Synthesis and pharmacological evaluation of 18,19-Dehydrobuprenorphine

Article abstract of DOI:10.3987/com-00-s(i)46

18,19-Dehydrobuprenorphine (2) was prepared in five steps starting from thevinone (3) which is readily available from thebaine by Diels-Alder reaction. Grignard reaction with tert-BuMgBr afforded tert-butylthevinol (4) which was N-demethylated via N-cyano-tert-butylthevinol (5) using BrCN. Alkali treatment gave N-nor-tert-butylthevinol (6) which was alkylated with cyclopropylmethyl bromide to give N-cyclopropylmethyl-tert-butylthevinol (7), followed by ether cleavage with thiolate to yield 2. 18,19-Dehydrobuprenorphine displayed in opioid receptor binding studies very high affinity for μ receptors, while the affinity for κ and δ receptors was lower. In the tail-flick test in mice, compound (2) was 25 times more potent than morphine and ca. 2.5 times as potent as buprenorphine.

Full text of DOI:10.3987/com-00-s(i)46

HETEROCYCLES, Vol. 54, No. 2, pp.989-998, Received, 29th May, 2000
SYNTHESIS
AND
PHARMACOLOGICAL
EVALUATION
OF
#
18,19-DEHYDROBUPRENORPHINE
a
a
a
Johannes Schütz, Roland Krassnig, Helmut Schmidhammer,* Klaus
b
c
c
Wurst, Roberta Lattanzi, and Lucia Negri*
a: Division of Pharmaceutical Chemistry, Institute of Pharmacy, University of
Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
b: Institute of General, Inorganic and Theoretical Chemistry, University of
Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
c: Institute of Medical Pharmacology, University "La Sapienza", Piazza A. Moro
5
, I-00185 Rome, Italy
Abstract – 18,19-Dehydrobuprenorphine (2) was prepared in five steps starting
from thevinone (3) which is readily available from thebaine by Diels-Alder
reaction. Grignard reaction with tert-BuMgBr afforded tert-butylthevinol (4)
which was N-demethylated via N-cyano-tert-butylthevinol (5) using BrCN. Alkali
treatment gave N-nor-tert-butylthevinol (6) which was alkylated with
cyclopropylmethyl bromide to give N-cyclopropylmethyl-tert-butylthevinol (7),
followed by ether cleavage with thiolate to yield 2. 18,19-Dehydrobuprenorphine
displayed in opioid receptor binding studies very high affinity for µ receptors,
while the affinity for κ and δ receptors was lower. In the tail-flick test in mice,
compound 2 was 25 times more potent than morphine and ca. 2.5 times as
potent as buprenorphine.
1
Buprenorphine (1) is a strong and widely used analgesic with marked opioid agonist potency. It is a
2
partial agonist at µ opioid receptors and an antagonist at κ receptors. As a partial agonist, buprenorphine
produces limited opioid side effects and, thus, overdose is rare. Because of its affinity for µ receptors,
buprenorphine effectively prevents the effects of other opioids, thus reducing the likelyhood of heroin
#
th
This paper is dedicated to Prof. Dr. Sho Ito on the occasion of his 77 birthday and to Prof. Dr. Sándor Makleit
on the occasion of his 70 birthday.
th

