Synthesis and biological evaluation of some 6-arylamidomorphines as analogues of morphine-6-glucuronide

Article abstract of DOI:10.1016/j.bmc.2004.08.019

A series of 6-β-arylamidomorphines was synthesized and biologically evaluated. Various aryl substituents were introduced into the arylamidomorphines to examine substituent structure-activity relationships. Competition binding assays showed that compounds 10a-h bound to the μ opioid receptor with high affinity (0.2-0.6 nM). Functional assays showed that compounds 10a-h acted as full μ opioid receptor agonists. The ED₅₀ of compound 10e·HCl as an analgesic was 12.6 mg/kg in the tail flick latency test in the rat.

Full text of DOI:10.1016/j.bmc.2004.08.019

Bioorganic & Medicinal Chemistry 12 (2004) 5983–5990
Synthesis and biological evaluation of some
6-arylamidomorphines as analogues of morphine-6-glucuronide
James M. MacDougall,a,* Xiao-Dong Zhang,a Willma E. Polgar,b Taline V. Khroyan,b
Lawrence Tollb and John R. Cashmana,*
aHuman BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121-2804, USA
bSRI International, 333 Ravenswood Ave., Menlo Park, CA 94025-3493, USA
Received 29 July 2004; accepted 12 August 2004
Available online 11 September 2004
Abstract—A series of 6-b-arylamidomorphines was synthesized and biologically evaluated. Various aryl substituents were intro-
duced into the arylamidomorphines to examine substituent structure–activity relationships. Competition binding assays showed that
compounds 10a–h bound to the l opioid receptor with high affinity (0.2–0.6nM). Functional assays showed that compounds 10a–h
acted as full l opioid receptor agonists. The ED50 of compound 10eÆHCl as an analgesic was 12.6mg/kg in the tail flick latency test in
the rat.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Me
H
Me
H
N
O
N
O
We recently reported the synthesis and biological evalu-
ation of a small library of thiosaccharide analogues of
morphine-6-glucuronide (M6G) 1 with the objective of
finding a compound with improved pharmacological
activity (Fig. 1).1 The thiosaccharides were evaluated
as opioid receptor ligands by competition binding as-
says, functional assays and for in vivo antinociceptive
activity. Both the 6-b-S-glucuronide, 2 and the 6-b-S-
glucoside, 3 showed high affinity for the l opioid recep-
tor and functioned as potent agonists (Fig. 1). The
evaluation of the thiosaccharides were of interest due
to the high analgesic potency and reduced side effects
of M6G compared to morphine.2 However, due to the
lowbioavailability of M6G it was of interest to deter-
mine if simpler more hydrolytically stable analogues
could be developed.3 Two studies have appeared in the
literature related to this question. A series of M6G ana-
logues was reported in which the carbohydrate residue,
the N-substituent and the (O)-3 substituent and satura-
tion of the 7,8-double bond were varied.4 Only the 6-
b-glucoside or 6-b-glucuronide showed potent agonism.
A series of compounds in which the sugar residue of
O
O
HO
HOOC
HO
O
S
R
OH
OH
HO
HO
HO
HO
1
2: R = COOH
3: R = CH2OH
Me
N
H
O
HO
N H
O
(CH2)n
4
Ar
Figure 1. M6G, 1 and analogues 2, 3, and 4.
M6G was replaced with non-saccharide hydrophobic
groups introduced through Wittig reactions on dihydro-
codeinone was also reported.5 None of these compounds
showed improved analgesic properties relative to co-
deine. The objective of the present study was to further
Keywords: 6-Arylamidomorphines; Morphine-6-glucuronide; Analge-
sics; Opioids.
*
Corresponding authors. Tel.: +1 858 458 9305; fax: +1 858 458
9311; e-mail addresses: jmacdougall@hbri.org; jcashman@hbri.org
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.019

5984
J. M. MacDougall et al. / Bioorg. Med. Chem. 12 (2004) 5983–5990
investigate the role of the saccharide portion of M6G on
biological activity. In this study, we investigated whether
the saccharide portion of M6G could be replaced with
simpler functionality, as represented by the general
structure 4 (Fig. 1), while retaining a similar pharmaco-
logical potency and functional profile-higher affinity for
the l versus d and j receptors and l agonism. The syn-
thesis and biological evaluation of this series of com-
pounds is reported herein.
the product 7 by heating in warm ethanol with hydra-
zine hydrate to give the primary amine 8 (94%). Treat-
ment of 8 with a variety of aryl and heteroaryl acid
chlorides in the presence of triethylamine in dichloro-
methane gave amides 9a–h (60–82%), containing a TIPS
group attached to the oxygen atom at the 3-position.
Subsequent treatment of the silyl ethers 9a–h with
TBAF in THF then gave the desired 6-b-amidomor-
phines 10a–h (56–82%). Treatment of the methyl ester
10h with lithium hydroxide in THF/H2O gave the carb-
oxylic acid 11 (69%). A characteristic singlet (d 4.85–
4.62) for the C-5 phenanthrene proton was evident in
2. Chemistry
1
the H NMR spectra of the amides 10a–h and 11. This
indicated that the dihedral angle with the vicinal C-6
The morphine amides 10a–h and 11 used in these studies
were prepared in good yields according to the synthetic
method outlined in Scheme 1. Morphine sulfate 5 was
converted to its 6-b-phthalimide 6 in good yield with a
three step procedure involving selective acetylation of
the phenolic hydroxyl group utilizing a slight variation
of the Welsh procedure,6 Mitsunobu reaction to replace
the C-6 a-disposed hydroxyl group with a b-disposed
phthalimido group7 and then selective removal of the
phenolic acetate group by treatment with hydroxylam-
ine hydrochloride in hot ethanol.7 Triisopropylsilyation
of the phenolic hydroxyl group in 6 (76%) was followed
by removal of the C-6 phthalimide protecting group in
a-disposed proton was approximately 90ꢁ. This observa-
tion was in agreement with the H NMR spectral data
1
from C-6 b-substituted morphine compounds prepared
by others.1,8 The 1H and 13C NMR and HRMS spectral
data for the compounds 10a–h and 11 were in agreement
with the proposed structures.
