxylic acid
(9) and cis-2-amino-trans-5-phenoxycyclopentan-1-ol (10)
or its methoxy analogues 11 and 12. The latter two
compounds were synthesized by following an adapted
procedure reported for 10 (Scheme 1).16 The opening of
epoxide 13 by appropriate sodium phenoxides in DMF
at 100 °C gave the corresponding cyclopentanols 14-
16, which, upon treatment with 3 N HCl-methanol,
afforded amines 10-12 bearing the aryloxy moiety in
a trans relationship with both the hydroxy and the
Enantiomers (+)-9 and (-)-9 were separately ami-
dated with racemic 10 to give a mixture of diastereo-
meric amides (+)-17 and (+)-18 or (-)-17 and (-)-18,
respectively, which were isolated by flash chromatog-
raphy. These amides were reduced with borane methyl
sulfide complex to afford the corresponding (-)-3, (-)-
4, (+)-3, and (+)-4 (Scheme 2). The enantiomeric excess
determined by capillary electrophoresis employing an

4216 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Bolognesi et al.
Sch em e 2
acidic running buffer (0.1 M phosphoric acid-trietha-
nolamine (pH 3.0) containing 10 mM hydroxypropyl-â-
cyclodextrin (HPCD)) turned out to be 97.6% and 97.8%
for (-)-4 and (+)-4, respectively (Figure 2), and 97.4%
and 98.0% for (-)-3 and (+)-3, respectively.
The absolute configuration of compounds (+)-3, (-)-
3, (+)-4, and (-)-4 was assigned using a chiral building
block for the synthesis of (+)-17 starting from (+)-(2R)-
2,3-dihydro-1,4-benzodioxine-2-carboxylic acid ((+)-9)
and (1S,2S,5S)-2-amino-5-phenoxycyclopentan-1-ol ((+)-
10). The latter was synthesized from (1S)-2-cyclopenten-
1-amine19 (25) following the procedure reported for
racemic 10 (Scheme 3). Acylation of 25 gave N1-[(1S)-
2-cyclopentenyl]acetamide (26). Epoxidation of 26 with
m-chloroperbenzoic acid gave N1-[(1aS,2S,4aR)tetra-
(+)-17, respectively, have a (1R,2R,5R)(2R), (1S,2S,5S)-
(2R), (1R,2R,5R)(2S), and (1S,2S,5S)(2S) configuration.
Biology
F u n ction a l Stu d ies. The pharmacological profile of
racemates 3-8 and enantiomers (+)-3, (-)-3, (+)-4, and
(-)-4 was evaluated at R1-adrenoreceptors on different
isolated tissues using WB 4101 (1) and BMY-7378 as
reference compounds (Figure 3). R1-Adrenoreceptor
subtype blocking activity was assessed by antagonism
of (-)-noradrenaline-induced contraction of rat prostatic
12
20
vas deferens (R1A
)
or thoracic aorta (R1D
)
and by
antagonism of (-)-phenylephrine-induced contraction of
rat spleen (R1B).20 Furthermore, the agonist efficacy of
the enantiomers of 3 and 4 toward 5-HT1A receptors was
assessed by determining the induced stimulation of [35S]-
GTPγS binding in cell membranes from HeLa cells
transfected with human cloned 5-HT1A receptors21 using
8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT),
5-hydroxytryptamine (5-HT), and 5-carboxamidotryp-
tamine (5-CT) as reference compounds (Figure 3).
hydro-1aH-cyclopenta[b]oxiren-2-yl]acetamide
(27),
which, upon treatment with sodium phenoxide, afforded
N1-[(1S,2S,3S)-2-hydroxy-3-phenoxycyclopentyl]aceta-
mide (28). Hydrolysis of 28 gave amine (+)-10, which,
following a reaction with (+)-9, led to (+)-17. This amide
was identical to a sample obtained by separation
through flash chromatography of a diastereomeric mix-
ture of (+)-17 and (+)-18 as described above. Assuming
that reactions proceeded without racemization, it rea-
sonably follows that (+)-17 has a (1S,2S,3S)(2R) con-
figuration and, consequently, (+)-18, which is the other
diastereoisomer obtained through the reaction between
(+)-9 and racemic 10, has a (1R,2R,3R)(2R) configura-
tion. Diastereoisomers (-)-17 and (-)-18, which are the
enantiomers of (+)-17 and (+)-18, respectively, have
a (1R,2R,3R)(2S) and (1S,2S,3S)(2S) configuration.
Finally, it derives that (+)-3, (+)-4, (-)-4, and (-)-3,
which were obtained from (-)-17, (-)-18, (+)-18, and
Bin d in g Exp er im en ts. Receptor subtype selectivity
of selected WB 4101-related benzodioxanes was further
determined by employing receptor binding assays using
WB 4101 (1), BMY-7378, and 8-OH-DPAT as reference
compounds. [3H]Prazosin was used to label cloned
human R1-adrenoreceptors expressed in Chinese ham-
ster ovary (CHO) cells.22 Furthermore, [3H]rauwolscine,
[3H]spiperone, and [3H]ketanserin were used to label R2-
adrenoreceptors in rat cortex,23 D2 receptors in rat
striatum,24 and 5-HT2A receptors in rat cortex,25 respec-
tively, whereas [3H]8-OH-DPAT was the radioligand to

WB 4101-Related Compounds
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4217
F igu r e 3. Chemical structures of the reference compounds
8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 5-hy-
droxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), and
BMY-7378.
Ta ble 1. Antagonist Affinities, Expressed as Apparent pKb
Values, of 1, 3-8, and BMY-7378 at R1-Adrenoreceptors in
Isolated Rat Prostatic Vas Deferens (R1A), Spleen (R1B), and
Thoracic Aorta (R1D
)
a
pKb
R1B
no.b
R
R1A
R1D
1
3
4
5
6
7
8
9.36 ( 0.04 8.21 ( 0.02 8.60 ( 0.02
5.48 ( 0.03 6.45 ( 0.03 7.66 ( 0.04
5.70 ( 0.02 6.47 ( 0.05 7.85 ( 0.01
5.68 ( 0.10 6.24 ( 0.03 7.15 ( 0.01
6.56 ( 0.02 6.80 ( 0.01 7.30 ( 0.03
H
H
F igu r e 2. Analytical capillary electrophoresis enantiosepa-
ration of (-)-4 (a), (+)-4 (b), and racemic mixture (()-4 (c).
Conditions: fused-silica capillary 48.5 cm in length (40 cm
effective length) × 50 mm i.d.; applied voltage 25 kV; detection
wavelength 220 nm; temperature 15 °C; hydrodynamic injec-
tion (pressure 5 kPa) 10 s; running buffer 10 mM hydroxypro-
pyl-â-cyclodextrin in 100 mM phosphoric acid adjusted to pH
3.0.
2-MeO
2-MeO
2,6-(MeO)2 5.89 ( 0.02 6.27 ( 0.01 7.32 ( 0.03
2,6-(MeO)2 5.79 ( 0.09 6.17 ( 0.01 6.94 ( 0.04
6.94 ( 0.02 7.55 ( 0.10 8.34 ( 0.08
BMY-7378
a
Apparent pKb values ( SE were calculated according to
Arunlakshana and Schild35 with the following equation: pKb
)
-log Kb ) log (DR - 1) - log [B]. The log (DR - 1) was calculated
from 2 to 3 different antagonist concentrations, and each concen-
tration [B] of antagonist was tested four times. Dose-ratio (DR)
values represent the ratio of the potency of the agonist (EC50) in
Sch em e 3
b
the presence of the antagonist and in its absence. The relation-
ship among the substituents linked to the cyclopentane ring was
always cis between the hydroxy group at position 1, taken as the
reference atom (1r), and the amine function at position 2 (2c) and
trans between both hydroxy and amine moieties and the phenoxy
group at position 5 (5t).
subtypes, whereas the enantiomers of 3 and 4 proved
to be agonists at 5-HT1A receptors.
