Synthesis of 7-amino-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazole derivatives displaying combined α2-adrenoceptor antagonistic and 5-HT reuptake inhibiting activities

Article abstract of DOI:10.1016/j.bmc.2006.02.043

Following a program searching for dual 5-HT reuptake inhibitors and α₂-adrenoceptor antagonists started at Johnson & Johnson Pharmaceutical Research & Development, we now report on the synthesis of a series of 7-amino-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazole derivatives, some of which proved to be the most potent α₂-adrenoceptor blockers within this chemical class of tricyclic isoxazolines, while keeping potent 5-HT reuptake inhibiting activity.

Full text of DOI:10.1016/j.bmc.2006.02.043

Bioorganic & Medicinal Chemistry 14 (2006) 4361–4372
Synthesis of 7-amino-3a,4-dihydro-3H-[1]benzopyrano
[4,3-c]isoxazole derivatives displaying combined a2-adrenoceptor
antagonistic and 5-HT reuptake inhibiting activities
a,
a
a
a
a
*
´
´
´
´
J. Ignacio Andres, Jesus Alcazar, Jose M. Alonso, Ana I. De Lucas, Laura Iturrino,
Ilse Biesmansb and Anton A. Megensb
aJohnson & Johnson Pharmaceutical Research and Development, Division of Janssen-Cilag,
Medicinal Chemistry Department, Jarama 75, 45007 Toledo, Spain
bJohnson & Johnson Pharmaceutical Research and Development, Division of Janssen Pharmaceutica N.V.,
Turnhoutseweg 30, B2340 Beerse, Belgium
Received 30 September 2005; revised 19 February 2006; accepted 24 February 2006
Available online 15 March 2006
Abstract—Following a program searching for dual 5-HT reuptake inhibitors and a2-adrenoceptor antagonists started at Johnson &
Johnson Pharmaceutical Research & Development, we now report on the synthesis of a series of 7-amino-3a,4-dihydro-3H-[1]ben-
zopyrano[4,3-c]isoxazole derivatives, some of which proved to be the most potent a2-adrenoceptor blockers within this chemical
class of tricyclic isoxazolines, while keeping potent 5-HT reuptake inhibiting activity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
As we have explained in our previous papers,8–10 com-
bining 5-HT reuptake inhibition with a2-adrenoceptor
blockade is expected to be more effective than 5-HT
reuptake inhibition alone, by enhancing monoaminergic
transmission in the brain. Recent studies suggest that
the addition of the a2-antagonist yohimbine to the SSRI
fluoxetine hastens the antidepressant response and, fur-
thermore, there is also a trend suggesting an increased
percentage of responders to the combined treatment at
the end of a 6-week trial.11 As the pharmacological effect
of this approach has not been fully analyzed yet, the
optimal balance of both activities remains unknown.
In our previous papers, we have described the discovery
of a new series of tricyclic isoxazolines displaying this
dual activity.8–10 Amongst the most interesting com-
pounds within that series the methylcinnamyl deriva-
tives 1 and 2 were identified. We now report on the
synthesis and primary pharmacological activity of a ser-
ies of 7-amino-substituted derivatives, of generic formu-
la I, within this chemical class (Fig. 1).
Depression is a severe mental disorder characterized by
exaggerated and pervasive feelings of sadness, loss of
interest, and decreased energy that affect up to 10% of
the population. This disorder is linked with a significant
mortality and its lifetime prevalence is 19%.1,2
The monoaminergic hypothesis for depression assumes
that it is mainly caused by a deficit of monoamines, sero-
tonin (5-HT) and norepinephrine (NE), in corticolimbic
synaptic clefts3 and this hypothesis has been used to ex-
plain the efficacy of existing antidepressant therapies.
Among these available therapies, selective serotonin
reuptake inhibitors (SSRIs) have become the standard
treatment for depression. However, there are some lim-
itations associated with the use of SSRIs including a de-
layed onset of action (2–4 weeks), partial treatment
response (60–70%), excitation during early treatment
response, nausea, and sexual dysfunction.4–7
2. Chemistry
Keywords: Isoxazolines; 5-HT reuptake inhibitors; a2-Antagonists;
Antidepressants.
*
In order to synthesize the 7-amino-3a,4-dihydro-3H-
[1]benzopyrano[4,3-c]isoxazole derivatives, the scaffolds
Corresponding author. Tel.: +34 925245780; fax: +34 925245771;
e-mail: iandres@prdes.jnj.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.043

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J. I. Andres et al. / Bioorg. Med. Chem. 14 (2006) 4361–4372
4362
nucleophilic character of primary amines. Preparation
of 8 was carried out in two steps for the parallel syn-
thesis of amides and ureas at position 7. Palladium-
catalyzed amination of 6b with tert-butyl carbamate
in the presence of palladium(II) acetate/Xantphos as
catalytic complex, using 1,4-dioxane as solvent and
cesium carbonate as base,13 followed by deprotection
of the tert-butoxycarbonyl group using a mixture of
trifluoroacetic acid in dichloromethane, afforded 8 in
91% overall yield. Acylation of this compound with
different acyl chlorides or reaction with the corre-
sponding isocyanates, under standard reaction condi-
tions, yielded the desired final products 9a–m.
Finally, due to the low nucleophilic character of aro-
matic amines, palladium-catalyzed coupling reactions
were also used for the introduction of such moieties.
In this case, parallel synthesis of compounds 10a–f
was carried out with palladium(II) acetate/BINAP as
catalyst and toluene as solvent.
Figure 1.
6a,b bearing fluorine or bromine atoms suitable for fur-
ther derivation were prepared (Scheme 1). The ester
intermediates 4a,b were obtained following essentially
the method that we previously described for the synthe-
sis of similar analogues.10 Thus, alkylation of 3a,b12
with ethyl 4-bromocrotonate followed by reaction with
hydroxylamine gave the corresponding oximes, from
which nitrile oxides were prepared using sodium hypo-
chlorite. Subsequent ring closure by intramolecular
1,3-dipolar cycloaddition, in the presence of triethyl-
amine, afforded cycloadducts 4a,b. The stereochemistry
of positions 3 and 3a of the tricyclic system was pre-de-
termined by the trans-alkene fragment and was unequiv-
ocally assigned by NMR. This assignment was based on
their respective 1H-coupling constants in compounds
4a,b (see Section 5) as well as on the NOE observed be-
tween the proton at position 3a and the methylene group
of the side chain at position 3, in compounds 5a and 5b.
Reduction of the ester with sodium borohydride in a
mixture of tetrahydrofuran and water yielded the corre-
sponding hydroxymethyl derivatives, which were con-
verted into the mesylates 5a,b by standard procedures.
Reaction of these mesylates with excess N-(2-methyl-3-
phenyl-2(E)-propen-1-yl)piperazine afforded the key
intermediates 6a,b.
Following the procedures described above, different
nitrogenated functions were introduced at position 7
of the 3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazole
scaffold: alkyl and arylamines, alkyl and arylamides,
and alkyl and arylureas.
3. Results and discussion
Results of the primary pharmacological evaluation of
selected compounds are reported in Table 1. In vitro
binding affinities at a2A- and a2C-adrenoceptors, and
at 5-HT transporter (5-HTT) site were considered as
the primary screening assays. Their affinities for other
serotonergic, adrenergic, and dopaminergic receptors,
as well as at the dopaminergic and noradrenergic uptake
sites, were evaluated as well. None of those compounds
showed relevant affinity for any other of those receptors
or transporter sites. Antagonism of medetomidine-in-
duced loss of righting and antagonism of p-chloroam-
phetamine (pCA)-induced excitation, in rats, were used
as primary assays for their in vivo evaluation as a2-adre-
noceptor blockers and 5-HT reuptake inhibitors,
respectively.10
The synthesis of the 7-nitrogenated derivatives 7–10
was accomplished from compounds 6a,b (Scheme 2).
Aromatic nucleophilic substitution of the fluorine
atom in compound 6a, by reaction with secondary
amines in pyridine or with neat primary amines, gave
compounds 7a–f in moderate yields. Potassium
fluoride was used in order to enhance the lower
Scheme 1. Reagents and conditions: (i) Ethyl 4-bromocrotonate, K2CO3, DMF, 0 ꢁC to rt, 5 h; (ii) NH2OHÆHCl, AcONa, EtOH, 0 ꢁC, 2 h; (iii) (a)
NaClO, CH2Cl2, 0 ꢁC to rt, 2 h; (b) Et3N, rt, 24 h; (iv) NaBH4, THF, H2O, 0 ꢁC to rt, 24 h; (v) CH3SO2Cl, Et3N, CH2Cl2, 0 ꢁC, 1 h; (vi) KI, K2CO3,
MIK, reflux, 24 h.

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J. I. Andres et al. / Bioorg. Med. Chem. 14 (2006) 4361–4372
4363
Scheme 2. Reagents and conditions: (i) HNR1R2, pyridine, 150 ꢁC, 24–48 h; (ii) H2NR1, KF, 150 ꢁC, 4–5 days; (iii) (a) H2N-BOC, Pd(OAc)2,
Xantphos, Cs2CO3, 1,4-dioxane, 100 ꢁC, 24 h; (b) TFA, CH2Cl2, rt, 5 h; (iv) RCOCl, Et3N, CHCl3, 0 ꢁC to rt, 24 h; (v) RNCO (R = Ar), THF, rt,
24 h or RNCO (R = alk.), toluene, 75 ꢁC, 24 h; (vi) ArNH2, Pd(OAc)2, BINAP, Cs2CO3, toluene, 100 ꢁC, 24 h.