3. Results and discussion
The IC50 values obtained from competition binding as-
says for compounds 10a–h and 11 were converted into
Me
Me
N
Me
N
H
N
H
H
H
iv
i-iii
2-
SO4
72%
O
76%
O
O
O
O
HO
OH
HO
N
TIPSO
N
2
O
O
7
5
6
Me
N
Me
Me
N
N
H
H
H
vii
vi
v
94%
56-85%
49-81%
O
R
O
R
O
O
O
TIPSO
N H
H
TIPSO
N
H
HO
10a-h
N
H
8
9a-h
10e
R =
a
b
c
e
OMe
CO2Me
69% viii
Me
OMe
f
N
NO2
Cl
H
g
O
O
Cl
OMe
HO
11
N
H
S
COOH
d
h
Scheme 1. Reagents and conditions: (i) Ac2O, NaHCO3, H2O; (ii) PPh3, i-PrO2CN@NCO2i-Pr, phthalimide; (iii) NH2OHÆHCl, EtOH, 55ꢁC;
(iv) TIPSCl, imidazole, DMF; (v) NH2NH2, EtOH, 55ꢁC; (vi) RC(O)Cl, NEt3, CH2Cl2; (vii) TBAF, THF; (viii) LiOH, H2O, AcOH.

J. M. MacDougall et al. / Bioorg. Med. Chem. 12 (2004) 5983–5990
5985
Ki values as described in Section 5 and are listed in Table
2. In the binding assays the following radioligands were
used: [3H]DAMGO (l opioid receptor agonist);
[3H]U69593 (j opioid receptor agonist); [3H]DPDPE
(d opioid receptor agonist). Ki values were determined
by measuring the inhibition of binding of these radio-
ligands to the receptor by the test compounds 10a–h
and 11.9 With the exception of compound 11
(Ki = 19.92nM), each of the test compounds 10a–h
showed high affinity (Ki = 0.20–0.59nM) for the l opi-
oid receptor. Compounds 10a–h possessed 21–64-fold
higher affinity for the l receptor compared with M6G
and 1.9–5.5-fold higher affinity for the l receptor com-
pared with morphine. The rank order of affinity for
the most potent compounds at the l receptor was
10g = 10f = 10e = 10c > 10h = 10a > 10d > 10b ꢀ 11. In
general, the compounds 10a–h showed significantly
more l versus d receptor selectivity than l versus j
receptor selectivity as indicated by the ratio of d/l and
j/l values (Table 1). An exception to this trend was
compound 11, prepared in order to provide a simple mi-
mic of M6G. Interestingly, the affinity of compound 11
for the l receptor was reduced by a factor of 87 relative
to its methyl ester 10c. The l versus d and l versus j
selectivity of compound 11 paralleled that observed for
M6G, suggesting that a negatively charged group pro-
duces a highly unfavorable interaction in the j binding
pocket.10 Compounds 10a, 10g, and 10h also showed
very high affinity for the j receptor (0.58–0.96nM).
With the exception of compound 10g (Ki = 0.94nM),
all compounds examined showed significantly less
affinity for the d receptor. Compound 10g was the least
selective of all compounds evaluated, at all three
opioid receptors examined.
In order to evaluate the opioid receptor-mediated acti-
vation of its associated G protein, test compounds were
evaluated using the [35S]GTPcS assay.11 In this assay, a
compoundꢀs potency or affinity for the receptor is de-
fined by its EC50 for stimulating [35S]GTPcS binding.
Agonist efficacy is defined as the degree to which the
compound maximally stimulates [35S]GTPcS binding
relative to control. The EC50 value represents the con-
centration of a compound that produced 50% maximal
stimulation of [35S]GTPcS binding. Full agonists stimu-
late [35S]GTPcS binding to a maximal extent and partial
agonists cause a reduced level of binding. Table 2 gives
the Emax and EC50 values for stimulation of [35S]GTPcS
binding at human l, d, and j opioid receptors. The
[35S]GTPcS binding data showed that compounds 10a
and 10c–h were full l agonists, and that compounds
10b and 11 were partial l agonists. Compounds 10e–g
were found to be full d agonists, whereas the remaining
compounds were found to be partial d agonists. Com-
pounds 10a–f and 10h were found to be full j agonists,
whereas compounds 10g and 11 showed decreased effi-
cacy and were therefore characterized as partial j agon-
ists. The overall rank order of the EC50 values for the
functional assay correlated with the Ki values derived
from the binding experiments.
Table 1. Ki inhibition values of l, d, and j opioid binding to CHO membranes by compounds 10a–h and 11
Compound
Ki ( nM) SEM
d/l
j/l
l
d
j
Morphine
M6G
10a
1.1 0.1
140
2
46.9 14
127
13
27
15
43
62
15
90
5
42
316
2.7
12.85 0.95
0.35 0.08
0.59 0.19
0.21 0.07
0.40 0.03
0.23 0.05
0.20 0.04
0.20 0.04
0.33 0.02
19.92 4.29
160.96 0.73
9.5 2.6
8.89 2.1
4058.75 230
0.96 0.2
2.84 0.96
2.65 1.25
4.13 1.21
1.53 0.2
2.63 1.1
0.75 0.2
0.58 0.17
>10K
10b
10c
5
13
10
8.96 1.95
24.95 2.33
3.39 0.03
18.0 5.63
0.94 0.02
14.42 1.26
50.78 8.86
10d
10e
6.7
10f
10g
13
4
10h
11
44
2
>500
2.5
Table 2. Stimulation of [35S]GTPcS binding by amides 10a–h and 11 mediated by l, d, and j opioid receptors
Compound
l
d
j
EC50
Emax
EC50
Emax
EC50
Emax
M6G
10a
10b
10c
10d
10e
10f
72.3 26.7
1.7 0.2
4.8 2.1
2.8 0.2
5.5 0.9
2.4 0.3
1.9 0.2
0.1 0.0
6.0 2.1
65.1 21.8
45.0 5.0
99.0 4.0
72.5 14.5
87.5 6.5
90.5 9.5
98.1 7.9
95.5 7.5
95.5 10.5
95.8 19.3
71.0 8.0
190.35 22.9
18.8 2.2
25.2 3.0
37.8 1.9
62.7 24.0
6.20 0.42
24.5 3.0
1.3 0.3
80.0
0
>10K
Flat
51.0 4.0
59.5 3.5
65.5 5.5
61.3 2.3
99.63 2.19
94.1 10.9
106.5 1.5
57.3 2.8
69.5 3.5
4.8 0.1
5.0 1.0
9.8 0.02
5.3 1.3
11.22 3.51
1.2 0.1
0.03 0.02
4.5 0.2
5096
89.0 6.0
81.5 1.5
86.0 0.0
81.1 8.9
88.6 8.6
90.3 4.8
78.5 1.5
98.0 1.0
71 12
10g
10h
11
32.4 2.1
74.0 12.7

5986
100
J. M. MacDougall et al. / Bioorg. Med. Chem. 12 (2004) 5983–5990
100
30-min
60-min
+
+
*
+
*
30-min
*
60-min
80
80
60
40
20
0
60
*
*
40
20
0
Vehicle
0.1
1.0
3.0
10.0
30.0
Vehicle
3.0
10.0
30.0
-20
Dose of 10e (s.c., mg/kg)
Dose of Morphine (s.c., mg/kg)
Figure 2. The two highest doses of 10e produced analgesic effects.
