Synthesis and in vitro biological evaluation of a carbon glycoside analogue of morphine-6-glucuronide

Article abstract of DOI:10.1016/j.bmcl.2005.01.072

Attachment of a glucose moiety to 6-β-aminomorphine afforded compound 3, where the glucose moiety was linked to the C-6 nitrogen atom by a two-carbon bridge. The synthesis of 3 was accomplished in eight steps from 3-triisopropylsilyl-6-β-aminomorphine and 2,3,4,6-tetra-O-benzyl-d-glucose. The C-glycoside 3 was prepared with the objective of examining a metabolically stable analogue of morphine-6-glucuronide and determining the potency and selectivity of opioid receptor binding. Competition binding assays showed that 3 bound to the μ opioid receptor with a K_i value of 3.5 nM. The C-glycoside 3 exhibited δ/μ and κ/μ selectivity ratios of 76 and 165, respectively. The synthetic intermediate (i.e., benzyl precursor, compound 11) bound to the μ opioid receptor with a K_i value of 0.5 nM, was less selective for the μ opioid receptor. The [³⁵S] GTPγS assay was used to evaluate the functional properties of compounds 3 and 11. Compound 3 was determined to be a full agonist at the μ opioid receptor, whereas compound 11 was found to be a partial agonist. Compound 3 was determined to be very stable in the presence of human liver S9, and rat and monkey liver microsomes: no detectable loss of 3 was observed up to 90 min. Compound 3 was also very stable at pH 2 and pH 7.4, suggesting that 3 possessed properties for sustained duration of action.

Full text of DOI:10.1016/j.bmcl.2005.01.072

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1583–1586
Synthesis and in vitro biological evaluation of a carbon
glycoside analogue 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 28 October 2004; revised 27 January 2005; accepted 28 January 2005
Abstract—Attachment of a glucose moiety to 6-b-aminomorphine afforded compound 3, where the glucose moiety was linked to the
C-6 nitrogen atom by a two-carbon bridge. The synthesis of 3 was accomplished in eight steps from 3-triisopropylsilyl-6-b-amino-
morphine and 2,3,4,6-tetra-O-benzyl-D-glucose. The C-glycoside 3 was prepared with the objective of examining a metabolically sta-
ble analogue of morphine-6-glucuronide and determining the potency and selectivity of opioid receptor binding. Competition
binding assays showed that 3 bound to the l opioid receptor with a Ki value of 3.5 nM. The C-glycoside 3 exhibited d/l and j/l
selectivity ratios of 76 and 165, respectively. The synthetic intermediate (i.e., benzyl precursor, compound 11) bound to the l opioid
receptor with a Ki value of 0.5 nM, was less selective for the l opioid receptor. The [35S]GTPcS assay was used to evaluate the func-
tional properties of compounds 3 and 11. Compound 3 was determined to be a full agonist at the l opioid receptor, whereas com-
pound 11 was found to be a partial agonist. Compound 3 was determined to be very stable in the presence of human liver S9, and rat
and monkey liver microsomes: no detectable loss of 3 was observed up to 90 min. Compound 3 was also very stable at pH 2 and
pH 7.4, suggesting that 3 possessed properties for sustained duration of action.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Morphine-6-glucuronide (M6G) is a phase II metabolic
conjugate of morphine with significantly greater analge-
sic potency than morphine.1–3 M6G is currently in late
stage clinical trials for the treatment of postoperative
pain.4 The oral bioavailability of M6G is only 11%5
and improvement of the chemical and metabolic stabil-
ity of M6G could possibly increase its effectiveness as
a potential pain medication. A general strategy for
improving the in vivo metabolic stability of glycoconju-
gates involves the replacement of the glycosidic oxygen
atom with carbon, nitrogen, or sulfur atoms.6,7 We pre-
viously employed this strategy in designing a library of
glucosyl and glucuronosyl analogues of M6G in which
the glycosidic oxygen atom was replaced with a sulfur
atom.8 The 6-b sulfur analogues of M6G showed mod-
est improvement in l opioid receptor affinity and func-
tional efficacy, but showed less selectivity for l versus
d and j opioid receptors. Reported herein is an exten-
sion of this work detailing the results of an eight-step
synthesis and in vitro biological and chemical evaluation
of a novel, l-selective, amide-linked carbon glycoside
analogue of M6G (Fig. 1).
