Syntheses of potential metabolites of a potent κ-opioid receptor agonist, TRK-820

Article abstract of DOI:10.1248/cpb.52.670

Chemical syntheses of three kinds of potential metabolites of TRK-820, a potent κ-opioid receptor agonist, were described. One of the potential metabolites 2, 17-N-dealkylated TRK-820, was synthesized starting from noroxycodone through 8 steps in 21% tota

Full text of DOI:10.1248/cpb.52.670

670
Chem. Pharm. Bull. 52(6) 670—674 (2004)
Vol. 52, No. 6
k
Syntheses of Potential Metabolites of a Potent -Opioid Receptor Agonist,
TRK-820
Kuniaki KAWAMURA,* Hiromasa HORIKIRI, Jun HAYAKAWA, Chie SEKI, Ken-ichi YOSHIZAWA,
Hideo UMEUCHI, and Hiroshi NAGASE
Pharmaceutical Research Laboratories, Toray Industries, Inc.; 1111 Tebiro, Kamakura, Kanagawa 248–8555, Japan.
Received December 11, 2003; accepted March 6, 2004
Chemical syntheses of three kinds of potential metabolites of TRK-820, a potent k-opioid receptor agonist,
were described. One of the potential metabolites 2, 17-N-dealkylated TRK-820, was synthesized starting from
noroxycodone through 8 steps in 21% total yield. Glucuronidation of intermediate 10 and compound 1, the free
base of TRK-820, was carried out stereoselectively to give 3-O-b-D-glucuronides 15 and 16 in good yields, respec-
tively. Syntheses of potential conjugated metabolites 3 and 4 were accomplished through 10 steps and 2 steps in
11% and 43% total yields, respectively. Among the potential metabolites of TRK-820, compounds 2 and 4 were
identified as metabolites in human hepatocytes. The results of pharmacological studies of compounds 2, 3, and 4
are described.
Key words k-opioid receptor agonist; TRK-820; metabolite; glucuronide
1
amino group was elucidated to be 6b-configuration by H-
NMR spectral analysis of the 5-proton, which was observed
at dꢀ4.75 with an axial–axial coupling of 7.1 Hz between
the 5- and 6-protons. Compound 7 was converted into amide
8 by debenzylation under hydrogenolysis conditions, fol-
lowed by acylation with (E)-3-(3-furyl)acryloyl chloride in
61% yield (2 steps). The next step was deprotection of the 3-
O-methyl and Boc groups of compound 8 to obtain the first
target compound 2. For this purpose, the 17-N-Boc group of
compound 8 was first converted to a 9H-fluorenylmethoxy-
carbonyl group (Fmoc) in 96% (2 steps). When compound 6
was directly treated with 1.0 M BBr3 for removal of the 3-O-
methyl and 17-N-Boc groups, the desired compound was ob-
tained in ca. 50% yield concomitant with a significant
amount of a 17-N-methylated compound due to side reaction
with the resulting methyl bromide under the reaction condi-
tions. Thus, in the case of compound 8, we had to replace the
17-N-Boc group with a 17-N-Fmoc group which is stable in
Lewis acidic conditions. The reason why an Fmoc group was
not used in the first protection step was due to instability of
the Fmoc group in the presence of intramolecular basic
amine at the 6-position (6-N(CH3)Bn or 6-NHCH3) in the
We have described the design and synthesis of (E)-N-[17-
(cyclopropylmethyl)-4,5a-epoxy-3,14-dihydroxymorphinan-
6b-yl]-3-(furan-3-yl)-N-methylprop-2-enamide monohydro-
chloride, TRK-820 (Fig. 1), and its pharmacological activ-
ity.1) TRK-820 exhibited a high k-opioid agonistic activity
and effective antipruritic actions in histamine resistant pruri-
tus models using mice.2,3) Now clinical trials of TRK-820 are
in progress (Phase III in Europe and Phase IIb in Japan) as an
antipruritic agent for uraemic pruritus.
In order to investigate pharmacokinetics of TRK-820, it is
necessary to synthesize potential metabolites of TRK-820 as
standard materials. In general, 17-N-dealkylation and conju-
gation (3-O-b-D-glucuronide formation) are well known as
major metabolic pathways for detoxification in 4,5-epoxy-
morphinans.4) Therefore, we supposed three kinds of
metabolites 2, 3, and 4 of TRK-820 would occur in metabo-
lism (Fig. 2). It is also important to investigate whether these
compounds are active or inactive metabolites in pharmaceuti-
cal research. Herein we wish to report the syntheses of com-
pounds 2, 3, and 4, and the results of their pharmacological
studies and in vitro metabolism on TRK-820.
Results and Discussion
Noroxycodone 5, which is a 17-N-dealkylated derivative
with a 14b-hydroxy-4,5a-epoxymorphinan skeleton, was
used as the starting material to synthesize compound 2
(Chart 1). Protection of the 17-amino group in noroxycodone
5 as a tert-butoxycarbonyl group (Boc), followed by reduc-
tive amination of the resulting compound 6 with N-benzyl-
methylamine using NaBH3CN5) gave compound 7 in 72%
yield (2 steps). Stereochemistry of the newly introduced
Fig. 2. Potential Metabolites of TRK-820
Fig. 1. Structure of TRK-820
∗ To whom correspondence should be addressed. e-mail: Kuniaki_Kawamura@nts.toray.co.jp
© 2004 Pharmaceutical Society of Japan

June 2004
671
Reagents and conditions: (a) 13, BF3·OEt2, MS4A, CH2Cl2, room temperature, 3 h
(92%); (b) 12, CdCO3, PhCH3, reflux, 16 h (27%); (c) 14, BF3·OEt2, MS4A, CH2Cl2,
ꢂ78 °C, 1 h (32%).