Data are mean %MPE ( SEM). Asterisks represent significant
differences from vehicle controls (Student Newman–Keuls, p < 0.05).
Plus signs represent a significant difference between 10 and 30mg/kg
10e (Student Newman–Keuls, p < 0.05).
Figure 3. Dose-dependent analgesia induced by administration of
morphine. Data are maximum possible antinociceptive effect (%MPE)
( SEM). Asterisks represent significant differences from vehicle
controls (Student Newman–Keuls, p < 0.05). Plus signs represent a
significant difference between 10 and 30mg/kg morphine (Student
Newman–Keuls, p < 0.05).
We evaluated compound 10eÆHCl for its analgesic effect
on tail flick latency in mice because 10e possessed a
broad spectrum of potency for l, d, and j opioid recep-
tors.12 Administration of 10eÆHCl produced an increase
in tail flick latency that was evident at 30min and con-
tinued to be present at 60min (Fig. 2). The overall
the effect on pharmacological activity observed by
replacing the saccharide portion of M6G with arylamide
groups.
5. Experimental
5.1. Chemistry
ANOVA indicated
a
significant effect of dose
[F(5,72) = 14.86, P < 0.0001]. Averaged across post-
injection time, the two highest doses of 10eÆHCl pro-
duced a significant increase in tail flick latency relative
to controls (Student Newman–Keuls, p < 0.05). The
analgesic effects of 30mg/kg dose of 10eÆHCl was
twofold greater than that produced by the 10mg/kg
dose (Student Newman–Keuls, p < 0.05). Compound
10eÆHCl appeared to have a slower onset of action, with
significantly increased potency at 60min over 30min.
Considering its very great potency at l receptors
(Ki = 0.23nM) and full agonist activity at the l receptor
with respect to stimulation of [35S]GTPcS binding
(EC50 = 2.4nM), compound 10eÆHCl had relatively
weak antinociceptive activity, with ED50 values of 30.2
and 12.6mg/kg at 30 and 60min, respectively. The rela-
tively poor ED50 value of 10eÆHCl may indicate poor
bioavailability relative to morphine that had an ED50
of approximately 3mg/kg under the same experimental
conditions (Fig. 3). We speculate that in vivo hydrolysis
of the methyl ester in 10e to the less active carboxylic
acid confounds the functional activity.
5.1.1. General information. All reactions were conducted
under a positive pressure of dry nitrogen with magnetic
stirring at ambient temperature using oven-dried glass-
ware unless otherwise indicated. Air- and moisture-sen-
sitive liquids were transferred via syringe through rubber
septa. The term brine refers to a saturated solution of
sodium chloride. Silica gel (230–400mesh) was used
for column chromatography. DMF was dried through
a column of neutral alumina and stored over activated
˚
4A molecular sieves under nitrogen prior to use. CH2Cl2
and THF were distilled from CaH2 immediately prior to
use. All other solvents and reagents were used as re-
1
ceived. H NMR spectra were recorded at 18ꢁC on a
500MHz Varian NMR (NuMega Resonance, San Die-
go, CA). Chemical shifts were reported in ppm (d) rela-
tive to CDCl3 at 7.26ppm unless indicated otherwise.
Lowresolution mass spectra was done on an Agilent
1100 MSD (HT Labs, San Diego, CA). High-resolution
mass spectra was done on a VG 7070 spectrometer with
Opus V3.1 and DEC 3000 Alpha Station data system at
the University of California Riverside. Melting points
were reported uncorrected. Where combustion analyses
are not specified, analytical purities were determined by
straight phase HPLC using a Hitachi L74 liquid chro-
matograph with a D7500 integrator and a Hamilton
PRP-I stainless steel column (250mm · 4.6mm id).
For determination of analytical purities: mobile phase
A = 60/40/0.02 MeOH/2-propanol/HClO4 (v:v); mobile
phase B = 55/45/0.018 MeOH/2-propanol/HClO4 (v:v).
4. Conclusion
In summary, a series of nine morphine arylamides was
synthesized by the reaction of the 6-b-aminomorphine,
8 with aryl and heteroaryl acid chlorides. Compounds
10a–h were found to bind to the l opioid receptor with
significant potency (0.20–0.59nM). Functional assays
showed that compounds 10a–h were full l opioid
receptor agonists. The carboxylic acid derivative, 11
possessed significantly diminished affinity for the l
receptor and functional efficacy. Compounds 10a, 10g,
and 10h also showed high affinity and functioned as full
agonists at the j opioid receptor. These studies showed
5.1.2. 3-O-Triisopropylsilyl-6-b-phthalimidomorphine 7.