3
abuse. As well as being a maintenance agent, buprenorphine has been prescribed for rapid detoxification
due to its reduced tendency to cause withdrawal effects and its ability to block the effects of other
4
5
opioids. A recent study demonstrated that buprenorphine was able to suppress ethanol intake in rats. It
was of interest to prepare the 18,19-dehydro analogue (2) of buprenorphine and to compare it biologically
and pharmacologically with buprenorphine.
Figure
N
1
8
CH3
1
9
20
OH
O
HO
OCH3
1
2
Buprenorphine
18,19-Dehydrobuprenorphine: ∆
1
8,19
RESULTS AND DISCUSSION
Synthesis
The synthesis of 18,19-dehydrobuprenorphine (2) has been disclosed earlier in two patent applications
6
,7
without any proof of the structure. We prepared compound (2) by a partly different route compared to
the routes described (outlined below). For structure elucidation of 2, NMR, IR, MS, elemental analysis
and X-Ray diffraction analysis (performed with intermediate (4)) were used.
8
Thevinone (3), which was readily available from thebaine by Diels-Alder reaction, underwent Grignard
9
reaction with tert-BuMgBr in tert-butylmethyl ether to yield tert-butylthevinol (4). The absolute
configuration of 4 was established by X-Ray diffraction analysis. N-Demethylation was accomplished by
6
using BrCN in CHCl to give cyanamide (5), which was treated with KOH in diethylene glycol at 170°C
3
6
6
to afford secondary amine (6). N-Alkylation with cyclopropylmethyl bromide gave compound (7),
which was 3-O-demethylated using sodium ethanethiolate in DMF to yield 18,19-dehydrobuprenorphine
6
,7
(
2). In ref. [6] the secondary amine (6) was acylated with cyclopropylcarbonyl chloride prior to
reduction of the resulting amide to amine (7). In ref. [7], cyanamide (5) was treated with KOH in

diethylene glycol at 200°C, the resulting 3-O-demethylated secondary amine acylated with
cyclopropylcarbonyl chloride and the corresponding amide reduced to 18,19-dehydrobuprenorphine (2)
with lithium aluminium hydride.
Scheme
CH3
R1
N
N
O
CH3
OH
CH3
OCH3
O
O
R O
OCH3
H CO
2
3
3
Thevinone
2 R = CPM, R = H
1 2
4
5
6
7
R = R = CH
1 2 3
R = CN, R = CH
3
1
2
R = H, R = CH
3
1
2
R = CPM, R = CH
3
1
2
CPM = cyclopropylmethyl
Pharmacological Evaluation
18,19-Dehydrobuprenorphine (2) was evaluated in comparison to buprenorphine (1) and morphine in
3
3
opioid receptor binding studies employing [ H]DAMGO (µ agonist), [ H]deltorphin II (δ agonist) and
3
[
H]U69593 (κ agonist) as radioligands (Table). Compound (2) displayed ca. three fold higher affinity for
µ opioid receptors than buprenorphine (1) but lower affinities for κ and δ receptors, thus exhibiting clear
preference for µ receptors. The µ/δ and µ/κ selectivity of compound (2) is about one order of magnitude
higher than that of buprenorphine (1).
1
0
Compound (2) produced dose-related antinociceptive effects in mice after subcutaneous administration
Table). Compound (2) was ca. twice as potent as buprenorphine (1) and 25 times more potent than
morphine. The antinociceptive effect was antagonized by naltrexone (5 mg/kg, s.c.).
(

Table
Opioid Receptor Binding Assay (ORBA) and Antinociceptive Potencies in the Tail-flick Assay
(TFA) of Compounds 1, 2 and Morphine
a)
ORBA (K , nM)
TFA
i
b)
Compound
µ
κ
δ
AD50 (µmol/kg)
(
1)
2.0
0.57
11.0
8.5
32.0
500
2.5
8.5
-
0.21
0.13
3.2
(2)
morphine
a) K = inhibition constant
i
b) AD = median antinoceptive dose
5
0
EXPERIMENTAL
General Details
Melting points: Kofler melting-point microscope; uncorrected. NMR Spectra: Varian Gemini 200
spectrometer; δ in ppm rel. to SiMe as internal reference, J in Hz. IR Spectra: Mattson Galaxy Series
4
FTIR 3000. Mass Spectra: Varian MAT 44 S apparatus. Elemental Analyses were performed at the
Institute of Physical Chemistry of the University of Vienna.
tert-Butylthevinol (= 4,5α−Epoxy-6α,14α-etheno-7α-(1-hydroxy-1,2,2-trimethylpropyl)-
3
,6β-dimethoxy-17-methylmorphinan; 4). 100 mL of 2 M tert-BuMgCl solution in diethyl ether was
evaporated in a 1 L three necked flask, 100 mL of tert-butyl methyl ether added and again evaporated.
This operation was repeated with 50 mL of tert-butyl methyl ether to give a colorless solid which was
dissolved in 350 mL tert-butyl methyl ether and refluxed under N . A Soxhlet apparatus containing 15.0 g
2
of thevinone ((3); 39.32 mmol) was placed onto the three necked flask and thevinone was slowly
dissolved by the refluxing tert-butyl methyl ether (3 h). After cooling to rt, the mixture was poured on
4
00 mL of saturated NH Cl solution. The organic phase was separated and the H O phase extracted with
4
2
Et O (2 x 80 mL, 2 x 50 mL). The combined organic layers were washed with H O (100 mL) and brine
2
2
(
100 mL), dried (Na SO ) and evaporated to give 16.49 g of a slightly yellow solid which was treated
2
4

