Design, chemical synthesis, and biological evaluation of thiosaccharide analogues of morphine- and codeine-6-glucuronide

Article abstract of DOI:10.1021/jm049554t

A series of 6-β-thiosaccharide analogues of morphine-6-glucuronide (M6G) and codeine-6-glucuronide (C6G) were synthesized and evaluated with the objective of preparing an analogue of M6G with improved biological activity. The affinity of the thiosaccharide analogues of M6G and C6G was examined by competitive binding assays at μ, δ, and κ opioid receptors. The thiosaccharide compounds in the morphine series 5b, 5e, 6a, and 6c showed 1.5-2.4-fold higher affinity for the μ receptor than M6G, but were generally less selective than M6G. The functional activity of the M6G and C6G analogues was examined with the [³⁵S]GTP-γ-S assay. Compounds 5b and 5e were determined to be full μ agonists, whereas compounds 6a and 6c were partial μ agonists. The in vivo antinociceptive activity of compound 5b was evaluated by the tail flick latency test, giving an ED₅₀ of 2.5 mg/kg.

Full text of DOI:10.1021/jm049554t

J. Med. Chem. 2004, 47, 5809-5815
5809
Design, Chemical Synthesis, and Biological Evaluation of Thiosaccharide
Analogues of Morphine- and Codeine-6-Glucuronide
James M. MacDougall,*,† Xiao-Dong Zhang,† Willma E. Polgar,‡ Taline V. Khroyan,‡ Lawrence Toll,‡ and
John R. Cashman*,†
Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, California 92121-2804, and SRI International,
333 Ravenswood Ave., Menlo Park, California 94025-3493
Received June 7, 2004
A series of 6-â-thiosaccharide analogues of morphine-6-glucuronide (M6G) and codeine-6-
glucuronide (C6G) were synthesized and evaluated with the objective of preparing an analogue
of M6G with improved biological activity. The affinity of the thiosaccharide analogues of M6G
and C6G was examined by competitive binding assays at µ, δ, and κ opioid receptors. The
thiosaccharide compounds in the morphine series 5b, 5e, 6a, and 6c showed 1.5-2.4-fold higher
affinity for the µ receptor than M6G, but were generally less selective than M6G. The functional
activity of the M6G and C6G analogues was examined with the [35S]GTP-γ-S assay. Compounds
5b and 5e were determined to be full µ agonists, whereas compounds 6a and 6c were partial
µ agonists. The in vivo antinociceptive activity of compound 5b was evaluated by the tail flick
latency test, giving an ED50 of 2.5 mg/kg.
Introduction
Chronic pain represents a major health problem
throughout the world. It is estimated that 22% of the
primary care patients between the ages of 18 and 65
years suffer from chronic pain at any given time.1 In
the United States there are more than 50 million people
who experience chronic pain, and approximately one-
half of the population seeks medical help for pain at
some point in their lives.1 Narcotic analgesics in the
morphine class remain the accepted treatment for pain
Figure 1. M6G 1 and the thiosaccharide analogue 2.
due to trauma, surgery, cancer, and for terminal pa-
tients. Morphine elicits a number of pharmacological
actions mediated by the µ opioid receptor including
analgesia, respiratory depression, and inhibition of
gastrointestinal transit.2 The adverse side-effects and
concerns due to abuse liability have limited morphine
availability and optimal use. Considerable effort has
therefore been put forward to develop and understand
the appropriate use of narcotic analgesics and to develop
new ones with more favorable pharmacological proper-
ties.
Morphine-6-glucuronide (M6G, 1, Figure 1) is pro-
duced by the UDP glucuronosyl transferase-catalyzed
reaction of morphine with UDP glucuronic acid in the
liver and is the second most abundant metabolite of
morphine in man, accounting for 15% of the total
morphine dose.3 The analgesic potency of M6G is
approximately 100 times greater than that of morphine
in animals with significantly reduced respiratory de-
pression, nausea, and sedation.4-7 The reduction in side-
effects and enhanced potency observed for M6G make
it a promising drug candidate for the treatment of
cancer-related pain in man.8
bioavailability of orally administered M6G is only 11 (
3%,3 and this severely limits M6G development as a
drug candidate. The hydrolysis of M6G in the gut
significantly reduces the available dose. The objective
of this work was to test the hypothesis that new pain
medications with improved in vivo stability formulated
for oral administration could be developed. The S-
glycoside linkage imparts significantly greater in vivo
stability relative to the corresponding O-glycoside link-
age in metabolic conjugates.9,10 To determine whether
the 6-â-S-glycosides (2) of M6G and codeine-6-glucu-
ronide (C6G) possess more favorable pharmacological
properties than M6G, the compounds 5 and 6 were
prepared and tested in vitro and in vivo. The synthesis
and the biological evaluation of the resulting compounds
are reported herein.
Chemistry
The starting compounds for the preparation of the
thiosaccharides 5a-f and 6a-d were 6-O-tosylmorphine
3a, 3-O-acetyl-6-tosylmorphine 3b, and 3-O-tosylcodeine
3c.11 The tosylates 3a-c were prepared in good yield
by the reaction of either 3-O-acetylmorphine or codeine
with p-toluenesulfonyl chloride in pyridine at 3 °C
overnight.12 6-O-Tosylmorphine 3a was prepared in 81%
yield by stirring the corresponding acetate 3b in metha-
nol for 2 h at room temperature with 1 equiv of
potassium carbonate. The key step in the synthesis of
Despite this promise, compared with iv M6G, the
* To whom correspondence should be addressed. Phone: 858.458.9305.
Fax:
858-458-9311. E-mail:
jmacdougall@hbri.org (J.M.M.),
jcashman@hbri.org (J.R.C.).
† Human BioMolecular Research Institute.
‡ SRI International.
10.1021/jm049554t CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/08/2004

5810 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
MacDougall et al.
Scheme 1. Synthesis of M6G Thiosaccharide Analogues
5a-f and 6a-d
Figure 2. Perspective drawings of 3-O-acetyl-6-â-O-tosylmor-
phine 7 and 3-O-acetyl-6-O-tosylmorphine 3b.
Scheme 2. Synthesis of Thiols 4a and 4b
Table 1. Ki Inhibition Values of µ, δ, and κ Opioid Binding to
CHO Membranes by Compounds 5a-f and 6a-d
Ki (nM) ( SEM
selectivity
entry
µ
δ
κ
δ/µ
κ/µ
5a-f was the attachment of the thiosaccharide to the
phenanthrene nucleus by an SN2 displacement reaction
(Scheme 1). Thus, to prepare the protected sulfur
analogue of M6G 5a, a 0 °C DMF solution of 3-O-acetyl-
6-O-tosylmorphine 3b was added to 2.7 equiv of a 0 °C
DMF solution of the thiolate corresponding to 4a. The
mixture was warmed to room temperature, providing
5a in 63% overall yield after aqueous workup and
purification. In analogous fashion, the morphine deriva-
tive 5b (56%) and the codeine derivative 5c (77%) were
prepared from the tosylates 3a and 3c, respectively. It
was found that a slight excess of the thiol 4a (2.7 equiv)
relative to sodium hydride (2.6 equiv) was required to
decrease loss of the ester protecting groups in the
glucuronic acid moiety or the 3-position acetate of 5a.