An inspection of the results reveals that the inclusion
of the ethylene chain separating the amine and the
phenoxy moieties of 1 into a cyclopentanol unit (Figure
1), affording 3-8, caused a dramatic effect in the affinity
profile for R1-adrenoreceptor subtypes and 5-HT1A re-
ceptors. The two diastereomeric racemates 3 and 4
displayed a similar selectivity profile, which was, how-
ever, remarkably different from that of 1 at R1-adreno-
receptor subtypes. Compounds 3 and 4 displayed a
significant selectivity toward the R1D-adrenoreceptor
label cloned human 5-HT1A receptors which were ex-
pressed in HeLa cells.26, 27
Resu lts a n d Discu ssion
The biological activity, expressed as pKb and pD2
values, at R1-adrenoreceptor subtypes and 5-HT1A re-
ceptors, respectively, of compounds used in the present
study is shown in Tables 1 and 2. Prototype 1 and the
reference compounds BMY-7378, 8-OH-DPAT, 5-HT,
and 5-CT were included for comparison. All the com-
pounds behaved as antagonists at R1-adrenoreceptor
while being weak antagonists for both R1A- and R1B
-
subtypes, though affinity was a bit higher at the latter.

4218 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Bolognesi et al.
Ta ble 2. Antagonist Affinities and Agonist Efficacies, Expressed as pKb and pD2 Values, Respectively, of the Enantiomers of 3 and 4
at R1-Adrenoreceptors in Isolated Rat Prostatic Vas Deferens (R1A), Spleen (R1B), and Thoracic Aorta (R1D) and at 5-HT1A Receptors in
HeLa Cells (binding [35S]GTP) in Comparison to 8-OH-DPAT, 5-HT, 5-CT, and BMY-7378
R1A
R1B
R1D
5-HT1A
ERb
a
a
a
c
no.
(-)-3
config
pKb
ERb
3
pKb
ERb
11
pKb
ERb
7
pD2
% max
82
(1S,2S,5S)(2S)
5.65 ( 0.06
5.79 ( 0.03
6.92 ( 0.01
7.30
27
(+)-3
(-)-4
(1R,2R,5R)(2R)
(1R,2R,5R)(2S)
6.10 ( 0.09
6.55 ( 0.05
6.83 ( 0.06
7.13 ( 0.01
7.78 ( 0.08
8.17 ( 0.08
5.87
5.87
23
100
>35
49
33
123
(+)-4
8-OH-DPAT
5-HT
5-CT
BMY-7378
(1S,2S,5S)(2R)
<5d
5.44 ( 0.01
6.65 ( 0.02
7.96
7.60
7.30
8.45
9.27
96
100
100
96
e
-
-
-
-
-
-
-
-
-
6.94 ( 0.02
7.55 ( 0.10
8.34 ( 0.08
26
a
Apparent pKb values ( SE were calculated according to Arunlakshana and Schild35 with the following equation: pKb ) -log Kb ) log
(DR - 1) - log [B]. The log (DR - 1) was calculated from three different antagonist concentrations, and each concentration [B] of antagonist
was tested four times. Dose-ratio (DR) values represent the ratio of the potency of the agonist (EC50) in the presence of the antagonist
b
and in its absence. The eudismic ratio (ER) is the antilog of the difference between the pKb and pD2 values of the eutomers and the
corresponding distomers. c pD2 values are the negative logarithm of the agonist concentration required to obtain 50% of the maximal
stimulation of [35S]GTPγS binding and were calculated from 2 to 3 experiments, which agreed within (20%. Each experiment was performed
d
in triplicate. Inactive up to a 10 µM concentration. e Not tested.
On the other hand, prototype 1 was about 4500-7500-,
55-58-, and 6-8-fold more potent than 3 and 4 at R1A-,
R
1B-, and R1D-adrenoreceptors, respectively, displaying
selectivity for the R1A-subtype.
Next, we investigated the effect of methoxy groups
on the biological profile of 5-8 as it is known that two
methoxy groups at positions 2 and 6 of the phenoxy unit
confer optimum affinity in prototype 1.28 Surprisingly,
the insertion of one, as in 5 and 6, or two as in 7 and 8,
methoxy groups did not modify the selectivity profile
at R1-adrenoreceptor subtypes. Instead, it caused a
decrease in the affinity for the R1D-subtype relative to
the unsubstituted compounds 3 and 4. This finding
clearly suggests that the methoxy groups play a differ-
ent role in 1 and related cyclopentane-bearing com-
pounds.
To verify whether the stereochemistry of the cyclo-
pentane unit may be important, we investigated the two
couples of enantiomers (-)-3 and (+)-3, and (-)-4 and
(+)-4 at both R1-adrenoreceptor subtypes and 5-HT1A
receptors. It has already been demonstrated that the
2S enantiomer of 1 is significantly more potent than the
2R enantiomer at R1-adrenoreceptors14,15 and at 5-HT1A
receptors as well.9 The results shown in Table 2, though
not in total disagreement with previous finding, deserve
comment. Epimers (+)-3 and (-)-4 have the opposite
configuration at the carbon at position 2 of the benzo-
dioxan unit, namely R and S, respectively, while having
the same configuration at the cyclopentane nucleus.
They are more potent at all R1-adrenoreceptor subtypes
and remarkably less potent at 5-HT1A receptors, as
compared with their corresponding enantiomers (-)-3
and (+)-4. This finding suggests clearly that the ster-
eochemistry of the cyclopentane unit has a greater
influence on affinity than that of the benzodioxane
moiety. Interestingly enough, a 1R configuration, as in
(+)-3 and (-)-4, conferred higher affinity at R1-adreno-
receptors, whereas a 1S configuration produced higher
affinity for 5-HT1A receptors, which indicates that the
F igu r e 4. Affinity constants (pKb) in rat prostatic vas deferens
(R1A), spleen (R1B), and aorta (R1D) R1-adrenoreceptor subtypes
of stereoisomers (-)-3, (+)-3, (-)-4, and (+)-4 in comparison
with BMY-7378.
two receptor systems have different stereochemical
requirements. Enantioselectivity was more pronounced
for enantiomers (-)-4 and (+)-4 than for (-)-3 and (+)-3
at R1-adrenoreceptors and 5-HT1A receptors as well
(Table 2).
It is evident that stereoisomers (-)-3, (+)-3, (-)-4, and
(+)-4 all have, though to a different extent, the same
selectivity profile, that is R1D > R1B > R1A, which is
similar to that displayed by BMY-7378. Among them,
(-)-4 displayed a significant R1D selectivity versus the
other subtypes, which was slightly higher than that of
BMY-7378 as graphically shown in Figure 4. Its selec-
tivity versus the 5-HT1A receptor is more than 2 log
units.
Finally, epimers (-)-3 and (+)-4 proved to be agonists
at 5-HT1A receptors with a potency comparable to or
even higher than that of 5-HT as revealed by their pD2
value (Table 2), while being 27- and 123-fold more
potent than the corresponding enantiomers (+)-3 and
(-)-4. Enantiomers (+)-4 and (-)-4 behaved as full
agonists, whereas (+)-3 and (-)-3 showed to be partial
agonists, in analogy with BMY-7378.