6-b-Phthalimidomorphine,7 6 (1.89g, 4.55mmol) and
imidazole (0.74g, 10.9mmol) were dissolved in CH2Cl2
(20mL) and TIPSCl (1.17mL, 5.45mmol) was added

J. M. MacDougall et al. / Bioorg. Med. Chem. 12 (2004) 5983–5990
5987
by syringe. After 2h, the solution was diluted with
CH2Cl2 (25mL) and poured into saturated aqueous
NaHCO3 (25mL). The CH2Cl2 layer was removed and
the aqueous layer was extracted with additional CH2Cl2
(2 · 10mL). The combined CH2Cl2 layers were washed
with brine (10mL), dried over Na2SO4, filtered, and con-
centrated. Flash chromatography of the residue (20:1
CH2Cl2/MeOH) provided 7 as a white foam (1.98g,
and concentrated. Flash chromatography (20:1 CHCl3/
MeOH) provided 10a as a white solid (38mg, 81%):
Rf = 0.14 (20:1 CHCl3/MeOH); mp = 144ꢁC; H NMR
d 7.24 (J = 7.9Hz, 1H, Ar-H), 6.82–6.79 (m, 3H, Ar-
1
H), 6.6
4 (d, J = 8.2Hz, 1H, Ar-H), 6.49 (d,
J = 8.2Hz, 1H, Ar-H), 5.67 (ddd, J = 3.6, 6.1, 9.1Hz,
1H, C7H), 5.58–5.53 (m, 2H, C8H, NH), 4.65 (s, 1H,
C5H), 4.37 (t, J = 6.4Hz, 1H, C6H), 3.79 (s, 3H,
OMe), 3.54 (s, 2H), 3.34 (m, 1H), 3.00 (d, J = 18.6Hz,
1H), 2.89 (s, 1H), 2.61 (dd, J = 4.0, 12.2Hz, 1H), 2.43
(s, 3H, NMe), 2.38–2.29 (m, 2H), 1.99 (dt, J = 9.6,
12.2Hz, 1H), 1.76 (dd, J = 1.9, 12.6Hz, 1H); 13C
NMR d 171.1, 160.2, 144.5, 138.9, 136.2, 132.8, 130.3,
129.9, 128.6, 125.8, 121.7, 119.6, 117.1, 115.1, 113.1,
93.0, 59.3, 55.4, 50.5, 47.3, 44.1, 43.9, 43.0, 39.8, 35.5,
20.5; MS (ESI) m/z 433 [M + H]+; HRMS (ESI) calcd
for C26H29N2O4 433.2127, found 433.2115 [M + H]+;
the average purity of 10a was found to be P 99% by
analytical HPLC giving tR = 4.38min (mobile phase A)
and tR = 5.09min (mobile phase B).
1
76%): Rf = 0.3 (20:1 CH2Cl2/MeOH); mp = 84.9ꢁC; H
NMR d 7.85–7.83 (m, 2H, Pht-H), 7.72–7.70 (m, 2H,
Pht-H), 6.66 (d, J = 8.1Hz, 1H, Ar-H), 6.51 (d,
J = 8.1Hz, 1H, Ar-H), 5.70–5.68 (m, 1H, C7H), 5.48–
5.46 (m, 1H, C8H), 4.98 (s, 1H, C5H), 4.88–4.86 (m,
1H, C6H), 3.42 (m, 1H), 3.32–3.30 (m, 1H), 3.02 (d,
J = 18.4Hz, 1H), 2.61–2.58 (m, 1H), 2.46 (s, 3H,
NMe), 2.36–2.25 (m, 3H), 1.72–1.69 (m, 1H), 1.29 (hep-
tet, J = 7.7Hz, 3H), 1.12–1.08 (18H); MS (ESI) m/z 571
[M + H]+.
5.1.3. 3-O-Triisopropylsilyl-6-b-aminomorphine 8. The
phthalimide 7 (1.72g, 3.03mmol) was dissolved in 95%
aqueous EtOH (26mL) and hydrazine hydrate
(0.7mL) was added by syringe. The solution was heated
at 55ꢁC for 80min, during which time a white precipi-
tate formed. The mixture was concentrated. Water
(50mL) and 3% aqueous NH4OH (4mL) were added
and the mixture was extracted with CHCl3
(5 · 50mL). The CHCl3 extracts were dried over
Na2SO4, filtered, and concentrated to a yellowoil. Flash
chromatography (SiO2, 15:1 CH2Cl2/MeOH with 0.1%
5.1.5. 6-b-(40-Methoxy)benzylamidomorphine 10b. Com-
pound 10b was prepared according to the general proce-
dure described for 10a, from 8 (78.4mg, 0.178mmol),
NEt3 (72lL, 0.517mmol), and 4-methoxyphenylacetyl
chloride (80lL, 0.523mmol) giving 9b as a white foam
(70mg, 67%): Rf = 0.11 (20:1 CHCl3/MeOH); MS
(ESI) m/z 589 [M + H]+. The intermediate silyl ether
9b (65mg, 0.11mmol) and TBAF (0.18mL, 0.18mmol)
gave 10b (29mg, 61%) as a white solid: Rf = 0.14 (20:1
CHCl3/MeOH); mp = 247ꢁC (dec); 1H NMR d 7.15
(d, J = 8.6Hz, 2H, Ar-H), 6.86 (d, J = 8.6Hz, 2H, Ar-
H), 6.64 (d, J = 8.1Hz, 1H, Ar-H), 6.48 (d, J = 8.1Hz,
1H, Ar-H), 5.67 (ddd, J = 3.0, 5.6, 9.5Hz, 1H, C7H),
5.56 (dd, J = 1.5, 9.5Hz, 1H, C8H), 5.45 (d, J = 6.4Hz,
1H, NH), 4.65 (s, 1H, C5H), 4.36 (t, J = 6.3Hz, 1H,
C6H), 3.79 (s, 3H, OMe), 3.50 (s, 2H), 3.34 (m, 1H),
3.00 (d, J = 18.6Hz, 1H), 2.87 (s, 1H), 2.61 (dd,
J = 3.8, 11.9Hz, 1H), 2.44 (s, 3H, NMe), 2.38–2.28 (m,
2H), 1.98 (dt, J = 4.9, 12.5Hz, 1H), 1.77 (d,
J = 10.7Hz, 1H); 13C d 171.5, 158.8, 144.4, 138.7,
132.6, 130.4, 129.7, 128.3, 126.5, 125.6, 119.3, 117.0,
114.5, 92.8, 59.1, 55.3, 50.3, 47.0, 43.9, 42.9, 42.7, 39.6,
35.4, 20.2; MS (ESI) m/z 433 [M + H]+; HRMS (ESI)
calcd for C26H29N2O4 433.2127, found 433.2136
[M + H]+; the average purity of 10b was found to be
P 99% by analytical HPLC giving tR = 4.44min (mobile
phase A) and tR = 5.24min (mobile phase B).
NEt3) provided
8 as a yellowoil (1.24, 94%):
1
Rf = 0.16; H NMR d 6.60 (d, J = 8.2Hz, 1H, Ar-H),
6.44 (d, J = 8.2Hz, 1H, Ar-H), 5.84 (ddd, J = 3.0, 5.4,
9.4Hz, 1H, C7H), 5.45 (dd, J = 1.8, 9.4Hz, 1H, C8H),
4.56 (s, 1H, C6H), 3.43 (d, J = 5.4Hz, 1H), 3.32 (dd,
J = 3.2, 5.5Hz, 1H), 3.02 (s, 1H), 2.98 (s, 1H), 2.59
(dd, J = 4.1, 12.1Hz, 1H), 2.45 (s, 3H, NMe), 2.39–
2.31 (m, 2H), 2.07 (dt, J = 5.0, 12.4Hz, 1H), 1.80 (dd,
J = 2.0, 12.6Hz, 1H), 1.27–1.20 (heptet, J = 7.6Hz,
3H), 1.09–1.06 (3 · d, J = 7.6Hz, 18H); MS (ESI) m/z
441 [M + H]+.