The use of stoichiometric amounts of the thiol 4a and
sodium hydride in the preparation of compounds 5a-c
gave significantly diminished yields.
morphine 1.1 ( 0.1
140 ( 2
47 ( 14
4060 ( 230 12.5 316
189 ( 14
11.0 ( 0.1
499 ( 7
127
42
M6G
5a
5b
5c
5d
5e
12.9 ( 0.9 170 ( 1
100 ( 2
5.4 ( 0.1
64 ( 1
305 ( 66
7.8 ( 0.5
165 ( 28
8.7 ( 0.9
463 ( 57
5.4 ( 0.8
127 ( 23
528 ( 47
15.6 ( 2.4
438 ( 5
762 ( 167
19.5 ( 0.3
466 ( 119
31.4 ( 2.3
2680 ( 200 >10 000
56.2 ( 2.2 136 ( 17
1140 ( 350 2480 ( 540
5.3
2.9
6.8
1.9
2.0
7.8
4.5
381
13.2
33
1390 ( 200 2.5
2970 ( 950 2.5
2190 ( 880 2.8
5f
6a
6b
6c
288 ( 12
3.6
5.8
10.4 25.4
9
>21
6d
19.4
by others.14 We also attempted to prepare the C-6
R-epimer of compound 5a. The SN2 displacement reac-
tion of 3-O-acetyl-6-â-O-tosylmorphine 7 (Figure 2) with
the thiolate of 4a did not result in sulfide bond forma-
tion, as indicated by analysis of the 1H NMR spectrum
of the crude reaction mixture. One explanation for this
observation is that nucleophilic attack at the C-6
position of 3-O-acetyl-6-â-O-tosylmorphine 7 via an SN2
process is prevented by steric interactions.12,15 This is
more clearly illustrated by examining the perspective
drawings of the tosylates 7 and 3b (Figure 2). These
drawings suggest that the SN2 displacement reaction
of the C-6 tosyl group in the â-epimer 7 is less favorable
than in the R-epimer 3b due to steric interactions that
would occur upon approach of a nucleophile to the
bottom face of 7. The thiosaccharides 4a (59%) and 4b
(76%) were prepared from the known bromides 8a and
8b, respectively (Scheme 2).16 The syntheses of either
4a or 4b was accomplished by a two-step procedure
involving an initial SN2 displacement reaction with
potassium thioacetate in DMF, followed by the selective
thiolysis of the sulfur acetate group in the intermediate
upon treatment with sodium methylthiolate in chloro-
form/methanol (4a) or dichloromethane/methanol (4b)
at 0 °C.17
To expand the scope of these studies, the 6-â-thioglu-
cose conjugates of 3-O-acetylmorphine 5d (74%) and
morphine 5e (15%), as well as codeine analogue 5f
(82%), were prepared by the reaction of the thiolate of
4b with 3b or 3c, respectively. It was found that
sufficient amounts of the phenol 5e could be obtained
for biochemical studies as a side product in the prepara-
tion of 5d, which precluded the preparation of 5d in a
separate reaction. The esters 5a-f were desired for
biological evaluation, because they can potentially func-
tion as prodrugs analogous to opioids such as heroin.
The increased hydrophobicity of the esters 5a-f should
impart improved central nervous system availability
relative to the deprotected congeners 6a-d. The re-
moval of the ester protecting groups in compounds
5a,c,d,f was accomplished by treatment with 5% aque-
ous sodium hydroxide, followed by an acidic workup,
giving the deprotected compounds 6a-d in modest
yields.13 A characteristic singlet (δ 5.03-5.22) for the
1
C-5 phenanthrene proton was evident in the H NMR
Pharmacological Results and Discussion
spectra of the thiosaccharides 5a-f and 6a-d. This
indicated that the dihedral angle with the vicinal C-6
R-disposed proton was approximately 90°. This observa-
Table 1 contains the Ki values for compounds 5 and
6. In the opioid receptor binding assays the following
radioligands were used: [3H]DAMGO (µ opioid receptor
agonist), [3H]U69593 (κ opioid receptor agonist), and
1
tion was in agreement with the H NMR spectral data
from C-6 â-substituted morphine compounds prepared

Thiosaccharide Analogues of M6G and C6G
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5811
Table 2. Stimulation of [35S]-GTP-γ-S Binding by Compounds 5a-f and 6a-d Mediated by the µ, δ, and κ Opioid Receptors
µ
δ
κ
entry
EC50
Emax
93 ( 3
EC50
NDb
Emax
NDb
80.0 ( 1.0
46.8 ( 2.0
104 ( 20
64.3 ( 8.3
39.3 ( 0.8
45.5 ( 1.5
44.9 ( 1.2
78.7 ( 0.9
44.2 ( 5.4
51.5 ( 6.5
27.7 ( 3.3
EC50
NDb
Emax
morphine
M6G
5a
15.6 ( 0.5
72.3 ( 26.7
1420 ( 240
43.7 ( 16.5
732 ( 47
NDb
45.0 ( 5.0
37.8 ( 6.7
88.3 ( 19.5
19.3 ( 0.3
44.1 ( 4.9
76.0 ( 3.0
41.9 ( 2.1
46.6 ( 10.1
NAa
190 ( 205
1320 ( 300
80.2 ( 0.2
1010 ( 180
3660 ( 1510
63.8 ( 14.6
872 ( 242
50.1 ( 36.7
1330 ( 20
192 ( 15
>10 000
NAa
NAa
NAa
5b
29.1 ( 8.2
2670 ( 60
1720 ( 630
52.0 ( 4.9
>10 000
NAa
23.5 ( 0.9
24.3 ( 4.2
36.9 ( 1.9
26.5 ( 2.5
NAa
5c
5d
4790 ( 1620
33.7 ( 11.3
994 ( 13
5e
5f
6a
90.6 ( 22.9
NAa
6b
NAa
NAa
NAa
6c
91.5 ( 23.4
1060 ( 20
64.5 ( 0.5
42.0 ( 6.0
321 ( 93
4630 ( 1770
42.5 ( 2.5
21.0 ( 1.0
6d
1740 ( 310
a No effect observed. b No data available.
[3H]DPDPE (δ opioid receptor agonist). Ki values were
determined by measuring the inhibition of binding of
these radioligands to the receptor by the ligands 5 and
6.18 As with M6G, each ligand tested showed a higher
affinity for the µ than the δ or κ receptor. The rank order
of affinity for the most active compounds at the µ
receptor was 6c ) 5b > 5e > 6a > M6G. Each of these
compounds possessed a free phenolic hydroxyl group at
the C-3 position. Compared to the affinity of M6G, the
affinity of the most active compounds 5b, 5e, 6a, 6c was
increased 1.5-2.4-fold at µ, 2.9-10.3-fold at δ, and 1.4-
369-fold at the κ receptor. Thus, both the S-glucuronide
6a and the S-glucoside 6c possessed slightly higher
affinity for the µ opioid receptor versus M6G. The µ
versus δ and the µ versus κ affinity of the S-glucuronide
6a was decreased 3.5- and 9.6-fold, respectively, com-
pared to M6G, indicating that the stereochemistry at
the C-6 position may play a significant role in receptor
interactions. The glucose analogue of M6G has been
shown to possess moderately higher affinity (5-fold) for
the µ opioid receptor compared to M6G,19 which is
consistent with our observations. Compounds 5b and
5a that possessed a carbomethoxy group at the C-5′
position showed 1.7-2.7-fold higher affinity for the κ
receptor versus the δ receptor. Each of the other ligands
showed higher affinity for the δ receptor versus the κ
receptor. The replacement of the carbomethoxy group
at C-5′ in compound 5b with an acetoxymethyl group
in compound 5e decreased the affinity for the κ receptor
270-fold. The affinity of the remaining compounds for
the µ, δ, and κ receptors possessing either 3-acetoxy
groups or 3-methyl ether groups was significantly
decreased when compared to M6G. In each instance,
removal of the ester functional group in the sugar
moiety greatly diminished affinity of the ligands for the
κ receptor, while having a less pronounced effect on the
affinity of the ligands for the µ and δ receptors.
agonists caused a reduced level of binding. As shown
in Table 2, with the exception of compound 6b, all of
the compounds examined stimulated [35S]GTP-γ-S bind-
ing at the µ and δ receptors. Generally, the compounds
were much less efficacious at the κ receptor. Stimulation
of [35S]GTP-γ-S binding by M6G was also selective for
the µ and δ receptors. The most efficacious compounds
at the µ receptor were compounds 5b (88.0%), 5e
(76.0%), 6c (64.5%), and 6a (46.6%). Each of these
compounds possessed a free phenolic hydroxyl group.