Most of the compounds showed nanomolar or sub-
nanomolar affinities for the a2-adrenoceptors and
nanomolar affinity for the 5-HTT site. Compound 7a
and its direct O-analogue previously described 28
showed similar in vitro potency. The N-ethyl analogue
7b presented comparable affinity but the two benzyl
derivatives 7c and 7d proved to decrease binding
potency. The presence of a heteroatom, either oxygen
or nitrogen, in the alkyl chain afforded very potent
derivatives such as 7e and 7f. We observed these same
features in the 7-O-alkyl series described in our previ-
ous article.10 As a matter of fact compound 7f was the
most potent dual-acting compound in vitro identified
within this series. The unsubstituted amino analogue
8 was 2-fold less potent than 7a at the 5-HTT site,
although the affinity for a2 receptors of both deriva-
tives was comparable. N-Acyl as well as N-carbamoyl
derivatives showed also quite potent binding affinities
for the three targets. Thus, N-acyl compounds 9a–f
showed subnanomolar potency at a2-adrenoceptors
and affinities at 5-HTT ranging from 3.5 to 19 nM.
Interestingly, the aliphatic or aromatic nature of the
substituents did not have much influence on potency.
Alkylurea derivatives 9g–j maintained high binding
affinities, but arylureas such as 9k and 9l showed a
dramatic drop in activity at the 5-HTT site. Quite sur-
prisingly, introduction of two methoxy groups in the
aromatic ring resulted in high potency at the 5-HTT
site again, as can be seen from the in vitro results
of compound 9m. Finally, arylamino derivatives
10a–f showed lower affinities at both a2-adrenoceptors
as well as at the 5-HTT site. The only exception was
the 3-cyano derivative 10d that also showed activity in
the nanomolar range.
Although most of the synthesized compounds showed
nanomolar or subnanomolar binding values, there was
not a clear correlation with their respective in vivo
activities. The N-methyl derivative 7a was significantly
potent in the medetomidine-induced loss of righting as
well as in the pCA-induced excitation assays, at lower
doses than its O-substituted analogue 2. The N-ethyl
derivative 7b maintained the in vivo potency as a2 block-
er but its activity as 5-HT reuptake inhibitor decreased,
although the in vitro affinities for the 5-HTT of both
compounds were similar. The in vivo activity of the
benzyl derivative 7c also dropped very significantly.
The introduction of a terminal hydroxy function in the
alkyl chain, such as in 7e, showed a good correlation
between in vivo activity and receptor binding values.
This derivative was the most potent compound in the
medetomidine assay and one of the most potent
compounds in the pCA assay. The replacement of the
hydroxy function by a dimethylamino group, exempli-
fied by compound 7f, showed also an interesting combi-
nation of activities, affording one of the most potent
derivatives in the medetomidine test and the most potent
one in the pCA test together with 7a. Interestingly,
although 7e and 7f showed comparable binding values
for both subtypes of a2 receptors, 7f was 15-fold less
active than 7e in the medetomidine test.
The aniline derivative 8 showed also in vivo activity as
an a2 antagonist but its activity as a 5-HT reuptake
inhibitor decreased dramatically, what was in correla-
tion with its lower affinity for the 5-HTT site. Acylation
of the nitrogen with small acyl groups, such as in 9a–d,
afforded derivatives showing comparable activities to 8

Table 1. In vitro binding affinities of 7-amino-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazole derivatives and their in vivo activities for antagonism of medetomidine-induced loss of righting and pCA-
induced excitation
N
O
N
N
R2
N
O
R1
5-HTTa Ki (nM)
Medetomidine (sc)b ED50 (mg/kg)
pCA (sc)b ED50 (mg/kg)
a
a
Compound
R1
R2
a2A Ki (nM)
a2C Ki (nM)
1
2
—
—
H
—
—
0.8
0.5
0.2
0.2
2.3
19
1.0
2.5
1.5
>2.5
1.3 (0.64–2.5)
>2.5
>2.5
nt
7a
7b
7c
7d
7e
7f
CH3
CH3CH2
0.66
1.4
0.27
0.11
0.68
2.9
17
14
0.32 (0.16–0.62)
0.32 (0.16–0.62)
2.5 (0.96–6.5)
H
H
C6H5CH2
C6H5CH2
3.4
9.3
16
45
CH3
CH3
CH3
H
nt
0.04 (0.03–0.06)
HOCH2CH2
(CH3)2NCH2CH2
H
0.31
0.27
0.66
0.68
0.77
0.39
0.36
1.3
0.10
0.09
0.39
0.24
0.10
0.08
0.06
0.31
0.12
0.12
0.16
0.54
0.13
1.3
8.5
1.6
34
1.8 (0.68–4.6)
1.3 (0.64–2.5)
>2.5
>2.5
>2.5
nt
0.63 (0.24–1.6)
0.16 (0.11–0.22)
8
9a
9b
9c
9d
9e
9f
H
CH3CO
(CH3)3CCO
C3H5CO
19
0.63 (0.24–1.6)
0.32 (0.16–0.62)
H
5.0
5.5
8.7
8.7
3.5
3.3
5.6
11
H
1.3 (0.35–4.5)
0.63 (0.24–1.6)
nt
H
CH2@CHCO
C6H5CO
>2.5
nt
H
H
3-Pyridoyl
Et-NHCO
(CH3)3CNHCO
C6H11NHCO
C2H5O(CO)CH2NHCO
C6H5NHCO
3-F-C6H4NHCO
3,4-Dimethoxy-C6H4NHCO
Ph
0.48
2.0
P2.5
nt
>2.5
nt
9g
9h
9i
H
H
2.0
6.2
P2.5
>2.5
>2.5
nt
>2.5
>2.5
>2.5
nt
H
9j
H
1.9
3.0
9.7
72
9k
9l
H
H
20
2.7
>100
11
16
>2.5
>2.5
P2.5
>2.5
>2.5
P2.5
>2.5
nt
>2.5
>2.5
>2.5
>2.5
>2.5
>2.5
>2.5
nt
9m
10a
10b
10c
10d
10e
10f
H
3.7
3.4
0.44
0.94
2.2
H
CH3
H
Ph
4-F-C6H4
5.5
3.8
50
37
1.5
1.6
H
3-CN-C6H4
2-Me-C6H4
3,4-DiMe-C6H4
3.7
5.0
5.0
27
H
1.9
3.6
H
15
97
nt, not tested.
a The activity of compounds was confirmed in an independent experiment. A difference in pIC50 up to 0.6 (SD < 0.5) was considered as reproducible and therefore accepted. SEM values were <0.4.
b ED50 values and corresponding 95% confidence limits were determined according to the modified Spearman–Kaerber estimate using theoretical probabilities instead of empirical ones. This modification
allows one to tabulate the ED50 and its confidence interval as a function of the slope of the log dose–response curve.

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4365
Table 2. In vitro binding affinities and in vivo activities for antagonism of medetomidine-induced loss of righting and pCA-induced excitation of
compound 7e and its enantiomers
5-HTTa Ki (nM)
Medetomidine (sc), ED50 (mg/kg)b
pCA (sc), ED50 (mg/kg)b
a
a
Compound
a2A Ki (nM)
a2C Ki (nM)
7e
(ꢀ)-7e
(+)-7e
0.31
150
0.10
3.5
8.5
3.7
21
0.04 (0.03–0.06)
>2.5
0.63 (0.24–1.6)
1.8 (0.68–4.6)
0.32 (0.16–0.62)
>2.5
0.43
0.03
a The activity of compounds was confirmed in an independent experiment. A difference in pIC50 up to 0.6 (SD < 0.5) was considered as reproducible
and therefore accepted. SEM values were <0.4.
b ED50 values and corresponding 95% confidence limits were determined according to the modified Spearman–Kaerber estimate using theoretical
probabilities instead of empirical ones. This modification allows one to tabulate the ED50 and its confidence interval as a function of the slope of the
log dose–response curve.
in both in vivo assays. In the case of arylamides, such as
derivative 9f, a drastic decrease in in vivo potency was
observed, although binding affinities were in the nano-
molar range. Finally, ureas 9g–m and arylamino deriva-
tives 10a–f did not show significant in vivo activity in
any of both assays.
compounds with different a2/5-HTT balance is in pro-
gress and results will be reported elsewhere.
5. Experimental
5.1. Chemistry
In view of the results showed above we decided to
resynthesize the hydroxyethylamino derivative 7e, in
order to isolate and evaluate its two enantiomers. Sur-
prisingly and in contrast to what we had previously ob-
served with the 7-O-substituted derivatives,10 both
enantiomers showed dissociation of activities, as can
be deduced from data gathered in Table 2. Thus, com-
pound(ꢀ)- 7e kept the same range of affinity for the 5-
HTT as its parent racemate, whereas its affinity for the
a2-adrenoceptors dropped dramatically, especially for
the a2A subtype. On the contrary, the other enantiomer
(+)-7e retained the potency as a2 blocker while showing
a 3-fold decrease in potency at the 5-HTT site. These
findings were confirmed in the in vivo assays. As can
be seen in Table 2, compound (ꢀ)-7e was not active
at 2.5 mg/kg in the medetomidine test but was even
more potent than its parent racemate in the pCA test.
On the other hand, compound (+)-7e was active in the
medetomidine test, although less potent than the race-
mate, whereas it did not show activity at 2.5 mg/kg in
the pCA assay.
Melting points were determined in open capillary tubes
on a Mettler FP62 apparatus and are uncorrected.
