Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1677
3.93 (dd, 1H, J ) 14.2, 2.2 Hz, H-5), 3.62 (s, 3H, 3-OCH3), 2.38
(s, 3H, H-18); MS m/z (EI) 317 (M+).
2H, H-20 and H-21), 0.19 (m, 2H, H-20 and H-21); MS m/z
(EI) 430 (M+). Anal. (C31H36N3O9‚0.5H2O) C, H, N.
6,7-Did eh yd r o-4,14-d ih yd r oxy-3-m et h oxy-17-m et h yl-
6,7:2′,3′-in d olom or p h in a n (16). Following the procedure
described for 11, compound 15 (700 mg, 2.2 mmol) was treated
with phenylhydrazine‚HCl (1.1 equiv) and TsOH‚H2O (1 equiv)
for 2 h. The crude product was purified by column chroma-
tography (SiO2, CHCl3:MeOH:NH3, 19:1:0.1) and then con-
Biologica l Assa ys. 1. Recep tor Bin d in g Assa ys. µ Bind-
ing sites were labeled using [3H]DAMGO (2.0 nM) as previ-
ously described44 with several modifications. Rat membranes
were prepared each day using a partially thawed-frozen rat
brain which was polytroned in 10 mL/brain of ice-cold 10 mM
Tris-HCl, pH 7.0. Membranes were then centrifuged twice at
30000g for 10 min each centrifugation. After the second
centrifugation, the membranes were resuspended in 50 mL/
brain of 25 °C 50 mM Tris-HCl, pH 7.4. Incubations proceeded
for 2 h at 25 °C in 50 mM Tris-HCl, pH 7.4, along with a
protease inhibitor cocktail. The nonspecific binding was de-
termined using 20 µM levallorphan. δ Binding sites were
labeled using [3H]DADLE (2.0 nM) as previously described45
with several modifications. Rat membranes were prepared
each day using a partially thawed-frozen rat brain which was
polytroned in 10 mL/brain of ice-cold 10 mM Tris-HCl, pH 7.0.
Membranes were then centrifuged twice at 30000g for 10 min
each centrifugation. After the second centrifugation, the
membranes were resuspended in 50 mL/brain of 25 °C 50 mM
Tris-HCl, pH 7.4. Incubations proceeded for 2 h at 25 °C in 50
mM Tris-HCl, pH 7.4, containing 100 mM choline chloride, 3
mM MnCl2, 100 nM DAMGO to block µ binding sites, and a
protease inhibitor cocktail. Nonspecific binding was deter-
mined using 20 µM levallorphan. κ 1 binding sites were labeled
using [3H]U69,593 (2.0 nM) as previously described46 with
several modifications. Guinea pig brain membranes were
prepared each day using a partially thawed guinea pig brain
which was polytroned in 10 mL/brain of ice-cold 10 mM Tris-
HCl, pH 7.0. The membranes were then centrifuged twice at
30000g for 10 min each centrifugation. After the second
centrifugation, the membranes were resuspended in 75 mL/
brain of 25 °C 50 mM Tris-HCl, pH 7.4. Incubations proceeded
for 2 h at 25 °C in 50 mM Tris-HCl, pH 7.4, containing 1 µg/
mL captopril and a protease inhibitor cocktail. Nonspecific
binding was determined using 1 µM U69,593. Each tritiated
ligand was displaced by 10 concentrations of test drug, two
times. All drug dilutions were done in 10 mM Tris-HCl, pH
7.4, containing 1 mg/mL bovine serum albumin. The IC50 and
slope factor (N) were obtained by using the program MLAB.
IC50 values were converted to Ki values according to the
equation Ki ) IC50/(1 + [L]/Kd).
2. GP I a n d MVD Bioa ssa ys.47 Electrically induced smooth
muscle contractions of mouse vas deferens and strips of guinea
pig ileum longitudinal muscle myenteric plexus were used.