Compounds 5b, 5e, 6c, and 6a were all more efficacious
than M6G in the [35S]GTP-γ-S assay. The same general
trend was observed at the δ receptor with compounds
possessing a free phenolic hydroxyl showing increased
efficacy. The most efficacious compounds at the δ
receptor were 5b (104.4%), 6a (78.70%), 5c (64.3%), and
6c (51.5%). Protection of the phenolic hydroxyl group
with an acetate group or a methyl group diminished the
efficacy and potency at each opioid receptor. Compounds
that effected maximal stimulation may be viewed as
agonists at a given receptor. Compounds that elicited
less than maximal binding may be viewed as partial
agonists or antagonists if no stimulation was observed.
Thus, compounds 5b and 5e may be viewed as full µ
agonists, whereas compounds 5a, 5c, 5d, 5f, 6a, 6c, and
6d are partial µ agonists. Compounds 5b and 6a were
also full δ agonists. The remaining compounds 5a, 5c,
5d, 5e, 5f, 6b, 6c, and 6d are all partial agonists at the
δ receptor. Compounds 5b, 5c, 5d, 5e, 6c, and 6d are
partial κ agonists. The compounds 5a, 5f, 6a, and 6b
did not stimulate [35S]GTP-γ-S binding at the κ receptor.
The overall rank order of the EC50 values for the
functional assays correlated with the Ki values derived
from the binding experiments.
On the basis of the results of the functional assay and
Ki values, the HCl salt of compound 5b was evaluated
for its effect on the tail-flick latency.21 Administration
of 5b‚HCl produced a dose-dependent increase in tail-
flick latency, indicative of the analgesic effects of the
compound (Figure 3). The overall ANOVA indicated a
significant effect of dose [F(5,64) ) 37.26, P < 0.0001].
Averaged across postinjection time, the 3-30 mg/kg
doses of compound 5b produced a significant increase
in tail-flick latency relative to controls (Student New-
man-Keuls, p < 0.05). The two highest doses (10 and
30 mg/kg) of 5b produced a greater amount of analgesia
relative to a 3 mg/kg dose (Student Newman-Keuls, p
< 0.05) of morphine (Figure 4). The ED50 of compound
5b‚HCl for tail flick latency (i.e., 2.5 mg/kg) was slightly
To evaluate the opioid receptor mediated activation
of its associated G protein, the compounds 5 and 6 were
evaluated using the [35S]GTP-γ-S assay.20 In this assay,
the compound’s potency or affinity for the receptor
(defined by its EC50 for stimulating [35S]GTP-γ-S bind-
ing) was examined in vitro. Agonist efficacy (Emax) was
defined as the degree to which the compound maximally
stimulated [35S]GTP-γ-S binding relative to control. The
EC50 value represented the concentration of a compound
that produced 50% maximal stimulation of [35S]GTP-
γ-S binding by that compound. Full agonists stimulated
[35S]GTP-γ-S binding to a maximal extent and partial

5812 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
MacDougall et al.
receptor relative to M6G. The phenol 5e, possessing a
peracetylated thioglucose moiety, showed a 2.5-fold
higher affinity for the µ versus δ receptor and a 382-
fold higher affinity for the µ versus κ receptor. Func-
tional studies showed that compounds 5b and 5e were
full µ agonists, whereas compounds 6a and 6c were
partial µ agonists. The ED50 of compound 5b in the tail
flick latency test was 2.5 mg/kg. It can therefore be
concluded that the replacement of the C-6 R-disposed
glycosidic oxygen atom in M6G and its congeners with
a â-disposed sulfur atom is a viable strategy for improv-
ing the pharmacological properties of these compounds.
Experimental Section
Figure 3. Dose-dependent analgesia induced by administra-
tion of 5b in tail flick assay. Data are mean maximum possible
antinociceptive effect (%MPE) ((SEM). Asterisks represent
significant differences from vehicle controls (Student New-
man-Keuls, p < 0.05). Plus signs represent a significant
difference from 3 mg/kg 5b (Student Newman-Keuls, p<0.05).
Synthetic Chemistry. All reactions were run under a
positive pressure of nitrogen with magnetic stirring at ambient
temperature using oven-dried glassware unless otherwise
indicated. Air- and moisture-sensitive liquids were transferred
via syringe through rubber septa. Silica gel (230-400 mesh)
was used for column chromatography. DMF was dried by
filtration through a column of neutral alumina and stored over
activated 4 Å molecular sieves under nitrogen prior to use.
All other solvents and reagents were used as received. 1H NMR
spectra were recorded at 500 MHz using a Varian NMR
(NuMega Resonance, San Diego, CA). Chemical shifts were
reported in ppm (δ) relative to CDCl3 at 7.26 ppm. Melting
points were reported uncorrected. Low resolution mass spectra
was done on an Agilent 1100 MSD (HT Labs, San Diego, CA).
High-resolution mass spectra was done on a VG 7070 spec-
trometer with an Opus V3.1 and DEC 3000 Alpha Station data
system at the University of CaliforniasRiverside. Where
combustion analyses are not specified, analytical purities were
determined by straight phase HPLC using a Hitachi L74 liquid
chromatograph with a L-7400 UV detector, a D7500 integrator,
and a Hamilton PRP-I stainless steel column (250 mm × 4.6
mm i.d.).
Figure 4. Dose-dependent analgesia induced by administra-
tion of morphine in tail flick assay. Data are maximum possible
antinociceptive effect (%MPE) ((SEM). Asterisks represent
significant differences from vehicle controls (Student New-
man-Keuls, p < 0.05). Plus signs represent a significant
difference from 3 mg/kg morphine (Student Newman-Keuls,
p < 0.05).
Methyl 2,3,4-tri-O-acetyl-1-â-thio-D-glucopyranosidu-
ronate 4a. The bromide 8a (2.7 g, 6.8 mmol) was dissolved in
DMF (20 mL), and KSAc (0.92 g, 8.1 mmol) was added all at
once. The flask was closed and stirred at room temperature
for 3.5 h. The DMF was removed by distillation under high
vacuum (0.25 mmHg) with the aid of a water bath (55 °C).
The residue was dissolved in CHCl3 (100 mL) and washed with
water (3 × 10 mL) and brine (10 mL), dried over anhydrous
Na2SO4, filtered, and concentrated to a red-brown oil. Absolute
EtOH (30 mL) was added to the oil and the mixture was heated
to boiling and then slowly cooled to room temperature and then
to 0 °C. The resulting crystals were collected on a fritted funnel
and washed with EtOH (0 °C, 5 × 4 mL) and dried under high
vacuum to provide the intermediate thioacetate as white
crystals (1.46 g, 65%): Rf ) 0.61 (1:1 hexane/EtOAc); mp )
less than that for morphine (i.e., 3.0 mg/kg) (Figure 4).