Elemental analyses are within 0.4% of the theoretical
values. Chiral preparative chromatography was per-
formed on a Waters Delta Prep 4000 with a 5 cm id Pro-
chrom D.A.C. column. The enantiomeric excess was
determined by HPLC using a Waters Alliance 2690
instrument with chiral columns (Chiralcel OD, Chiralcel
OJ, Chiralpak AD, and Chiralpak AS, Daicel 10 lm).
Optical rotations were measured on a Perkin-Elmer
341 polarꢂimeter with a sodium lamp and reported as fol-
t
C
lows: ½aꢁk (c g/100 mL, solvent). 1H NMR spectra were
recorded on a Bruker DPX-400 and on a Bruker AC-
200 spectrometer with standard pulse sequences, operat-
ing at 400 and 200 MHz, respectively. Chemical shifts
(d) are reported in parts per million (ppm) downfield
from tetramethylsilane (TMS), which was used as inter-
nal standard. HPLC-MS analyses were done with an
Agilent Technologies 1100 series consisting of a quater-
nary pump with degasser, autosampler, column oven,
and DAD detector. A generic gradient: 80:10:10
AcONH4 0.05%/MeOH/CH3CN to 50:50 CH3CN/
MeOH in 6 min to 100% CH3CN in 1.5 min was per-
formed on a Zorbax XDB C-18 30 · 4.6 mm id 3.5 lm
from Agilent Technologies. Low-resolution mass spec-
tra were obtained on an HPLC-MS system, an Agi-
lent-Micromass HP1100 Platform spectrometer with
electrospray ionization (ES). High-resolution mass spec-
tra were recorded on an Agilent-Micromass LCT Time-
of-Flight mass spectrometer with electrospray ionization
and Lockmass device for mass calibration. Thin-layer
chromatography (TLC) was carried out on silica gel
60 F254 plates (Merck) using reagent grade solvents.
Flash chromatography was performed on silica gel, par-
ticle size 60 , mesh = 230–400 (Merck).
4. Conclusions
In conclusion, the introduction of a nitrogen atom at
position 7 of the 3a,4-dihydro-3H-[1]benzopyrano-
[4,3-c]isoxazole scaffold was achieved. Diverse substitu-
tion patterns and functionalities have been introduced,
in order to establish a SAR and to get a more complete
picture of this modification. All the compounds synthe-
sized showed higher affinity for the a2 receptor subtypes,
especially for the a2C than for the 5-HTT site. From this
exploration alkylamino derivatives proved to be the
most interesting compounds according to their in vitro
and in vivo potencies, especially 7a, 7e, and 7f, which
showed different levels of activity as a2-adrenoceptor
blockers and 5-HT reuptake inhibitors. The enantiomers
of 7e were isolated and evaluated proving that in this
case, in contrast to the 7-O-substituted analogues, a2
antagonistic activity and 5-HTT inhibitory activity
were dissociated. Further work trying to understand
these findings and to evaluate the potential of these
5.1.1.
Ethyl
7-bromo-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazole-3-carboxylate (4b). A mixture of 5-
bromosalicylaldehyde12 3b (3 g, 14.9 mmol), K2CO3
(4.12 g, 29.8 mmol), and (E)-ethyl 4-bromocrotonate
(3 mL, 22.3 mmol) in anhydrous dimethylformamide
(17 mL) was stirred at room temperature for 4 h.

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J. I. Andres et al. / Bioorg. Med. Chem. 14 (2006) 4361–4372
4366
When the TLC analysis showed the disappearance of
starting material, the crude reaction mixture was filtered
through a CELITE pad and the filtrate was concentrat-
ed in vacuo. The residue was diluted with water (17 mL)
and extracted with dichloromethane (3· 17 mL). The
organic phase was dried and concentrated in vacuo.
The residue was precipitated with diisopropylether
affording 3.11 g (61% yield) of ethyl 4-(2-formyl-5-
bromophenoxy)but-2(E)-enoate. To a solution of previ-
ously prepared ester (3.11 g, 9.9 mmol) in absolute etha-
nol (25 mL), hydroxylamine hydrochloride (0.83 g,
11.9 mmol) and sodium acetate (1.22 g, 14.8 mmol) were
added. After 2 h at room temperature, the TLC analysis
showed the absence of starting material. The solvent was
evaporated in vacuo, and the residue was dissolved in
water (20 mL) and extracted with dichloromethane (3·
30 mL). The organic layer was dried (Na2SO4) and
concentrated at reduced pressure to yield 3.95 g (quanti-
tative yield) of ethyl 4-[2-(hydroxyiminomethyl)-5-
bromophenoxy]but-2(E)-enoate used in the next
reaction step without further purification. To a solution
of previously synthesized oxime (3.8 g, 11.6 mmol) in
dichloromethane (47 mL), 4% aqueous solution of sodi-
um hypochlorite (40 mL, 23.1 mmol) was added por-
tionwise and the reaction mixture was stirred for 2 h
at room temperature. Then, triethylamine (2.4 mL,
17.4 mmol) was added dropwise at 0 ꢁC. The reaction
mixture was stirred overnight at room temperature.
The organic layer was separated, dried over anhydrous
Na2SO4, and filtered, and the solvent evaporated. The
residue was purified by column chromatography
(dichloromethane), affording 1.71 g of 4b as an oil.
4.72 (m, 2H), 4.76 (dd, J = 10.5 and 5.9 Hz, 1H), 7.24
(dd, J = 8.3 and 1.9 Hz, 1H), 7.30 (d, J = 1.9 Hz, 1H),
7.60 (d, J = 8.3 Hz, 1H). MS m/z 349 (MH+).
5.1.3. 7-Bromo-3-[4-(2-methyl-3-phenyl-2(E)-propen-1-
yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazole (6b). To a solution of 5b (17 g,
46.9 mmol) and N-methylcinnamylpiperazine (15.23 g,
70.4 mmol) in methylisobutylketone (220 mL) were
added K2CO3 (6.48 g, 46.9 mmol) and KI (7.79 g,
46.9 mmol). This mixture was refluxed for 24 h. Inorganic
salts were then filtered out, and the solvent was removed
under reduced pressure. The resulting oil was dissolved
in dichloromethane, washed several times with water,
and dried (Na2SO4). The dichloromethane was removed
under reduced pressure. The residue was purified by col-
umn chromatography (dichloromethane/methanol 99:1)
and precipitated with diisopropylether affording 10.4 g
of compound 6b as a foam. Yield: 46%. 1H NMR
(400 MHz, CDCl3) d ppm 1.90 (d, J = 1.2 Hz, 3H), 2.47
(br s, 4H), 2.61 (br s, 4H), 2.82 (dd, J = 13.2 and
5.60 Hz, 1H), 2.89 (dd, J = 13.0 and 6.0 Hz, 1H), 3.01
(s, 2H), 3.68 (dt, J = 12.4 and 5.8 Hz, 1H), 4.10 (dd,
J = 12.5 and 10.5 Hz, 1H), 4.40–4.48 (m, 1H), 4.65 (dd,
J = 10.4 and 6.0 Hz, 1H), 6.42 (s, 1H), 7.14–7.18 (m,
2H), 7.17–7.23 (m, 1H), 7.24–7.36 (m, 4H), 7.63 (d,
J = 8.7 Hz, 1H). MS m/z 483 (MH+).
The following compound was prepared analogously
starting from 5-fluorosalicylaldehyde 3a:
5.1.4. 7-Fluor-3-[4-(2-methyl-3-phenyl-2(E)-propen-1-
yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyr-
1
Yield: 45%. H NMR (400 MHz, CDCl3) d ppm 1.36
1
ano[4,3-c]isoxazole (6a). Overall yield: 12%. H NMR
(t, J = 7.1 Hz, 3H), 4.06 (dt, J = 12.2 and 5.3 Hz, 1H),
4.16 (dd, J = 12.6 and 10.2 Hz, 1H), 4.34 (q,
J = 7.3 Hz, 2H), 4.73 (d, J = 11.8 Hz, 1H), 4.75 (dd,
J = 9.1 and 5.2 Hz, 1H), 7.14–7.18 (m, 2H), 7.63 (d,
J = 8.7 Hz, 1H). MS m/z 327 (MH+).
(400 MHz, CDCl3) d ppm 1.90 (d, J = 1.2 Hz, 3H),
2.47 (br s, 4H), 2.61 (br s, 4H), 2.82 (dd, J = 13.2 and
5.60 Hz, 1H), 2.89 (dd, J = 13.0 and 6.0 Hz, 1H), 3.01
(s, 2H), 3.68 (dt, J = 12.4 and 5.8 Hz, 1H), 4.10 (dd,
J = 12.5 and 10.5 Hz, 1H), 4.40–4.48 (m, 1H), 4.65
(dd, J = 10.4 and 6.0 Hz, 1H), 6.42 (s, 1H), 6.66 (dd,
J = 9.9 and 2.5 Hz, 1H), 6.73 (dt, J = 8.5 and 2.5 Hz,
1H), 7.17–7.23 (m, 1H), 7.24–7.36 (m, 4H), 7.76 (dd,
J = 8.7 and 6.4 Hz, 1H). MS m/z 422 (MH+).
5.1.2.
Ethyl
7-bromo-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazol-3-yl methanesulfonate (5b). To a solu-
tion of 4b (3.3 g, 10.1 mmol) in THF (77 mL) and water
(7 mL) stirred at room temperature, sodium borohy-
dride (0.96 g, 25.3 mmol) was added portionwise and
the reaction mixture was stirred for 24 h at room tem-
perature. Then, a saturated solution of ammonium chlo-
ride (20 mL) was added and the mixture was extracted
with dichloromethane. The organic layer was separated,
dried (Na2SO4), and filtered, and the solvent evaporat-
ed, affording 3.01 g of crude alcohol, which was used
as such without further purification. To a solution of
previously prepared alcohol (3.01 g, 10.1 mmol) and tri-
ethylamine (2.1 mL, 15.1 mmol) in dichloromethane
(45 mL) stirred at 0 ꢁC under nitrogen atmosphere,
methanesulfonyl chloride (0.86 mL, 11.1 mmol) was
added dropwise. The reaction mixture was stirred at
0 ꢁC for 2 h. Then, saturated solution of sodium bicar-
bonate (40 mL) was added. The organic layer was sepa-
rated, dried with anhydrous Na2SO4, and filtered, and
the solvent evaporated, affording 4.06 g of 5b as a foam.