The binding affinities, expressed as pKi values, at
human cloned R1-adrenoreceptor subtypes and 5-HT1A

WB 4101-Related Compounds
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4219
Ta ble 3. Affinity Constants, Expressed as pKi (-log Ki, M), of Compounds 1, 3-8, 8-OH-DPAT, and BMY-7378 for Human
Recombinant R1-Adrenoreceptor Subtypes and 5-HT1A Receptors and for Rat Native R2, D2, and 5-HT2 Receptorsa
pKi, human cloned receptors
pKi, native receptors (rat brain)
no.
R1a
R1b
R1d
5-HT1A
R2
D2
5-HT2
1
9.37
6.41
7.11
7.44
6.02
8.0
5.74
7.03
7.33
<6
-
9.29
6.70
7.79
8.10
6.03
-
8.68
9.08
7.65
8.26
8.43
7.61
7.57
7.30
7.47
9.03
8.46
7.83
6.23
6.27
6.05
6.21
-
6.91
5.62
5.21
5.62
5.95
-
6.0
<6
-
(-)-3
(+)-3
(-)-4
<6
-
(+)-4
b
5
6
7
8
-
-
-
-
-
-
-
-
-
-
-
7.16
<6
-
-
8.84
<6
-
-
5.93
6.08
-
-
7.31
<6
-
-
-
BMY-7378
8-OH-DPAT
6.42
<6
6.31
<6
a
Equilibrium dissociation constants (Ki) were derived from IC50 values using the Cheng-Prusoff equation.37 The affinity estimates
were derived from displacement of [3H]prazosin, [3H]8-hydroxy-2-(di-n-propylamino)tetralin, [3H]rauwolscine, [3H]ketanserin, and
[3H]spiperone binding for R1-adrenoreceptors, 5-HT1A receptors, R2-adrenoreceptors, 5-HT2 receptors, and D2 receptors, respectively. Each
experiment was performed in triplicate. Ki values were from 2 to 3 experiments, which agreed within (20%. Not determined.
b
receptors and at native R2-adrenoreceptors, D2, and
5-HT2 receptors of selected compounds are shown in
Table 3 together with those of prototype 1 and reference
compounds BMY-7378 and 8-OH-DPAT.
With regard to the 5-HT1A receptor, enantiomer (+)-4
proved functionally the most selective, in analogy with
(-)-4 at the R1D subtype.
It can be seen that the insertion of methoxy groups
in the phenoxy moiety, as in 5-8, did not increase the
affinity for 5-HT1A receptors, following the trend ob-
served in functional experiments at R1-adrenoreceptors.
In conclusion, a most intriguing finding of the present
investigation was the observation that the replacement
of the carbon chain separating the amine and the
phenoxy groups of 1 with a cyclopentane ring afforded
stereoisomers (-)-3, (+)-3, (-)-4, and (+)-4 that retained
good affinity for R1-adrenoreceptor subtypes or 5-HT1A
receptors according to the configuration of the cyclo-
pentane unit. In particular for the enantiomers of 4, a
1R configuration imparted high affinity and selectivity
for R1D-adrenoreceptors, whereas the reverse applied for
high affinity and selectivity at 5-HT1A receptors. The
trend noted above might help in developing relevant
structure-activity relationships to differentiate and to
understand the structural elements which confer selec-
tive interaction at R1-adrenoreceptor subtypes and at
the 5-HT1A receptor.
It can be seen that, while binding affinities of refer-
ence compound BMY-7378 are qualitatively and quan-
titatively comparable with pKb values derived from
functional experiments, those observed for (-)-3, (+)-3,
(-)-4, and (+)-4 are not in complete agreement in
particular for the R1a-subtype, where functional affini-
ties were lower. Very recently,29 we discussed the
possibility that if an antagonist does not adhere per-
fectly to the concept of neutral antagonism in the
interaction with the receptor but behaves as an inverse
agonist, then a discrepancy between affinity values
estimated in functional assays and in binding experi-
ments may not represent a surprise, because the
estimated affinities of inverse agonists are system-
dependent. Interestingly, the prototype of the present
study, WB 4101 (1), was shown to be an inverse agonist
in a vascular model containing R1D-adrenoreceptors.30
Thus, the 5-fold difference observed for 1 between
binding and functional affinity at R1d/D-adrenoreceptors
might be explained by the fact that 1 is an inverse
agonist at this subtype and, as a consequence, its
affinity is not system-independent. It derives that a
similar explanation might apply for the discrepancy
observed between binding and functional affinities for
stereoisomers (-)-3, (+)-3, (-)-4, and (+)-4 at R1a-
adrenoreceptors.
A further analysis of the results reveals that stere-
oisomers (-)-3, (+)-3, (-)-4, and (+)-4 displayed a lower
affinity not only at all R1-adrenoreceptor subtypes but
also at R2-adrenoreceptors and D2 receptors as well in
comparison to 1.
Similarly to the trend observed at R1a-adrenorecep-
tors, stereoisomers (-)-3, (+)-3, (-)-4, and (+)-4 dis-
played a radioreceptor binding affinity higher than that
observed in functional assays at 5-HT1A receptors.
Furthermore, the eudismic ratio between (-)-3 and
(+)-3 enantiomers was identical to that observed in
functional assays, whereas for (-)-4 and (+)-4 it was
much lower. However, a difference in potency observed
for the agonists in different assays is not surprising,
since the affinity is system-dependent, as assumed by
theory.
Exp er im en ta l Section
Ch em istr y. Melting points were taken in glass capillary
tubes on a Bu¨chi 530 apparatus and are uncorrected. Electron
impact (EI) mass and 1H NMR spectra were recorded on VG
7070E and Varian VXR 300 instruments, respectively. Chemi-
cal shifts are reported in parts per million (ppm) relative to
tetramethylsilane (TMS), and spin multiplicities are given as
s (singlet), br s (broad singlet), d (doublet), dd (double doublet),
t (triplet), or m (multiplet). Optical activity was measured at
20 °C with a Perkin-Elmer 241 polarimeter. The elemental
compositions of the compounds agreed to within (0.4% of the
calculated value. When the elemental analysis is not included,
crude compounds were used in the next step without further
purification. Chromatographic separations were performed on
silica gel columns (Kieselgel 40, 0.040-0.063 mm; Merck) by
flash chromatography. Petroleum ether refers to the fraction
with a boiling point of 40-60 °C. The term “dried” refers to
the use of anhydrous sodium sulfate. All the electrophoretic
separations were performed on a HP 3D capillary electro-
phoresis system with real-time UV-visible diode array detec-
tion (Hewlett-Packard GmbH, Waldbronn, Germany). The
system was controlled by a HP Vectra 486 PC using the 3DCE-
ChemStation software and macro programs based on estab-
lished algorithms.
N1-[2-cis-Hyd r oxy-3-tr a n s-(2-m eth oxyp h en oxy)cyclo-
p en tyl]a ceta m id e (15). This was synthesized following an

4220 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Bolognesi et al.
adapted procedure described for 14.16 A solution of 2-methoxy-
phenol (5.14 g, 41.4 mmol) in dry DMF (8 mL) was added to a
cooled (5 °C) suspension of 60% NaH in mineral oil (0.86 g,
21.2 mmol) in dry DMF (8 mL) followed, after the gas evolution
was ceased, by the addition of a solution of 1316 (3.0 g, 21.2
mmol) in dry DMF (15 mL). The resulting mixture was heated
at 100 °C for 16 h. After cooling, excess NaH was destroyed
by cautious addition of water. Extraction with EtOAc, followed
by washing with aqueous saturated NaCl solution, drying, and
evaporation of the extracts gave 15: 30% yield; mp 118-120
°C; 1H NMR (CDCl3) δ 1.51-1.69 (m, 1), 1.79-1.96 (m, 1), 2.01
(s, 3), 2.15-2.34 (m, 2), 2.62 (br s, 1, exchangeable with D2O),
3.83 (s, 3), 4.23-4.33 (m, 1), 4.36-4.50 (m, 1), 4.52-4.59 (m,
1), 5.92 (br s, 1, exchangeable with D2O), 6.80-7.00 (m, 4).