In contrast to morphine, which appeared to be more
effective at the 30 min time point, the analgesia of
compound 5b at the 60 min time point was greater than
or equal to the analgesia observed at the 30 min time
point. It is possible that 5b has a greater duration of
action than morphine. Although compound 5b had
slightly lower binding affinity than morphine, it pos-
sessed similar [35S]GTP-γ-S potency and percent stimu-
lation, indicating that the bioavailability of 5b was also
similar to that of morphine. Because this compound is
rather nonselective with respect to each of the opioid
receptors, it is not certain at this point which receptor
mediates the antinociceptive activity. However, the fact
that this is a partial agonist at the κ receptors suggests
that the antinociceptive activity is likely mediated by
the µ receptor.
1
162 °C; H NMR (CDCl3) δ 5.32-5.28 (m, 2H), 5.17-5.13 (m,
2H), 4.15 (d, J ) 9.8 Hz, 1H), 3.71 (s, 3H), 2.37 (s, 3H), 2.03
(s, 3H), 2.02 (s, 3H), 2.01 (s, 3H); MS (ESI) m/z 415 [ M +
Na]+.
The thioacetate (100 mg, 0.26 mmol) was dissolved in 1:1
CHCl3/MeOH (2 mL) and cooled to 0 °C. Nitrogen was bubbled
through the solution for 5 min, followed by the addition of
NaSMe (18 mg, 0.26 mmol). After 5 min, the mixture was
poured into 1% concentrated aqueous HCl (10 mL) and
extracted with CH2Cl2 (2 × 10 mL). The combined CH2Cl2
layers were washed with brine (5 mL), dried over anhydrous
sodium sulfate, filtered, and concentrated to an orange-white
solid that was recrystallized from boiling absolute EtOH (2
mL). After cooling to 0 °C, the crystals were collected on a
fritted funnel and washed with small portions of 0 °C EtOH
(2 mL total) and dried under high vacuum to give 4a as white
crystals (80 mg, 90%): Rf ) 0.16 (1:1 hexane/EtOAc); mp )
Conclusion
In summary, a series of 10 6-â-thiosaccharide ana-
logues of M6G and C6G were synthesized. The key
synthetic step utilized an SN2 displacement reaction of
glucose and glucuronic acid derived 1-â-thiols with 6-O-
tosylmorphines or 6-O-tosylcodeine. The phenols 5b, 5e,
6a, and 6c showed increased affinity for the µ opioid
1
125 °C; H NMR (CDCl3) δ 5.24-5.22 (m, 2H), 4.99 (m, 1H),
4.57 (t, J ) 10.1 Hz, 1H), 4.04 (d, J ) 9.3 Hz, 1H), 3.76 (s,
3H), 2.38 (d, J ) 10.3 Hz, 1H), 2.08 (s, 3H), 2.02 (s, 6H); 13C
NMR (CDCl3) δ 170.1, 169.7, 169.5, 166.9, 79.1, 77.5, 73.4, 73.0,
+
69.5, 53.2, 20.9, 20.7, 20.6; MS (ESI) m/z 373 [M + H]
.

Thiosaccharide Analogues of M6G and C6G
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5813
2,3,4,6-Tetra-O-acetyl-1-â-thio-D-glucopyranose 4b.
2,3,4,6-Tetra-O-acetyl-1-S-acetyl-â-D-glucopyranose10 (496 mg,
1.2 mmol) was dissolved in 1:2 MeOH/CH2Cl2 (6 mL) and
cooled to 0 °C. Nitrogen was bubbled through the solution for
5 min, followed by the addition of NaSMe (85 mg, 1.2 mmol)
in a single portion. The solution was stirred for 5 min under
nitrogen, at which time TLC analysis (2:1 hexane/EtOAc)
indicated that the reaction was complete. The solution was
transferred to a separatory funnel containing 10 mL of 1%
aqueous HCl and 10 mL of H2O and extracted with CH2Cl2 (4
× 20 mL). The combined CH2Cl2 layers were washed with H2O
(5 mL) and brine (5 mL), dried (Na2SO4), filtered, and
concentrated. The residue was purified by chromatography (2:1
hexane/EtOAc) to provide 4b as an orange gel (336 mg, 76%):
scribed for 5b, 3c (231 mg, 0.51 mmol), 4a (483 mg, 1.38
mmol), and NaH (1.33 mmol) gave 5c as an off-white solid (249
mg, 77%): Rf ) 0.30, 20:1 CH2Cl2/EtOH); mp ) 172 °C; 1H
NMR (CDCl3) δ 6.64 (d, J ) 8.1 Hz, 1H), 6.54 (d, J ) 8.1 Hz,
1H), 5.82 (ddd, J ) 3.0, 6.0, 9.2 Hz, 1H), 5.53 (dd, J ) 1.8, 9.4
Hz, 1H), 5.27-5.22 (m, 2H), 5.14 (s, 1H), 4.99 (t, J ) 2.1 Hz,
1H), 4.71 (d, J ) 10.3 Hz, 1H), 4.02 (d, J ) 7.3 Hz, 1H), 3.83
(s, 3H), 3.79 (d, J ) 6.0 Hz, 1H), 3.73 (s, 3H), 3.29 (dd, J )
3.3, 5.8 Hz, 1H), 3.05-3.00 (d, J ) 19.0 Hz, 1H over m, 1H),
2.55 (dd, J ) 4.3, 12.2 Hz, 1H), 2.42 (s, 3H), 2.35 (td, J ) 3.4,
12.4 Hz, 1H), 2.29 (dd, J ) 6.0, 19.0 Hz, 1H), 2.15 (td, J ) 5.0,
12.4 Hz, 1H), 2.02 (s, 3H), 2.016 (s, 3H), 2.00 (s, 3H), 1.77 (dd,
J ) 1.5, 12.3 Hz, 1H); 13C NMR δ 170.2, 169.5, 169.4, 166.9,
146.3, 142.2, 133.1, 130.7, 127.9, 127.7, 119.1, 112.8, 93.6, 85.2,
76.1, 73.4, 70.3, 69.3, 59.1, 56.4, 53.0, 47.0, 45.5, 44.7, 43.3,
40.0, 36.2, 20.9, 20.8, 20.7, 20.6; MS (ESI) m/z 632 [M + H]+.
Anal. (C31H37NO11S) C, H, N, S.