5.1.4. General procedure for the synthesis of 6-b-amido-
morphines. 6-b-(30-Methoxy)benzylamidomorphine 10a.
The amine 8 (80mg, 0.18mmol) was dissolved in
CH2Cl2 (4mL) and NEt3 (72lL, 0.52mmol) and then
3-methoxyphenylacetyl chloride (80lL, 0.51mmol) were
added by syringe. The resulting pale yellowsolution was
stirred at rt in a closed vial. After 2h, the solution was
concentrated and the residue was purified by flash chro-
matography (20:1 CHCl3/MeOH) to provide 9a as a
white foam (78mg, 73%): Rf = 0.18 (20:1 CH2Cl2/
MeOH); MS (ESI) m/z 589 [M + H]+. Compound 9a
(64mg, 0.11mmol) was dissolved in 5% aqueous THF
(2.2mL). TBAF (0.18mL, 1.0M solution in THF) was
added by syringe and the pale yellowsolution was stir-
red at rt for 2h. The solution was concentrated and
1% HCl (2mL) was added. The mixture was stirred
for 2min and then transferred to a separatory funnel
with the aid of water (20mL). The mixture was made
basic (pH8.5) with solid NaHCO3 and extracted with
CHCl3 (5 · 10mL). The combined CHCl3 extract was
washed with brine (4mL), dried (Na2SO4), filtered,
5.1.6. 6-b-(30-Methoxy)benzamidomorphine 10c. Com-
pound 10c was prepared according to the general proce-
dure described for 10a, from 8 (82mg, 0.185mmol),
NEt3 (76lL, 0.545mmol), and anisoyl chloride (71lL,
0.521mmol) giving 9c as a white foam (88mg, 82%);
Rf = 0.28 (10:1 CH2Cl2/MeOH); MS (ESI) m/z 575
[M + H]+. The intermediate silyl ether 9c (73mg,
0.174mmol) and TBAF (0.21mL, 0.21mmol) then gave
10c as a white solid (42.5mg, 58%): Rf = 0.15 (15:1
1
DCM/MeOH); mp = 212.9ꢁC (dec); H NMR d 7.34–
7.33 (m, 1H, Ar-H), 7.27–7.23 (m, 2H, Ar-H), 7.02–
7.00 (m, 1H, Ar-H), 6.68 (d, J = 8.1Hz, 1H, Ar-H),
6.51 (d, J = 8.1Hz, 1H, Ar-H), 6.27 (d, J = 6.8Hz, 1H,
NH), 5.84 (ddd, J = 3.0, 5.8, 9.2Hz, 1H, C7H), 5.68

5988
J. M. MacDougall et al. / Bioorg. Med. Chem. 12 (2004) 5983–5990
(dd, J = 1.2, 9.9Hz, 1H, C6H), 4.85 (s, 1H, C5H), 4.59 (t,
J = 6.4Hz, 1H, C6H), 3.81 (s, 3H, OMe), 3.47 (dd,
J = 3.0, 5.0Hz, 1H), 3.19–3.16 (m, 1H), 3.04 (d,
J = 18.5Hz, 1H), 2.68 (dd, J = 4.0, 12.0Hz, 1H), 2.49
(s, 3H, NMe), 2.43–2.38 (m, 1H), 2.11 (dt, J = 4.8,
12.6Hz, 1H), 1.80 (d, J = 10.9Hz, 1H); 13C NMR d
167.5, 160.0, 144.7, 139.1, 135.6, 132.7, 129.9, 129.8,
128.9, 125.5, 119.7, 119.0, 118.2, 117.4, 112.6, 93.0,
59.5, 55.7, 50.9, 47.4, 44.1, 43.0, 39.9, 35.4, 20.6; MS
(ESI) m/z 419 [M + H]+; HRMS (ESI) calcd for
C25H27N2O4 419.1971, found 419.1984 [M + H]+; the
average purity of 10c was found to be P 99% by analyt-
ical HPLC giving tR = 4.41min (mobile phase A) and
tR = 5.05min (mobile phase B).
166.2, 144.3, 138.7, 134.2, 133.0, 132.7, 131.9, 130.5,
129.8, 128.9, 128.3, 127.6, 125.7, 119.5, 117.0, 92.9,
59.1, 52.4, 50.9, 47.1, 44.0, 42.9, 39.9, 35.4, 20.2; MS
(ESI) m/z 447 [M + H]+; HRMS (ESI) calcd for
C26H27N2O5 447.1920, found 447.1930 [M + H]+; the
average purity of 10e was found to be P 99% by analyt-
ical HPLC giving tR = 4.39min (mobile phase A) and
tR = 5.06min (mobile phase B).
5.1.9. 6-b-(30-Nitro)benzamidomorphine 10f. Compound
10f was prepared according to the general procedure de-
scribed for 10a, from 8 (80mg, 0.18mmol), NEt3 (76lL,
0.55mmol), and 3-nitrobenzoyl chloride (95mg,
0.51mmol) giving 9f as a white foam (87mg, 81%);
Rf = 0.17 (20:1 CHCl3/MeOH); MS (ESI) m/z 590
[M + H]+. The intermediate silyl ether 9f (73mg,
0.168mmol) and TBAF (0.20mL, 0.20mmol) then gave
10f as a light yellowsolid (36mg, 49%): Rf = 0.11 (20:1
5.1.7. 6-b-Benzamidomorphine 10d. Compound 10d was
prepared according to the general procedure described
for 10a, from 8 (82mg, 0.23mmol), NEt3 (76lL,
0.55mmol), and benzoyl chloride (61lL, 0.52mmol)
giving 9d as a white foam (79mg, 78%); Rf = 0.23
(20:1 CHCl3/MeOH); MS (ESI) m/z 545 [M + H]+.