N1-[3-tr a n s-(2,6-Dim eth oxyp h en oxy)-2-cis-h yd r oxycy-
clop en tyl]a ceta m id e (16). This was synthesized from 13 (3.0
g, 21.2 mmol) and 2,6-dimethoxyphenol (6.38 g, 41.4 mmol)
following the procedure described for 15: yield 35%; mp 152-
155 °C; 1H NMR (CDCl3) δ 1.52-1.69 (m, 1), 1.81-1.95 (m,
1), 1.98 (s, 3), 2.10-2.32 (m, 2), 2.81 (br s, 1, exchangeable
with D2O), 3.82 (s, 6), 4.20-4.28 (m, 1), 4.30-4.39 (m, 1), 4.41-
4.54 (m, 1), 5.98 (br s, 1, exchangeable with D2O), 6.59 (d, 2),
7.01 (t, 1).
2-cis-Am in o-5-tr a n s-(2-m eth oxyp h en oxy)cyclop en ta n -
1-ol Hyd r och lor id e (11). This was synthesized following an
adapted procedure described for 10.16 A solution of 15 (1.70 g,
6.4 mmol) in MeOH (25 mL) and 3 N HCl (20 mL) was heated
to reflux for 15 h under a stream of dry nitrogen. After cooling
to room temperature, the formed solid was collected and then
dissolved in water. Resulting solution was washed with ether,
made basic with NaOH pellets, and extracted with CHCl3.
Removal of dried solvent gave 11 as the free base, which was
transformed into the hydrochloride salt: yield 56%; mp 190-
191 °C; 1H NMR (DMSO-d6) δ 1.57-1.78 (m, 2), 1.99-2.17 (m,
1), 2.19-2.36 (m, 1), 3.46-3.60 (m, 1), 3.75 (s, 3), 4.10-4.15
(m, 1), 4.56-4.58 (m, 1), 6.04 (br s, 1, exchangeable with D2O),
6.87-7.26 (m, 4), 8.01 (br s, 3, exchangeable with D2O).
The second fraction was 20: 43% yield; 1H NMR (CDCl3) δ
1.62-1.85 (m, 2), 2.15-2.31 (m, 2), 2.99 (br s, 1, exchangeable
with D2O), 3.84 (s, 3), 4.16-4.22 (m, 1), 4.34 (m, 1), 4.43-4.56
(m, 3), 4.66 (dd, 1), 6.86-6.98 (m, 8), 7.09 (d, 1, exchangeable
with D2O).
Dia ster eom er ic N2-[3-tr a n s-(2,6-Dim eth oxyp h en oxy)-
2-cis-h yd r oxycyclop en tyl]-2,3-d ih yd r o-1,4-ben zod ioxin e-
2-ca r boxa m id es (21 a n d 22). These were synthesized from
acid 9 and amine 12 as described for 17 and 18. The first
fraction was 21: 40% yield; mp 194-195 °C; 1H NMR (CDCl3)
δ 1.83-1.98 (m, 2), 2.30-2.43 (m, 2), 3.87 (s, 6), 4.17-4.24
(m, 2), 4.35-4.43 (m, 1), 4.54-4.65 (m, 2), 4.70 (dd, 1), 6.59-
6.62 (m, 2), 6.87-7.06 (m, 5 + 1, tha latter was exchangeable
with D2O).
1
The second fraction was 22 as an oil: 44% yield; H NMR
(CDCl3) δ 1.83-1.92 (m, 2), 2.10-2.36 (m, 2), 2.68 (br s, 1,
exchangeable with D2O), 3.87 (s, 6), 4.09-4.39 (m, 5), 4.57 (m,
1), 5.11 (br s, 1, exchangeable with D2O), 6.58-6.62 (m, 2),
6.83-7.05 (m, 5).
Dia ster eom er ic 2-cis-[(2,3-Dih yd r o-1,4-ben zod ioxin -2-
ylm eth yl)a m in o]-5-tr a n s-p h en oxycyclop en ta n -1-ol Hy-
d r och lor id es (3 a n d 4). A solution of 10 M BH3‚CH3SCH3
(3.6 mL) in dry diglyme (10 mL) was added dropwise at room
temperature to a solution of 17 (0.71 g, 2.0 mmol) in dry
diglyme (20 mL) with stirring under a stream of dry nitrogen.
When the addition was completed, the reaction mixture was
heated at 100 °C for 7 h. After cooling at 0 °C, excess borane
was destroyed by cautious dropwise addition of MeOH (5 mL).
The resulting mixture was left to stand overnight at room
temperature, cooled at 0 °C, treated with HCl gas for 10 min,
and then heated at 120 °C for 3 h. After cooling, the solid was
filtered and washed with ether to give 3: 90% yield; mp 227-
1
230 °C; H NMR (CD3OD) δ 1.79-2.09 (m, 2), 2.12-2.51 (m,
2), 3.31-3.46 (m, 2), 3.79-3.89 (m, 1), 4.01-4.07 (m, 1), 4.30-
4.37 (m, 2), 4.53-4.61 (m, 1), 4.67-4.69 (m, 1), 6.84-6.97 (m,
7), 7.24-7.30 (m, 2); EI MS m/z 341 (M+). Anal. (C20H24ClNO4)
C, H, N.
Diastereomeric 4 was synthesized from 18 following the
procedure described for 3: 80% yield; mp 207-209 °C; 1H NMR
(CD3OD) δ 1.78-2.04 (m, 2), 2.22-2.56 (m, 2), 3.25-3.49 (m,
2), 3.80-3.90 (m, 1), 3.99-4.08 (m, 1), 4.29-4.37 (m, 2), 4.52-
4.65 (m, 1), 4.69-4.71 (m, 1), 6.81-6.98 (m, 7), 7.25-7.32 (m,
2); EI MS m/z 341 (M+). Anal. (C20H24ClNO4) C, H, N.
Dia ster eom er ic 2-cis-[(2,3-Dih yd r o-1,4-ben zod ioxin -2-
ylm eth yl)a m in o]-5-tr a n s-(2-m eth oxyp h en oxy)cyclop en -
ta n -1-ol Hyd r och lor id es (5 a n d 6). These were synthesized
from 19 and 20, respectively, following the procedure described
for 3. Compound 5: 56% yield; mp 230-233 °C; 1H NMR (CD3-
OD) δ 1.82-2.03 (m, 2), 2.26-2.45 (m, 2), 3.30-3.49 (m, 2),
3.80 (s, 3), 3.90-3.98 (m, 1), 4.02-4.09 (m, 1), 4.32-4.37 (m,
2), 4.55-4.63 (m, 1), 4.66 (d, 1), 6.84-6.94 (m, 4), 6.96-7.02
(m, 4). Anal. (C21H26ClNO5) C, H, N.
2-cis-Am in o-5-tr a n s-(2,6-d im eth oxyp h en oxy)cyclop en -
ta n -1-ol Hyd r och lor id e (12). This was synthesized from 16
(1.86 g, 6.4 mmol) following the procedure described for 11:
1
yield 52%; mp 180-181 °C; H NMR (DMSO-d6) δ 1.54-1.77
(m, 2), 1.99-2.17 (m, 2), 3.62-3.75 (m, 1), 3.73 (s, 6), 3.98-
4.01 (m, 1), 4.40-4.45 (m, 1), 6.66 (d, 2), 7.0 (t, 1), 8.0 (br s, 3,
exchangeable with D2O).