Chart 2
Reagents and conditions: (a) (Boc)2O, Na2CO3, CHCl3, H2O, room temperature, 3 h
(quant.); (b) BnNHCH3, PhCO2H, PhH reflux, 19 h; NaBH3CN, CH3OH, 0 °C, 2 h
(72%); (c) H2, 10% Pd–C, o-phthalic acid, CH3OH, room temperature, 25 h; (d) (E)-3-
(3-furyl)acryloyl chloride, Na2CO3, THF, H2O, 0 °C to room temperature, 14 h (61% 2
steps); (e) CF3CO2H, CH2Cl2, 0 °C to room temperature, 1 h; (f) FmocCl, Na2CO3, 1,4-
dioxane, H2O, room temperature, 2 h (96% 2 steps); (g) BBr3, CH2Cl2, 0 °C to room
temperature, 3 h (65%); (h) NaOH, H2O, THF, room temperature, 16 h (77%).
Reagents and conditions: (a) 13, BF3·OEt2, MS4A, CH2Cl2, room temperature, 19 h
(82%); (b) 11, NaH, 15-crown-5-ether, DMF, room temperature, 2 h (0%); (c) 12,
CdCO3, PhCH3, 110 °C, 20 h (0%).
Chart 3
Chart 1
Treatment of compound 10 with 2 equivalents of
trichloroacetimidate 13 in the presence of 2.3 equivalents of
BF3·OEt2 in CH2Cl2 at room temperature for 75 min afforded
the glucuronated derivative 15 in 92% yield after chromato-
graphic purification. The 1H-NMR spectrum of compound 15
showed signals indicative of a methyl 2,3,4-tri-O-acetylglu-
curonate function at dꢀ1.96 (3H, s), 1.99 (3H, s), 2.00 (3H,
s), and 3.72 (3H, s) and also showed signals indicative of an
N-methyl amide function at dꢀ3.14 (3H, s). The electro-
spray ionization mass (ESI-MS) spectrum showed the molec-
ular ion peak at m/zꢀ961 ([MꢁH]ꢁ).
In the case of glucuronidation on compound 1, the free
base of TRK-820, the use of epoxide 11 and bromide 12 was
unsuccessful and the desired glucuronated derivative 16 was
not produced. The reason why compound 16 was not ob-
tained is unclear. The only point of difference between 1 and
10 is that the substrate 1 has a basic nitrogen. It has been re-
ported that reproducibility of the Koenig-Knorr reaction
using bromide 12 was poor.13,14)
Fig. 3. Glycosyl Donors
compound derived from reductive amination reaction. Then,
compound 9 was treated with 1.0 M BBr3 at 0 °C to room
temperature to give phenol 10 in 65% yield. Deprotection of
the Fmoc group of compound 10 under basic conditions gave
the first target compound 2 in 77% yield. The chemical struc-
ture of compound 2 was confirmed by 1H-NMR and electron
1
ionization mass (EI-MS) spectra. H-NMR analysis showed
that the characteristic 5-proton of 4,5-epoxymorphinan was
observed at dꢀ4.55 (0.7H, d, Jꢀ7.8 Hz) and 4.63 (0.3H, d,
Jꢀ7.8 Hz) with an axial–axial coupling between the 5- and
6-position and the (E)-olefinic protons of the amide side
chain at dꢀ6.38 (0.7H, d, Jꢀ15.2 Hz), 6.89 (0.3H, d,
Jꢀ15.2 Hz), 7.20 (0.7H, d, Jꢀ15.2 Hz), and 7.36 (0.3H, d,
Jꢀ15.2 Hz) with trans coupling as a mixture of rotation iso-
mers on the amide bond axis. The EI-MS spectrum showed
the molecular ion peak at m/zꢀ422 ([M]ꢁ).
Similarly, trichloroacetimidate 13 was an appropriate gly-
cosyl donor in this case (Chart 3). Treatment of compound 1
with 1.5 equivalents of trichloroacetimidate 13 in the pres-
ence of 1.8 equivalents of BF3·OEt2 in CH2Cl2 at room tem-
perature afforded the glucuronated derivative 16 in 82% yield
1
after chromatographic purification. The H-NMR spectrum
The next step was to establish glucuronidation methods on
compounds 10 and 1 to synthesize compounds 3 and 4. First,
we prepared glucuronic acid derivatives 11,6) 12,7) 13,8—11)
and 1412) in accordance with literature to investigate the reac-
tivity with compounds 10 and 1 (Fig. 3).
Treatment of compound 10 with glycosyl donor 12 or 14
gave the desired glucuronated derivative 15 in low yields
(27—32%). Unfortunately, our efforts to improve the yield
by using 12 or 14 were in vain. We finally found that
trichloroacetimidate 13 was the best glycosyl donor for the
glucuronidation on compound 10 (Chart 2).
showed signals indicative of a methyl 2,3,4-tri-O-acetylglu-
curonate function at dꢀ1.95 (3H, s), 1.98 (3H, s), 2.00 (3H,
s), and 3.71 (3H, s) and also showed signals indicative of an
N-methyl amide function at dꢀ3.16 (3H, s). The ESI-MS
spectrum showed the molecular ion peak at m/zꢀ793
([MꢁH]ꢁ).