3-O-Acetyl-6-â-S-(2′,3′,4′,5′-tetra-O-acetyl-â-D-glucopy-
ranosyl)morphine 5d and 6-â-S-(2′,3′,4′,5′-Tetra-O-acetyl-
â-D-glucopyranosyl)morphine 5e. Following the general
procedure described for 5b, the thiol 4b (500 mg, 1.37 mmol),
NaH (53 mg, 1.32 mmol), and tosylate 3b (246 mg, 0.51 mmol)
gave 5d as a white foam (255 mg, 74%) together with 5e (47
mg, 14.6%). Data for 5d: Rf ) 0.2 (20:1 CH2Cl2/MeOH); mp )
151.9 °C; 1H NMR (CDCl3) δ 6.73 (d, J ) 8.2 Hz, 1H), 6.58 (d,
J ) 8.2 Hz, 1H), 5.83-5.79 (m, 1H), 5.50 (dd, J ) 1.8, 9.7 Hz,
1H), 5.24-5.17 (m, 2H), 5.04 (t, J ) 9.9 Hz, 1H), 4.96 (t, J )
9.9 Hz, 1H), 4.69 (d, J ) 10.2 Hz, 1H), 4.19 (d, J ) 4.2 Hz,
2H), 3.79-3.75 (m, 1H), 3.71 (d, J ) 6.0 Hz, 1H), 3.30 (dd, J
) 3.3, 5.6 Hz, 1H), 3.06 (s, 1H), 3.04 (d, J ) 18.8 Hz, 1H), 2.57
(dd, J ) 4.0, 12.2 Hz, 1H), 2.42 (s, 3H), 2.34-2.29 (m, 2H),
2.14-2.11 (m, 1H), 2.07 (s, 6H), 2.05 (s, 6H), 2.00 (s, 3H), 1.80-
1.77 (m, 1H); 13C NMR (CDCl3) δ 170.8, 170.1, 169.5, 169.3,
168.5, 148.8, 132.8, 132.1, 131.7, 131.6, 127.9, 121.6, 119.3,
94.4, 85.1, 75.9, 73.8, 70.3, 68.5, 62.4, 58.8, 46.7, 45.8, 44.3,
43.0, 39.7, 35.6, 20.7, 20.6, 20.5; MS (ESI) m/z 674 [M + H]+;
HRMS (ESI) calcd for C33H40NO12S 674.2271, found 674.2238;
the average purity of 5d was found to be g98% by analytical
HPLC giving tR ) 4.86 min (60/40/0.02 MeOH/2-propanol/
HClO4 (v:v)) and tR ) 5.66 min (55/45/0.02 MeOH/2-propanol/
HClO4 (v:v)). Analytical data for compound 5e: Rf ) 0.08 (20:1
CH2Cl2/MeOH); mp ) 137.3 °C; 1H NMR δ 6.63 (d, J ) 8.2
Hz, 1H), 6.51 (d, J ) 8.2 Hz, 1H), 5.79 (ddd, J ) 3.6, 5.8, 9.6
Hz, 1H), 5.50 (dd, J ) 1.7, 9.6 Hz, 1H), 5.22 (t, J ) 9.3 Hz,
1H), 5.09-4.99 (m, 3H), 4.69 (d, J ) 10.2 Hz, 1H), 4.27-4.19
(m, 2H), 3.78-3.74 (m, 1H), 3.67 (d, J ) 5.8 Hz, 1H), 3.32 (m,
1H), 3.01 (d, J ) 18.9 Hz, 1H) over bs (1H), 2.61-2.58 (m,
1H), 2.44 (s, 3H), 2.39-2.27 (m, 2H), 2.16-2.11 (m, 2H), 2.08
(s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.78-1.75 (m,
1H); 13C NMR (CDCl3) δ 171.1, 170.4, 169.69, 169.66, 144.6,
130.3, 131.8, 130.6, 128.3, 126.9, 119.7, 116.7, 94.2, 84.9, 76.1,
74.0, 70.4, 68.7, 62.5, 59.2, 47.2, 45.8, 44.5, 43.2, 39.8, 35.8,
21.0, 20.83, 20.80, 20.6; MS(ESI) m/z 632 [M + H]+; HRMS
(ESI) calcd for C31H38NO11S 632.2166, found 632.2136; the
average purity of 5e was found to be g99% by analytical
HPLC, giving tR ) 3.98 min (60/40/0.02 MeOH/2-propanol/
HClO4 (v:v)) and tR ) 4.40 min (55/45/0.02 MeOH/2-propanol/
HClO4 (v:v)).
1
Rf ) 0.2 (2:1 hexane/EtOAc); H NMR (CDCl3) δ 5.19 (t, J )
9.5 Hz, 1H), 5.10 (t, J ) 9.9 Hz, 1H), 4.97 (t, J ) 9.6 Hz, 1H),
4.54 (t, J ) 9.9 Hz, 1H), 4.24 (dd, J ) 4.8, 12.5 Hz, 1H), 4.12
(dd, J ) 2.2, 8.5 Hz, 1H), 3.72 (ddd, J ) 2.2, 4.8, 7.1 Hz, 1H),
2.30 (d, J ) 9.9 Hz, 1H), 2.09 (s, 3H), 2.08 (s, 3H), 2.02 (s,
3H), 2.00 (s, 3H); MS (ESI) m/z 365 [M + H]+.
General Procedure for the Reaction of Thiosaccha-
rides with Morphine or Codeine Tosylates. 6-â-S-(Methyl
2′,3′,4′-tri-O-acetyl-â-D-glucopyranosyluronate)mor-
phine 5b. A 60% dispersion of NaH (46 mg, 1.2 mmol) was
added to a 0 °C DMF solution of 4a (424 mg, 1.2 mmol) and
the resulting red mixture was stirred for 10 min. A 0 °C DMF
(8 mL) solution of 3a (183 mg, 0.42 mmol) was added by
syringe over 1 min and the resulting solution was stirred for
4 h while warming to room temperature. The reaction was
poured into 20 mL of 0.5% aqueous HCl and the pH was raised
to 9 by the addition of solid NaHCO3. The mixture was
extracted with CH2Cl2 (3 × 50 mL), and the combined organic
layers were washed with H2O (10 mL) and brine (10 mL), dried
over anhydrous Na2SO4, filtered, and concentrated to provide
a gray solid, which was purified by flash chromatography
(SiO2, 60:1 to 20:1 CH2Cl2/EtOH) to provide an off-white solid
(144 mg, 56%). An analytical sample of 5b was obtained by
recrystallization from absolute EtOH as a white solid: Rf )
0.10 (20:1 CH2Cl2/MeOH); mp ) 135 °C (dec); 1H NMR δ 6.63
(d, J ) 7.9 Hz, 1H), 6.50 (d, J ) 7.9 Hz, 1H), 5.81 (ddd, J )
3.0, 5.6, 9.1 Hz, 1H), 5.52 (dd, J ) 8.6, 10.3 Hz, 1H), 5.29-
5.21 (m, 2H), 5.05 (d, J ) 9.2 Hz, 1H), 5.03 (s, 1H), 4.70 (d, J
) 9.6 Hz, 1H), 4.06 (d, J ) 9.6 Hz, 1H), 3.74 (s, 3H), 3.71 (d,
J ) 5.6 Hz, 1H), 3.31 (m, 1H), 3.03-2.99 (m, 2H), 2.62-2.59
(m, 1H), 2.58 (dd, J ) 11.7, 4.0 Hz, 1H), 2.43 (s, 3H), 2.38-
2.28 (m, 2H), 2.15-2.09 (m, 2H), 2.05 (s, 3H), 2.022 (s, 3H),
2.016 (s, 3H), 1.76 (dd, J ) 12.2, 1.8 Hz, 1H); 13C NMR (CDCl3)
δ 170.3, 169.6, 169.5, 167.0, 144.7, 138.5, 131.8, 130.4, 128.5,
126.7, 119.7, 116.8, 93.7, 84.6, 76.3, 73.4, 70.0, 69.4, 59.2, 53.1,
47.2, 45.0, 44.5, 43.1, 39.8, 35.6, 20.9, 20.8, 20.7; HRMS (ESI)
calcd for C30H36NO11S 618.2009, found 618.2011; the average
purity of 5b was found to be g99% by analytical HPLC, giving
tR ) 3.47 min (55:45:0.01 MeOH/2-propanol/HClO4) and tR
3.98 min (45/55/0.01 MeOH/2-propanol/HClO4).
