Synthesis of 3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles, displaying combined 5-HT uptake inhibiting and α2-adrenoceptor antagonistic activities: A novel series of potential antidepressants

Article abstract of DOI:10.1016/S0960-894X(03)00525-0

The synthesis of a series of novel 3-piperazinylmethyl-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles as novel dual 5-HT reuptake inhibitors and α₂-adrenoceptor antagonists is described. Their affinity at the three different human α₂-adrenoceptor subtypes and the 5-HT transporter site is reported. The in vivo activity of the compounds was measured in two different assays: (1) inhibition of pCA-induced excitation, which evaluates the ability to block the central 5-HT transporter, and (2) inhibition of xylazine-induced loss of righting, which evaluates the ability to block central α₂-adrenoceptors.

Full text of DOI:10.1016/S0960-894X(03)00525-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2719–2725
Synthesis of 3a,4-Dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles,
Displaying Combined 5-HTUptake Inhibiting and
ꢀ2-Adrenoceptor Antagonistic Activities: A Novel Series of
Potential Antidepressants
J. Ignacio Andres,a,* Jesus Alcazar,a Jose M. Alonso,a Rosa M. Alvarez,a Jose M. Cid,a
Ana I. De Lucas,a Javier Fernandez,a Sonia Martınez,a Carmen Nieto,a Joaquın Pastor,a
Margot H. Bakker,b,y Ilse Biesmans,b Lieve I. Heylenb and Anton A. Megensb
aJohnson & Johnson Pharmaceutical Research & Development, Division of Janssen-Cilag, Medicinal Chemistry Department,
Jarama s/n, 45007 Toledo, Spain
bJohnson & Johnson Pharmaceutical Research & Development, Division of Janssen Pharmaceutica N.V., Turnhoutseweg 30,
B2340Beerse, Belgium
Received 13 March 2003; revised 28 April 2003; accepted 22 May 2003
Abstract—The synthesis of a series of novel 3-piperazinylmethyl-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles as novel dual 5-
HT reuptake inhibitors and a2-adrenoceptor antagonists is described. Their affinity at the three different human a2-adrenoceptor
subtypes and the 5-HT transporter site is reported. The in vivo activity of the compounds was measured in two different assays:
(1) inhibition of pCA-induced excitation, which evaluates the ability to block the central 5-HT transporter, and (2) inhibition of
xylazine-induced loss of righting, which evaluates the ability to block central a2-adrenoceptors.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Currently, the most commonly used pharmacological
treatments for depression are the selective serotonin
(SER) reuptake inhibitors, the SSRIs. Their mood lift-
ing effects are the current gold standard in the treatment
of depression. However, limitations of the available
treatments include a 2–4-week delay for the onset of
action, partial treatment response, excitation during
early treatment response, nausea and reduced sexual
function. The period of several weeks typically required
for the onset of antidepressant action with such agents
has been attributed to a feedback inhibition, via pre-
synaptic autoreceptors,4,5 of further noradrenaline (NE)
and/or SER release caused by an acute rise in synaptic
neurotransmitter concentrations following inhibition of
metabolism or reuptake. Newer drugs also specifically
inhibit NE reuptake, have a dual mechanism of action
inhibiting both SER and NE reuptake, or inhibit
monoamine breakdown by monoamine oxidase inhibi-
tion. In addition a number of ‘atypical antidepressants’
are available on the market (nefazodone, mirtazapine,
bupropion), which act by several other mechanisms of
action. Enhancement of multiple monoaminergic system
The monoaminergic hypothesis for depression, initially
put forward to explain the antidepressant effect of tri-
cyclic antidepressants (TCAs) and monoamine oxidase
inhibitors (MAOIs), assumes that depression is caused
by a functional deficit of monoamines (noradrenaline
and/or serotonin) in corticolimbic synaptic clefts.1 This
biogenic amine hypothesis showed that increased nor-
adrenergic and/or serotonergic neurotransmission
results in improvement of mood, and was based on
observed induction of depressive symptoms reported to
occur as a side effect of treatment with the anti-
hypertensive agent reserpine, which results in the deple-
tion of monoaminergic stores.2,3
*Corresponding author. Tel.: +34-925-245-771; e-mail: iandres@
prdes.jnj.com
yPresent address: Abbott GmbH & Co. KG, D-67008 Ludwigshafen,
Germany.