Deprotection of both compounds 15 and 16 was accom-
plished by treatment with lithium hydroxide followed by neu-
tralization using 1 M HCl to produce the target compounds 3
and 4, respectively (Chart 4). Because both compounds 3 and
4 have high polarity, they were distributed into water layer on

672
Vol. 52, No. 6
the scratching behavior at low dose.3) These results indicated
that compounds 2, 3, and 4 were substantially inactive on the
above experimental conditions.
Conclusion
Chemical syntheses of three kinds of potential metabolites
of TRK-820 were accomplished. Compound 2 was prepared
from noroxycodone 5 in 21% total yield (8 steps). Stereose-
lective glucuronidation of compounds 1 and 10 succeeded by
using trichloroacetimidate methods. Compound 3 was pre-
pared from noroxycodone 5 in 11% total yield (10 steps),
and compound 4 was prepared from compound 1 in 43%
yield (2 steps). Among the potential metabolites of TRK-
820, compounds 2 and 4 were indeed confirmed to occur in
human hepatocytes, and the synthesized compounds 2, 3, and
4 are unlikely to have antipruritic activity like TRK-820. The
availability of these metabolites by chemical synthesis
should facilitate further clinical studies on TRK-820.
Reagents and conditions: (a) LiOH·H2O, THF, CH3OH, H2O, room temperature,
18 h for 15 (43%); (b) LiOH·H2O, THF, H2O, room temperature, 25 h for 16 (54%).
Chart 4
work-up. Therefore, their purification was carried out as fol-
lows. The water layer containing compound was washed with
an organic solvent, and the resulting water layer was directly
concentrated in vacuo. To the residue which contained the
desired compound and inorganic salts was added an alco-
holic solvent to precipitate the desired compound and inor-
ganic salts, and the resulting precipitation was purified
through Sep-Pak Vac® filled with ODS using water and
Experimental
General Methods All reactions were proceeded under protection from
light and the reactions requiring anhydrous conditions were conducted under
atmosphere of dry argon. The progress of reactions and purity of intermedi-
CH3OH as eluent. As the results of the above purification ates were monitored on Merck analytical silica gel TLC plates 60 F254. For
column chromatography Merck silica gel 60 (0.063—0.200 mm) was used.
methods, pure compounds 3 and 4 were obtained in 43% and
Melting points were measured on Yanaco MP-500D melting point apparatus
54% yields, respectively. The chemical structure of com-
without correction. 1H-NMR spectra were measured (TMS internal stan-
1
pound 3 was confirmed by H-NMR and ESI-MS spectra.
dard) on a Varian Gemini 2000 (300 MHz) spectrometer or on a JEOL JNM-
AL 400 (400 MHz) spectrometer. IR spectra were measured on JASCO
FT/IR-410. Electron ionization mass spectra (EI-MS) and high-resolution
electron ionization mass spectra (HR-EI-MS) were measured on JEOL DX-
303. Electrospray ionization mass spectra (ESI-MS) and high-resolution
electrospray ionization mass spectra (HR-ESI-MS) were measured on micro-
mass LCT.
The anomeric a-proton was observed as a doublet signal at
dꢀ5.09 with an axial–axial coupling of 7.4 Hz. One- and
two-position protons were observed at dꢀ7.13 (1H, d,
Jꢀ8.2 Hz) and 6.79 (1H, d, Jꢀ8.2 Hz), and the (E)-olefinic
protons of the amide side chain at dꢀ6.87 (1H, d, Jꢀ
15.0 Hz) and 7.64 (1H, d, Jꢀ15.0 Hz) with a trans coupling.
The ESI-MS spectrum showed the molecular ion peak at
m/zꢀ599 ([MꢁH]ꢁ).
17-N-(tert-Butoxycarbonyl)-4,5a-epoxy-14b-hydroxy-3-methoxymor-
phinan-6-one (6) To a stirred suspension of noroxycodone hydrochloride
5 (102.45 g, 303.29 mmol) and NaHCO3 (55.35 g, 658.8 mmol) in 900 ml of
water and 1050 ml of CHCl3 was added dropwise (Boc)2O (73.41 g,
336.3 mmol) at room temperature, and the suspension became a clear solu-
tion soon. The solution was stirred at room temperature for 3 h, and the or-
ganic layer was separated. The water layer was extracted with CHCl3 and the
combined organic layer was dried over Na2SO4, and concentrated in vacuo to
give 149.42 g of compound 6 as an amorphous solid.