The C-b-glycopyranosyl acyl chloride 9 was prepared by
a five step procedure from commercially available
2,3,4,6-tetrabenzylglucopyranose 4 (Scheme 1). Com-
pound 4 was oxidized with DMSO/Ac2O to provide
the corresponding lactone 5 (94%).9 Addition of 5 to a
À78 ꢁC THF solution of lithium ethyl acetate,10 fol-
lowed by acidic aqueous workup afforded the hemiketal
6, that was formed by a stereoselective aldol reaction
(91%).11 The reduction of 6 with triethylsilane in the
presence of BF3ÆOEt2 in acetonitrile at 0 ꢁC gave the
known ethyl ester 7 (86%).11,12 Hydrolysis of the ethyl
ester group of 7 with LiOH in 1:1 THF/H2O at reflux
and subsequent acidic aqueous workup gave the carbox-
ylic acid 8 (87%). The conversion of 8 to the correspond-
ing acyl chloride 9 (100%, crude yield) was accomplished
by stirring in neat thionyl chloride for 18 h. Addition of
1.5 equiv of the crude acid chloride 9 to 3-triisopropylsil-
yl-6-b-aminomorphine13 in CH2Cl2 in the presence of
2.0 equiv of Et3N gave the protected morphine glucose
analogue 10 (93%). The triisopropylsilyl protecting
Keywords: Morphine-6-glucuronide; Pain; Morphine.
*
Corresponding author. Tel.: +1 858 458 9305; fax: +1 858 458
9311; e-mail: jmacdougall@hbri.org
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.072

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J. M. MacDougall et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1583–1586
receptor selective. Compared to M6G, compound 11
Me
H
Me
H
N
N
possessed 27-fold greater potency than M6G for the l
opioid receptor. The selectivity of compound 11 for
the l versus d and l versus j receptors were 10-fold
and 34-fold, respectively. Compared to M6G, com-
pound 3 showed 3.7-fold greater potency for the l opi-
oid receptor. This result was consistent with previous
studies that have shown that saturation of the 7,8-dou-
ble bond provided analogues of M6G with increased
analgesic potency.17 The selectivity of compound 3 for
the l versus d and l versus j receptors was 77- and
166-fold, respectively. It is noteworthy that the l versus
d but not l versus j receptor selectivity of compound 3
was considerably improved relative to the value for
M6G (i.e., 12.5- and 316-fold selectivity, respectively).
Compound 3 showed slightly greater potency toward
the l receptor compared to thiosaccharides 2a (2.5-fold)
and 2b (1.6-fold) and significantly improved d/l and j/l
receptor selectivity ratios (Table 1).
O
O
HO
HO
O
O
S
HOOC
O
R
OH
HO
OH
HO
HO
HO
Me
N
1
2a: R = COOH
2b: R = CH2OH
H
O
HO
HO
HO
NH
O
O
OH
HO
3
Figure 1. Compound 1 is M6G and 2 and 3 are metabolically stable
analogs.