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00525-0

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2720
activities in parallel is expected to increase the efficacy
of antidepressant treatment, and broaden the number of
parameters on which the therapy will be effective.6
compound 5 showed more activity than compound 4 in
some of our in vivo tests in rats.23 Both compounds
showed much less affinity or no affinity at all for other
serotonergic, dopaminergic and adrenergic receptors. We
synthesized as well the monomethoxy-substituted deriva-
tives 6 and 7 (Fig. 1). While the 7-methoxy analogue
showed comparable in vitro and in vivo activity to that
of the dimethoxy compound 5, the 8-methoxy derivative
kept the in vitro activity but exhibited less in vivo
potency (see Table 1).
Blockade of a2-adrenoceptors in the brain prevents the
negative feedback NE exerts on its own synthesis, neu-
ronal firing and release, resulting in enhanced NEergic
neurotransmission.7,8 a2-adrenoceptor blockade also
increases extracellular dopamine,9 acetylcholine10 and
SER levels11 in vivo in the rat and human. Adding a2-
adrenoceptor blocking activity to SER reuptake inhibi-
tion is therefore expected to be more effective and to
more broadly enhance monoaminergic activity in the
brain. The immediate effect on monoamine release of
autoreceptor blockade, in contrast to the initial inhibi-
tion of activation by reuptake inhibitors through their
activation of feedback mechanisms, may reduce the
onset of action of such a compound compared to the
currently available drugs. In addition, a2-adrenoceptor
antagonism improves sexual function as shown by
treatment with the a2-adrenoceptor antagonist yohim-
bine,12 and enhancement of NEergic neurotransmission
improves social function more effectively than
SSRI’s.13,14 Furthermore, combination therapy of
depressive patients with drugs with an a2-adrenoceptor
antagonistic component (mianserin, mirtazapine or
yohimbine) in addition to an SSRI, have repeatedly
shown increased efficacy and effectiveness on treatment
resistant patients.15ꢀ19
As a part of the subsequent Lead Optimisation pro-
gramme, we synthesised a large number of compounds
with several different moieties replacing the cinnamyl
fragment present in our lead 5. This paper describes the
synthesis and structure–activity relationship of only
some of those compounds, which were prepared within
this series.
Chemistry
The ethyl 3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isox-
azole-3-carboxylates 11a–d were obtained following
essentially the method previously described for the
synthesis of the methyl ester analogue of 11a.24 Thus,
alkylation of salicylaldehydes 8a–d with ethyl 4-bromo-
crotonate, in presence of potassium carbonate as base
and DMF as solvent, afforded intermediates 9a–d.
These aldehydes were transformed into the oximes 10a–d
by reaction with hydroxylamine. The generation of the
required nitrile oxides and subsequent ring closure
by intramolecular 1,3-dipolar cycloaddition to the
cycloadducts 11a–d, was carried out using sodium
hypochlorite and triethylamine, in yields ranging from
42 to 85% from the corresponding oximes.25 The ste-
reochemistry of positions 3 and 3a of the tricyclic sys-
tem was predetermined by the trans-alkene fragment
and was unequivocally assigned by NMR. Reduction of
the four esters with NaBH4 in THF/H2O as solvent
afforded the corresponding hydroxymethyl derivatives
in good yields, which were converted into the mesylates
12a–d by standard procedures. These mesylates were
In the past years a few compounds have been described
which combine SER reuptake inhibition and a2-adre-
noceptor blockade: Sterling-Winthrop’s napamezole
(1),20 Abbott’s A-80426 (2)21 or Servier’s S-34324 (3)22
(Fig. 1). In recent years we started a programme at
Johnson & Johnson Pharmaceutical Research & Devel-
opment, searching for compounds combining both
activities. As a result of the biological screening the tri-
cyclic isoxazoline derivative 4 was identified as a hit.
The introduction of two methoxy groups in positions 7
and 8 of the tricyclic system slightly enhanced the in
vitro potency for the SER transporter, while decreased
the affinity at the a2-receptors. But most interestingly,
Figure 1.