)
3-O-Acetyl-6-â-S-(methyl 2′,3′,4′-tri-O-acetyl-â-D-glu-
copyranosyluronate)morphine 5a. According to the general
procedure described for 5b, NaH (53 mg, 1.33 mmol), 4a (484
mg, 1.38 mmol), and 3b (246 mg, 0.51 mmol) provided 5a as
an off-white powder (211 mg, 63%): Rf ) 0.33 (20:1 CH2Cl2/
EtOH); mp ) 194 °C (dec); 1H NMR (CDCl3) δ 6.74 (d, J ) 8.2
Hz, 1H), 6.58 (d, J ) 8.2 Hz, 1H), 5.83-5.80 (m, 1H), 5.53 (dd,
J ) 1.5, 10.2 Hz, 1H), 5.29-5.21 (m, 2H), 5.09 (s, 1H), 5.00 (t,
1H), 4.72 (d, J ) 10.3 Hz, 1H), 4.06 (d, J ) 9.3 Hz, 1H), 3.76-
3.73 (4H), 3.33 (m, 1H), 3.07-3.03 (2H), 2.60-2.58 (m, 1H),
2.44 (s, 3H), 2.37-2.20 (s, 3H over m, 2H), 2.15 (dt, J ) 3.7,
11.8 Hz, 1H), 2.04 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.80 (d, J
) 11.6 Hz, 1H); 13C NMR (CDCl3) δ 170.3, 169.5, 169.4, 168.8,
167.0, 149.0, 133.0, 132.6, 132.0, 131.8, 128.0, 121.8, 119.5,
94.5, 85.1, 76.2, 73.3, 70.3, 69.4, 59.0, 53.0, 46.9, 45.6, 44.5,
43.2, 40.0, 35.7, 20.93, 20.90, 20.8, 20.7; MS (ESI) m/z 660 [M
+ H]+. Anal. Calcd (C32H37NO12S): C, 58.26; H, 5.65; N, 2.12;
S, 4.86. Found: C, 57.94; H, 5.75; N, 2.07; S, 4.97.
6-â-S-(2′,3′,4′,5′-Tetra-O-acetyl-â-D-glucopyranosyl)co-
deine, 5f. Compound 5f was prepared according to the general
procedure described for 5b, from thiol 4b (440 mg, 1.21 mmol),
NaH (47 mg, 1.16 mmol), and 3c (203 mg, 0.448 mmol).
Purification was accomplished by flash chromatography (SiO2,
20:1 CH2Cl2/EtOH) to provide 5f as an off-white foam (237 mg,
82%). An analytical sample was obtained by recrystallization
from boiling hexanes: Rf ) 0.26 (20:1 CH2Cl2/EtOH); mp )
142.5 °C; 1H NMR (CDCl3) δ 6.65 (d, J ) 8.2 Hz, 1H), 6.55 (d,
J ) 8.2 Hz, 1H), 5.85-5.81 (m, 1H), 5.52 (dd, J ) 1.9, 9.6 Hz,
1H), 5.22-5.19 (m, 2H), 5.06 (t, J ) 9.8 Hz, 1H), 4.97 (dd, J )
9.5, 10.1 Hz, 1H), 4.69 (d, J ) 10.1 Hz, 1H), 4.23-4.16 (m,
2H), 3.83 (s, 3H), 3.76 (d, J ) 6.0 Hz, 1H), 3.76-3.71 (m, 1H),
3.29 (dd, J ) 3.3, 5.8 Hz, 1H), 3.06-3.01 (m, 2H), 2.56 (dd, J
) 4.0, 12.2 Hz, 1H), 2.42 (s, 3H), 2.38-2.30 (m, 2H), 2.18-
2.15 (m, 1H), 2.12 (s, 3H), 2.09 (s, 6H), 2.00 (s, 3H), 1.99-1.80
6-â-S-(Methyl 2′,3′,4′-tri-O-acetyl-â-D-glucopyranosyl-
uronate)codeine 5c. Following the general procedure de-

5814 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
MacDougall et al.
(m, 1H); 13C NMR (CDCl3) δ 171.0, 170.4, 169.6, 169.5, 146.3,
142.2, 132.9, 130.7, 127.9, 127.6, 119.1, 112.7, 93.8, 85.5, 76.1,
74.0, 70.5, 68.5, 62.5, 59.1, 56.4, 47.0, 46.1, 44.6, 43.3, 40.0,
36.3, 20.93, 20.92, 20.8, 20.5; MS (ESI) m/z 646 [M + H]+;
HRMS (ESI) calcd for C32H40NO11S 646.2322, found 646.2304;
the average purity of 5f was found to be g99% by analytical
HPLC, giving tR ) 5.00 min (60/40/0.02 MeOH/2-propanol/
HClO4 (v:v)) and tR ) 5.71 min (55/45/0.02 MeOH/2-propanol/
HClO4 (v:v)).
Morphine 6-â-S-D-glucuronide 6a. To a solution of 5a
(50.0 mg, 0.08 mmol) in MeOH (1.6 mL) was added 5% aqueous
NaOH (0.12 mL). The golden solution was stirred at room
temperature for 18.5 h, acidified to a pH of 5 with 10 drops of
glacial AcOH, and concentrated. The resulting white, amor-
phous solid was purified by flash chromatography on silica gel
(16 × 1 cm, 5:1 to 1:1 CH3CN/MeOH) and then dried for 4 h
at 100 °C (0.25 mmHg) to provide 5b as a white powder (19.4
mg, 54%): Rf ) 0.26 (1:1 CH3CN/MeOH with 0.2% AcOH); mp
> 300 °C (dec); 1H NMR (D2O) δ 6.65 (d, J ) 8.1 Hz, 1H), 6.61
(d, J ) 8.1 Hz, 1H), 5.92 (m, 1H), 5.61 (d, J ) 9.6 Hz, 1H),
5.19 (s, 1H), 4.69 (d, J ) 9.9 Hz, 1H), 3.87 (d, J ) 5.9 Hz, 1H),
3.77 (d, J ) 9.0 Hz, 1H), 3.64 (m, 1H), 3.57-3.51 (m, 2H), 3.38
(t, J ) 9.2 Hz, 1H), 3.13-3.09 (m, 2H), 2.83 (dd, J ) 3.7, 12.3
Hz, 1H), 2.59-2.54 (s, 3H over m, 2H), 2.19 (dt, J ) 8.5, 13.2
Hz, 1H), 1.87 (d, J ) 12.0 Hz, 1H); 13C NMR (D2O) δ 174.6,
144.3, 139.6, 129.8, 128.8, 127.7, 123.3, 119.0, 116.9, 91.7, 85.0,
79.3, 76.1, 71.3, 70.7, 58.0, 45.6, 43.3, 42.6, 40.3, 37.2, 33.0,
19.7; MS (ESI) m/z 478 [M + H]+. Anal. Calcd (C23H27NO8S):
C, 57.85; H, 5.70; N, 2.93; S, 6.71. Found: C, 57.75; H, 5.95;
N, 2.73; S, 6.58.
(60/40/0.02 MeOH/2-propanol/HClO4 (v:v)) and tR ) 4.42 min
(55/45/0.02 MeOH/2-propanol/HClO4 (v:v)).
Codeine 6-â-S-D-Glucose 6d. 5f (120 mg, 0.19 mmol) was
dissolved in MeOH (4 mL) and 5% aqueous NaOH (0.6 mL)
was added. The mixture was stirred at room temperature for
18 h and then treated with saturated aqueous NH4Cl (1 mL).