Dia ster eom er ic N2-[2-cis-Hyd r oxy-3-tr a n s-p h en oxy-
cyclop e n t yl]-2,3-d ih yd r o-1,4-b e n zod ioxin e -2-ca r b ox-
a m id es (17 a n d 18). Ethyl chlorocarbonate (0.47 mL, 4.96
mmol) was added dropwise to a cooled (5 °C) solution of
racemic 931 (0.89 g, 4.96 mmol) and Et3N (0.69 mL, 4.96 mmol)
in anhydrous dioxane (60 mL) followed, after stirring for 15
min, by the addition of a solution of racemic 1016 (0.96 g, 4.96
mmol) in anhydrous dioxane (40 mL). After standing overnight
at room temperature, the solvent was evaporated to give a
residue, which was purified by chromatography eluting with
cyclohexanes-EtOAc (13:3). The first fraction was 17: 0.71 g
(40% yield); mp 137-138 °C (from EtOAc/cyclohexane); 1H
NMR (CDCl3) δ 1.69-1.85 (m, 2), 2.22-2.40 (m, 2 + 1
exchangeable with D2O), 4.20-4.30 (m, 2), 4.46-4.61 (m, 3),
4.75 (dd, 1), 6.83-7.05 (m, 7 + 1, the latter is exchangeable
with D2O), 7.20-7.31 (m, 2).
1
Compound 6: 60% yield; mp 188-192 °C; H NMR (CD3-
OD) δ 1.85-1.99 (m, 2), 2.31-2.47 (m, 2), 3.30-3.48 (m, 2),
3.80 (s, 3), 3.92-3.98 (m, 1), 4.01-4.07 (m, 1), 4.32-4.36 (m,
2), 4.56-4.63 (m, 1), 4.67 (d, 1), 6.81-6.95 (m, 5), 6.97-6.99
(m, 3); EI MS m/z 371 (M+). Anal. (C21H26ClNO5) C, H, N.
Dia ster eom er ic 2-cis-[(2,3-Dih yd r o-1,4-ben zod ioxin -2-
ylm eth yl)a m in o]-5-tr a n s-(2,6-d im eth oxyp h en oxy)cyclo-
p en ta n -1-ol Hyd r och lor id es (7 a n d 8). These were synthe-
sized from 21 and 22, respectively, following the procedure
1
The second fraction was 18: 0.76 g (43% yield); mp 134-
described for 3. Compound 7: 16% yield; mp 173-175 °C; H
1
135 °C (from EtOAc/cyclohexane); H NMR (CDCl3) δ 1.68-
NMR (CD3OD) δ 1.86-1.97 (m, 2), 2.17-2.40 (m, 2), 3.35-
3.48 (m, 2), 3.82 (s, 6), 4.04-4.13 (m, 2), 4.27-4.30 (m, 1),
4.34-4.40 (m, 1), 4.56-4.65 (m, 2), 6.56-6.59 (d, 2), 6.87-
6.92 (m, 3), 6.99-7.08 (m, 2). Anal. (C22H28ClNO6) C, H, N.
1.85 (m, 2), 2.12-2.33 (m, 2), 2.40 (br s, 1, exchangeable with
D2O), 4.15-4.22 (m, 1), 4.31 (m, 1), 4.41-4.52 (m, 2), 4.54-
4.58 (m, 1), 4.71 (dd, 1), 6.86-7.04 (m, 7 + 1, the latter was
exchangeable with D2O), 7.25-7.32 (m, 2); EI MS m/z 355 (M+).
1
Compound 8: 22% yield; mp 214-216 °C; H NMR (CD3-
OD) δ 1.83-1.98 (m, 2), 2.21-2.48 (m, 2), 3.48-3.72 (m, 2),
3.82 (s, 6), 4.07-4.14 (m, 2), 4.31-4.33 (m, 1), 4.40 (dd, 1),
4.58-4.65 (m, 2), 6.70 (d, 2), 6.95-6.98 (m, 3), 7.0-7.09 (m,
2). Anal. (C22H28ClNO6) C, H, N.
(3R)-4,4-Dim eth yl-2-oxotetr a h yd r o-3-fu r a n yl (2R)-2,3-
Dih yd r o-1,4-ben zod ioxin e-2-ca r boxyla te (23) a n d (3R)-
4,4-Dim eth yl-2-oxotetr ah ydr o-3-fu r an yl (2S)-2,3-Dih ydr o-
1,4-ben zodioxin e-2-car boxylate (24). 1-Ethyl-3-[3-(dimethyl-
Dia ster eom er ic N2-[2-cis-Hydr oxy-3-tr a n s-(2-m eth oxy-
phenoxy)cyclopentyl]-2,3-dihydro-1,4-benzodioxine-2-carbox-
a m id es (19 a n d 20). These were synthesized from acid 9 and
amine 11 as described for 17 and 18. The first fraction was
19: 40% yield; 1H NMR (CDCl3) δ 1.65-1.87 (m, 2), 2.23-
2.35 (m, 2), 3.0 (d, 1, exchangeable with D2O), 3.81 (s, 3), 4.13-
4.21 (m, 2), 4.45-4.55 (m, 3), 4.66 (dd, 1), 6.85-6.98 (m, 8),
7.11 (d, 1, exchangeable with D2O).

WB 4101-Related Compounds
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4221
amino)propyl]carbodiimide hydrochloride (EDCI; 1.89 g, 9.85
mmol) was added to a stirred and cooled (0 °C) solution of
racemic 9 (1.50 g, 9.02 mmol), N,N-(dimethylamino)pyridine
(0.60 g, 4.9 mmol), and (-)-(R)-pantolactone (1.47 g, 11.3
mmol). After stirring 2 h at 0 °C and overnight at room
temperature, the mixture was added with water and the
organic layer was separated and washed with aqueous NaH-
CO3 saturated solution and finally with water. Removal of
dried solvent gave a residue, which was purified by chroma-
tography with methylene chloride as the eluting solvent. The
first fraction was 23: 42% yield; mp 99-101 [R]D +27.2° (c 1,
136-138 °C; Rf 0.25 (EtOAc-cyclohexane, 7:3); [R]D -25.9° (c
0.5, MeOH). The 1H NMR was identical to that of (+)-18.
(-)-(1S,2S,5S)-2-{[(2S)-2,3-Dih yd r o-1,4-ben zod ioxin -2-
ylm eth yl]a m in o}-5-p h en oxycyclop en ta n -1-ol Hyd r och lo-
r id e ((-)-3). This was synthesized from (+)-17 following the
procedure described for racemic 3: 53% yield; mp 228-230
°C (from MeOH/2-PrOH); [R]D -26.2° (c 0.5, MeOH); 1H NMR
(CD3OD) δ 1.81-1.94 (m, 1), 1.96-2.06 (m, 1), 2.23-2.33 (m,
1), 2.37-2.51 (m, 1), 3.35-3.48 (m, 2), 3.81-3.89 (m, 1), 4.03-
4.09 (m, 1), 4.32-4.39 (m, 2), 4.57-4.63 (m, 1), 4.69-4.71 (m,
1), 6.86-6.98 (m, 7), 7.26-7.31 (m, 2); EI MS m/z 341 (M+).
Anal. (C20H24ClNO4) C, H, N.