with boiling MeOH (20 mL) to yield 10.03 g (58%) of 4 which contains a little amount of thevinone
TLC). This material was used for the next step without further purification. An analytical sample was
obtained the following way: 1.58 g of 4 were recrystallized three times from MeOH/CH Cl (6:1) to
(
2
2
give 1.08 g of 4 which contained very little thevinone (TLC). Flash chromatography of this material
silica gel; elution with CH Cl /MeOH/conc.NH OH 250:2:0.5) afforded 790 mg of crystalline 4 which
(
2
2
4
9
was treated with 3 mL of MeOH to yield 720 mg of pure 4. mp 220-222°C (lit., 216°C (EtOH)). IR
1
(
KBr): 3438 (OH). H-NMR (CDCl ): 6.62 (d, J = 8.1, 1 arom. H); 6.51 (d, J = 8.1, 1 arom. H); 5.99 (d,
3
J = 8.7, 1 olef. H); 5.62 (s, OH-C(20)); 5.43 (d, J = 8.7, 1 olef. H); 4.56 (s, H-C(5)); 3.82 (s, CH O-C(3));
3
1
3
3
1
.77 (s, CH O-C(6)); 2.36 (s, CH N); 1.01 (s, (CH ) C-C(20)); 0.99 (s, CH -C(20)). C-NMR (CDCl ):
3 3 3 3 3 3
48.73 (arom. C); 142.38 (arom. C); 135.96 (olef. CH(18)); 135.19 (arom. C); 129.14 (arom. C); 125.52
(olef. CH(19)); 119.91 (arom. CH(1)); 114.49 (arom CH(2)); 99.47 (CH(5)); 85.02 (C(6) or C(20); 79.02
(
C(6) or C(20); 60.60 (CH(9) or CH O-C(3) or CH O-C(6)); 57.47 (CH(9) or CH O-C(3) or CH O-C(6));
3
3
3
3
55.84 (CH(9) or CH O-C(3) or CH O-C(6)); 47.01 (quart. C); 46.49 (CH); 46.10 (CH ); 44.18 (N-CH );
3 3 2 3
4
3.79 (quart. C); 40.32 (quart. C); 34.40 (CH ); 32.68 (CH ); 27.28 ((CH ) C-C(20)); 22.95 (CH ); 20.30
2
2
3 3
2
+
(
CH -C(20)). EI-MS: 439 (M ). Anal. Calcd for C H NO ⋅ 0.3 MeOH: C 73.00, H 8.57, N 3.12. Found:
3 27 37 4
C 72.96, H 8.43, N 3.11.
Crystal Structure Data of 4: C H NO , M 439.58; orthorhombic, P2 2 2 , a = 10.693(2),
2
7
33
4
1 1 1
3
3
b = 12.094(2), c = 17.999(4) Å, V = 2327.7(7) Å (λ 0.71073), Z = 4, D = 1.254 g/cm , F(000) = 952;
calc
-1
µ = 0.083 mm , crystal size 0.9 x 0.7 x 0.35 mm. Data were collected at 213(2) K in the θ range
2.5 - 24.0° on a Bruker P4 diffractometer. Data were measured via ω-scans and corrected for Lorentz and
polarisation effects, but not for absorption. The structure was solved by direct methods (SHELXS-86) and
2
refined by full matrix least-squares against F (SHELXL-93). All non-hydrogen atoms were refined with
anisotropic displacement parameters. All hydrogen atoms were located by difference Fourier methods
and refined at the hydroxyl group, the others were generated geometrically and refined with isotropic
displacement parameters 1.2 and 1.5 times higher than U(eq) of the attached C atoms. In the final least-
squares refinement cycles the model converged at R = 0.0306, wR = 0.0773, and GOF = 1.067 for 3464
1
2
reflections with F ≥ 4σ(F ) and 301 parameters. Copies of the crystallographic data are available on
o
o
application to the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK
fax: +44(1223)336033; e-mail: deposit@ccdc.cam.ac.uk) citing the deposition No CCDC-135525.
(