The chemical structure of compound 4 was also confirmed
1
by H-NMR and ESI-MS spectra. Protons of the cyclopro-
pylmethyl part were observed at dꢀ0.40—0.50 (2H, m),
0.65—0.90 (2H, m), and 1.00—1.20 (1H, m), and the
anomeric a-proton was observed as a doublet signal at
dꢀ5.06 with an axial–axial coupling of 7.4 Hz. One-position
proton was observed at dꢀ7.20 (1H, d, Jꢀ8.2 Hz), and the
1H-NMR (400 MHz, CDCl3) d: 1.50 (9H, s), 1.59—1.72 (3H, m), 1.89—
1.94 (1H, m), 2.30 (1H, dt, Jꢀ14.6, 3.0 Hz), 2.33—2.45 (1H, m), 2.60—
2.80 (2H, m), 2.90 (1H, d, Jꢀ18.3 Hz), 2.98—3.08 (2H, m), 3.90 (3H, s),
(E)-olefinic proton of the amide side chain at dꢀ7.66 (1H, d, 4.30—4.60 (1H, m), 4.67 (1H, s), 6.64 (1H, d, Jꢀ8.3 Hz), 6.73 (1H, d,
Jꢀ8.3 Hz). IR (KBr) cmꢂ1: 3437, 1724, 1681. HR-EI-MS m/z Mꢁ Calcd for
C22H27NO6: 401.1838. Found: 401.1855.
6b-(N-Benzyl)methylamino-17-(tert-butoxycarbonyl)-4,5a-epoxy-3-
methoxymorphinan-14b-ol (7) To a stirred solution of compound 6
Jꢀ15.6 Hz) with a trans coupling. The ESI-MS spectrum
showed the molecular ion peak at m/zꢀ653 ([MꢁH]ꢁ).
To confirm the presence of compounds 2, 3 and 4 as the
metabolites of TRK-820, radio-chromatograms of an incuba-
tion mixture of [3H]TRK-820 with cryopreserved human he-
patocytes were compared with UV-chromatograms of com-
pounds 2, 3 and 4.15) Metabolite peaks in the incubation mix-
ture were detected at retention times of 9.4 and 8.0 min on
radio-chromatograms, corresponding to those of compound 2
and 4 simultaneously analyzed. These results indicated that
compounds 2 and 4 were the metabolites derived from TRK-
820 in human hepatocytes.
(21.50 g, 53.55 mmol) in 500 ml of benzene were added N-benzylmethyl-
amine (14.2 ml, 110 mmol) and benzoic acid (6.68 g, 54.7 mmol). This mix-
ture was stirred under reflux conditions for 19 h, using a Dean-Stark appara-
tus. After removal of benzene, the crude products were dissolved in 100 ml
of CH3OH, and NaBH3CN (4.11 g, 65.4 mmol) was added to the resulting
solution, and the mixture was stirred at 0 °C. After stirring for 2 h, the solu-
tion was concentrated and the crude products were partitioned in EtOAc and
saturated aq. NaHCO3 solution. The organic layer was separated and the
water layer was extracted with EtOAc. The combined organic layer was
washed successively with saturated aq. NaHCO3 solution and saturated aq.
NaCl solution, dried over Na2SO4 and concentrated in vacuo to give 33.70 g
of crude products. This was chromatographed on silica gel. Elution with n-
hexane/EtOAc (10/1—1/1) gave 19.65 g (72%) of compound 7 as an amor-
phous solid.
Pharmacological studies of compounds 2, 3, and 4 were
carried out to investigate their antipruritic activity.16) Scratch-
ing behavior was induced by the intradermal injection of
Substance P, a pruritogen, in 5-weeks-old male ddY mice.3)
Subcutaneous administration of compounds 2, 3, and 4 (1,
10, 100, 1000 mg/kg each) did not inhibit the scratching be-
1H-NMR (400 MHz, CDCl3) d: 1.35—1.54 (3H, m), 1.48 (9H, s), 1.62—
1.65 (3H, m), 1.90—2.01 (1H, m), 2.17—2.28 (1H, m), 2.32 (3H, s), 2.57—
2.64 (1H, m), 2.65—2.80 (1H, m), 2.86 (1H, d, Jꢀ17.8 Hz), 3.01—3.08
(1H, m), 3.71 (1H, d, Jꢀ13.6 Hz), 3.76 (1H, d, Jꢀ13.6 Hz), 3.89 (3H, s),
havior induced by Substance P, though TRK-820 inhibited 4.75 (1H, d, Jꢀ7.1 Hz), 6.59 (1H, d, Jꢀ8.3 Hz), 6.72 (1H, d, Jꢀ8.3 Hz),

June 2004
673
7.18—7.44 (5H, m). IR (KBr) cmꢂ1: 3432, 1669, 1606. HR-EI-MS m/z Mꢁ
Calcd for C30H38N2O5: 506.2781. Found: 506.2790.
N-methylprop-2-enamide (2) To a stirred solution of compound 10
(655 mg, 1.01 mmol) in 10 ml of THF was added dropwise a solution of
2.5 M aq. NaOH (2.0 ml, 5.0 mmol). The mixture was stirred for 16 h at room
temperature, and then a solution of 1 M aq. HCl was added to neutralize (pH
7). This solution was extracted with EtOAc to remove Fmoc residue and the
water layer was concentrated in vacuo to give crude products. This was puri-
fied by Sep-Pak Vac® with water and CH3OH as eluent to remove inorganic
salts to give 338 mg (77%) of compound 2 as a white powder.