1
CH2Cl2); H NMR (CDCl3) δ 0.86 (s, 3), 1.10 (s, 3), 4.0 (s, 2),
4.38 (dd, 1), 4.57 (dd, 1), 5.04 (m, 1), 5.39 (s, 1), 6.86-6.94 (m,
3), 7.0-7.04 (m, 1).
(+)-(1R,2R,5R)-2-{[(2R)-2,3-Dih yd r o-1,4-ben zod ioxin -2-
ylm eth yl]a m in o}-5-p h en oxycyclop en ta n -1-ol Hyd r och lo-
r id e ((+)-3). This was synthesized from (-)-17 following the
procedure described for racemic 3: 88% yield; mp 207-210
°C (from MeOH/2-PrOH); [R]D +26.9° (c 0.5, MeOH). The 1H
NMR was identical to that of (-)-3. Anal. (C20H24ClNO4) C,
H, N.
(-)-(1R,2R,5R)-2-{[(2S)-2,3-Dih yd r o-1,4-ben zod ioxin -2-
ylm eth yl]a m in o}-5-p h en oxycyclop en ta n -1-ol Hyd r och lo-
r id e ((-)-4). This was synthesized from (+)-18 following the
procedure described for racemic 3: 68% yield; mp 209-212
°C (from MeOH/2-PrOH); [R]D -59.4° (c 0.5, MeOH); 1H NMR
(CD3OD) δ 1.83-2.01 (m, 2), 2.29-2.37 (m, 1), 2.41-2.52 (m,
1), 3.36-3.47 (m, 2), 3.83-3.90 (m, 1), 4.01-4.07 (m, 1), 4.31-
4.37 (m, 2), 4.57-4.63 (m, 1), 4.70-4.72 (m, 1), 6.82-6.99 (m,
7), 7.27-7.32 (m, 2); EI MS m/z 341 (M+). Anal. (C20H24ClNO4)
C, H, N.
(+)-(1S,2S,5S)-2-{[(2R)-2,3-Dih yd r o-1,4-ben zod ioxin -2-
ylm eth yl]a m in o}-5-p h en oxycyclop en ta n -1-ol Hyd r och lo-
r id e ((+)-4). This was synthesized from (-)-18 following the
procedure described for racemic 3: 73% yield; mp 232-235
°C (from MeOH/2-PrOH); [R]D +60.2° (c 0.5, MeOH). The 1H
NMR was identical to that of (-)-4. Anal. (C20H24ClNO4) C,
H, N.
(-)-N1-[(1S)-2-Cyclop en ten yl]a ceta m id e (26). This was
synthesized from 2519 (0.44 g, 5.3 mmol) following the proce-
dure described for racemic 2632 and purified by chromatogra-
phy. Eluting with petroleum ether-EtOAc (3:7) gave 26: 0.53
g (80% yield); [R]D -92.9° (c 0.5, CHCl3).
The second fraction was 24 as an oil: 1H NMR (CDCl3) δ
1.12 (s, 3), 1.20 (s, 3), 4.05 (s, 2), 4.38 (dd, 1), 4.55 (dd, 1), 5.09
(m, 1), 5.44 (s, 1), 6.88-6.94 (m, 3), 7.01-7.04 (m, 1).
(+)-(2R)-2,3-Dih ydr o-1,4-ben zodioxin e-2-car boxylic Acid
((+)-9). A solution of 23 (1.06 g, 3.62 mmol) and 6 N HCl (32
mL) in THF (24 mL) was heated at 60-65 °C for 2.5 h. After
THF removal under reduced pressure, aqueous solution was
made basic with saturated K2CO3 solution, washed with
methylene chloride to remove nonacidic materials, acidified
with concentrated HCl, and finally extracted with methylene
chloride. Removal of dried solvent gave (+)-9: 75% yield; mp
104-105 °C; [R]D +63° (c 1, MeOH); 1H NMR (CDCl3) δ 4.37-
4.49 (m, 2), 4.90-4.95 (m, 1), 6.89-7.03 (m, 4), 10.33 (br s, 1,
exchangeable with D2O).
(-)-(2S)-2,3-Dih ydr o-1,4-ben zodioxin e-2-car boxylic Acid
((-)-9). This was synthesized from 24 following the procedure
described for (+)-9: 78% yield; mp 105-107 °C; [R]D -63.5°
1
(c 1, MeOH); H NMR (CDCl3) δ 4.37-4.49 (m, 2), 4.90-4.95
(m, 1), 6.89-7.03 (m, 4), 10.33 (br s, 1, exchangeable with D2O).
(+)-N2-[(1S,2S,3S)-2-Hyd r oxy-3-p h en oxycyclop en tyl]-
(2R)-2,3-d ih yd r o-1,4-ben zod ioxin e-2-ca r boxa m id e ((+)-
17) a n d (+)-N2-[(1R,2R,3R)-2-Hyd r oxy-3-p h en oxycyclo-
p e n t yl]-(2R )-2,3-d ih yd r o-1,4-b e n zod ioxin e -2-ca r b ox-
a m id e ((+)-18). These were synthesized from (+)-9 (0.49 g,
2.72 mmol) and racemic 10 (0.96 g, 4.96 mmol) following the
procedure described for racemic 17 and 18 and separated by
chromatography (eluting system: cyclohexanes-ETOAc, 13:
3). The first fraction was (+)-17, which was further purified
by crystallization from EtOAc-cyclohexane: 0.18 g (19%
yield); mp 141-142 °C; Rf 0.36 (EtOAc-cyclohexane, 7:3); [R]D
+54.7 (c 0.5, MeOH); 1H NMR (CDCl3) δ 1.63-1.79 (m, 2), 2.07
(br s, 1, exchangeable with D2O), 2.12-2.34 (m, 2), 4.13 (m,
1), 4.18-4.24 (m, 1), 4.38-4.48 (m, 2), 4.50-4.53 (m, 1), 4.67
(dd, 1), 6.79-6.96 (m, 7 + 1, the latter was exchangeable with
D2O).
The second fraction was (+)-18, which was further purified
by crystallization from EtOAc-cyclohexane: 0.2 g (21% yield);
mp 137-140 °C; Rf 0.25 (EtOAc-cyclohexane, 7:3); [R]D +25.4
(c 0.5, MeOH); 1H NMR (CDCl3) δ 1.67-1.82 (m, 2), 2.13-
2.35 (m, 2), 2.48 (br s, 1, exchangeable with D2O), 4.16-4.22
(m, 1), 4.31 (m, 1), 4.41-4.51 (m, 2), 4.54-4.59 (m, 1), 4.70
(dd, 1), 6.87-7.04 (m, 7 + 1, the latter was exchangeable with
D2O), 7.25-7.31 (m, 2).
(-)-N1-[(1a S,2S,4a R)Tetr a h yd r o-1a H-cyclop en t[b]ox-
ir en -2-yl]a ceta m id e (27). This was synthesized from 26
following the procedure described for racemic 2732 and purified
by chromatography. Eluting with EtOAc-EtOH (9.8:0.2) gave
27: 60% yield; mp 89-91 °C; [R]D -98.0° (c 0.5, CHCl3).
N1-[(1S,2S,3S)-2-Hyd r oxy-3-p h en oxycyclop en tyl]a ce-
ta m id e (28). This was synthesized from 27 following the
procedure described for racemic 1416 and purified by chroma-
tography. Eluting with EtOAc-CHCl3 (1.5:8.5) gave 28: 17%
1
yield; H NMR (CDCl3) δ 1.86-2.07 (m, 2), 2.26 (s, 3), 2.39-
2.61 (m, 2), 3.33 (br s, 1, exchangeable with D2O), 4.47-4.49
(m, 1), 4.60-4.69 (m, 1), 4.82-4.85 (m, 1), 6.35 (br s, 1,
exchangeable with D2O), 7.12-7.21 (m, 3), 7.49-7.54 (m, 2).