ORTEP plot of the molecular structure of 4 (with 30% probability ellipsoids)
N-Cyano-tert-Butylthevinol (= 17-Cyano-4,5α-epoxy-6α,14α-etheno-7α-(1-hydroxy-
1,2,2-trimethylpropyl)-3,6β-dimethoxymorphinan; 5). A mixture of 4 (10.05 g, 22.86 mmol), BrCN
(
4.70 g, 44.37 mmol) and CHCl (45 mL) was refluxed for 18 h and then evaporated. The crystalline
3
residue (9.61 g) was recrystallized from 30 mL of EtOH to afford 9.06 g (89%) of slightly impure 5
which was not further purified for the next step. Flash chromatography of 2.0 g of this slightly impure 5
(see above) gave 840 mg of crystalline 5 which was treated with 5 mL of MeOH to yield 600 mg of pure
6
1
5
. mp 265-267°C (lit., 254-258°C (EtOH)). IR (KBr): 3457, 2202 (OH, NCN). H-NMR (CDCl ): 6.67
3
(
(
(
(
(
d, J = 8.2, 1 arom. H); 6.56 (d, J = 8.2, 1 arom. H); 6.09 (d, J = 8.9, 1 olef. H); 5.47 (s, OH-C(20)); 5.37
d, J = 8.9, 1 olef. H); 4.56 (d, J = 1.0, H-C(5)); 3.83 (s, CH O-C(3)); 3.77 (s, CH O-C(6)); 1.01 (s,
3
3
13
CH ) C-C(20)); 0.99 (s, CH -C(20)). C-NMR (CDCl ): 148.93 (arom. C); 143.03 (arom. C); 133.38
3
3
3
3
olef. CH(18)); 133.01 (arom. C); 127.07 (olef. CH(19)); 126.19 (arom. C); 120.52 (arom. CH(1)); 118.61
NCN); 115.26 (arom CH(2)); 98.95 (CH(5)); 84.56 (C(6) or C(20); 78.77 (C(6) or C(20); 59.02 (CH(9)