(E)-N-[17-(tert-Butoxycarbonyl)-4,5a-epoxy-14b-hydroxy-3-methoxy-
morphinan-6b-yl]-3-(furan-3-yl)-N-methylprop-2-enamide (8) o-Ph-
thalic acid (2.97 g, 17.87 mmol) and 10% Pd–C (2.23 g, 2.09 mmol) were
added to a solution of compound 7 (17.07 g, 33.69 mmol) in 70 ml of
CH3OH. The mixture was stirred under hydrogen atomosphere (1 atm) at
room temperature for 25 h. The Pd–C catalyst was then removed by filtration
with hyflo super-cell® and washed with CH3OH. The filtrate was concen-
trated in vacuo to give 16.64 g of crude debenzylated compound. To a stirred
solution of the crude products in 100 ml of THF and 50 ml of water was
added Na2CO3 (2.83 g, 26.7 mmol) followed by (E)-3-(3-furyl)acryloyl chlo-
ride (7.96 g, 50.8 mmol) at 0 °C, and the temperature was gradually raised to
room temperature. After stirring for 14 h, the organic layer was separated
and the water layer was extracted with EtOAc. The combined organic layer
was concentrated and dissolved in EtOAc again, and washed with a saturated
aq. NaHCO3 solution and saturated aq. NaCl solution, dried over Na2SO4
and concentrated in vacuo to give 13.72 g of crude products. This was chro-
matographed on silica gel (250 g) with CHCl3/CH3OH (50/1) as eluent to
give 11.03 g (61% 2 steps) of compound 8 as an amorphous solid.
1H-NMR (400 MHz, DMSO-d6) d: 1.00—1.15 (1H, m), 1.16—1.40 (4H,
m), 1.40—1.50 (1H, m), 2.00—2.15 (2H, m), 2.30—2.40 (1H, m), 2.45—
2.60 (2H, m), 2.70—3.00 (3H, m), 2.86 (2H, s), 3.09 (1H, s), 3.50—3.60
(0.7H, m), 4.15—4.25 (0.3H, m), 4.55 (0.7H, d, Jꢀ7.8 Hz), 4.63 (0.3H, d,
Jꢀ7.8 Hz), 6.38 (0.7H, d, Jꢀ15.2 Hz), 6.51—6.73 (2.7H, m), 6.89 (0.3H, d,
Jꢀ15.2 Hz), 6.99 (0.3H, s), 7.20 (0.7H, d, Jꢀ15.2 Hz), 7.36 (0.3H, d,
Jꢀ15.2 Hz), 7.66 (0.7H, s), 7.72 (0.3H, s), 7.91 (0.7H, s), 8.02 (0.3H, s). IR
(KBr) cmꢂ1: 3399, 1647, 1591. HR-EI-MS m/z Mꢁ Calcd for C24H26N2O5:
422.1842. Found: 422.1832. mp 286 °C (dec.).
Methyl {4,5a-Epoxy-17-(9H-fluorenylmethoxycarbonyl)-14b-hydroxy-
6b-[(E)-N-methyl-3-(3-furyl)-2-propenamido]morphinan-3-yl-2ꢀ,3ꢀ,4ꢀ-
tri-O-acetyl-1ꢀb-D-glucopyranosid}uronate (15) To a stirred solution of
compounds 13 (2.61 g, 5.45 mmol) and 10 (1.75 g, 2.72 mmol) in 27 ml of
dry CH2Cl2 was added 3.42 g of MS4A, and the mixture was stirred at room
temperature for 2.5 h. To this suspension was added dropwise BF3·OEt2
(0.780 ml, 6.15 mmol) at room temperature, and the mixture was stirred for
75 min. Then, a saturated aq. NaHCO3 solution was added to the suspension,
and the mixture was filtered through hyflo super-cell® and washed with
CHCl3. The organic layer was separated and the water layer was extracted
with CHCl3. The combined organic layer was dried over Na2SO4 and con-
centrated in vacuo to give 4.52 g of crude products. This was chromato-
graphed on silica gel (130 g) with n-hexane/EtOAc (1/1) as eluent to give
2.41 g (92%) of compound 15 as a white powder.
1H-NMR (400 MHz, CDCl3) d: 1.40—1.70 (6H, m), 1.49 (9H, s), 2.17—
2.30 (2H, m), 2.35—2.80 (2H, m), 2.85—3.11 (2H, m), 3.00 (2H, s), 3.14
(1H, s), 3.82 (2H, s), 3.85 (1H, s), 4.20—4.50 (1H, m), 4.61 (0.7H, d,
Jꢀ8.0 Hz), 4.76 (0.3H, d, Jꢀ8.0 Hz), 6.45 (0.7H, d, Jꢀ15.6 Hz), 6.47 (0.7H,
s), 6.56—6.66 (0.9H, m), 6.70 (0.7H, d, Jꢀ8.3 Hz), 6.76 (0.3H, d,
Jꢀ8.3 Hz), 6.83 (0.7H, d, Jꢀ8.3 Hz), 7.38 (0.7H, s), 7.42 (0.3H, s), 7.48
(1H, d, Jꢀ15.6 Hz), 7.57 (0.7H, s), 7.61 (0.3H, s). IR (KBr) cmꢂ1: 3383,
1652, 1591. HR-EI-MS m/z Mꢁ Calcd for C30H36N2O7: 536.2523. Found:
536.2541.
(E)-N-[4,5a-Epoxy-17-(9H-fluorenylmethoxycarbonyl)-14b-hydroxy-
3-methoxymorphinan-6b-yl]-3-(furan-3-yl)-N-methylprop-2-enamide (9)
Trifluoroacetic acid (16 ml, 207 mmol) was added dropwise to a stirred solu-
tion of compound 8 (9.46 g, 17.6 mmol) in 60 ml of CH2Cl2 at 0 °C. After
stirring at room temperature for 1 h, the solution was concentrated in vacuo.