The mixture was concentrated and the residue was purified
by preparative TLC (SiO2, 200:40:5:0.05 CH2Cl2/MeOH/water/
13% concentrated NH4OH). After drying for 4 h at 100 °C, 6d
was obtained as a white powder (23 mg, 26%): Rf ) 0.17; mp
) 201.6 °C; 1H NMR (CD3OD) δ 6.79 (d, J ) 8.3 Hz, 1H), 6.68
(d, J ) 8.3 Hz, 1H), 6.02 (ddd, J ) 3.0, 5.9, 9.1 Hz, 1H), 5.55
(dd, J ) 1.8, 9.8 Hz, 1H), 5.36 (s, 1H), 4.57 (d, J ) 9.8 Hz,
1H), 4.10 (dd, J ) 3.0, 6.2 Hz, 1H), 3.91 (dd, J ) 1.6, 12.1 Hz,
1H), 3.83 (d, J ) 6.1 Hz, 1H), 3.81 (s, 3H), 3.70 (dd, J ) 5.1,
12.0 Hz, 1H), 3.39-3.23 (complex m, 6H), 3.01-3.00 (m, 1H),
2.98 (s, 3H), 2.90 (dd, J ) 6.6, 19.8 Hz, 1H), 2.41 (dt, J ) 4.8,
13.8 Hz, 1H), 2.04 (dd, J ) 2.7, 14.0 H, 1H); 13C NMR (CD3-
OD) δ 147.8, 144.5, 131.7, 130.1, 128.9, 125.3, 121.1, 116.2,
94.5, 88.3, 82.3, 79.9, 79.6, 74.8, 71.6, 63.1, 62.3, 57.3, 45.7,
44.3, 41.9, 38.8, 34.8, 23.0; MS (ESI) m/z 478 [M + H]+; HRMS
(ESI) calcd for C24H32NO7S 478.1899, found 478.1886; the
average purity of 6d was found to be g99% by analytical
HPLC, giving tR ) 4.79 min (60/40/0.02 MeOH/2-propanol/
HClO4 (v:v)) and tR ) 5.64 min (55/45/0.02 MeOH/2-propanol/
HClO4 (v:v)).
Receptor Binding. Binding to cell membranes was con-
ducted in a 96-well format, as described previously.22 Cells
were removed from the plates by scraping with a rubber
policeman, homogenized in Tris buffer using a Polytron
homogenizer, and then centrifuged once and washed by an
additional centrifugation at 27 000g for 15 min. The pellet was
resuspended in 50 mM Tris, pH 7.5, and the suspension
incubated with [3H]DAMGO, [3H]DPDPE, or [3H]U69593, for
binding to µ, δ, or κ opioid receptors, respectively. The total
volume of incubation was 1.0 mL, and samples were incubated
for 60-120 min at 25 °C. The amount of protein in the binding
reaction varied from approximately 15 to 30 µg. 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 calculated using Graphpad/Prism (ISI,
San Diego, CA). Ki values were determined by the method of
Cheng and Prusoff.23
Codeine 6-â-S-D-glucuronide 6b. Compound 6b was
prepared according to the procedure described for 6a. 5c (60.4
mg, 0.095 mmol), MeOH (2 mL), and 5% aqueous NaOH (0.28
mL) provided 6b (38 mg, 79%) as a white solid: Rf ) 0.08 (1:1
1
MeCN/MeOH with 0.2% AcOH); mp > 300 °C (dec); H NMR
(D2O) 6.82 (d, J ) 8.2 Hz, 1H), 6.69 (d, J ) 8.2 Hz, 1H), 5.83
(ddd, J ) 2.8, 6.0, 9.5 Hz, 1H), 5.22 (s, 1H), 4.66 (d, J ) 9.9
Hz, 1H), 3.84 (d, J ) 6.0 Hz, 1H), 3.81 (s, 3H), 3.76 (d, J ) 6.0
Hz, 1H), 3.57-3.50 (m, 2H), 3.42 (dd, J ) 3.3, 5.7 Hz, 1H),
3.40-3.34 (m, 1H), 3.08 (d, J ) 19.0 Hz, 1H), 3.01 (m, 1H),
2.60 (dd, J ) 4.0, 12.4 Hz, 1H), 2.43-2.37 (s, 3H overlapping
with m, 1H), 2.30 (td, J ) 3.4, 12.6 Hz, 1H), 2.12 (td, J ) 4.8
Hz, 13.0, 1H), 1.75 (d, J ) 12.0 Hz, 1H); 13C NMR (D2O) δ
174.5, 144.4, 140.5, 131.0, 129.5, 127.3, 127.1, 119.0, 112.9,
92.8, 85.1, 79.3, 76.1, 71.3, 70.8, 57.1, 55.7, 45.1, 43.2, 43.0,
40.5, 37.7, 33.8, 19.3; HRMS (ESI) calcd for C24H28NO8S
490.1536, found 490.1525; the average purity of 6b was found
to be >99% by analytical HPLC, giving tR ) 1.62 min (70:30
CH3CN/MeOH (v:v)) and tR ) 1.46 min (MeOH).
[
35S]GTP-γ-S Binding. [35S]GTP-γ-S binding was con-
ducted basically as described by Traynor and Nahorski.20 Cells
were scraped from tissue culture dishes into 20 mM Hepes, 1
mM EDTA and then centrifuged at 500g for 10 min. Cells were
resuspended in this buffer and homogenized using a Polytron
homogenizer. The homogenate was centrifuged at 27 000g for
15 min and the pellet resuspended in buffer A, containing 20
mM Hepes, 10 mM MgCl2, 100 mM NaCl, pH 7.4. The
suspension was recentrifuged at 27 000g and suspended once
more in buffer A. For the binding assay, membranes (8-15
µg protein) were incubated with [35S]GTP-γ-S (50 pM), GDP
(10 µM), and the appropriate compound, in a total volume of
1.0 mL, for 60 min at 25 °C. Samples were filtered over glass
fiber filters and counted as described for the binding assays.
Statistical analysis was conducted using the program Prism.
Morphine 6-â-S-D-Glucose, 6c. To an amber solution of
5d (50 mg, 0.074 mmol) in MeOH (6 mL) was added 5%
aqueous NaOH (0.4 mL). A white precipitate formed within 5
min. The mixture was stirred for 18 h and then AcOH (1.5 N,
15 drops to a pH of 7) was added. The solution was stirred for
5 min and then saturated NaHCO3 (15 drops to a pH of 8.5)
was added. The mixture was concentrated and the residue was
dissolved in 0.5 mL of water with 3 drops of 13% NH4OH and
purified by preparative TLC (SiO2, 200:40:5:0.05 CH2Cl2/
MeOH/H2O/13% NH4OH). After drying for 4 h at 100 °C (0.25
mmHg), 6c was obtained as a white powder (20 mg, 59%): Rf
1
) 0.21; mp > 300 °C (dec); H NMR (CD3OD) δ 6.55 (d, J )
8.0 Hz, 1H), 6.46 (d, J ) 8.0 Hz, 1H), 5.90 (ddd, J ) 2.9, 5.8,
9.0 Hz, 1H), 5.52 (dd, J ) 1.6, 9.7 Hz, 1H), 5.22 (s, 1H), 4.54
(d, J ) 9.8 Hz, 1H), 3.90 (d, J ) 11.2 Hz, 1H), 3.77 (d, J ) 6.0
Hz, 1H), 3.70 (dd, J ) 5.2, 11.9 Hz, 1H), 3.42 (dd, J ) 3.2, 5.6
Hz, 1H), 3.42-3.41 (m, 3H), 3.24 (dd, J ) 8.4, 9.6 Hz, 1H),
3.07 (bs, 1H), 3.05 (d, J ) 18.7 Hz, 1H), 2.68 (dd, J ) 4.0, 12.3
Hz, 1H), 2.49 (s, 3H), 2.49-2.39 (m, 2H), 2.18 (dt, J ) 7.9,
12.8 Hz, 1H), 1.81-1.79 (m, 1H); 13C NMR (CD3OD) δ 146.3,
140.4, 131.5, 131.4, 130.3, 126.7, 120.4, 117.9, 95.3, 88.4, 82.3,
79.9, 74.8, 71.6, 63.2, 60.7, 48.2, 46.3, 45.3, 43.0, 40.4, 36.7,
24.2; MS (ESI) m/z 464 [M + H]+; HRMS (ESI) calcd for C23H30-
NO7S 464.1743, found 464.1727; the average purity of 6c was
found to be 97.0% by analytical HPLC, giving tR ) 3.94 min
Determination of Analgesic Activity of Morphine
Analogues 5b‚HCl.21 Male ICR mice weighing 20-25 g at
the start of the experiment were used. Animals were group-
housed under standard laboratory conditions and were kept
on a 12:12 h day-night cycle (lights on at 08:00). Animals were
handled for 1-2 days prior to conducting the experiments.