Tissues came from male ICR mice weighing 25-40 g and male
Hartley guinea pigs weighing 250-500 g. The tissues were
tied to gold chain with suture silk, suspended in 20-mL baths
containing 37 °C oxygenated (95% O2, 5% CO2) Krebs bicar-
bonate solution (magnesium free for the MVD), and allowed
to equilibrate for 15 min. The tissues were then stretched to
optimal length previously determined to be 1 g tension (0.5 g
for MVD) and allowed to equilibrate for 15 min. The tissues
were stimulated transmurally between platinum wire elec-
trodes at 0.1 Hz, 0.4-ms pulses (2-ms pulses for MVD), and
supramaximal voltage. An initial dose-response curve of
DPDPE or PL-017 was constructed at the start of each assay
to establish tissue effects, allowing each tissue to be used as
its own control. Tissues not producing typical results were not
used. Experimental compounds were added to the baths in 14-
60-µL volumes. Succeeding doses of agonist were added
cumulatively to the bath at 3-min intervals to produce a
concentration-response curve. The tissues were then washed
extensively with fresh buffer until the original contraction
height was reestablished. Agonist effects of the compounds at
1 µM were measured as percent inhibition of contraction height
10 min after addition to the bath. Antagonist effects to DPDPE
and PL-017 were assayed after incubation of the tissues with
1 µM concentration of the compound in the bath for 30 min.
The tissues were then washed with fresh buffer for 30 min,
and the agonist dose-response curve was repeated. Rightward
shifts in the dose-response curves were calculated by dividing
the antagonized dose-response curve IC50 value by the un-
i
verted to the fumaric acid salt from PrOH/MeOH (250 mg,
22%): mp >270 °C dec; 1H NMR (CDCl3) δ 7.75 (s, 1H, H-1′),
7.28 (m, 1H, indole ring), 7.22 (m, 1H, indole ring), 7.02 (m,
2H, indole ring), 6.58 (m, 2H, H-1 and H-2), 5.95 (s, 1H, 4-OH),
4.52 (d, 1H, J ) 15.2 Hz, H-5), 3.73 (s, 3H, 3-OCH3), 2.40 (s,
3H, H-18); MS m/z (EI) 390 (M+). Anal. (C28H30N2O7‚0.5MeOH)
C, H, N.
14-Hyd r oxy-17-n or d ih yd r oth eba in on e (18). A mixture
of noroxyoxycodone‚HI (17)41,42 (4.0 g, 9.3 mmol), NH4Cl (5.0
g, 93 mmol), zinc dust (4.0 g, 62 mmol), and EtOH (95%, 60
mL) was heated at reflux for 2.5 h. After cooling, the mixture
was filtered and the solids was washed with NH4OH (8 mL).
The combined filtrate and washings were concentrated, redis-
solved in a mixture of water (12 mL) and NH4OH (8 mL), and
extracted with CHCl3 (25 mL, then 6 × 15 mL). The organic
extracts were dried (MgSO4), and the solvent was removed to
i
give the crude product. Crystallization from PrOH gave 18
1
(1.71 g, 61%): mp 203-205 °C dec; H NMR (CDCl3) δ 6.68
(d, 1H, J ) 8.8 Hz, H-2), 6.57 (d, 1H, J ) 8.8 Hz, H-1), 3.89 (d,
1H, J ) 13.2 Hz, H-5), 3.82 (s, 3H, 3-OCH3); MS m/z (EI) 303
(M+). Anal. (C17H21NO4‚0.2H2O) C, H, N.