The excess trifluoroacetic acid in the mixture was removed azeotropically by
using CH3OH, C2H5OH followed by CHCl3. The crude products (9.75 g)
were then dissolved in 50 ml of 1,4-dioxane, and a solution of 10% aq.
Na2CO3 (50 ml, 47.1 mmol) was added with stirring. Then, a solution of
FmocCl (5.53 g, 21.37 mmol) in 20 ml of 1,4-dioxane was added dropwise to
the solution at room temperature. After stirring for 2 h, the mixture was ex-
tracted with EtOAc and the combined organic layer was concentrated in
vacuo to give 13.69 g of crude products. This was chromatographed on silica
gel (360 g) with n-hexane/EtOAc (1/1—0/1) as eluent to give 11.02 g (96%
2 steps) of compound 9 as an amorphous solid.
1H-NMR (300 MHz, CDCl3) d: 1.30—1.50 (3H, m), 1.96 (3H, s), 1.99
(3H, s), 2.00 (3H, s), 2.03—2.21 (4H, m), 2.50—2.90 (2H, m), 3.14 (3H, s),
3.72 (3H, s), 3.74—3.77 (1H, m), 4.20—4.30 (1H, m), 4.40—4.63 (4H, m),
4.65 (1H, d, Jꢀ10.4 Hz), 5.14—5.24 (3H, m), 5.33—5.43 (2H, m), 6.50—
6.70 (2H, m), 6.67 (1H, d, Jꢀ15.4 Hz), 6.95—7.05 (1H, m), 7.25—7.44
(5H, m), 7.55—7.61 (2H, m), 7.70 (1H, d, Jꢀ15.4 Hz), 7.70—7.85 (3H, m).
IR (KBr) cmꢂ1: 3443, 1756, 1690, 1653, 1606. HR-ESI-MS m/z [MꢁH]ꢁ
Calcd for C52H53N2O16: 961.3395. Found: 961.3401. mp 157 °C.
Methyl {17-Cyclopropylmethyl-4,5a-epoxy-14b-hydroxy-6b-[(E)-N-
methyl-3-(3-furyl)-2-propenamido]morphinan-3-yl-2ꢀ,3ꢀ,4ꢀ-tri-O-acetyl-
1ꢀb-D-glucopyranosid}uronate (16) To a stirred solution of compounds
13 (2.53 g, 5.28 mmol) and 1 (1.65 g, 3.46 mmol) in 26 ml of dry CH2Cl2
was added 7.95 g of MS4A, and the mixture was stirred at room temperature
for 2 h. To the suspension was added dropwise BF3·OEt2 (0.800 ml,
6.31 mmol), and the mixture was stirred at room temperature for 17 h. Then,
a saturated aq. NaHCO3 solution was added to the suspension, and the mix-
ture was filtered through hyflo super-cell® and washed with CHCl3. The or-
ganic layer was separated and the water layer was extracted with CHCl3. The
combined organic layer was dried over Na2SO4 and concentrated in vacuo to
give 4.48 g of crude products. This was chromatographed on silica gel
(158 g) with EtOAC/CH3OH (10/0—10/1) as eluent to give 2.26 g of a mix-
ture. This was chromatographed on silica gel (113 g) with CHCl3/CH3OH
(10/1) as eluent to give 2.25 g (82%) of compound 16 as an amorphous
solid.
1H-NMR (300 MHz, CDCl3) d: 1.40—1.50 (3H, m), 1.55—1.80 (3H, m),
2.10—2.40 (2H, m), 2.60—2.75 (1H, m), 2.80—2.90 (1H, m), 3.00 (2H, s),
3.15 (1H, s), 3.75—4.00 (2H, m), 3.81 (2H, s), 3.85 (1H, s), 4.20—4.30
(1H, m), 4.40—4.80 (4H, m), 6.40—6.80 (4H, m), 7.26—7.62 (9H, m), 7.77
(2H, broad s). IR (KBr) cmꢂ1: 3426, 1690, 1652, 1604. HR-ESI-MS m/z
[MꢁH]ꢁ Calcd for C40H39N2O7: 659.2757. Found: 659.2784.
(E)-N-[4,5a-Epoxy-17-(9H-fluorenylmethoxycarbonyl)-3,14b-dihy-
droxymorphinan-6b-yl]-3-(furan-3-yl)-N-methylprop-2-enamide (10)
To a stirred solution of compound 9 (10.98 g, 16.66 mmol) in 100 ml of
CH2Cl2 was added dropwise BBr3 (1.0 M in CH2Cl2, 180 ml, 180 mmol) at
0 °C during 1 h, and the temperature was gradually raised to room tempera-
ture. After stirring for 2 h, the flask was cooled to 0 °C again and the mixture
was quenched by adding crushed ice and slowly adding 20 ml of water. After
that, a solution of 10% aq. Na2CO3 was slowly added until the solution
showed pH 8, and then the mixture was extracted with CHCl3. The organic
layer was washed with water and a saturated aq. NaCl solution, and the or-
ganic layer was concentrated in vacuo to give 10.50 g of crude products.
This was chromatographed on silica gel (360 g) with EtOAc as eluent to give
6.99 g (65%) of compound 10 as an amorphous solid.