Yield: 99%. 1H NMR (400 MHz, DMSO-d6) d ppm 3.27
(s, 3H), 3.83 (dt, J = 12.2 and 6.0 Hz, 1H), 4.25 (dd,
J = 12.4 and 10.6 Hz, 1H), 4.51–4.57 (m, 1H), 4.62–
5.2. General procedure for the synthesis of compounds
7a–f
5.2.1. Method A (primary amines)
5.2.1.1. Benzyl-3-[4-(2-methyl-3-phenyl-2(E)-propen-1-
yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyrano-
[4,3-c]isoxazol-7-ylamine (7c). A mixture of 6a (1 g,
2.4 mmol), potassium fluoride (0.14 g, 2.4 mmol), and
benzylamine (6.3 mL, 57.6 mmol) was heated at 150 ꢁC
for 5 days in a sealed tube. Then, water was added
and the mixture was extracted with dichloromethane.
The organic layer was separated, dried with anhydrous
Na2SO4, and filtered, and the solvent evaporated. The
residue was purified by column chromatography
(dichloromethane/ethyl acetate 2:1) and treated with
diisopropylether affording 0.89 g of 7c as a foam. Yield:
73%. 1H NMR (400 MHz, CDCl3) d ppm 1.90 (d,
J = 1.0 Hz, 3H), 2.47 (br s, 4H), 2.59 (br s, 2H), 2.63
(br s, 2H), 2.80 (dd, J = 13.1 and 5.4 Hz, 1H), 2.87

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4367
(dd, J = 13.1 and 6.2 Hz, 1H), 3.01 (s, 2H), 3.60 (dt,
J = 12.5 and 5.7 Hz, 1H), 4.04 (dd, J = 12.4 and
10.4 Hz, 1H), 4.31–4.41 (m, 4H), 4.56 (dd, J = 10.2 and
5.8 Hz, 1H), 6.12 (d, J = 2.3 Hz, 1H), 6.30 (dd, J = 8.5
and 2.3 Hz, 1H), 6.42 (s, 1H), 7.17–7.23 (m, 1H), 7.25–
7.39 (m, 9H), 7.56 (d, J = 8.5 Hz, 1H). MS m/z 509
(MH+). Anal. Calcd for C32H36N4O2: C, 75.56; H,
7.13; N, 11.01. Found: C, 75.40; H, 7.25; N, 10.84.
NMR (400 MHz, CDCl3) d ppm 1.63 (s, 1H), 1.90 (d,
J = 1.2 Hz, 3H), 2.47 (br s, 4H), 2.59 (br s, 2H), 2.63
(br s, 2H), 2.81 (dd, J = 13.1 and 5.2 Hz, 1H), 2.87
(dd, J = 13.1 and 6.2 Hz, 1H), 3.01 (s, 2H), 3.03 (s,
3H), 3.53 (t, J = 5.7 Hz, 2H), 3.60 (dt, J = 12.5 and
5.7 Hz, 1H), 3.83 (t, J = 5.6 Hz, 2H), 4.04 (dd, J = 12.4
and 10.4 Hz, 1H), 4.32–4.39 (m, 1H), 4.57 (dd,
J = 10.2 and 5.8 Hz, 1H), 6.22 (d, J = 2.5 Hz, 1H),
6.41–6.46 (m, 2H), 7.18–7.23 (m, 1H), 7.26–7.35 (m,
4H), 7.60 (d, J = 8.9 Hz, 1H). MS m/z 477 (MH+).
The following compounds were prepared analogously:
5.2.1.2. Methyl-3-[4-(2-methyl-3-phenyl-2(E)-propen-
1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazol-7-ylamine (7a). Crystallized as
dihydrochloride salt in 2-propanol. Yield: 19%, mp
5.2.2.3. N,N-Dimethyl-N0-{3-[4-(2-methyl-3-phenyl-
2(E)-propen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-
[1]benzopyrano[4,3-c]isoxazol-7-yl}-ethane-1,2-diamine
1
(7f). Yield: 76%. Foam. H NMR (400 MHz, CDCl3) d
1
>300 ꢁC (dec). H NMR (400 MHz, DMSO-d6) d ppm
ppm 1.90 (d, J = 1.2 Hz, 3H), 2.29 (s, 6H), 2.42–2.53 (m,
6H), 2.58 (br s, 2H), 2.63 (br s, 2H), 2.81 (dd, J = 13.2
and 5.4 Hz, 1H), 2.87 (dd, J = 12.8 and 6.2 Hz, 1H),
2.98 (s, 3H), 3.01 (s, 2H), 3.42–3.51 (m, 2H), 3.60 (dt,
J = 12.6 and 5.8 Hz, 1H), 4.05 (dd, J = 12.4 and
10.4 Hz, 1H), 4.31–4.38 (m, 1H), 4.57 (dd, J = 10.4
and 5.8 Hz, 1H), 6.16 (d, J = 2.5 Hz, 1H), 6.38 (dd,
J = 9.1 and 2.5 Hz, 1H), 6.42 (s, 1H), 7.17–7.23 (m,
1H), 7.26–7.35 (m, 4H), 7.60 (d, J = 9.1 Hz, 1H). MS
m/z 504 (MH+). Anal. Calcd for C30H41N5O2: C,
71.54; H, 8.20; N, 13.90. Found: C, 71.40; H, 8.35; N,
13.74.
1.90 (d, J = 1.2 Hz, 3H), 2.63 (s, 3H), 2.73–3.29 (m,
10H), 3.59 (dt, J = 12.5 and 6.0 Hz, 1H), 3.68 (br s,
2H), 3.99 (dd, J = 12.4 and 10.8 Hz, 1H), 4.32–4.40
(m, 1H), 4.56 (dd, J = 10.6 and 5.6 Hz, 1H), 5.99 (d,
J = 2.1 Hz, 1H), 6.27 (dd, J = 8.5 and 2.3 Hz, 1H),
6.67 (s, 1H), 7.23–7.41 (m, 6H), 9.5–10.75 (br s, 1H).
MS m/z 433 (MH+).
5.2.1.3. Ethyl-3-[4-(2-methyl-3-phenyl-2(E)-propen-1-
yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazol-7-ylamine (7b). Yield: 27%. Foam. 1H
NMR (400 MHz, CDCl3) d ppm 1.26 (t, J = 7.1 Hz,
3H), 1.90 (d, J = 1.2 Hz, 3H), 2.47 (br s, 4H), 2.58 (br s,
2H), 2.63 (br s, 2H), 2.80 (dd, J = 13.3 and 5.4 Hz, 1H),
2.87 (dd, J = 13.3 and 6.2 Hz, 1H), 3.01 (s, 2H), 3.11–
3.21 (m, 2H), 3.60 (dt, J = 12.5 and 5.7 Hz, 1H), 3.92 (t,
J = 4.9 Hz, 1H), 4.05 (dd, J = 12.4 and 10.4 Hz, 1H),
4.30–4.39 (m, 1H), 4.57 (dd, J = 10.4 and 5.8 Hz, 1H),
6.08 (d, J = 2.3 Hz, 1H), 6.24 (dd, J = 8.5 and 2.3 Hz,
1H), 6.42 (s, 1H), 7.17–7.23 (m, 1H), 7.24–7.36 (m, 4H),
7.55 (d, J = 8.5 Hz, 1H). MS m/z 447 (MH+).