(+)-(1S,2S,5S)-2-Am in o-5-ph en oxycyclopen tan -1-ol ((+)-
10). This was synthesized from 28 following the procedure
described for racemic 10: 43% yield; [R]D +16.6 (c 0.5, MeOH).
Ca p illa r y Electr op h or esis. Deter m in a tion of Ster eo-
ch em ica l P u r ity. Fused-silica capillaries (Supelco, Milan,
Italy) 48.5 cm in length (40 cm effective length) × 50 µm i.d.
were used. The applied voltage was held constant at 25 kV,
and the detection wavelength was adjusted to 220 nm. All the
electrophoretic runs were realized at 15 °C; the samples were
introduced hydrodynamically for 10 s (injection pressure 5
kPa). The sample solutions were prepared in water at a
concentration of 0.1 mg/mL and stored at ambient tempera-
ture. All carrier electrolytes were filtered through 0.45-mm
Millex-HV filter units (Millipore, Milford, MA). The capillary
was rinsed (3 min) between runs with the separation electro-
lyte. Chiral analysis of diastereoisomers (+)-3, (-)-3, (+)-4,
and (-)-4 was performed using as running buffer 0.1 M
phosphoric acid solution adjusted to pH 3.0 with triethanola-
mine and containing 10 mM HPCD.33 The enantiomeric purity
control of (+)-9 and (-)-9 was realized using, as running buffer,
Amide (+)-17 was synthesized also starting from (+)-9 and
(+)-10. It was identical to that obtained by a different method
(see above).
(-)-N2-[(1R,2R,3R)-2-Hyd r oxy-3-p h en oxycyclop en tyl]-
(2S)-2,3-d ih yd r o-1,4-ben zod ioxin e-2-ca r boxa m id e ((-)-
17) a n d (+)-N2-[(1S,2S,3S)-2-Hyd r oxy-3-p h en oxycyclo-
p e n t yl]-(2S )-2,3-d ih yd r o-1,4-b e n zod ioxin e -2-ca r b ox-
a m id e ((-)-18). These were synthesized from (-)-9 and
racemic 10 following the procedure described for racemic 17
and 18. The first fraction was (-)-17, which was further
purified by crystallization from EtOAc-cyclohexane: 21%
yield; mp 142-143 °C; Rf 0.36 (EtOAc-cyclohexane, 7:3); [R]D
-55.5° (c 0.5, MeOH). The 1H NMR spectra was identical to
that of (+)-17.
The second fraction was (-)-18, which was further purified
by crystallization from EtOAc-cyclohexane: 20% yield; mp

4222 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Bolognesi et al.
a 25 mM citric acid solution adjusted to pH 8.0 with Tris base
and containing 20 mM DMCD. The quantification limit for
both couples of enantiomers, expressed as the signal-to-noise
ratio of 10, was evaluated by progressive dilution of enanti-
omer solutions (over 98% purity) and it corresponded to 0.2
µg/mL.
Tyrode solution. Strips were secured at one end to Plexiglas
hooks and connected to transducer for monitoring changes in
isometric contraction. After at least a 1-h equilibration period
under an optimal tension of 1 g, cumulative (-)-noradrenaline
concentration-response curves were recorded at 30-min in-
tervals: the first two being discarded and the third one taken
as control. The antagonist was allowed to equilibrate with the
tissue for 30 min before the generation of the fourth cumulative
concentration-response curve to (-)-noradrenaline.
Biology. F u n ction a l An ta gon ism in Isola ted Ra t Tis-
su es. Male Sprague-Dawley rats (Charles River, Italy) were
killed by cervical dislocation under ketamine anesthesia, and
the organs required were isolated, freed from adhering con-
nective tissue, and set up rapidly under a suitable resting
tension in 15-mL organ baths containing physiological salt
solution kept at appropriate temperature (see below) and
aerated with 5% CO2:95% O2 at pH 7.4. Concentration-
response curves were constructed by cumulative addition of
agonist. The concentration of agonist in the organ bath was
increased approximately 5-fold at each step, with each addition
being made only after the response to the previous addition
had attained a maximal level and remained steady. Contrac-
tions were recorded by means of a force displacement trans-
ducer (FT.03 Grass and 7003 Basile) connected to a four-
channel pen recorder (Battaglia-Rangoni KV 380). In addition
parallel experiments in which tissues did not receive any
antagonist were run in order to check any variation in
sensitivity.
Va s Defer en s P r osta tic P or tion . This tissue (from rats
of 200-230 g) was used to assess R1A-adrenergic antagonism.12
Prostatic portions of 2-cm length were mounted under 400-
450 g tension at 37 °C in Tyrode solution of the following
composition (mM): NaCl, 130.0; KCl, 2.0; CaCl2‚2H2O, 1.8;
MgCl2, 0.89; NaHCO3, 25.0; NaH2PO4‚2H2O, 0.42; glucose, 5.6;
desipramine hydrochloride (0.01 µM) was added to prevent the
neuronal uptake of (-)-noradrenaline. The preparations were
equilibrated for 45-60 min. During this time the bathing
solution was changed every 10 min. Concentration-response
curves for isotonic contractions in response to (-)-noradrena-
line were obtained at 30-min intervals: the first one being
discarded and the second one taken as control. The antagonist
was allowed to equilibrate with the tissue for 30 min, then a
new concentration-response curve to the agonist was ob-
tained. (-)-Noradrenaline solutions contained 0.05% Na2S2O5
to prevent oxidation.
Sp leen . This tissue (from rat of 250-300 g) was employed
to determine R1B-adrenoreceptor antagonist potency.20 The
spleen was removed and bisected longitudinally into two strips
which were suspended in tissue baths containing Krebs
solution of the following composition (mM): NaCl, 118.4; KCl,
4.7; CaCl2, 1.9; MgSO4, 1.2; NaHCO3, 25.0; NaH2PO4, 1.2;
glucose, 11.7; desipramine hydrochloride (0.01 µM) and (()-
propranolol hydrochloride (1 µM) were added to prevent the
neuronal uptake of (-)-phenylephrine and to block â-adreno-
receptors, respectively. The spleen strips were placed under 1
g resting tension and equilibrated for 1 h. The cumulative
concentration-response curves to phenylephrine were mea-
sured isometrically and obtained at 30-min intervals, the first
one being discarded and the second one taken as control. The
antagonist was allowed to equilibrate with the tissue for 30
min, and then a new concentration-response curve to the
agonist was constructed.