1H-NMR (300 MHz, CDCl3) d: 0.12—0.14 (2H, m), 0.51—0.55 (2H, m),
0.78—0.90 (1H, m), 1.20—1.70 (3H, m), 1.95 (3H, s), 1.98 (3H, s), 2.00
(3H, s), 2.04—2.15 (3H, m), 2.36 (2H, d, Jꢀ6.3 Hz), 2.55—2.70 (2H, m),
3.00—3.10 (3H, m), 3.16 (3H, s), 3.71 (3H, s), 4.53 (1H, d, Jꢀ7.6 Hz),
4.60—4.75 (1H, m), 4.67 (1H, d, Jꢀ9.6 Hz), 5.14—5.23 (2H, m), 5.35—
5.45 (2H, m), 6.59 (1H, d, Jꢀ8.2 Hz), 6.66 (1H, s), 6.68 (1H, d, Jꢀ15.2 Hz),
6.96 (1H, d, Jꢀ8.2 Hz), 7.43 (1H, s), 7.70 (1H, d, Jꢀ15.2 Hz), 7.78 (1H, s).
IR (KBr) cmꢂ1: 3426, 1756, 1652, 1605. HR-ESI-MS m/z [MꢁH]ꢁ Calcd
for C41H49N2O14: 793.3184. Found: 793.3201.
4,5a-Epoxy-14b-hydroxy-6b-[(E)-N-methyl-3-(3-furyl)-2-prope-
namido]morphinan-3-yl-b-D-glucopyranosiduronic acid (3) To a stirred
suspension of compound 15 (2.37 g, 2.46 mmol) in 50 ml of THF and 10 ml
of CH3OH was added a solution of LiOH·H2O (808 mg, 19.3 mmol) in
20 ml of water, and the mixture was stirred at room temperature. After stir-
ring for 18 h, a solution of 1 M aq. HCl was added to neutralize, and then the
mixture was concentrated to remove THF and CH3OH. The resulting water
layer was washed with EtOAc and concentrated in vacuo. A mixture of
water and 2-propnol (1/4) was added to the residue to precipitate the desired
compound and inorganic salts to give 0.80 g of a solid after drying. This
1H-NMR (400 MHz, DMSO-d6) d: 1.20—1.40 (4H, m), 1.45—1.60 (1H,
m), 1.95—2.15 (1H, m), 2.20—2.40 (1H, m), 2.45—2.65 (1H, m), 2.80—
3.00 (1H, m), 2.86 (2H, s), 3.10 (1H, s), 3.56 (0.7H, broad s), 3.75—3.85
(1H, m), 4.09 (0.3H, broad s), 4.15—4.50 (4H, m), 4.60—4.65 (1H, m),
4.91—5.08 (1H, m), 6.30—6.40 (0.7H, m), 6.50—6.65 (2.3H, m), 6.75—
6.80 (0.7H, m), 6.89 (0.3H, d, Jꢀ15.2 Hz), 6.99 (0.3H, s), 7.21 (0.7H, d,
Jꢀ15.2 Hz), 7.30—7.45 (4H, m), 7.61—7.72 (3H, m), 7.85—7.89 (2H, m),
7.91 (0.7H, s). 8.03 (0.3H, s), 9.09 (0.3H, broad s), 9.50 (0.7H, broad s). IR
(KBr) cmꢂ1: 3398, 1678, 1649, 1593. HR-ESI-MS m/z [MꢁH]ꢁ Calcd for
C39H37N2O7: 645.2601. Found: 645.2600.
(E)-N-[4,5a-Epoxy-3,14b-dihydroxymorphinan-6b-yl]-3-(furan-3-yl)-

674
Vol. 52, No. 6
solid was further purified through Sep-Pak Vac® with water and CH3OH as
eluent to give 0.63 g (43%) of compound 3 as a pale yellow powder.
1H-NMR (300 MHz, CD3OD) d: 1.21—1.60 (3H, m), 1.65—1.80 (1H,
m), 2.10—2.30 (1H, m), 2.40—2.60 (2H, m), 3.00—3.10 (1H, m), 3.15—
3.24 (5H, m), 3.38—3.64 (4H, m), 4.00 (1H, d, Jꢀ9.6 Hz), 4.30—4.50 (1H,
m), 4.81 (1H, d, Jꢀ8.8 Hz), 5.09 (1H, d, Jꢀ7.4 Hz), 6.79 (1H, d, Jꢀ8.2 Hz),
6.87 (1H, d, Jꢀ15.0 Hz), 6.88 (1H, s), 7.13 (1H, d, Jꢀ8.2 Hz), 7.57 (1H, s),
7.64 (1H, d, Jꢀ15.0 Hz), 7.89 (1H, s). IR (KBr) cmꢂ1: 3409, 1707, 1648,
1602. HR-ESI-MS m/z [MꢁH]ꢁ1 Calcd for C30H35N2O11: 599.2241. Found:
599.2221. mp 239 °C (dec.).