or CH O-C(3) or CH O-C(6)); 57.37 (CH(9) or CH O-C(3) or CH O-C(6)); 55.96 (CH(9) or CH O-C(3)
3
3
3
3
3
or CH O-C(6)); 46.47 (quart. C); 46.42 (CH); 42.61 (CH ); 42.51 (quart. C); 40.32 (quart. C); 32.43
3
2
(
CH ); 27.23 ((CH ) C-C(20)); 20.18 (CH -C(20)). EI-MS: 450 (M
+
). Anal. Calcd for C27
H
34
N
2
O4 ·
2
3 3
3
0.1 MeOH: C 71.73, H 7.64, N 6.17. Found: C 71.78, H 7.73, N 6.14.
N-nor-tert-Butylthevinol (= 4,5α-Epoxy-6α,14α-etheno-7α-(1-hydroxy-1,2,2-trimethylpropyl)-
,6β-dimethoxymorphinan; 6). A mixture of KOH (10.0 g, 178.22 mmol), 5 (10.0 g, 22.19 mmol) and
diethylene glycol (70 mL) was stirred at 165-175°C (bath temperature) for 80 min, cooled to rt and
poured on 550 mL of ice water. This mixture was extracted with CH Cl (1 x 150 mL, 3 x 50 mL), the
3
2
2
combined organic layers washed with H O (100 mL) and brine (100 mL), dried (Na SO ) and
2
2
4
evaporated to give 8.22 g of a slightly yellow crystalline residue. Treatement with boiling MeOH (20 mL)
yielded 7.07 g (75%) of slightly impure 6 which was not further purified for the next step. Flash
chromatography of 1.87 g of this slightly impure 6 (see above) gave 1.63 g of crystalline 6 which was
6
treated with 5 mL of MeOH to yield 1.31 g of pure 6. mp 204-206°C (lit., 186-190°C). IR (KBr): 3382,
1
3
316 (OH, NH). H-NMR (CDCl ): 6.64 (d, J = 8.3, 1 arom. H); 6.52 (d, J = 8.3, 1 arom. H); 6.03 (d,
3
J = 9.1, 1 olef. H); 5.59 (s, OH-C(20)); 5.41 (d, J = 9.1, 1 olef. H); 4.55 (d, J = 1.4, H-C(5)); 3.83 (s,
1
3
CH O-C(3)); 3.78 (s, CH O-C(6)); 1.02 (s, (CH ) C-C(20)); 1.00 (s, CH -C(20)). C-NMR (CDCl ):
3
3
3 3
3
3
148.78 (arom. C); 142.53 (arom. C); 135.50 (olef. CH(18)); 134.85 (arom. C); 128.68 (arom. C); 126.12
(olef. CH(19)); 120.01 (arom. CH(1)); 114.67 (arom CH(2)); 99.81 (CH(5)); 84.81 (C(6) or C(20); 78.95
(
C(6) or C(20); 57.45 (CH(9) or CH O-C(3) or CH O-C(6)); 55.88 (CH(9) or CH O-C(3) or CH O-C(6));
3
3
3
3
5
3.54 (CH(9) or CH O-C(3) or CH O-C(6)); 47.75 (quart. C); 46.59 (CH); 42.85 (quart. C); 40.34 (quart.
3 3
C); 37.88 (CH ); 34.70 (CH ); 33.82 (CH ); 32.59 (CH ); 27.29 ((CH ) C-C(20)); 20.27 (CH -C(20)).
2
2
2
2
3 3
3
+
EI-MS: 425 (M ). Anal. Calcd for C H NO ⋅ 0.1 MeOH: C 73.11, H 8.32, N 3.27. Found: C 73.06,
2
6
35
4
H 8.35, N 3.25.
N-Cyclopropylmethyl-tert-Butylthevinol (= 17-Cyclopropylmethyl-4,5α-epoxy-6α,14α-etheno-
α-(1-hydroxy-1,2,2-trimethylpropyl)-3,6β-dimethoxymorphinan; 7). A mixture of 6 (10.0 g, 23.5
7
mmol), NaHCO (12.0 g, 142.84 mmol), cyclopropylmethyl bromide (3.0 mL, 30.0 mmol) and
3
anhydrous DMF (75 mL) was stirred at 80° (bath temperature) for 50 min, cooled to rt and filtered. The
filtrate was diluted with H O (500 mL) and extracted with CH Cl (2 x 100 mL, 3 x 50 mL). The
2 2
2
combined organic layers were washed with H O (3 x 200 mL) and brine (150 mL), dried (Na SO ) and
2
2
4
evaporated to give 11.12 g of a crystalline solid which was treated with boiling MeOH (20 mL) to afford
0.23 g (91%) of slightly impure 7 which was not further purified for the next step. Flash chromatography
1