The functional activity of compounds 3 and 11 was eval-
uated using the [35S]GTPcS assay (Table 2).18 The
[35S]GTPcS assay measures the ability of the test
compound to activate the G protein associated with
either the l, d, or j opioid receptor. In this assay, the
compoundꢀs potency or affinity for the receptor (defined
by its EC50 for stimulating [35S]GTPcS binding) was
examined in vitro. Agonist efficacy (Emax) was defined
as the degree to which the compound maximally stimu-
lated [35S]GTPcS binding relative to control. The EC50
value represented the concentration that produced 50%
maximal stimulation of [35S]GTPcS binding by that
compound. Full agonists stimulated [35S]GTPcS binding
to a maximal extent and partial agonists caused a de-
creased level of binding. Based on the Emax values for
stimulating [35S]GTPcS binding, compound 3 was deter-
mined to be a full agonist at the l and d receptors and a
partial agonist at the j receptor. Compound 11 was
determined to be a full agonist at the d receptor and a
partial agonist at the l and j receptors. The efficacy
(Emax) of compound 3 at the l receptor (75%) was sub-
stantially higher than M6G (45%) and the thiosaccha-
ride analogues 2a and 2b (i.e., 46.6% and 36.0%,
respectively).
group of 10 was removed by the addition of TBAF in
THF to give the phenol 11 (90%). Subsequent catalytic
hydrogenation of 11 with activated 10% Pd/C in acidic
methanol gave the amide-linked C-b-glycopyranoside
analogue of M6G, compound 3 (52%). Hydrogenolysis
of 11 with Pd/C in the absence of HCl resulted in the
reduction of the phenanthrene carbon double bond
and afforded 12 in quantitative yield, but did not result
in hydrogenolysis of the glucose benzyl ether groups.
Under these conditions, the basic morphinan nitrogen
atom in 11 likely inhibited O-debenzylation.14 The spec-
tral data for all the synthetic compounds was in full
agreement with the assigned structures or their literature
values.15
The IC50 values obtained from competition binding as-
says with l, d, and j opioid receptors for compounds
3 and 11 were converted into Ki values as described in
the Experimental section. The Ki values for the test com-
pounds and reference materials were listed in Table 1. 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 recep-
tor agonist). Ki values were determined by measuring the
inhibition of binding of these radioligands to the recep-
tor by the test compounds 3 and 11.16 The benzyl deriv-
ative 11 and the deprotected congener 3 were both l
In order to evaluate metabolic stability, test compounds
3, 11, and 12 were incubated with hepatic microsome
preparations from rat (RLM) and monkey (MLM) sup-
plemented with NADPH.15 In addition, stability in the
Table 1. Competitive inhibition of l, d, and j opioid receptors by compounds 2a, 2b, 3, and 11
Entry
Ki (nM) SEMa
Receptor selectivity
j/l
l
d
j
d/l
M6Gb
2ab
2bb
3
12.9 0.9
8.7 0.9
5.4 0.8
3.5 0.4
0.5 0.2
170
1
4060 230
288 12
136 17
12.5
3.6
316
33
31.4 2.3
56.2 2.2
266.3 47.3
7.2 1.2
10.4
76.7
10.3
25.4
166
33.7
574.9 7.8
15.8 0.4
11
a SEM, standard error of the mean. Each value is the mean of at least three independent determinations SEM.
b Data taken from Ref. 6. The assay conditions used in the evaluation of test compounds 3 and 11 were the same as those used in the evaluation of
M6G, 2a, and 2b.

J. M. MacDougall et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1583–1586
1585
Table 2. Stimulation of [35S]GTPcS binding by compounds 2a, 2b, 3, and 11 mediated by the l, d, and j opioid receptors
Entry
l
d
j
a
b
EC50
Emax
EC50
190 20
Emax
EC50
Emax
M6Gd
2ad
2bd
3
72.3 26.7
90.6 22.9
91.5 23.4
37.2 0.5
45.0 5.0
46.6 10.1
64.5 0.5
75.2 3.6
36.0 11.2
80.0 1.0
78.7 0.9
51.5 6.5
73.9 7.8
65.4 1.9
>10 K
NDc
NDc
NDc
50.1 36.7
192 15
321 93
1717.5 24.5
1.8 0.6
42.5 2.5
38.5 5.5
44.5 16.3
334.9 123.2
2.2 1.1
11
622.6 9.2
a EC50 (nM): The EC50 value represents the concentration of compound that produced 50% stimulation of [35S]GTPcS binding.