The intermediate silyl ether 9d (65mg, 0.110mmol)
and TBAF (0.18mL, 0.18mmol) then gave 10d as a
white solid (29mg, 61%): Rf = 0.15 (15:1 CH2Cl2/
1
CHCl3/MeOH); mp 208.3ꢁC (dec); H NMR d 8.54 (t,
J = 1.4Hz, 1H, Ar-H), 8.26 (dd, J = 1.4, 8.2Hz, 1H,
Ar-H), 8.11 (d, J = 8.0Hz, 1H, Ar-H), 7.55 (t,
J = 8.0Hz, 1H, Ar-H), 6.85 (d, J = 6.4Hz, 1H, NH),
6.65 (d, J = 8.1Hz, 1H, Ar-H), 6.51 (d, J = 8.1Hz, 1H,
Ar-H), 5.84 (ddd, J = 3.0, 5.5, 9.2Hz, 1H, C7H), 5.71
(d, J = 10.3Hz, 1H, C8H), 4.88 (s, 1H, C5H), 4.63 (t,
J = 6.3Hz, 1H, C6H), 3.45 (t, J = 3.2Hz, 1H), 3.18 (s,
1H), 3.04 (d, J = 18.6Hz, 1H), 2.68 (dd, J = 3.8,
11.8Hz, 1H), 2.47 (s, 3H, NMe), 2.41–2.34 (m, 2H),
2.15 (dt, J = 7.6, 12.4Hz, 1H), 1.78 (d, J = 11.5Hz,
1H); 13C NMR d 165.2, 148.0, 144.4, 138.9, 135.5,
133.3, 132.7, 129.8, 129.7, 128.2, 126.1, 125.4, 122.0,
119.5, 117.3, 92.5, 59.2, 51.1, 47.1, 43.9, 42.8, 39.6,
35.1, 20.3; MS (ESI) m/z 434 [M + H]+; HRMS (ESI)
calcd for C24H24N3O5 434.1716, found 434.1719
[M + H]+; the average purity of 10f was found to be
P 99% by analytical HPLC giving tR = 4.11min (mo-
bile phase A) and tR = 4.78min (mobile phase B).
1
MeOH); mp = 184.4ꢁC (dec); H NMR d 7.78 (m, 2H,
Ar-H), 7.53–7.50 (m, 1H, Ar-H), 7.42–7.41 (m, 1H,
Ar-H), 6.72 (d, J = 8.1Hz, 1H, Ar-H), 6.55 (d,
J = 8.1Hz, 1H, Ar-H), 6.18 (d, J = 6.2Hz, 1H, NH),
5.89 (ddd, J = 3.0, 5.3, 9.5Hz, 1H, C7H), 5.69 (dd,
J = 1.5, 9.5Hz, 1H, C8H), 4.86 (s, 1H, C5H), 4.64 (t,
J = 6.3Hz, 1H, C6H), 3.54 (br s, 1H), 3.31 (br s, 1H),
3.06 (d, J = 18.7Hz, 1H), 2.77 (m, 1H), 2.57 (s, 3H,
NMe), 2.50–2.48 (m, 3H), 2.23–2.19 (m, 1H), 1.86 (dd,
J = 2.2, 12.9Hz, 1H); 13C NMR d 167.4, 144.5, 138.8,
133.9, 132.9, 131.7, 129.8, 128.6, 128.6, 127.0, 125.7,
119.4, 117.1, 92.8, 59.1, 50.6, 47.0, 44.0, 42.9, 39.9,
35.5, 20.5; MS (ESI) m/z 389 [M + H]+; HRMS (ESI)
calcd for C24H25N2O3 389.1865, found 389.1867
[M + H]+; the average purity of 10d was found to be
99% by analytical HPLC giving tR = 4.43min (mobile
phase A) and tR = 5.16min (mobile phase B).
5.1.10. 6-b-(30,40-Dichloro)benzamidomorphine 10g.
Compound 10g was prepared according to the general
procedure described for 10a, from 8 (81mg, 0.18mmol),
NEt3 (77lL, 0.55mmol), and 3,4-dichlorobenzoyl chlo-
ride (108mg, 0.52mmol) giving 9g as a white foam
(96mg, 85%); Rf = 0.2 (20:1 CH2Cl2/MeOH); MS
(ESI) m/z 614 [M + H]+. The intermediate silyl ether
9g (81mg, 0.18mmol) and TBAF (0.22mL, 0.22mmol)
then gave 10g as a white solid (48mg, 49%): Rf = 0.17
(15:1 CH2Cl2/MeOH); mp = 228.5ꢁC (dec); 1H NMR
d 7.82 (d, J = 2.1Hz, 1H, Ar-H), 7.54 (dd, J = 2.1,
8.4Hz, 1H, Ar-H), 7.39 (d, J = 8.4Hz, 1H, Ar-H), 6.67
(d, J = 8.0Hz, 1H, Ar-H), 6.56 (d, J = 6.2Hz, 1H, Ar-
H), 6.52 (d, J = 8.0Hz, 1H, Ar-H), 5.82 (ddd, J = 2.9,
5.3, 9.5Hz, 1H, C7H), 5.68 (dd, J = 1.2, 9.5Hz, 1H,
C8H), 4.83 (s, 1H, C5H), 4.58 (t, J = 6.3Hz, 1H, C6H),
3.47 (dd, J = 3.0, 5.3Hz, 1H), 3.19 (m, 1H), 3.05 (d,
J = 18.6Hz, 1H), 2.70 (dd, J = 3.8, 11.9Hz, 1H), 2.50
(s, 3H, NMe), 2.43–2.37 (m, 2H), 1.77 (d, J = 11.0Hz,
1H), 13C NMR d 165.4, 144.4, 138.9, 136.1, 133.7,
133.0, 132.5, 130.5, 129.6, 129.3, 128.4, 126.3, 125.2,
119.5, 117.4, 92.5, 59.2, 52.4, 50.8, 47.2, 43.8, 42.7,
39.5, 35.1, 29.7, 25.8, 20.3, 20.5, 13.6; MS (ESI) m/z
457 [M + H]+; HRMS (ESI) calcd for C24H23Cl2N2O3
457.1086, found 457.1087 [M + H]+; the average purity
5.1.8. 6-b-(30-Carbomethoxy)benzamidomorphine 10e.