of 1.8 g of this slightly impure 7 (see above) gave 1.7 g of an oil which was crystallized from a mixture
of MeOH (3 mL) and CH Cl (1 mL) to yield 1.53 g of 7. Recrystallization from a mixture of MeOH (5
2
2
6
mL) and CH Cl (1 mL) afforded 910 mg of pure 7. mp 165-168°C (lit., 145-153°C). IR (KBr): 3463
2
2
1
(
OH). H-NMR (CDCl ): 6.61 (d, J = 8.2, 1 arom. H); 6.48 (d, J = 8.2, 1 arom. H); 5.99 (d, J = 8.9, 1 olef.
3
H); 5.63 (s, OH-C(20)); 5.44 (d, J = 8.9, 1 olef. H); 4.56 (d, J = 1.2, H-C(5)); 3.82 (s, CH O-C(3)); 3.77
3
13
(
s, CH O-C(6)); 1.01 (s, (CH ) C-C(20), CH -C(20)); 0.51 (m, CH (cp)); 0.15 (m, CH (cp)). C-NMR
3 3 3 3 2 2
(
CDCl ): 148.74 (arom. C); 142.33 (arom. C); 136.18 (olef. CH(18)); 135.41 (arom. C); 129.15 (arom.
3
C); 125.44 (olef. CH(19)); 119.89 (arom. CH(1)); 114.44 (arom CH(2)); 99.62 (CH(5)); 85.13 (C(6) or
C(20); 79.01 (C(6) or C(20); 60.17 (N-CH -cp); 57.46 (CH(9) or CH O-C(3) or CH O-C(6)); 55.84
2
3
3
(
(
(
CH(9) or CH O-C(3) or CH O-C(6)); 47.73 (quart. C); 46.48 (CH); 44.72 (CH ); 43.71 (quart. C); 40.32
3
3
2
quart. C); 34.61 (CH ); 32.81 (CH ); 27.28 ((CH ) C-C(20)); 23.76 (CH ); 20.25 (CH -C(20)); 10.13
2
2
3 3
2
3
+
CH(cp)); 4.94 (CH (cp)); 3.82 (CH (cp)). EI-MS: 479 (M ). Anal. Calcd for C H NO ⋅ 0.8 MeOH:
2
2
30 41
4
C 73.21, H 8.82, N 2.77. Found: C 73.17, H 8.70, N 2.76.
1
1
8,19-Dehydrobuprenorphine (= 17-Cyclopropylmethyl-4,5α-epoxy-6α,14αetheno-7α-(1-hydroxy-
,2,2-trimethylpropyl)-6β-methoxymorphinan-3-ol; 2). A mixture of 7 (9.5 g, 19.8 mmol), sodium
ethanethiolate (6.66 g, 79.18 mmol) and anhydrous DMF (150 mL) was stirred under N at 130° (bath
2
temeperature) for 3.5 h, cooled to rt and poured on saturated NH Cl solution (700 mL). The mixture was
4
alkalinized with conc. NH OH, extracted with CH Cl (3 x 100 mL, 3 x 50 mL), the combined organic
2 2
4
layers washed with H O (3 x 200 mL) and brine (100 mL), dried (Na SO ) and evaporated to yield
2
2
4
10.4 g of a yellowish crystalline solid which was recrystallized from MeOH (20 mL) to give 6.89 g (75%)
of slightly impure 2. Flash chromatography of 850 mg of this slightly impure 2 (see above) gave 650 mg
6
colorless crystals of 2 which was recrystallized from MeOH to yield 530 mg pure 2. mp 245-247°C (lit.,
7
1
2
38-240°C (MeOH), lit., 237.5-239° (MeOH)). IR (KBr): 3423, 3280 (OH-C(20), OH-C(3)). H-NMR
(
CDCl ): 6.59 (d, J = 8.1, 1 arom. H); 6.45 (d, J = 8.1, 1 arom. H); 5.96 (d, J = 8.9, 1 olef. H); 5.58 (s,
3
OH-C(20)); 5.43 (d, J = 8.9, 1 olef. H); 4.59 (d, J = 1.4, H-C(5)); 3.76 (s, CH O-C(6)); 1.01 (s, (CH ) C-
3
3 3
1
3
C(20), CH -C(20)); 0.52 (m, CH (cp)); 0.15 (m, CH (cp)). C-NMR (CDCl ): 147.30 (arom. C); 138.06
3
2
2
3
(
arom. C); 136.35 (olef. CH(18)); 135.16 (arom. C); 128.56 (arom. C); 125.12 (olef. CH(19)); 120.39
arom. CH(1)); 116.83 (arom CH(2)); 99.98 (CH(5)); 85.19 (C(6) or C(20); 79.32 (C(6) or C(20); 60.20
N-CH -cp); 57.48 (CH(9) or CH O-C(6)); 55.85 (CH(9) or CH O-C(6)); 48.07 (quart. C); 46.44 (CH);
(
(
2
3
3
4
4.77 (CH ); 43.81 (quart. C); 40.34 (quart. C); 34.56 (CH ); 32.81 (CH ); 27.30 ((CH ) C-C(20)); 23.84
2 2 2 3 3
+
(
CH ); 20.29 (CH -C(20)); 10.13 (CH(cp)); 4.95 (CH (cp)); 3.84 (CH (cp)). EI-MS: 465 (M ). Anal.
2 3 2 2
Calcd for C H NO ⋅ 0.1 MeOH: C 74.55, H 8.47, N 2.99. Found: C 74.55, H 8.72, N 2.97.
2
9
39
4