5.2.3. Resolution of racemic mixture of 7e
5.2.3.1. (ꢀ)-2-{3-[4-(2-Methyl-3-phenyl-2(E)-propen-
1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazole-7ylamino}-ethanol ((ꢀ)-7e) and (+)-
2-{3-[4-(2-methyl-3-phenyl-2(E)-propen-1-yl)piperazin-1-yl-
methyl]-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazole-
7ylamino}-ethanol ((+)-7e). The racemate 2-{3-[4-(2-
methyl-3-phenyl-2(E)-propen-1-yl)piperazin-1-ylmethyl]-
3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazole-7ylami-
no}-ethanol 7e (0.83 g, 1.7 mmol) was separated into its
enantiomers by preparative HPLC (ethanol/acetonitrile
100:0 to 80:20 at 100 mL/min; column: CHIRALPAK
AD 1000 20 lm Daicel). Two pure fractions were col-
lected, the solvents were evaporated yielding 0.48 g of
(ꢀ)-7e as a free base from diisopropylether as a white
solid: mp 116.9 ꢁC; 99% ee; 1H NMR (400 MHz,
CDCl3) d ppm 1.7 (br s, 1H), 1.90 (s, 3H), 2.47 (br s,
4H), 2.58 (br s, 2H), 2.63 (br s, 2H), 2.81 (dd, J = 13.1
and 5.2 Hz, 1H), 2.87 (dd, J = 13.3 and 6.2 Hz, 1H),
3.01 (s, 2H), 3.03 (s, 3H), 3.53 (t, J = 5.7 Hz, 2H), 3.60
(dt, J = 12.4 and 5.8 Hz, 1H), 3.83 (t, J = 5.6 Hz, 2H),
4.04 (dd, J = 12.1 and 10.5 Hz, 1H), 4.30–4.39 (m,
1H), 4.57 (dd, J = 10.2 and 5.8 Hz, 1H), 6.22 (d,
5.2.2. Method B (secondary amines). 5.2.2.1. Benzylm-
ethyl-3-[4-(2-methyl-3-phenyl-2(E)-propen-1-yl)piperazin-
1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxa-
zol-7-ylamine (7d). To a solution of 6a (0.2 g, 0.5 mmol)
in pyridine (2 mL), benzylmethylamine (1.5 mL,
11.4 mmol) was added and the mixture was heated at
150 ꢁC for 4 days in a sealed tube. Then, the solvent
was evaporated and the residue was purified by column
chromatography (dichloromethane/methanol 99:1) and
crystallized as dihydrochloride salt in 2-propanol afford-
ing 22 mg of 7d. Yield: 10%, mp >300 ꢁC (dec). 1H
NMR (400 MHz, DMSO-d6)
d
ppm 1.90 (d,
J = 0.8 Hz, 3H), 2.95–3.10 (m, 5H), 3.04 (s, 3H), 3.17–
3.26 (m, 5H), 3.62 (dt, J = 12.5 and 5.6 Hz, 1H), 3.73
(s, 2H), 3.99 (dd, J = 12.4 and 10.8 Hz, 1H), 4.37–4.44
(m, 1H), 4.51–4.61 (m, 3H), 6.10 (d, J = 2.5 Hz, 1H),
6.45 (dd, J = 9.1 and 2.5 Hz, 1H) 6.68 (s, 1H), 7.12 (d,
J = 7.0 Hz, 2H), 7.20 (t, J = 7.3 Hz, 1H), 7.25–7.33 (m,
5H), 7.37 (t, J = 8.5 Hz, 3H). MS m/z 423 (MH+).
J = 2.1 Hz, 1H), 6.40–6.47 (m, 2H), 7.20 (t, J = 7.1 Hz,
20
D
1H), 7.24–7.36 (m, 4H), 7.60 (d, J = 8.7 Hz, 1H). ½aꢁ
ꢀ45.80 (c 0.44, MeOH); HRMS Calcd for
C28H37N4O3 (M+1): 477.2848. Found: 477.2866 and
0.40 g of (+)-7e precipitated as a dihydrochloride acid
salt and crystallized in methanol as a light yellow solid:
mp >300 ꢁC (dec); 99% ee; 1H NMR (400 MHz,
DMSO-d6) d ppm 2.05 (s, 3H), 2.96 (s, 3H), 3.39–3.46
(m, 2H), 3.46–3.72 (m, 12H), 3.72–3.82 (m, 2H), 3.92
(s, 2H), 4.07 (dd, J = 12.2 and 10.6 Hz, 1H), 4.63–4.73
(m, 2H), 6.20 (d, J = 2.5 Hz, 1H), 6.47 (dd, J = 9.0 and
2.4 Hz, 1H), 6.82 (s, 1H), 7.29–7.34 (m, 1H), 7.34–7.39
The following compounds were prepared analogously:
5.2.2.2. 2-{3-[4-(2-Methyl-3-phenyl-2(E)-propen-1-yl)pip-
erazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]-
isoxazole-7ylamino}-ethanol (7e). Yield: 62%. Foam. H
1

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4368
20
(m, 2H), 7.39–7.46 (m, 2H), 10.5–12.5 (br s, 1H). ½aꢁ
7.26–7.31 (m, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.40 (d,
J = 1.7 Hz, 1H), 7.56 (d, J = 8.7 Hz, 1H), 10.15 (s,
1H). MS m/z 461 (MH+). Anal. Calcd for
C27H32N4O3: C, 70.41; H, 7.00; N, 12.16. Found: C,
69.98; H, 7.27; N, 11.92.
D
+71.30 (c 0.44, MeOH); HRMS Calcd for
C28H37N4O3 (M+1): 477.2859. Found: 477.2866.
5.2.3.2. 7-Amino-3-[4-(2-methyl-3-phenyl-2(E)-propen-
1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazole (8). A dried flask was charged with
6b (6 g, 12.4 mmol), palladium acetate (0.028 g,
0.12 mmol), Xantphos (0.11 g, 0.19 mmol), tert-butyl
carbamate (1.75 g, 14.9 mmol), and cesium carbonate
(6.06 g, 18.6 mmol) in previously deoxygenated 1,4-di-
oxane (40 mL). The mixture was stirred at 100 ꢁC for
24 h until the starting aryl bromide had been completely
consumed as judged by TLC analysis. The reaction mix-
ture was then cooled to room temperature, diluted with
dichloromethane (400 mL), filtered, and concentrated in
vacuo. The crude material was purified by column chro-
matography (dichloromethane/methanol 99:1 and 98:2)
yielding 6.43 g of a carbamate intermediate, which was
treated with TFA (34 mL) in dichloromethane
(170 mL) and stirred at room temperature for 5 h. Then,
the solvent was evaporated and the residue was basified
with a saturated solution of sodium carbonate (150 mL)
and extracted with dichloromethane (3· 150 mL). The
organic phase was separated, dried with Na2SO4, and fil-
tered, and the solvent evaporated. The residue was crys-
tallized with diisopropylether affording 4.72 g of solid 8.
Yield: 91% (two steps), mp 134.1 ꢁC. 1H NMR
(400 MHz, DMSO-d6) d ppm 1.84 (d, J = 1.0 Hz, 3H),
2.37 (br s, 4H), 2.49 (br s, 4H), 2.69 (dd, J = 13.1 and
6.4 Hz, 1H), 2.78 (dd, J = 13.1 and 5.0 Hz, 1H), 2.97
(s, 2H), 3.52 (dt, J = 12.4 and 5.8 Hz, 1H), 4.02 (dd,
J = 12.2 and 10.6 Hz, 1H), 4.28 (dt, J = 12.1 and
5.9 Hz, 1H), 4.51 (dd, J = 10.4 and 5.8 Hz, 1H), 5.76
(br s, 2H), 6.08 (d, J = 2.1 Hz, 1H), 6.24 (dd, J = 8.5
and 2.1 Hz, 1H), 6.41 (s, 1H), 7.19–7.24 (m, 1H), 7.26–
7.30 (m, 3H), 7.34 (t, J = 7.5 Hz, 2H). MS m/z 419
(MH+). Anal. Calcd for C25H30N4O2: C, 71.74; H,
7.22; N, 13.39. Found: C, 71.84; H, 7.36; N, 13.19.
The following compounds were prepared analogously
following a parallel synthetic approach:
5.3.2. 2,2-Dimethyl-propionic acid 3-[4-(2-methyl-3-phen-
yl-2(E)-propen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-
3H-[1]benzopyrano[4,3-c]isoxazole-7-ylamide (9b). Yield:
1
62%. H NMR (400 MHz, CDCl3) d ppm 1.31 (s, 9H),
1.90 (d, J = 1.0 Hz, 3H), 2.48 (br s, 4H), 2.62 (br s,
4H), 2.81 (dd, J = 13.3 and 5.6 Hz, 1H), 2.89 (dd,
J = 13.3 and 6.2 Hz, 1H), 3.02 (s, 2H), 3.66 (dt,
J = 12.4 and 5.8 Hz, 1H), 4.07 (dd, J = 12.4 and
10.6 Hz, 1H), 4.38–4.47 (m, 1H), 4.63 (dd, J = 10.4
and 5.8 Hz, 1H), 6.42 (s, 1H), 6.97 (dd, J = 8.5 and
2.1 Hz, 1H), 7.17–7.23 (m, 1H), 7.24–7.38 (m, 5H),
7.45 (d, J = 2.1 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H). MS
m/z 503 (MH+).
5.3.3. Cyclopropanecarboxylic acid 3-[4-(2-methyl-3-
phenyl-2(E)-propen-1-yl)piperazin-1-ylmethyl]-3a,4-dihy-
dro-3H-[1]benzopyrano[4,3-c]isoxazole-7-ylamide
(9c).
1
Yield: 75%. H NMR (400 MHz, CDCl3) d ppm 0.87–
0.97 (m, 2H), 1.00–1.12 (m, 2H), 1.75–1.84 (m, 1H),
1.91 (d, J = 1.0 Hz, 3H), 2.48 (br s, 4H), 2.65 (br s,
4H), 2.80 (dd, J = 13.3 and 5.4 Hz, 1H), 2.91 (dd,
J = 13.3 and 6.2 Hz, 1H), 3.02 (s, 2H), 3.67 (dt,
J = 12.4 and 5.7 Hz, 1H), 4.10 (dd, J = 12.4 and
10.6 Hz, 1H), 4.36–4.46 (m, 1H), 4.60 (dd, J = 10.2
and 5.7 Hz, 1H), 6.43 (s, 1H), 7.06 (dd, J = 8.9 and
1.7 Hz, 1H), 7.16–7.30 (m, 5H), 7.37–7.43 (m, 2H),
10.30 (s, 1H). MS m/z 487 (MH+). Anal. Calcd for
C29H34N4O3: C, 71.58; H, 7.04; N, 11.51. Found: C,
71.26; H, 7.35; N, 11.17.