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J. I. Andres et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2719–2725
2721
reacted with commercially available cinnamylpiperazine
furnishing the target compounds 4–7 in 56 to 69%
yields (Scheme 1). The relative cis configuration between
the 3-exocyclic chain and the 3a-hydrogen atom was
kept unaltered during these last three steps in the four
cases.
yield. Alkylation of this compound with the different
required haloalkyl derivatives, or reductive amination
with the corresponding aldehydes afforded the desired
final products 15b–n (Scheme 2).
The synthesis of the cis-cinnamyl analogue 15a was
achieved by a different procedure. Piperazinyl derivative
14 was reacted with vinyltriphenylphosphonium bro-
mide yielding the phosphonium salt 16. This inter-
mediate was subjected to a Wittig reaction with
benzaldehyde furnishing the mixture of trans and cis
isomers in a 3/1 ratio, which was separated by pre-
parative HPLC affording compound 15a in 22% yield
(Scheme 3). The biological results led us to decide to
separate and isolate the enantiomers of four selected
compounds for pharmacological screening. The separa-
tion and isolation of those enantiomers was performed
by preparative chiral HPLC (Chiralpak AD 1000A, 20
Mesylate 12b served also as starting material for the
introduction of other moieties replacing the cinnamyl
fragment. In some cases we obtained the target com-
pounds by reaction of this mesylate with the corre-
sponding required piperazine derivatives, which had to
be previously synthesized. But for most of the com-
pounds we used a slightly modified and more con-
vergent strategy. Thus, reaction of 12b with excess N-
tert-butyloxycarbonylpiperazine afforded the N-Boc-
protected intermediate 13, which was deprotected with
CF3COOH to furnish the key derivative 14 in 90%
Scheme 1. Reagents and conditions: (i) K2CO3, DMF, rt, 2–4h; (ii) NH2OH2HCl, pyridine, CH3CH2OH, rt, 2 h, 63–75% (two steps); (iii) Et3N,
NaOCl (5%), CH2Cl2, rt, overnight, 42–85%; (iv) NaBH4, THF/H2O, rt, 2h, 84–99%; (v) CH3SO2Cl, Et3N , CHCl2, rt, 1 h, 78–90%;
2
(vi) N-cinnamylpiperazine (2 eq.), neat, 100ꢁC, 2h, 56-69%.
Scheme 2. Reagents and conditions: (i) 1-Boc-piperazine, KI, K2CO3, MIK, reflux, overnight, 90%; (ii) CF3COOH, CH2Cl2, rt, 3 h, 100%;
(iii) l-CH2-Hal, K2CO3, MIK, reflux, overnight; (iv) L-CHO, NaBH(AcO)3, CH3COOH (cat.), CH2Cl2, rt, 3 h-overnight.

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J. I. Andres et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2719–2725
2722
Scheme 3. Reagents and conditions: (i) CH2Cl2, rt, overnight, 90%; (ii) (a) PhCHO, MeONa, THF, ꢀ60ꢁC, 6 h, then rt, overnight; (b) preparative
HPLC separation, 22%.
mm, Diacel, using as eluents mixtures hexane/ethanol or
ethanol/acetonitrile with different gradients).
lower. As can be observed in Table 1, compound 15a,
the corresponding cis-analogue of 5, showed less affinity
at both a2-receptors and SER transporter site and
showed less activity in our pCA test. Reduction of the
double bond present in the cinnamyl moiety, exempli-
fied by compound 15d, did not substantially affect the in
vitro affinity for both targets, but resulted in a sig-
nificant decrease of the activity in the pCA test. Short-
ening of the methylene spacer between the phenyl and
the piperazine rings (15b,c) dramatically decreased the
receptor affinities. Replacement of the methylene group
adjacent to the phenyl ring in 15d by a carbonyl group
resulted in a compound about 10-fold less active at the
SER transporter site (15e).
Biological Results and Discussion
Molecules were evaluated in in vitro assays to define
their affinity at the three different human a2-adreno-
ceptor subtypes and the SER transporter site. Mem-
branes prepared from CHO cells, expressing the human
adrenergic a2A-, a2B- or a2C-receptors, were thawed and
incubated with [3H]rauwolscine (1 nM final concen-
tration) with or without competitor for 30 min at 25 ꢁC.