Aor ta . This tissue (from rats of 250-300 g) was used to
assess R1D-adrenoreceptor antagonist potency.20 Thoracic aorta
was cleaned from extraneous connective tissue and placed in
Krebs solution of the following composition (mM): NaCl, 118.4;
KCl, 4.7; CaCl2, 1.9; MgSO4 1.2; NaHCO3, 25.0; NaH2PO4, 1.2;
glucose, 11.7; desipramine hydrochloride (0.01 µM) and (()-
propranolol hydrochloride (1 µM) were added to prevent the
neuronal uptake of (-)-noradrenaline and to block â-adreno-
receptors, respectively. Two helicoidal strips (15 mm × 3 mm)
were cut from each aorta beginning from the end most
proximal to the heart. The endothelium was removed by
rubbing with filter paper: the absence of acetylcholine-induced
relaxation was taken as an indicator that vessel was denuded
successfully. Vascular strips were then tied with surgical
thread and suspended in a jacketed tissue bath containing
Ra d ioliga n d Bin d in g Assa ys. Na tive Recep tor s. Bind-
ing studies on native R2-adrenoreceptors, 5-HT2A, and D2
receptors were carried out in membranes of rat cerebral cortex
(R2 and 5-HT2A)
23,25 and striatum (D2).24 Male Sprague-Dawley
rats (200-300 g; Charles River, Italy) were killed by cervical
dislocation, and different tissues were excised and immediately
frozen and stored at -70 °C until use. For R2 and 5-HT2A
membrane preparations, cerebral cortex was homogenized (2
× 20 s) in 50 volumes of cold Tris-HCl buffer, pH 7.4, using a
Polytron homogenizer (speed 7). Homogenates were centri-
fuged at 49000g for 10 min, resuspended in 50 volumes of the
same buffer, incubated at 37 °C for 15 min, and centrifuged
and resuspended two more times. The final pellets were
suspended in 100 volumes of Tris-HCl buffer, pH 7.4, contain-
ing 10 µM pargiline and 0.1% ascorbic acid. Membranes were
incubated in a final volume of 1 mL for 30 min at 25 °C with
0.5-1.5 nM [3H]rauwolscine (R2-adrenoreceptors) or for 20 min
at 37 °C with 0.7-1.3 nM [3H]ketanserin (5-HT2A receptors),
in the absence or presence of competing drugs. For D2
membrane preparations, rat striata were homogenized (2 ×
20 s) in 30 volumes of cold Tris-HCl buffer, pH 7.4, using a
Polytron homogenizer (speed 7) and centrifuged at 49000g for
10 min. The final pellets were suspended in 200 volumes of
Tris-HCl incubation buffer containing 10 µM pargiline, 0.1%
ascorbic acid, and the following saline concentrations: NaCl,
120 mM; KCl, 5 mM; CaCl2, 2 mM; MgCl2, 1 mM; then
membranes were incubated for 15 min at 37 °C with 0.2-0.6
nM [3H]spiperone. Nonspecific binding was determined in the
presence of 10 µM phentolamine (R2-adrenoreceptors), 2 µM
ketanserin (5-HT2A receptors), and 1 µM (+)-butaclamol (D2
receptors). The incubation was stopped by addition of ice-cold
Tris-HCl buffer and rapid filtration through 0.2% poly(ethyl-
enimine)-pretreated Whatman GF/B or Schleicher & Schuell
GF52 filters. The filters were then washed with ice-cold buffer,
and the radioactivity retained on the filters was counted by
liquid scintillation spectrometry.
Clon ed Recep tor s. Binding to cloned human R1-adreno-
receptor subtypes was performed in membranes from CHO
(chinese hamster ovary) cells transfected by electroporation
with DNA expressing the gene encoding each R1-adrenorecep-
tor subtype. Cloning and stable expression of the human R1-
adrenoreceptor gene was performed as previously described.22
CHO cell membranes (30 µg of protein) were incubated in 50
mM Tris-HCl, pH 7.4, with 0.1-0.4 nM [3H]prazosin, in a final
volume of 1.02 mL for 30 min at 25 °C, in the absence or
presence of competing drugs (1 pM-10 µM). Nonspecific
binding was determined in the presence of 10 µM phentola-
mine. The incubation was stopped by addition of ice-cold Tris-
HCl buffer and rapid filtration through 0.2% poly(ethylenimine)-
pretreated Whatman GF/B or Schleicher & Schuell GF52
filters. Genomic clone G-21 coding for the human 5-HT1A
receptor was stably transfected in a human cell line (HeLa).26
HeLa cells were grown as monolayers in Dulbecco’s modified
Eagle’s medium (DMEM), supplemented with 10% fetal calf
serum and gentamicin (100 µg/mL), 5% CO2 at 37 °C. Cells
were detached from the growth flask at 95% confluence by a
cell scraper and were lysed in ice-cold Tris 5 mM and EDTA
5 mM buffer (pH 7.4). Homogenates were centrifuged at
40000g for 20 min, and pellets were resuspended in a small
volume of ice-cold Tris (5 mM) and EDTA (5 mM) buffer (pH
7.4) and immediately frozen and stored at -70 °C until use.
On the experimental day, cell membranes were resuspended
in binding buffer: 50 mM Tris-HCl (pH 7.4), 2.5 mM MgCl2,
10 µM pargiline.27 Membranes were incubated in a final
volume of 1 mL for 30 min at 30 °C with 0.7-1.4 nM [3H]8-

WB 4101-Related Compounds
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4223
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Lefkowitz, R. J .; Minneman, K. P.; Molinoff, P. B.; Ruffolo, R.
R., J r.; Trendelenburg, U. IV. International union of pharmacol-
ogy nomenclature of adrenoceptors. Pharmacol. Rev. 1994, 46,
121-136. (b) Hieble, J . P.; Bylund, D. B.; Clarke, D. E.;
Eikenburg, D. C.; Langer, S. Z.; Lefkowitz, R. J .; Minneman, K.
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OH-DPAT, in the absence or presence of competing drugs.
Nonspecific binding was determined in the presence of 10 µM
5-HT. The incubation was stopped by addition of ice-cold Tris-
HCl buffer and rapid filtration through 0.2% poly(ethylen-
imine)-pretreated Whatman GF/B or Schleicher & Schuell
GF52 filters.
Stim u la tion of [35S]GTP γS Bin d in g a t Clon ed 5-HT1A
Recep tor s. The effects of the compounds tested with [35S]-
GTPγS binding were evaluated according to the method of
Stanton and Beer21 with minor modifications. On the experi-
mental day, cell membranes from HeLa cells transfected with
human cloned 5-HT1A receptors, prepared as above-described,
were resuspended in buffer containing 20 mM HEPES, 3 mM
MgSO4, and 120 mM NaCl (pH 7.4). The membranes were
incubated with 30 µM GDP and decreasing concentrations of
test drugs (from 100 µM to 0.1 nM) or decreasing concentra-
tions of 5-HT, from 100 µM to 0.1 nM (reference curve), for 20
min at 30 °C in a final volume of about 0.5 mL. Samples were
then transferred to ice, added with [35S]GTPγS (150-250 pM),
and then incubated for a further 30 min at 30 °C. Nonspecific
binding was determined in the presence of 10 µM GTPγS. The
incubation was stopped by addition of ice-cold HEPES and
rapid filtration on Schleicher & Schuell GF52 filters, using a
Brandel cell harvester. The filters were washed three times
with a total of 5 mL of the same buffer. Radioactivity was
counted by liquid scintillation spectrometry at an efficiency
of >90%. All assays were carried out in triplicate on 2-3
separate sessions.
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Da ta An a lysis. In functional studies responses were ex-
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the agonist concentration-response curve taken as a control.
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pharmacological computer programs.34 pKb values were cal-
culated according to Arunlakshana and Schild35 by the for-
mula: pKb ) log([B]/(DR - 1)), where B is the antagonist
concentration and the dose ratio (DR) is the ratio of the potency
of the agonist (EC50) in the presence of the antagonist and in
its absence. DR values were obtained for 2-3 different
antagonist concentrations, and each concentration was tested
four times.
Binding data were analyzed using the nonlinear curve-
fitting program Allfit.36 Scatchard plots were linear in all
preparations. All pseudo-Hill coefficients (nH) were not sig-
nificantly different from unity (p > 0.05). Equilibrium inhibi-
tion constants (Ki) were derived from the Cheng-Prusoff
equation:37 Ki ) IC50/(1 + L/Kd), where L and Kd are the
concentration and the equilibrium dissociation constant of the
radioligand. pKi values (Table 2) are the mean ( SE of 2-3
separate experiments performed in triplicate.
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