5.3.4. Acrylic acid 3-[4-(2-methyl-3-phenyl-2(E)-propen-
1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H -[1]benzopyr-
ano[4,3-c]isoxazole-7-ylamide (9d). Yield: 60%. 1H NMR
(400 MHz, CDCl3) d ppm 1.91 (d, J = 1.0 Hz, 3H), 2.48
(br s, 4H), 2.65 (br s, 4H), 2.80 (dd, J = 13.3 and 5.4 Hz,
1H), 2.91 (dd, J = 13.3 and 6.2 Hz, 1H), 3.02 (s, 2H),
3.67 (dt, J = 12.4 and 5.7 Hz, 1H), 4.10 (dd, J = 12.4
and 10.6 Hz, 1H), 4.36–4.46 (m, 1H), 4.60 (dd,
J = 10.2 and 5.7 Hz, 1H), 5.77 (dd, J = 10.4 and
2.7 Hz, 1H), 6.23 (dd, J = 16.7 and 2.7 Hz, 1H), 6.43
(s, 1H), 6.83 (dd, J = 16.7 and 10.4 Hz, 1H), 7.2–7.45
(m, 8H), 10.30 (s, 1H). MS m/z 473 (MH+).
5.3. General procedure for the synthesis of compounds
9a–f
5.3.1. Acetic acid 3-[4-(2-methyl-3-phenyl-2(E)-propen-1-
yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazole-7-ylamide (9a). To a solution of 8
(0.1 g, 0.2 mmol) in CHCl3 (3 mL), triethylamine
(0.1 mL, 0.9 mmol) and acetyl chloride (0.06 mL,
0.9 mmol) were added. The mixture was stirred at room
temperature for 24 h. Then, saturated solution of sodi-
um bicarbonate (3 mL) was added. The organic layer
was separated, dried over anhydrous Na2SO4, and
filtered, and the solvent evaporated. The residue was
purified by column chromatography (dichloromethane/
methanol 99:1) and treated with DIPE affording 80 mg
of 9a as a foam. Yield: 73%. 1H NMR (400 MHz,
DMSO-d6) d ppm 1.84 (d, J = 1.2 Hz, 3H), 2.05 (s,
3H), 2.38 (br s, 4H), 2.50 (br s, 4H), 2.72 (dd, J = 13.3
and 6.2 Hz, 1H), 2.81 (dd, J = 12.8 and 5.0 Hz, 1H),
2.97 (s, 2H), 3.66 (dt, J = 12.4 and 5.8 Hz, 1H), 4.14
(dd, J = 12.4 and 10.4 Hz, 1H), 4.38–4.46 (m, 1H),
4.60 (dd, J = 10.6 and 6.0 Hz, 1H), 6.41 (s, 1H), 7.13
(dd, J = 8.7 and 2.1 Hz, 1H), 7.21 (t, J = 7.3 Hz, 1H),
5.3.5. Benzoic acid 3-[4-(2-methyl-3-phenyl-2(E)-propen-
1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazole-7-ylamide (9e). Yield: 80%. 1H NMR
(400 MHz, CDCl3) d ppm 1.91 (d, J = 1.0 Hz, 3H), 2.48
(br s, 4H), 2.63 (br s, 4H), 2.82 (dd, J = 13.3 and 5.4 Hz,
1H), 2.89 (dd, J = 13.3 and 6.2 Hz, 1H), 3.01 (s,
2H), 3.66 (dt, J = 12.4 and 5.8 Hz, 1H), 4.09 (dd,
J = 12.4 and 10.6 Hz, 1H), 4.36–4.46 (m, 1H), 4.61
(dd, J = 10.2 and 5.7 Hz, 1H), 6.43 (s, 1H), 7.18–7.37
(m, 9H), 7.38–7.43 (m, 2H), 7.48–7.53 (m, 2H), 10.49
(s, 1H). MS m/z 523 (MH+).

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4369
5.3.6. Nicotinic acid 3-[4-(2-methyl-3-phenyl-2(E)-pro-
pen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzo-
pyrano[4,3-c]isoxazole-7-ylamide (9f). Yield: 58%. 1H
NMR (400 MHz, CDCl3) d ppm 1.90 (d, J = 1.0 Hz,
3H), 2.47 (br s, 4H), 2.64 (br s, 4H), 2.81 (dd, J = 13.3
and 5.4 Hz, 1H), 2.90 (dd, J = 13.3 and 6.2 Hz, 1H),
3.01 (s, 2H), 3.67 (dt, J = 12.4 and 5.8 Hz, 1H), 4.09 (dd,
J = 12.4 and 10.6 Hz, 1H), 4.36–4.45 (m, 1H), 4.61 (dd,
J = 10.2 and 5.8 Hz, 1H), 6.43 (s, 1H), 7.20–7.45 (m,
8H), 7.50 (dd, J = 7.2 and 4.6 Hz, 1H), 8.21 (dt, J = 7.2
and 1.7 Hz, 1H), 8.65 (dt, J = 4.6 and 1.7 Hz, 1H), 9.03
(br s, 1H), 10.40 (s, 1H). MS m/z 524 (MH+).
6.75 (dd, J = 8.5 and 2.1 Hz, 1H), 6.95 (s, 1H), 7.14–
7.23 (m, 2H), 7.23–7.36 (m, 4H), 7.59 (d, J = 8.7 Hz,
1H). MS m/z 544 (MH+).
5.4.1.4. Ethyl (3-{3-[4-(2-methyl-3-phenyl-2(E)-pro-
pen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzo-
pyrano[4,3-c]isoxazole-7-yl}-ureido)-acetate (9j). Yield:
1
72%. Foam. H NMR (400 MHz, CDCl3) d ppm 1.31
(t, J = 7.1 Hz, 3H), 1.90 (d, J = 0.8 Hz, 3H), 2.47 (br s,
4H), 2.61 (br s, 4H), 2.80 (dd, J = 13.2 and 5.6 Hz,
1H), 2.88 (dd, J = 13.3 and 6.2 Hz, 1H), 3.00 (s, 2H),
3.61 (dt, J = 12.4 and 5.8 Hz, 1H), 4.01–4.10 (m, 3H),
4.24 (q, J = 7.1 Hz, 2H), 4.36–4.45 (m, 1H), 4.57 (dd,
J = 10.3 and 5.9 Hz, 1H), 5.80 (t, J = 5.5 Hz, 1H), 6.42
(s, 1H), 6.73 (dd, J = 8.6 and 2.0 Hz, 1H), 7.10 (d,
J = 2.1 Hz, 1H), 7.16–7.36 (m, 6H), 7.52 (d,
J = 8.5 Hz, 1H). MS m/z 548 (MH+). Anal. Calcd for
C30H37N5 O5: C, 65.79; H, 6.81; N, 12.79. Found: C,
65.48; H, 7.03; N, 12.84.
5.4. General procedure for the synthesis of compounds
9g–m
5.4.1. Method C (alkylisocyanates)
5.4.1.1. 1-Ethyl-3-{3-[4-(2-methyl-3-phenyl-2(E)-pro-
pen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzo-
pyrano[4,3-c]isoxazole-7-yl}-urea (9g). To a solution of 8
(0.2 g, 0.5 mmol) in toluene (3 mL), ethylisocyanate
(0.1 mL, 1.3 mmol) was added and the mixture was stirred
at 75 ꢁC for 24 h. After cooling, the solvent was evaporat-
ed and the residue was purified by column chromatogra-
phy (dichloromethane/methanol 99:1 and 98:2)
5.4.2. Method D (arylisocyanates)
5.4.2.1. 1-{3-[4-(2-Methyl-3-phenyl-2(E)-propen-1-yl)-
piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyrano[4,3-
c]isoxazole-7-yl}-3-phenylurea (9k).
(0.27 g, 0.6 mmol), phenylisocyanate
A
mixture of 8
(0.08 mL,
1
affording 119 mg of 9 g as a sticky oil. Yield: 51%. H
0.7 mmol), and triethylamine (catalytic amount) in
THF was stirred at room temperature for 24 h. Then,
the solvent was evaporated and the residue was purified
by column chromatography (dichloromethane/methanol
98:2 and 96:4) and crystallized with diisopropylether
affording 0.16 g of solid 9k. Yield: 46%, mp 180.3 ꢁC.
1H NMR (400 MHz, DMSO-d6) d ppm 1.84 (s, 3H),
2.39 (br s, 4H), 2.50 (br s, 4H), 2.73 (dd, J = 13.1 and
6.4 Hz, 1H), 2.82 (dd, J = 13.3 and 5.0 Hz, 1H), 2.97 (s,
2H), 3.66 (dt, J = 12.4 and 5.8 Hz, 1H), 4.15 (dd,
J = 12.2 and 10.8 Hz, 1H), 4.37–4.46 (m, 1H), 4.61 (dd,
J = 10.5 and 5.9 Hz, 1H), 6.42 (s, 1H), 6.95–7.03 (m,
2H), 7.16–7.39 (m, 8H), 7.45 (d, J = 7.7 Hz, 2H), 7.54
(d, J = 8.5 Hz, 1H), 8.76 (s, 1H), 8.97 (s, 1H). MS m/z
538 (MH+). Anal. Calcd for C32H35N5O3: C, 71.49; H,
6.56; N, 13.03. Found: C, 71.40; H, 6.39; N, 12.88.
NMR (400 MHz, DMSO-d6) d ppm 1.05 (t, J = 7.1 Hz,
3H), 1.84 (d, J = 1.0 Hz, 3H), 2.39 (br s, 4H), 2.51 (br s,
4H), 2.72 (dd, J = 13.1 and 6.4 Hz, 1H), 2.81 (dd, J =
13.3 and 5.2 Hz, 1H), 2.97 (s, 2H), 3.07–3.14 (m, 2H),
3.63 (dt, J = 12.4 and 6.0 Hz, 1H), 4.12 (dd, J = 12.4
and 10.8 Hz, 1H), 4.39 (dt, J = 12.2 and 5.9 Hz, 1H), 4.59
(dd, J = 10.6 and 6.0 Hz, 1H), 6.21 (t, J = 5.6 Hz, 1H),
6.42 (s, 1H), 6.92 (dd, J = 8.7 and 2.1 Hz, 1H), 7.19–
7.26 (m, 2H), 7.26–7.30 (m, 2H), 7.31–7.37 (m, 2H), 7.48
(d, J = 8.5 Hz, 1H), 8.73 (s, 1H). MS m/z 490 (MH+).