Compound 10e was prepared according to the general
procedure described for 10a, from
8
(159mg,
0.36mmol), NEt3 (151lL, 1.09mmol), and 3-carbo-
methoxybenzoyl chloride (217mg, 1.01mmol) giving 9e
as a white foam (122mg, 56%); Rf = 0.22 (20:1 CHCl3/
MeOH); MS (ESI) m/z 603 [M + H]+. The intermediate
silyl ether 9e (102mg, 0.17mmol) and TBAF (0.47mL,
0.47mmol) then gave 10e as a white solid: Rf = 0.14
1
(30:1 DCM/MeOH); mp = 166.0ꢁC (dec); H NMR d
8.32 (s, 1H, Ar-H), 8.14 (d, J = 7.8Hz, 1H, Ar-H),
8.00 (d, J = 7.8Hz, 1H, Ar-H), 7.49 (t, J = 7.8Hz, 1H,
Ar-H), 6.68 (d, J = 8.1Hz, 1H, Ar-H), 6.52 (d,
J = 8.1Hz, 1H, Ar-H), 6.33 (d, J = 7.0Hz, 1H, NH),
5.85 (ddd, J = 9.4, 5.6, 3.1Hz, 1H, C7H), 5.73 (d,
J = 9.9Hz, 1H, C8H), 4.85 (s, 1H, C5H), 4.62 (t,
J = 6.2Hz, 1H, C6H), 3.93 (s, 3H, OMe), 3.42 (dd,
J = 3.2, 5.2Hz, 1H), 3.10 (br s, 1H), 3.05 (d,
J = 18.5Hz, 1H), 2.63 (dd, J = 12.1, 4.2Hz, 1H), 2.46
(s, 3H, NMe), 2.40–2.34 (m, 2H), 2.08 (dt, J = 12.5,
4.9Hz, 1H), 1.81 (d, J = 10.8Hz, 1H); 13C d 166.4,

J. M. MacDougall et al. / Bioorg. Med. Chem. 12 (2004) 5983–5990
5989
of 10g was found to be P 99% by analytical HPLC giv-
ing tR = 3.93min (mobile phase A) and tR = 4.43min
(mobile phase B).
6. Biological methods
6.1. Receptor binding
Binding to cell membranes was conducted in a 96-well
format, as described previously.9 Cells were removed
from the plates by scraping with a rubber policeman,
homogenized in Tris buffer using a Polytron homoge-
nizer, then centrifuged once and washed by an addi-
tional centrifugation at 27,000g for 15min. The pellet
was resuspended in 50mM Tris, pH7.5, and the suspen-
sion incubated with [3H]DAMGO, [3H]DPDPE, or
[3H]U69593, for binding to l, d, or j opioid receptors,
respectively. The total volume of incubation was
1.0mL and samples were incubated for 60–120min at
25ꢁC. The amount of protein in the binding reaction
varied from approximately 15 to 30lg. The reaction
was terminated by filtration using a Tomtec 96 harvester
(Orange, CT) with glass fiber filters. Bound radioactivity
was counted on a Pharmacia Biotech beta-plate liquid
scintillation counter (Piscataway, NJ) and expressed in
counts per minute. IC50 values were determined using
at least six concentrations of test compound, and calcu-
lated using Graphpad/Prism (ISI, San Diego, CA). Ki
values were determined by the method of Cheng and
Prusoff.13
5.1.11. 6-b-(Thiophen-20-yl)acetamidomorphine 10h.
Compound 10h was prepared according to the general
procedure described for 10a, from 8 (86mg, 0.2mmol),
2-thiopheneacetyl chloride (0.68lL, 0.55mmol), and
NEt3 (82lL, 0.59mmol) giving 9h as a white foam
(66mg, 60%); Rf = 0.23 (20:1 CHCl3/MeOH); MS
(ESI) m/z 565 [M + H]+. The intermediate silyl ether
9h (56mg, 0.1mmol) and TBAF (0.120mL, 0.12mmol)
then gave 10h as a white solid (32mg, 79%): Rf = 0.10
1
(20:1 CHCl3/MeOH); mp = 154.4ꢁC; H NMR d 7.18
(d, J = 4.5Hz, 1H, Ar-H), 6.94–6.91 (m, 2H, Ar-H),
6.64 (d, J = 8.1Hz, 1H, Ar-H), 6.49 (d, J = 8.1Hz, 1H,
Ar-H), 5.82 (d, J = 7.1Hz, 1H, NH), 5.69 (ddd,
J = 3.5, 5.5, 9.4Hz, 1H, C7H), 5.59 (dd, J = 1.2,
9.9Hz, 1H, C8H), 4.65 (s, 1H, C5H), 4.39 (t,
J = 6.4Hz, 1H, C6H), 3.77 (s, 2H), 3.37 (dd, J = 3.3,
5.8Hz, 1H), 3.01 (d, J = 18.5Hz, 1H), 2.93 (m, 1H),
2.64 (dd, J = 4.1, 12.1Hz, 1H), 2.45 (s, 3H, NMe),
2.39–2.31 (m, 2H), 2.02 (dt, J = 7.6, 12.6Hz, 1H), 1.77
(dd, J = 10.8, 1.7Hz, 1H); 13C NMR d 170.2, 144.6,
139.0, 136.0, 132.9, 129.9, 128.4, 127.6, 125.9, 125.6,
119.6, 117.3, 92.9, 59.2, 50.4, 47.3, 44.1, 43.0, 39.7,
37.7, 35.5, 20.5; MS (ESI) m/z 409 [M + H]+; HRMS
(ESI) calcd for C23H25N2O3S 409.1586, found
409.1572 [M + H]+; the average purity of 10h was found
to be P 99% by analytical HPLC giving tR = 4.31min
(mobile phase A) and tR = 5.02min (mobile phase B).
6.2. [35S]GTPcS binding
[35S]GTPcS binding was conducted basically as described
by Traynor and Nahorski.11 Cells were scraped from tis-
sue culture dishes into 20mM Hepes, 1mM EDTA, then
centrifuged at 500g for 10min. Cells were resuspended in
this buffer and homogenized using a Polytron homoge-
nizer. The homogenate was centrifuged at 27,000g for
15min and the pellet resuspended in buffer A, containing
20mM Hepes, 10mM MgCl2, 100mM NaCl, pH7.4. The
suspension was recentrifuged at 27,000g and suspended
once more in buffer A. For the binding assay, membranes
(8–15lg protein) were incubated with [35S]GTPcS
(50pM), GDP (10lM), and the appropriate compound,
in a total volume of 1.0mL, for 60min at 25ꢁC. Samples
were filtered over glass fiber filters and counted as de-
scribed for the binding assays. Statistical analysis was
conducted using the program Prism.