b Emax (% stimulation): Agonist efficacy is defined as the degree to which the compound maximally stimulates [35S]GTPcS binding relative to control.
c ND: No detectable activity.
d Data from Ref. 6. The assay conditions used in the evaluation of 2a and 2b were the same as those used in the evaluation of M6G, 3, and 11.
presence of the post-mitochondrial supernatant from
human liver (HLS9) was examined.15 HPLC was used
to evaluate the disappearance of starting material over
time. The benzyl derivative 11 possessed half-lives of
24.6 and 30.7 min in the presence of RLM and MLM,
respectively. Mass spectral analysis of metabolic extracts
of 11 showed a prominent metabolite ion that was con-
sistent with loss of a benzyl group. In the presence of
HLS9, no detectable metabolism of 11 was observed in
incubations run for 90 min. The C7/C8 dihydro deriva-
tive 12 possessed half-lives of 42.9 and 19.8 min in the
presence of HLS9 and RLM, respectively, but was com-
pletely stable for 90 min in the presence of MLM. The
glucose derivative 3 was quite stable in the presence of
all three hepatic preparations from these species and
no detectable loss of 3 was observed for 90 min. The
chemical stability of 3, 11, and 12 was examined at
pH 2.0 and pH 7.4 by HPLC analysis. No detectable
loss of 3 was observed at pH 7.4. At pH 7.4, compounds
11 and 12 had half-lives of 1419 and 1903 min, respec-
tively. At pH 2.0, compounds 3, 11, and 12 had half-
lives of 3311, 1229, and 1900 min, respectively.
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
91%
ii.
94%
i.
86%
iii.
O
O
O
OEt
BnO
BnO
BnO
OH
O
OH
O
4
5
6
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
87%
iv.
100%
v.
O
O
O
OEt
OH
Cl
BnO
BnO
BnO
7
8
9
O
O
O
Me
H
Me
H
N
N
90%
vii.
93%
vi.
O
O
TIPSO
BnO
NH
HO
NH
O
O
BnO
BnO
O
O
OBn
OBn
BnO
BnO
10
11
BnO
ix.
viii.
100%
52%
Me
Me
H
N
N
H
3
12
O
HO
NH
O
HO
NH
O
BnO
BnO
O
O
HO
HO
O
OBn
OH
BnO
HO
Scheme 1. Reagents and conditions: (i) CH3S(O)CH3, Ac2O; (ii) LiCH2CO2Et, THF then HCl, À78 ꢁC to rt; (iii) Et3SiH, BF3ÆOEt2,CH3CN, 0 ꢁC to
rt; (iv) NaOH, H2O, THF, reflux then HCl; (v) SOCl2; (vi) 3-O-triisopropylsilyl-6-b-aminomorphine, Et3N, CH2Cl2; (vii) Bu4NF, THF, H2O; (viii)
H2, 10% Pd–C, MeOH; (ix) H2, 10% Pd–C, MeOH, HCl.

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J. M. MacDougall et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1583–1586
13. MacDougall, J. M.; Zhang, X.-D.; Polgar, W. E.; Khro-
yan, T. V.; Toll, L.; Cashman, J. R. Bioorg. Med. Chem.
2004, 12, 5983–5990.
14. Czech, B. P.; Bartsch, R. A. J. Org. Chem. 1984, 49, 4076–
4078.
In summary, the C-glycoside 3 was prepared by an
eight-step convergent synthesis from 2,3,4,6-tetra-O-
benzyl-D-glucose, 4, and 3-triisopropylsilyl-6-b-amino-
morphine. Compound 3 showed a 3.7-fold greater
affinity for the l opioid receptor compared to M6G.
The selectivity ratios of compound 3 for the d versus l
and j versus l receptors were 76.7 and 166, respectively.