Human platelet membranes (Oceanix Biosciences Cor-
poration, Hanover, USA) were thawed and incubated
with [3H]paroxetine (0.5 nM final concentration) with or
without competitor for 60 min at 25 ꢁC as well. The
affinities of the compounds for other serotonergic as
well as dopaminergic and adrenergic receptors were also
measured by standard procedures. Their affinities at the
dopaminergic and noradrenergic uptake sites were eval-
uated as well. None of those compounds showed rele-
vant affinity at any other of those receptors or
transporter sites. p-Chloroamphetamine (pCA) induces
release of serotonin from serotonergic nerve terminals,
which results in typical behavioural effects in rats. As
pCA produces its biochemical and behavioural effects
only after its uptake into the serotonergic neurones by
the neuronal SER transporter, compounds that block
this transporter mechanism can inhibit its effects.
Antagonism of pCA-induced excitation in rats, in the
absence of overt sedative effects, is therefore a reliable in
vivo index of the ability of test compounds to block the
central serotonin transporter.26 Antagonism of the loss
of righting, induced by the a2-adrenoceptor agonist
xylazine in rats, is a reliable index for the central a2-
adrenoceptor blocking activity of test compounds, at
least when occurring without behavioural stimulant
effects.27 These two in vivo assays, the inhibition of
pCA-induced excitation and the inhibition of xylazine-
induced loss of righting, were selected as primary tests
to evaluate the in vivo activity of our compounds as
SER transporter inhibitors and a2-adrenoceptor
antagonists respectively (Table 1).
The introduction of a methyl group in either one or the
other position of the double bond of the cinnamyl moi-
ety afforded very interesting compounds. Compound
15f was over 10-fold more potent than 5 at the a2-
receptors and 3-fold more potent at the SER transpor-
ter. Even more interesting was its in vivo activity. It
showed comparable potency both in the pCA and the
xylazine assays. The other methyl-cinnamyl analogue
15g exhibited also more potency than 5 at the a2-recep-
tors while both compounds were equipotent at the SER
transporter. But interestingly this compound showed 4-
fold more potency in the pCA assay and the same
potency in the xylazine test when compared to 5.
The b-naphthyl derivative 15h was the most potent
compound at the SER transporter site and it showed as
well quite potent activity in the pCA test, while its
affinity for the a2-receptors was quite similar to that of
the cinnamyl lead compound 5. Some naphthyl-like
heterocyclic derivatives were synthesized as well (15i, j,
m, n) but all of them showed much less affinity at the a2-
receptors, while keeping the affinity for the SER trans-
porter site. The corresponding a-naphthyl analogue 15l
kept as well the affinity at the SER transporter site and
the activity in the pCA test, but did not show potent
affinity at the a2-receptors. Compound 15k, which was a
cyclic analogue of the methyl-cinnamyl derivative 15f,
showed also quite remarkable affinity at the SER trans-
porter site and high potency in the pCA assay, but its
affinity at the a2-receptors dropped again showing very
low activity in the xylazine test.
Compound A-80426 showed nanomolar affinities at
both a2-receptors and SER transporter site, but did not
show significant in vivo activity in our tests. On the
contrary S-34324, which is described as a NE reuptake
inhibitor as well,22 exhibited moderate activity in both
pCA and xylazine tests although its in vitro affinity was
The promising results of several compounds shown in
Table 1 encouraged us to select four of them for the
separation and screening of their corresponding enan-
tiomers. Our first aim was to determine if there was a

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J. I. Andres et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2719–2725
2723
Table 1. Active doses of tricyclic isoxazolines for binding to the a2A, a2B, a2C receptors and the 5-HT transporter in vitro and for antagonism of
pCA-induced excitation and xylazine-induced loss of righting in vivo (ED50 values; mg/kg)
5-HTTa
Ki (nM)
pCA (sc)b
ED50 (mg/kg)
Xylazine (sc)b
ED50 (mg/kg)
a
a
a
Compd
a2A
Ki (nM)
a2B
Ki (nM)
a2C
Ki (nM)
2
3
4
A-80426
S-34324
See Fig. 1
2.4
5.9
0.9
11
23
2
2.1
19
1.7
4
77
16
10
2
5
>10
2.7
2
5
8.8
42
6.2
8.3
0.76
2.5
6
7
See Fig. 1
See Fig. 1
0.5
0.8
16
12
1.9
2.3
19
14
1.4
5
2
2.5
n.t.