5.1.12. 6-b-(30-Carboxy)benzamidomorphine 11. Com-
pound 10e (31mg, 0.067mmol) was dissolved in 4mL
of 1:1 THF/H2O and LiOHÆH2O (27mg, 0.643mmol)
was added. The colorless solution was stirred at rt for
3.75h, glacial AcOH was added (15 drops), and the solu-
tion was concentrated. The residue was purified by flash
chromatography on SiO2 (5:1–1:1 CHCl3/MeOH). The
appropriate fractions were concentrated and the residue
was stirred with 10mL of 10:1 CHCl3/MeOH and fil-
tered through paper to remove residual SiO2. The filtrate
was concentrated to provide 11 as a white solid, 20mg
(69%): Rf = 0.17 (1:1 CHCl3/MeOH with 0.2% AcOH),
mp = 217.1ꢁC (dec); 1H NMR (CD3OD) d 8.41–8.40
(m, 1H, NH), 8.13–8.12 (m, 1H, Ar-H), 7.90–7.88 (m,
1H, Ar-H), 7.47 (t, J = 7.7Hz, 1H, Ar-H), 6.62 (d,
J = 8.2Hz, 1H, Ar-H), 6.55 (d, J = 8.2Hz, 1H, Ar-H),
5.81–5.78 (m, 1H, C7H), 5.69 (d, J = 9.9Hz, 1H, C8H),
4.82 (s, 1H, C5H), 4.51–4.50 (m, 1H), 3.65 (dd, J = 3.2,
5.4Hz, 1H), 3.27 (br s, 1H), 3.12 (d, J = 18.9Hz, 1H),
2.87 (dd, J = 4.0, 12.9Hz, 1H), 2.64 (s, 3H, NMe),
2.62–2.57 (m, 2H), 2.20 (dt, J = 4.8, 12.9Hz, 1H), 1.81
(dd, J = 2.4, 12.9Hz, 1H); 13C NMR (CD3OD) d
180.5, 170.2, 145.6, 141.2, 139.4, 135.5, 133.6, 131.0,
130.9, 130.5, 130.3, 129.5, 129.2, 125.6, 120.7, 118.5,
93.8, 61.3, 52.8, 42.6, 39.9, 35.1, 30.9, 22.1; MS (ESI)
m/z 431 (M ꢁ H)ꢁ; HRMS (ESI) calcd for
C25H25N2O5 433.1758, found 433.1749; the average pur-
ity of 11 was found to be 99% by analytical HPLC giv-
ing tR = 3.93min (mobile phase A) and tR = 4.43min
(mobile phase B).
6.3. Determination of analgesic activity of morphine
analogue 10eÆHCl12
Male ICR mice weighing 20–25g at the start of the
experiment were used. Animals were group-housed un-
der standard laboratory conditions and were kept on a
12:12h day–night cycle (lights on at 08:00). Animals
were handled for 1–2days prior to conducting the exper-
iments. MorphineÆHCl or 10eÆHCl were dissolved in
water. Drugs were injected at a volume of 0.1mL. Noci-
ception was assessed using the tail flick assay with an
analgesia instrument (Stoelting) that uses radiant heat.
This instrument is equipped with an automatic quantifi-
cation of tail flick latency, and a 15s cutoff to prevent
damage to the animalꢀs tail. During testing, the focused
beam of light was applied to the lower half of the ani-
malꢀs tail, and tail flick latency was recorded. Baseline

5990
J. M. MacDougall et al. / Bioorg. Med. Chem. 12 (2004) 5983–5990
3. Penson, R. T.; Joel, S. P.; Roberts, M.; Gloyne, A.;
Beckwith, S.; Slevin, M. L. Br. J. Clin. Pharmacol. 2002,
53, 347–354.
4. Stachulski, A. V.; Scheinmann, F.; Ferguson, J. R.; Law,
J. L.; Lumbard, K. W.; Hopkins, P.; Patel, N.; Clarke, S.;
Gloyne, A.; Joel, S. P. Bioorg. Med. Chem. Lett. 2003, 13,
1207–1214.
values for tail flick latency were determined before drug
administration in each animal. Basal tail flick latency
was between 3.2 and 8.0s (average 5.86
0.16 SEM).
Immediately after testing, animals were injected subcu-
taneously with the test compound or saline as a vehicle
control. Following injections, animals were tested for
tail flick latencies at 30-, and 60-min post-injection.
Antinociception was quantified by the following for-
5. Liu, M.; Mahom, M. F.; Sainsbury, M. J. Chem. Soc.,
Perkin. Trans. 1 1998, 9, 2943–2951.
mula: % antinociception = 100 [(test latency ꢁ baseline
6. Welsh, L. H. J. Org. Chem. 1954, 19, 1409.
7. Makleit, S.; Simo, C.; Hosztafi, S. Synth. Commun. 1991,
21, 407–412.
8. Yoshimura, H.; Natsuki, R.; Ida, S.; Oguri, K. Chemical
Reactivity of Morphine and Morphine-6-Conjugates and
their Binding to Rat Brain. Chem. Pharm. Bull. 1976, 24,
901–906.
9. Zaveri, N.; Polgar, W.; Olsen, C.; Kelson, A. B.; Grundt,
P.; Lewis, J. W.; Toll, L. Eur. J. Pharmacol. 2001, 428, 29–
36.
*
latency)/(15 ꢁ baseline latency)]. If the animal did not
respond prior to the 15s cutoff, the animal was assigned
a score of 100%. Behavioral results were analyzed using
ANOVAs with morphine, 10eÆHCl as between group
variables and post-drug treatment time (30, 60min) as
the repeated measure followed by Student Newman–
Keuls post-hoc tests where appropriate. The level of sig-
nificance was set at p < 0.05.
10. Stevens, W. C.; Jones, R. M.; Subramanian, G.; Metzger,
T.; Ferguson, D. M.; Portoghese, P. S. J. Med. Chem.
2000, 43, 2759–2769.
References and notes
11. Traynor, J. R.; Nahorski, S. R. Mol. Pharmacol. 1995, 47,
848–854.
1. MacDougall, J. M.; Zhang, X. D.; Polgar, W. E.;
Khroyan, T. V.; Toll, L.; Cashman, J. R. J. Med. Chem.,
submitted for publication.
2. Thompson, P. I.; Joel, S. P.; John, L.; Wedzicha, J. A.;
Maclean, M.; Slevin, M. L.. Br. J. Clin. Pharmacol. 1995,
40, 145–152.
12. Dewey, W. L.; Harris, L. S. The Tail Flick Test. In
Methods in Narcotics Research; Ehenpreis, S., Neidle, A.,
Eds.; Marcel Dekker: NewYork, 1975; pp 101–109.
13. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099–3108.