The following compounds were prepared analogously:
5.4.1.2. 1-Tertbutyl-3-{3-[4-(2-methyl-3-phenyl-2(E)-
propen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]ben-
zopyrano[4,3-c]isoxazole-7-yl}-urea (9h). Yield: 25%.
Foam. 1H NMR (400 MHz, CDCl3) d ppm 1.38 (s,
9H), 1.90 (s, 3H), 2.47 (br s, 4H), 2.60 (br s, 4H), 2.80
(dd, J = 13.3 and 5.6 Hz, 1H), 2.87 (dd, J = 13.1 and
6.0 Hz, 1H), 3.01 (s, 2H), 3.62 (dt, J = 12.4 and
5.8 Hz, 1H), 4.03 (dd, J = 12.3 and 10.5 Hz, 1H), 4.35–
4.43 (m, 1H), 4.58 (dd, J = 10.4 and 5.8 Hz, 1H), 4.92
(s, 1H), 6.42 (s, 1H), 6.71–6.77 (m, 2H), 7.15 (d,
J = 2.1 Hz, 1H), 7.20 (t, J = 7.1 Hz, 1H), 7.24–7.35 (m,
4H), 7.58 (d, J = 8.5 Hz, 1H). MS m/z 518 (MH+).
The following compounds were prepared analogously:
5.4.2.2. 1-(3-Fluorophenyl)-3-{3-[4-(2-methyl-3-phen-
yl-2(E)-propen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-
3H-[1]benzopyrano[4,3-c]isoxazole-7-yl}-urea (9l). Yield:
1
63%, mp 186.8 ꢁC. H NMR (400 MHz, DMSO-d6) d
ppm 1.85 (d, J = 1.0 Hz, 3H), 2.39 (br s, 4H), 2.51 (br s,
4H), 2.73 (dd, J = 13.3 and 6.4 Hz, 1H), 2.82 (dd,
J = 13.3 and 5.2 Hz, 1H), 2.98 (s, 2H), 3.66 (dt, J = 12.4
and 5.8 Hz, 1H), 4.16 (dd, J = 12.4 and 10.8 Hz, 1H),
4.38–4.47 (m, 1H), 4.62 (dd, J = 10.5 and 5.9 Hz, 1H),
6.42 (s, 1H), 6.81 (dt, J = 8.4 and 2.0 Hz, 1H), 7.01 (dd,
J = 8.7 and 2.1 Hz, 1H), 7.14 (dd, J = 8.3 and 1.0 Hz,
1H), 7.19–7.25 (m, 1H), 7.25–7.39 (m, 6H), 7.48 (dt,
J = 11.9 and 2.2 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 9.01
(s, 1H), 9.05 (s, 1H). MS m/z 556 (MH+).
5.4.1.3. 1-Cyclohexyl-3-{3-[4-(2-methyl-3-phenyl-2(E)-
propen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]ben-
zopyrano[4,3-c]isoxazole-7-yl}-urea (9i). Yield: 48%. Syr-
1
up. H NMR (400 MHz, CDCl3) d ppm 1.06–1.22 (m,
3H), 1.27–1.42 (m, 2H), 1.55–1.65 (m, 1H), 1.65–1.77
(m, 2H), 1.90 (d, J = 1.0 Hz, 3H), 1.92–2.01 (m, 2H),
2.47 (br s, 4H), 2.61 (br s, 4H), 2.80 (dd, J = 13.3 and
5.6 Hz, 1H), 2.87 (dd, J = 13.1 and 6.0 Hz, 1H), 3.00
(s, 2H), 3.57–3.70 (m, 2H), 4.03 (dd, J = 12.4 and
10.6 Hz, 1H), 4.34–4.43 (m, 1H), 4.59 (dd, J = 10.4
and 5.8 Hz, 1H), 5.04 (d, J = 7.9 Hz, 1H), 6.42 (s, 1H),
5.4.2.3. 1-(3,4-Dimethoxyphenyl)-3-{3-[4-(2-methyl-3-
phenyl-2(E)-propen-1-yl)piperazin-1-ylmethyl]-3a,4-dihy-
dro-3H-[1]benzopyrano[4,3-c]isoxazole-7-yl}-urea (9m).
Yield: 73%. Foam. 1H NMR (400 MHz, CDCl3) d

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ppm 1.90 (s, 3H), 2.46 (br s, 4H), 2.60 (br s, 4H), 2.79
(dd, J = 13.3 and 5.6 Hz, 1H), 2.88 (dd, J = 13.3 and
6.2 Hz, 1H), 3.00 (s, 2H), 3.61 (dt, J = 12.4 and
5.9 Hz, 1H), 3.84 (s, 3H), 3.85 (s, 3H), 4.01 (dd,
J = 12.4 and 10.4 Hz, 1H), 4.35–4.45 (m, 1H), 4.58
(dd, J = 10.4 and 5.8 Hz, 1H), 6.41 (s, 1H), 6.71–6.82
(m, 3H), 6.96–7.07 (m, 2H), 7.15–7.36 (m, 7H), 7.59
(d, J = 8.5 Hz, 1H). MS m/z 598 (MH+).
6.87 (dd, J = 8.3 and 2.4 Hz, 1H), 6.90 (t, J = 7.4 Hz,
1H), 7.01 (d, J = 8.3 Hz, 2H), 7.17–7.25 (m, 3H), 7.27–
7.36 (m, 4H), 7.5 (d, J = 8.3 Hz, 1H). MS m/z 509
(MH+).
5.4.3.4. 4-Fluorophenyl-3-[4-(2-methyl-3-phenyl-2(E)-
propen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]ben-
zopyrano[4,3-c]isoxazole-7-yl-amine (10c). Yield: 80%.
Foam. 1H NMR (400 MHz, CDCl3) d ppm 1.91 (d,
J = 1.0 Hz, 3H), 2.48 (br s, 4H), 2.63 (br s, 4H), 2.82
(dd, J = 13.3 and 5.4 Hz, 1H), 2.89 (dd, J = 13.3 and
6.2 Hz, 1H), 3.01 (s, 2H), 3.66 (dt, J = 12.4 and
5.8 Hz, 1H), 4.09 (dd, J = 12.4 and 10.6 Hz, 1H), 4.36–
4.46 (m, 1H), 4.61 (dd, J = 10.2 and 5.7 Hz, 1H), 6.08
(s, 1H), 6.43 (s, 1H), 6.74 (d, J = 2.4 Hz, 1H), 6.86
(dd, J = 8.3 and 2.4 Hz, 1H), 7.18–7.39 (m, 9H), 7.49
(d, J = 8.3 Hz, 1H). MS m/z 513 (MH+).
5.4.3. General procedure for the synthesis of compounds
10a–f
5.4.3.1. 3-[4-(2-Methyl-3-phenyl-2(E)-propen-1-yl)pip-
erazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyrano[4,3-
c]isoxazole-7-yl-amino benzonitrile (10d). A dried flask
was charged with 6b (0.2 g, 0.4 mmol), cesium carbonate
(0.19 g, 0.6 mmol), palladium acetate (0.002 g,
0.01 mmol), BINAP (0.011 g, 0.02 mmol), 3-amin-
obenzonitrile (0.06 g, 0.5 mmol), and toluene (2 mL),
and purged with nitrogen. The mixture was stirred at
100 ꢁC for 24 h. The mixture was cooled to room tem-
perature, diluted with dichloromethane, filtered, and
concentrated. The crude product was then purified by
column chromatography (dichloromethane/methanol
99:1) and treated with diisopropylether affording 0.14 g
of 10d as a foam. Yield: 67%.1H NMR (400 MHz,
CDCl3) d ppm 1.90 (d, J = 1.0 Hz, 3H), 2.48 (br s,
4H), 2.63 (br s, 4H), 2.82 (dd, J = 13.3 and 5.4 Hz,
1H), 2.89 (dd, J = 13.3 and 6.2 Hz, 1H), 3.01 (s, 2H),
3.66 (dt, J = 12.4 and 5.8 Hz, 1H), 4.09 (dd, J = 12.4
and 10.6 Hz, 1H), 4.36–4.47 (m, 1H), 4.62 (dd,
J = 10.2 and 5.8 Hz, 1H), 6.07 (s, 1H), 6.42 (s, 1H),
6.60–6.68 (m, 2H), 7.17–7.43 (m, 9H), 7.69 (d,
J = 8.3 Hz, 1H). MS m/z 520 (MH+). Anal. Calcd for
C32H33N5O2: C, 73.96; H, 6.40; N, 13.48. Found: C,
73.61; H, 6.76; N, 13.21.
5.4.3.5. 3-[4-(2-Methyl-3-phenyl-2(E)-propen-1-yl)pip-
erazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyrano[4,3-
c]isoxazole-7-yl-2-tolylamine (10e). Yield: 68%. Foam.