15a
23
n.t.
9.5
35
10
15b
15c
15d
15e
15f
15g
15h
15i
186
234
26
995
>1000
42
89
158
199
10
8
>10
>10
2.5
n.t.
1
89
10
1.9
5.3
0.2
0.6
3.1
13
5
12
n.t.
123
2.3
10
n.t.
1.5
0.2
0.79
n.t.
2.5
0.32
3.2
0.63
5
0.8
1.4
5
7.5
31
2.5
5
49
1.7
7.2
5
66
370
n.t.
>10
10
15j
98
>1000
315
89
15k
15l
22
4.7
89
4.1
3.9
8.1
14
234
117
34
>1000
721
>10
5
15m
36
15n
>1000
30
5
n.t., not tested.
aThe 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.
bED50 values and corresponding 95% confidence limits were determined according to the modified Spearman–Kaerber estimate using theoretical probabilities
instead of empirical ones.28 This modification allows to tabulate the ED50 and its confidence interval as a function of the slope of the log dose–response curve.

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2724
Table 2. Active doses of some selected pure enantiomers for binding to the a2A, a2B, a2C receptors and the 5-HT transporter in vitro and for
antagonism of pCA-induced excitation and xylazine-induced loss of righting in vivo (ED50 values; mg/kg)
a
a
a
Compd
a2A
Ki (nM)
a2B
Ki (nM)
a2C
Ki (nM)
5-HTTa
Ki (nM)
pCA (sc)b
ED50 (mg/kg)
Xylazine (sc)b
ED50 (mg/kg)
(ꢀ)-5
25
42
4.8
3.4
1.2
2.8
5.4
10
2.5
0.51
>10
0.82
>10
0.5
5
2.5
(+)-5
2.6
16
14
15
3
0.5
1.4
0.1
10
(ꢀ)-15f
(+)15f
(ꢀ)-15g
(+)-15g
(ꢀ)-15 h
(+)-15 h
>10
3.1
0.3
30
405
14
95
14
>10
0.32
10
0.3
93
0.2
9.5
0.8
4.5
1.3
1.7
1.5
1.3
0.08
6.1
aThe activity of compounds was confirmed in an independent experiment. A difference in pIC50 up to 0.6 (S.D. <0.5) was considered as reproducible
and therefore accepted.
bED50 values and corresponding 95% confidence limits were determined according to the modified Spearman–Kaerber estimate using theoretical
probabilities instead of empirical ones.28 This modification allows to tabulate the ED50 and its confidence interval as a function of the slope of the
log dose–response curve.
dissociation of affinities between the a2-adrenoceptors
and the SER transporter site. Our second purpose was,
in case the activity resided mainly in one of the enan-
tiomers, to biologically characterize the compounds
searching for a potential candidate for more in-depth
pharmacological evaluation. The selected compounds
were the lead 5, the two methyl-cinnamylpiperazinyl
analogues 15f and 15g and the naphthylmethylpiper-
azinyl derivative 15h. The primary biological results are
summarized in Table 2.
active at those receptors than their corresponding (ꢀ)-
pairs. Thus, compound (+)-5 was approximately equi-
potent at both binding targets, showing higher affinity
than racemic 5. However, this increase was not corre-
lated with an improvement of potency in our xylazine
assay. Compound (+)-15f showed binding values com-
parable to its corresponding racemate, while its activity
in the pCA and xylazine tests remained quite compar-
able as well. The methyl-cinnamyl analogue (+)-15g
showed the best balance of in vivo activities. It showed
comparable potency in both assays, keeping the potency
in the pCA test but increasing by 8-fold the potency in
the xylazine test of its parent racemate. The naphthyl
derivative (+)-15h exhibited higher affinity at the a2-
adrenoceptors than its parent racemate, showing mod-
erate activity in the xylazine test as well. Although the
affinity at the SER transporter site was of the same
order, this compound showed the highest potency in our
pCA test (ED50=0.08 mg/kg). It is worthy to note that
although the (+) and (ꢀ) isomers of 15f and 15g
showed only slight in vitro difference in their affinities at
As can be deduced from the data shown in Table 2, the
affinity at the SER transporter site was comparable for
each pair of enantiomers. In the case of the enantiomers
of 15g and 15h their potencies were very similar to that
of the racemate, while the enantiomers of 5 showed
higher affinity and those of 15f showed slightly less affi-
nity than the corresponding racemate. On the contrary,
the stereochemical configuration did influence the affi-
nity at the a2-adrenoceptors. As a matter of fact the
four (+)-enantiomers were very significantly more

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J. I. Andres et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2719–2725
2725
the SER uptake site, a large difference in their activity in
the pCA test was observed. We have not any convincing
explanation for these results. All these compounds are
being more in-depth pharmacologically evaluated.