Morphine‚HCl or 5b‚HCl were dissolved in water. Drugs were
injected at a volume of 0.1 mL. Nociception was assessed using
the tail flick assay with an analgesia instrument (Stoelting)
that uses radiant heat. This instrument is equipped with an
automatic quantification of tail flick latency and a 15 s cutoff
to prevent damage to the animal’s tail. During testing, the
focused beam of light was applied to the lower half of the

Thiosaccharide Analogues of M6G and C6G
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5815
(8) Osborne, R. J.; Joel, S.; Slevin, M. Morphine intoxication in renal
failure; the role of morphine-6-glucuronide. Br. Med. J. 1986,
292, 1548-1549.
animal’s tail, and tail flick latency was recorded. Baseline
values for tail flick latency were determined before drug
administration in each animal. Basal tail flick latency was
between 3.2 and 8.0 s (average 5.86 ( 0.16 SEM). Immediately
after testing, animals were injected subcutaneously with the
test compound or saline as a vehicle control. Following
injections, animals were tested for tail flick latencies at 30 and
60 min postinjection. Antinociception was quantified by the
following formula: % antinociception ) 100 × [(test latency
- baseline latency)/(15 - baseline latency)]. If the animal did
not respond prior to the 15 s cutoff, the animal was assigned
a score of 100%. Behavioral results were analyzed using
ANOVAs with morphine and 5b‚HCl as between-group vari-
ables and postdrug treatment time (30, 60 min) as the repeated
measure followed by Student Newman-Keuls post-hoc tests
where appropriate. The level of significance was set at p <
0.05.
(9) Knapp, S.; Myers, D. S. R-GlcNAc Thioconjugates. J. Org. Chem.
2001, 66, 3636-3638.
(10) Kiefel, M. J.; Thomson, R. T.; Radocanovi, M.; von Itzstein, M.
Synthesis of carbohydrates with an anomeric thiol moiety for
elaboration into metabolically stable thioglycosides. J. Carbo-
hydr. Chem. 1999, 18, 937-959.
(11) Welsh, L. H. Synthesis of 3-O-acetyl-morphine. J. Org. Chem.
1954, 19, 1409.
(12) Stork, G.; Clarke, F. H. The SN2′ reaction. III. Structure and
SN2′ reactions of the halocodides. J. Am. Chem. Soc. 1956, 78,
4619-4624.
(13) Rukhman, I.; Yudovich, L.; Nisnevich, G.; Gutman, A. L.
Selective synthesis of both isomers of morphine 6-â-D-glucu-
ronide and their analogues. Tetrahedron 2001, 57, 1083-1092.
(14) Yoshimura, H.; Natsuki, R.; Ida, S.; Oguri, K. Chemical Reactiv-
ity of morphine and morphine-6-conjugates and their binding
to rat brain. Chem. Pharm. Bull. 1976, 24, 901-906.
(15) Bognar, R.; Makleit, S.; Radics, L. Conversions of tosyl and mesyl
derivatives of the morphine group, VIII. Acta Chim. Acad. Sci.
Hung. 1971, 67, 63-69.
(16) Bollenback, G. N.; Long, J. W.; Benjamin, D. G.; Lindquist, J.
A. The synthesis of aryl-D-glucopyranosiduronic Acids. J. Org.
Chem. 1955, 77, 3310-3315.
(17) Wallace, O. B.; Springer, D. M. Mild, selective deprotection of
thioacetates using sodium thiomethoxide. Tetrahedron Lett.
1998, 2693-2694.
Acknowledgment. The authors thank Prof. Gavril
Pasternak for helpful suggestions regarding this work.
Supporting Information Available: Elemental analysis
of 5a, 5c, and 6a; HPLC purity results for 5b, 5d-f, 6b-d;
and HRMS data for 5b, 5d-f, 6b-d. This material is available
free of charge via the Internet at http://pubs.acs.org.
(18) Pasternak, G. W.; Wilson, H. A.; Snyder, S. H. Differential effects
of protein-modifying reagents on receptor binding of opiate
agonists and antagonists. Mol. Pharmacol. 1975, 11, 340-351.
(19) 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. Structure-Activity Relationships of Some Opiate
Glycosides. Bioorg. Med. Chem. Lett. 2003, 13, 1207-1214.
(20) Traynor, J. R.; Nahorski S. R. Modulation by mu-opioid agonists
of guanosine-5′-O-(3-[35S]thio)triphosphate binding to mem-
branes from human neuroblastoma SH-SY5Y cells. Mol. Phar-
macol. 1995, 47, 848-854.
(21) Dewey, W. L.; Harris, L. S. The tail flick test. In Methods in
Narcotics Research; Ehenpreis, S., Neidle, A., Eds.; Marcel
Dekker: New York, 1975; pp 101-109.
(22) Zaveri, N.; Polgar, W.; Olsen, C.; Kelson, A. B.; Grundt, P.; Lewis,
J. W.; Toll, L. Characterization of opiates, neuroleptics and
synthetic analogs at ORL1 and opioid receptors. Eur. J. Phar-
macol. 2001, 428, 29-36.
References
(1) Gureje, O.; Von Kkorff, M.; Simon, G. E.; Gater, R. Persistent
pain and well-being. J. Am. Med. Assoc. 1998, 280, 147-151.
(2) Ray, O. Drugs, Society and Human Behavior, 3rd ed.; The C. V.
Mosby Co.: St. Louis, MO, 1983.
(3) Penson, R. T.; Joel, S. P.; Roberts, M.; Gloyne, A.; Beckwith, S.;
Slevin, M. L. The bioavailability and pharmacokinetics of
subcutaneous, nebulized and oral morphine-6-glucuronide. Br.
J. Clin. Pharmacol. 2002, 53, 347-354.
(4) Yoshimura, H.; Ida, S.; Oguri, K.; Tsukamoto, H. Biochemical
basis for analgesic activity of morphine-6-glucuronide: penetra-
tion of morphine-6-glucuronide in the brain of rats. Biochem.
Pharmacol. 1973, 22, 1423-1430.
(5) Pasternak, G. W.; Bodnar, R. J.; Clark, J. A.; Inturkisi, C. E.
Morphine-6-glucuronide, a potent mu agonist. Life Sci. 1987, 41,
2845-2849.
(6) Peat, S. J.; Hanna, M. H.; Woodham, M. Morphine-6-glucuronide
effects on ventilation in normal volunteers. Pain 1991, 45, 101-
104.
(7) Thompson, P. I.; Joel, S. P.; John, L. Respiratory depression
following morphine and morphine-6-glucuronide in normal
subjects. Br. J. Clin. Pharmacol. 1995, 40, 145-152.
(23) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (I50) of an enzymatic reaction. Biochem. Phar-
macol. 1973, 22, 3099-3108.
JM049554T