6,7-Did eh yd r o-4,14-d ih yd r oxy-3-m eth oxy-6,7:2′,3′-in d o-
lom or p h in a n (19). Following the procedure described for 11,
compound 18 (1.35 g, 4.45 mmol) was treated with phenylhydra-
zine‚HCl (1.1 equiv) and TsOH‚H2O (1 equiv) for 2 h. The crude
product was purified as the hydrated hydrochloride salt from
water (1.05 g, 50%): mp >250 °C dec; 1H NMR (CDCl3) δ 7.80
(s, 1H, H-1′), 7.26 (m, 1H, indole ring), 7.22 (m, 1H, indole
ring), 7.00 (m, 2H, indole ring), 6.59 (m, 2H, H-1 and H-2),
4.59 (d, 1H, J ) 16.5 Hz, H-5), 3.77 (s, 3H, 3-OCH3); MS m/z
(EI) 376 (M+). Anal. (C23H25N2O3‚2.75H2O) C, H, N.
6,7-Did eh yd r o-17-eth yl-4,14-d ih yd r oxy-3-m eth oxy-6,7:
2′,3′-in d olom or p h in a n (20). A mixture of iodoethane (0.07
mL, 0.88 mmol), the hydrochloride salt of 19 (700 mg, 1.5
mmol), NaHCO3 (1 g, 11.9 mmol), and DMF (15 mL) was
stirred at room temperature for 2 h. After quenching with NH4-
OH (20 mL), the products were extracted into Et2O (3 × 30
mL); the organic extracts were washed with brine (3 × 50 mL),
dried, and concentrated to give the crude product. The product
was purified by column chromatography (SiO2, EtOAc), fol-
lowed by formation of the hydrochloride salt from water (245
mg, 59%): mp 210-212 °C dec; 1H NMR (CDCl3) δ 7.76 (s,
1H, H-1′), 7.26 (m, 1H, indole ring), 7.21 (m, 1H, indole ring),
7.02 (m, 2H, indole ring), 6.57 (m, 2H, H-1 and H-2), 5.96 (s,
1H, 4-OH), 4.50 (d, 1H, J ) 15.4 Hz, H-5), 3.77 (s, 3H, 3-OCH3),
2.58 (m, 2H, H-18), 1.13 (t, 3H, J ) 6.6 Hz, H-19); MS m/z
(EI) 404 (M+). Anal. (C25H29N2O3Cl‚2H2O) C, H, N.
17-Cyclop r op ylm eth yl-14-h yd r oxy-17-n or d ih yd r oth e-
ba in on e (22).55 Naltrexone 3-methyl ether‚HCl (21)43 (2.0 g,
5.1 mmol) was treated as 14 above. The crude product was
purified by column chromatography (SiO2, CHCl3:MeOH, 19:
1) to give 22 (1.4 g, 76%): 1H NMR (CDCl3) δ 6.67 (d, 1H, J )
8.3 Hz, H-2), 6.57 (d, 1H, J ) 8.3 Hz, H-1), 6.13 (s, 1H, 4-OH),
3.94 (d, 1H, J ) 12.9 Hz, H-5), 3.81 (s, 3H, 3-OCH3), 0.83 (m,
1H, H-19), 0.55 (m, 2H, H-20 and H-21), 0.18 (m, 2H, H-20
and H-21); MS m/z (EI) 357 (M+).
17-Cyclop r op ylm eth yl-6,7-d id eh yd r o-4,14-d ih yd r oxy-
3-m eth oxy-6,7:2′,3′-in d olom or p h in a n (23). Following the
procedure described for 11, compound 22 (1.4 g, 3.9 mmol) was
treated with phenylhydrazine‚HCl (1.1 equiv) and TsOH‚H2O
(1 equiv) for 2 h. The crude product was purified as the (+)-
i
tartaric acid salt from PrOH (700 mg, 31%): mp 210 °C dec;
1H NMR (CDCl3) δ 7.75 (s, 1H, H-1′), 7.28 (m, 1H, indole ring),
7.22 (m, 1H, indole ring), 6.98 (m, 2H, indole ring), 6.56 (m,
2H, H-1 and H-2), 5.96 (s, 1H, 4-OH), 4.50 (d, 1H, J ) 15.6
Hz, H-5), 3.77 (s, 3H, 3-OCH3), 0.92 (m, 1H, H-19), 0.59 (m,