The d/l selectivity for compound 3 (i.e., 76.7) was signif-
icantly improved relative to the value for M6G, which
was 12.5. Compound 3 was quite stable in the presence
of human liver S9, and rat and monkey liver microsomes
supplemented with NADPH. Compound 3 was also
very stable at pH 2 and pH 7.4. Together, the data sug-
gests that 3 has the properties necessary for sustained
pharmacological activity. Further work is in progress
to investigate the biological properties of these com-
pounds. Increasing the chemical and metabolic stability
of M6G may provide a new class of longer-lived, potent
opioid agonists with greater bioavailability.
15. New compounds 3, 10, 11, and 12 showed satisfactory 1H
NMR, 13C NMR, and MS data. Analytical purity for
compound 11 was determined by straight phase HPLC
using a Hitachi L74 liquid chromatograph with a L-7400
UV–vis detector (254 nm), a D7500 integrator, and an
Axxi-chrom silica column (250 mm · 4.6 mm, i.d.) (Rich-
ard Scientific, Novato, CA). For determination of analyt-
ical purities a mobile phase A = 60/40/0.02, MeOH/
2-propanol/HClO4 (v/v) or mobile phase B = 55/45/0.01,
MeOH/2-propanol/HClO4 (v/v) was used. The average
purity of 11 was found to be P99% by analytical HPLC
giving tR = 3.24 min (mobile phase A) and tR = 4.66 min
(mobile phase B). The purity of 3 was determined with
straight phase HPLC as described above. The purity of 3
was found to be P98% by analytical HPLC giving
tR = 3.41 min (mobile phase A) and tR = 3.74 min (mobile
phase B). Metabolic stability was evaluated by incubating
the test compound (0.4 mM) in the presence of MLM or
HLM (1.2 mg/mL) or HLS9 (3.2 mg/mL), 50 mM potas-
sium phosphate buffer (pH 7.4), 0.5 mM NADP+, 0.5 mM
glucose-6-phosphate, 5 IU/mL glucose-6-phosphate dehy-
drogenase, 1 mg/mL diethylenetriaminepentaacetic acid,
and 7 mM MgCl2 (0.1 mL total volume). Each time course
incubation was run for 0, 10, 20, 30, 60, or 90 min at
37 ꢁC. For compounds 11 and 12, the reaction was
stopped at the appropriate time by the addition of
CH2Cl2/2-propanol (1 mL, 3:1 v/v), 10 mg Na2CO3 and
mixed thoroughly. After centrifugation to separate the
layers, the organic solvent was removed under a stream of
argon. MeOH (0.2 mL) was added to the residue and the
resulting material was thoroughly mixed, centrifuged, and
an aliquot was analyzed by HPLC using the conditions
described above. The extraction efficiency for 11 and 12
was calculated to be 85%. For compound 3, a solid phase
extraction procedure was used. The metabolic incubations
were stopped by the addition of cold Na2CO3 (0.02 mL,
2 M) and centrifuged. The supernatant was placed on an
Oasis solid phase extraction cartridge (Waters Corp.,
Bedford, MA), washed with H2O (0.7 mL), followed by
5:95 MeOH/H2O (0.7 mL) and eluted with 80:20 CH3CN/
MeOH (0.7 mL). The organic solvent was removed under
a stream of argon. To the residue MeOH (0.2 mL) was
added and the resulting material was analyzed by HPLC
using the reverse phase conditions described above. The
extraction efficiency was 45%. Selected metabolic extracts
were also analyzed with an M-8000 ion trap mass
spectrometer (Hitachi, San Jose, CA) equipped with an
electrospray ionization source. Sample delivery was car-
ried out in the flow injection analysis mode using a
LaChrom separation module with an isocratic solvent
system of MeOH/water/HCOOH (50:50:0.17, v/v) running
at 0.2 mL/min.
Acknowledgments
We thank Andy Liu for synthesis of actone 5.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2005.01.072.
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