Opioid receptor binding assay
Binding to µ and δ opioid receptors was assayed on crude membrane preparations from adult male rat
brain (Wistar, 250-300 g) and binding to κ opioid receptors was assayed on crude membrane preparations
1
1
from adult male guinea-pig brain (450 g) as previously described. The inhibition constant (K ) was
i
1
2
calculated from competitive binding curves with the computer program Ligand. Data obtained from four
independent measurements are presented as the arithmetic mean.
Tail-flick assay
Male Swiss CD-1 mice (30-40 g) were used for the experiments. They were housed at 22 ± 2 °C, with
food and water ad libitum. A standard light/dark cycle was maintained with a time-regulated light period
from 6 h to 18 h. The IASP guidelines on ethical standards for investigations of experimental pain in
animals were followed. Compounds were dissolved in 10% DMSO and administered subcutaneously
-1
-1
(
s.c.) to the mice in a volume of 5 µL g of body weight. Control mice received vehicle at 5 µL g . Each
animal received one injection only. Every dose of each compound was evaluated in groups of 6-8
1
0
animals. Antinociception was assessed by exposing the mouse tail to radiant heat and the latency to a
rapid tail-flick was recorded with the baseline cutoff and the maximal possible latencies set at 4 sec and
1
2 sec, respectively. The degree of antinociception was expressed as percentage maximum possible effect
% MPE) according to the equation: % MPE = [(test latency - control latency)/(12 - control latency)] x
00. The tail-flick latency was measured before drug treatment (control) and every 15 min after drug
(
1
injections, during the first hour, and every 30 min thereafter, until antinociception disappeared. A
computer program (PRISM, GraphPad, CA, USA) was used to calculate the mean peak effect for each
dose. Doses that produced peak effects between 20% and 80% MPE were plotted into a log dose-response
1
3
curve and AD values were calculated as doses that produced 50% MPE.
5
0
ACKNOLEDGEMENT
We wish to thank Tasmanian Alkaloids Pty., Ltd.,Westbury, Tasmania, Australia, for the generous gift of
thebaine. We further thank Dr. D. Rakowitz (Division of Pharmaceutical Chemistry, Institute of
Pharmacy, University of Innsbruck) for performing the mass spectra. This work was in part supported by
the Österreichische Nationalbank (Jubiläumsfondsprojekt Nr. 5973/7).

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