17-Cyclopropylmethyl-4,5a-epoxy-14b-hydroxy-6b-[(E)-N-methyl-3-
(3-furyl)-2-propenamido]morphinan-3-yl-b-D-glucopyranosiduronic
acid (4) To a stirred suspension of compound 16 (1.89 g, 2.39 mmol) in
20 ml of THF was added a solution of LiOH·H2O (980 mg, 23.3 mmol) in
6 ml of water, and the mixture was stirred at room temperature. After stirring
for 25 h, a solution of 1 M aq. HCl was added to neutralize, and then the mix-
ture was concentrated to remove THF. The resulting water layer was washed
with CHCl3 and concentrated in vacuo. 2-Propanol was added to the residue
to precipitate the desired compound and inorganic salts to give 1.03 g of a
solid after drying. This solid was further purified with Sep-Pak Vac® with
water and CH3OH as eluent to give 0.85 g (54%) of compound 4 as a yellow
powder.
(Radiomatic FLO-ONE/beta Series A-505; Packard Co.) and HPLC (LC-
10Ai Liquid Chromatograph System; Shimadzu Co.). The HPLC conditions
were as follows: mobile phase, CH3OH (A) and 50 mM NaH2PO4 aq. solu-
tion (B); flow rate, 0.9 ml/min; column temperature, 40 °C. The gradient elu-
tion profile was 30 to 40% B for 0 to 20 min, 40 to 100% B for 20 to 25 min
and 100% B for 25 to 30 min.
References and Notes
1) Nagase H., Hayakawa J., Kawamura K., Kawai K., Takezawa Y., Ma-
tsuura H., Tajima C., Endo T., Chem. Pharm. Bull., 46, 366—369
(1998).
2) Togashi Y., Umeuchi H., Okano K., Ando N., Yoshizawa Y., Honda T.,
Kawamura K., Endoh T., Utsumi J., Kamei J., Tanaka T., Nagase H.,
Eur. J. Pharmacol., 435, 259—264 (2002).
3) Umeuchi H., Togashi Y., Honda T., Nakao K., Okano K., Tanaka T.,
Nagase H., Eur. J. Pharmacol., 477, 29—35 (2003).
4) Casy A. F., Parfitt R. T., “Opioid Analgesics Chemistry and Recep-
tors,” Plenum Press, New York, 1986, pp. 87—91.
5) Sayre L. M., Portoghese P. S., J. Org. Chem., 45, 3366—3368 (1980).
6) Halcomb R. L., Danishefsky S. J., J. Am. Chem. Soc., 111, 6661—
6666 (1989).
7) Koenig W., Knorr E., Ber. Dtsch. Chem. Ges., 34, 957—981 (1901).
8) Schmidt R. R., Grundler G., Synthesis, 1981, 885—887 (1981).
9) Schmidt R. R., Angew. Chem. Int. Ed. Engl., 25, 212—235 (1986).
10) Fischer B., Nudelman A., Ruse M., Herzig J., Gottlieb H. E., J. Org.
Chem., 49, 4988—4993 (1984).
1H-NMR (300 MHz, CD3OD) d: 0.40—0.50 (2H, m), 0.65—0.90 (2H,
m), 1.00—1.20 (1H, m), 1.40—1.50 (1H, m), 1.50—1.70 (2H, m), 1.70—
1.85 (1H, m), 2.20—2.40 (1H, m), 2.45—2.70 (2H, m), 2.70—2.90 (1H, m),
3.00—3.20 (2H, m), 3.25 (3H, s), 3.40—3.60 (5H, m), 3.70—4.00 (2H, m),
4.30—4.50 (1H, m), 4.80—5.00 (1H, m), 5.06 (1H, d, Jꢀ7.4 Hz), 6.81—
6.90 (3H, m), 7.20 (1H, d, Jꢀ8.2 Hz), 7.52—7.78 (1H, m), 7.66 (1H, d,
Jꢀ15.6 Hz), 7.90 (1H, s). IR (KBr) cmꢂ1: 3405, 1648, 1600. HR-ESI-MS
m/z [MꢁH]ꢁ Calcd for C34H41N2O11: 653.2710. Found: 653.2737. mp
206 °C (dec.).
11) Berrang B., Brine G. A., Carrol F. I., Synthesis, 1997, 1165—1168
(1997).
12) Hashimoto S., Umeo K., Sano A., Watanabe N., Nakajima M.,
Ikegami S., Tetrahedron Lett., 36, 2251—2254 (1995).
13) Lacy C., Sainsbury M., Tetrahedron Lett., 36, 3949—3950 (1995).
14) Brown R. T., Carter N. E., Lumbard K. W., Scheinmann F., Tetrahe-
dron Lett., 36, 8661—8664 (1995).
15) The use of human hepatocytes was in compliance with the Declaration
of Helsinki and approved by the Ethical Committee of Toray Research
Laboratories.
In Vitro Metabolic Experiment of TRK-820 [3H]TRK-820 was incu-
bated with cryopreserved human hepatocytes for 1, 2 and 4 h at 37 °C and
the metabolic reaction was terminated by the addition of ice-cold acetoni-
trile. The incubation mixture was centrifuged at 37 °C, 3000 rpm for 5 min
and the entire supernatant obtained was evaporated to dryness with a cen-
trifugal evaporator. The residue was dissolved in the mobile phase and in-
jected to an analytical column (YMC-Pack ODS-AM, S-5 mm, AM-302,
150ꢃ4.6 mm; YMC). The radio-HPLC system consisted of a radio detector
16) Pharmacological studies were conducted in accordance with the guide-
lines for Care and Use of Laboratory Animals in Toray Research Lab-
oratories.