1H NMR (400 MHz, CDCl3)
d ppm 1.91 (d,
J = 1.0 Hz, 3H), 2.25 (s, 3H), 2.48 (br s, 4H), 2.62 (br
s, 4H), 2.81 (dd, J = 13.3 and 5.4 Hz, 1H), 2.89 (dd,
J = 13.3 and 6.2 Hz, 1H), 3.02 (s, 2H), 3.68 (dt, J =
12.4 and 5.8 Hz, 1H), 4.10 (dd, J = 12.4 and 10.6 Hz,
1H), 4.36–4.46 (m, 1H), 4.62 (dd, J = 10.2 and 5.7 Hz,
1H), 6.08 (s, 1H), 6.43 (s, 1H), 6.60 (dt, J = 7.7 and
0.8 Hz, 1H), 6.75 (m, 2H), 6.87 (dd, J = 8.3 and
2.4 Hz, 1H), 7.02 (dt, J = 7.8 and 1.3 Hz, 1H), 7.14 (d,
J = 7.8 Hz, 1H), 7.17–7.24 (m, 1H), 7.27–7.36 (m, 4H),
7.5 (d, J = 8.3 Hz, 1H). MS m/z 509 (MH+).
5.4.3.6. 3,4-Dimethylphenyl-3-[4-(2-methyl-3-phenyl-
2(E)-propen-1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-
[1]benzopyrano[4,3-c]isoxazole-7-yl-amine (10f). Yield:
1
The following compounds were prepared analogously
following a parallel synthetic approach:
75%. Foam. H NMR (400 MHz, CDCl3) d ppm 1.90
(d, J = 1.0 Hz, 3H), 2.25 (s, 3H), 2.35 (s, 3H), 2.48 (br
s, 4H), 2.62 (br s, 4H), 2.81 (dd, J = 13.3 and 5.4 Hz,
1H), 2.90 (dd, J = 13.3 and 6.2 Hz, 1H), 3.02 (s, 2H),
3.68 (dt, J = 12.4 and 5.8 Hz, 1H), 4.11 (dd, J = 12.4
and 10.6 Hz, 1H), 4.36–4.47 (m, 1H), 4.61 (dd,
J = 10.2 and 5.7 Hz, 1H), 6.08 (s, 1H), 6.43 (s, 1H),
6.55 (d, J = 7.8 and 2.2 Hz, 1H), 6.60 (dt, J = 7.7 and
0.8 Hz, 1H), 6.87 (dd, J = 8.3 and 2.4 Hz, 1H), 6.99 (d,
J = 2.2 Hz, 1H), 7.07 (d, J = 7.8 Hz, 1H), 7.19–7.23
(m, 1H), 7.24–7.29 (m, 2H), 7.30–7.36 (m, 2H), 7.5 (d,
J = 8.3 Hz, 1H). MS m/z 523 (MH+).
5.4.3.2. 3-[4-(2-Methyl-3-phenyl-2(E)-propen-1-yl)pip-
erazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyrano[4,3-
c]isoxazole-7-yl-phenylamine (10a). Yield: 73%. Oil. H
1
NMR (400 MHz, CDCl3) d ppm 1.91 (d, J = 1.0 Hz,
3H), 2.48 (br s, 4H), 2.63 (br s, 4H), 2.82 (dd, J = 13.3
and 5.4 Hz, 1H), 2.89 (dd, J = 13.3 and 6.2 Hz, 1H),
3.01 (s, 2H), 3.66 (dt, J = 12.4 and 5.8 Hz, 1H), 4.09
(dd, J = 12.4 and 10.6 Hz, 1H), 4.36–4.46 (m, 1H),
4.61 (dd, J = 10.2 and 5.7 Hz, 1H), 6.08 (s, 1H), 6.43
(s, 1H), 6.74 (d, J = 2.4 Hz, 1H), 6.86 (dd, J = 8.3 and
2.4 Hz, 1H), 6.91 (t, J = 7.4 Hz, 1H), 7.01 (d,
J = 8.3 Hz, 2H), 7.18–7.25 (m, 3H), 7.27–7.35 (m, 4H),
7.49 (d, J = 8.3 Hz, 1H). MS m/z 495 (MH+).
5.5. Biology
5.5.1. Receptor binding assays. Frozen membranes of
CHO cells, stably transfected with either human adren-
ergic 2A or 2C receptors, were thawed on ice, briefly
homogenized with an Ultra Turrax homogenizer, and
then suspended in glycylglycine buffer (25 mM, pH
7.6) at an appropriate pre-determined protein concen-
tration (5–10 lg protein per incubation mixture). The
reaction was started by adding the membrane suspen-
sion to the reaction tube that contained the compound
of interest together with [3H]rauwolscine (1 nM) in a to-
tal volume of 500 lL. The mixture was incubated for
30 min at 25 ꢁC. Non-specific binding was determined
5.4.3.3. Methyl-3-[4-(2-methyl-3-phenyl-2(E)-propen-
1-yl)piperazin-1-ylmethyl]-3a,4-dihydro-3H-[1]benzopyr-
ano[4,3-c]isoxazole-7-yl-phenylamine (10b). Yield: 55%.
Oil. 1H NMR (400 MHz, CDCl3) d ppm 1.91 (d,
J = 1.0 Hz, 3H), 2.48 (br s, 4H), 2.62 (br s, 4H), 2.81
(dd, J = 13.3 and 5.4 Hz, 1H), 2.89 (dd, J = 13.3 and
6.2 Hz, 1H), 3.02 (s, 2H), 3.29 (s, 3H), 3.67 (dt,
J = 12.4 and 5.8 Hz, 1H), 4.09 (dd, J = 12.4 and
10.6 Hz, 1H), 4.36–4.47 (m, 1H), 4.61 (dd, J = 10.2
and 5.7 Hz, 1H), 6.43 (s, 1H), 6.75 (d, J = 2.4 Hz, 1H),

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4371
in the presence of oxymetazoline (1 lM) for the 2A sub-
type and spiroxatrine (1 lM) for 2C subtype. Free radi-
oligand was separated from the radioligand–receptor
complex by means of rapid vacuum filtration over GF/
B unifilter plates with a Packard Harvester Filtration
Unit. Filter plates were washed with ice-cold Tris–HCl
buffer (50 mM, pH 8.0) and dried overnight. Bound
counts were measured in a Topcount Scintillation
Counter in the presence of Microscint O. For 5-HTT
binding, frozen membranes of human platelets (Nova-
screen, Maryland, USA) were thawed on ice, briefly
homogenized, and resuspended in Tris–HCl buffer
(50 mM, pH 7.4) supplemented with NaCl (120 mM)
and KCl (5 mM) at a concentration of 50 lg protein
per incubation mixture. The membrane suspension was
added to the compound of interest together with [3H]par-
oxetine (0.5 nM) in a total volume of 250 lL and incu-
bated (60 min, 25 ꢁC). Non-specific binding was
determined in the presence of imipramine (1 lM). Filtra-
tion was done over pre-soaked GF/B unifilters (0.1%
PEI) and washed as above with the Tris salt buffer used
for the incubation. Specific binding was calculated and
sigmoidal curves were plotted by an internally developed
software program based on S-plus software. Ki values
were calculated using the Cheng–Prusoff equation.
variability and to minimize systematic errors. Control
injections of solvent were included in each experimental
session. All-or-none criteria for significant (p < 0.05)
effects were defined by analyzing a frequency distribu-
tion of a large series of historical control data. On the
basis of the thus obtained criteria, ED50 values and cor-
responding 95% confidence limits were determined
according to the modified Spearman–Kaerber estimate
using theoretical probabilities instead of empirical ones.
This modification allows one to tabulate the ED50 and
its confidence interval as a function of the slope of the
log dose–response curve.16
5.5.2.4. Tests: medetomidine-induced loss of righting in
rats. Medetomidine (0.10 mg/kg, iv)-induced loss of
righting was recorded in overnight-starved rats
(200–250 g), pre-treated with test compound or solvent.
Criterion for drug-induced reversal: absence of loss of
righting (1.0% false positive controls; n > 400).
5.5.2.5. p-Chloroamphetamine-induced behavior in
rats. p-Chloroamphetamine (pCA; 5 mg/10 mL/kg, sc)-
induced excitation was scored (0, 1, 2, or 3) over a
15-min interval starting 45 min after the pCA injection
in male rats (200–250 g) pre-treated with test compound
or solvent. The following all-or-nothing criteria were
selected to assess drug-induced inhibition: score for
excitation <2 (0.5% false positives; n > 200).
The affinity of the compounds for the remaining target
receptors and transporters was also determined by
means of several radioligand competition binding exper-
iments.14,15 In general, the compound of interest (or
control blank) together with the appropriate tritiated
or iodinated radioligand and a membrane suspension
with abundant target receptor/transporter were incubat-
ed under optimized experimental conditions. The reac-
tion was stopped by filtration as above, except for a1A
and D3, where a SPA-based assay was used (Scintilla-
tion Proximity Assay). All assays were carried out with
membranes from cell lines transfected with the human
target, except for DAT and NET, which were done with
the rat striatum and the rat cortex, respectively.
Acknowledgments
The authors thank Ms. Valle Ancos, Ms. Alcira Del
Cerro, and Ms. Encarna Matesanz for the synthesis of
some starting materials and Mr. Luis Miguel Font and
Ms. Carmen Nieto for their skilful analytical and
chromatographic assistance. They also acknowledge
Dr. Shirley Pullan and Ms. Ilse Lenaerts for their help
and assistance in binding experiments, to Mr. P.C.M.
Vermote and Mr. K.A. Hens for performing the pCA
and medetomidine tests, respectively, and Dr. Antonio
5.5.2. In vivo pharmacology
´
Gomez for his help in management and coordination
of all data generated.
5.5.2.1. Animals. Male Wistar rats (Charles River
Breeding Facilities) were used. They were housed in
individual cages in air-conditioned laboratories
(21 2 ꢁC; 65 15% relative humidity). They were fast-
ed overnight but tap water remained available ad libitum
except during the test period.
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