Naunyn Schmiedeberg’s Arch. Pharmacol. 1990, 342, 508.
12. Maggi, M.; Filipi, S.; Ledda, F.; Magini, A.; Forti, G.
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14. Schatzberg, A. F. J. Clin. Psychiatry 2000, 61, 38.
15. Lauritzen, L.; Clemmesen, L.; Klysner, R.; Loldrup, D.;
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16. Cappiello, A.; McDougle, C. J.; Malison, R. T.; Heninger,
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In summary, we have discovered a new series of 3-
piperazinylmethyl-3a,4-dihydro-3H-[1]benzopyrano[4,3-
c]isoxazoles as novel dual 5-HT uptake inhibitors and
a2-adrenoceptor antagonists. The combined activity
mainly resided in one of the enantiomers. Additional
chemical exploration of the most interesting structural
modifications is being carried out and will be published
elsewhere. The biological evaluation of the most pro-
mising compounds as potential new antidepressants will
be the subject for further publications.
18. Carpenter, L. L.; Jocic, Z.; Hall, J. M.; Rasmussen, S. A.;
Price, L. H. J. Clin. Psychiatry 1999, 60, 45.
19. Maes, M.; Libbrecht, I.; van Hunsel, F.; Campens, D.;
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Acknowledgements
21. Meyer, M. D.; Hancock, A. A.; Tietje, K.; Sippy, K. B.;
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23. Unpublished results.
24. Baraldi, P. G.; Bigoni, A.; Cacciari, B.; Caldari, C.; Man-
fredini, S.; Spalluto, G. Synthesis 1994, 1158.
25. For reviews of nitrile oxide-isoxazoline methodology
see: (a) Curran, D. P. In Advances in Cycloaddition; Cur-
ran, D. P., Ed.; JAI: Greenwich, CT, 1988; Vol. 1, p 129.
(b) Torssel, K. B. G. In Organic Nitro Chemistry Series. Nitrile
Oxides, Nitrones and Nitronates in Organic Synthesis. Novel
Strategies in Synthesis; Feuer, H., Ed.; VCH: Weinheim, 1988;
p 55.
The authors would like to thank Ms. Valle Ancos, Ms.
Alcira Del Cerro and Mr. Jos Leenaerts for their syn-
thetic chemistry work and Dr. Laura Iturrino and Mr.
Luis Font for their skilful analytical assistance. They
would also like to acknowledge Mr. Patrick C. M. Ver-
mote and Mr. Koen A. Hens for performing the pCA
and xylazine tests, respectively, and Dr. Antonio Gomez
and Mr. Willy Van Gorp for their help in the manage-
ment and coordination of all data generated.
References and Notes
26. p-Chloroamphetamine (pCA)-induced behaviour in rats:
p-Chloroamphetamine (pCA; 5 mg/10 mL/kg, sc)-induced
excitation was recorded over a 15-min interval starting 45 min
after the pCA injection in overnight-starved, male Wistar rats
(200–250 g; internal breeding facilities) pre-treated sub-
cutaneously with test compound or solvent 1 h earlier. A score
<2 was adopted as all-or-nothing criterion for drug-induced
inhibition of excitation (1.0% false positives; n>200).
27. Xylazine-induced loss of righting in rats: Xylazine (15.0
mg/kg, iv)-induced loss of righting reflex was recorded up to
120 min after injection in overnight-starved, male Wistar rats
(200–250 g; internal breeding facilities) pre-treated with test
compound or solvent 1 h earlier. Criterion for drug-induced
reversal: absence of loss of righting reflex (4.2% false positive
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