Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2319
Table 2. In Vivo Potency of 6-Desoxymorphine (7) in Micea
ED50 (sc, mg/kg)b
chromatography (silica gel, 5% MeOH/CH2Cl2), followed by
precipitation of the oxalate salt from acetone, resulted in 13.2 mg
(2.1%). Anal. (C20H25NO6 ·0.1H2O) C, H, N; mp 190–192 °C; 1H
NMR (500 MHz, CDCl3) δ 7.05 (t, J ) 8.0 Hz, 1H), 6.64 (d, J )
8.0 Hz, 2H), 4.68 (d, J ) 4.50 Hz, 1H), 3.47 (m, 1H), 3.38 (s,
3H), 3.09 (dd, J ) 6.27 and 2.85 Hz, 1H), 3.02 (d, J ) 18.82 Hz,
1H), 2.52 (dd, J ) 12.54 and 5.13 Hz, 1H), 2.40–2.46 (m, 1H),
2.41 (s, 3H), 2.29 (dd, J ) 12.58 and 3.99 Hz, 1H), 2.20–2.26 (m,
1H), 1.90 (td, J ) 12.58 and 1.13 Hz, 1H), 1.72 (m, 1H), 1.50–1.60
(m, 2H), 1.30 (m, 1H), 1.08 (m, 1H); m/z: 286 (M + H)+.
P-gp-Glo Assay. In a 96-well plate, recombinant human P-gp
were incubated with P-gp-Glo assay buffer (20 µL), verapamil (200
µM), sodium orthovanidate (100 µM), or 200 µM of test compound
(1-9). Each compound was loaded in four individual wells. The
reaction was initiated by addition of MgATP (10 mM) and quenched
40 min later by addition of 50 µL of firefly luciferase reaction
mixture (ATP detection reagent), which initiated an ATP-dependent
luminescence reaction. Signals were measured 60 min later using
an Lmax luminometer (Molecular Devices Corporation, Sunnyvale,
CA).
cmpd
TF
PPQ
HP
1 (morphine)c
7 (6-desoxymorphine) 0.2 (0.1-0.3)
1.92 (0.89-4.14) 0.4 (0.2-0.8)
0.85 (0.39-1.86)
0.03 (0.018-0.055) 0.33 (0.15-0.72)
a TF ) tail-flick assay; PPQ ) p-phenylquinone writhing assay; HP )
hot plate assay. b Effective dose 50% (95% C.L.); subcutaneous (sc)
administration. c Data from ref 29.
approximately 10- and 50-fold lower binding affinity to µ opioid
receptors compared to morphine and were, therefore, not chosen
for further study. In addition to showing the poorest opioid
binding affinity of all compounds analyzed, 5 would be 3- and
6-O-demethylated to heterocodeine (4) and morphine (1),
compounds which are both P-gp substrates. Compound 7 was
therefore selected for further analysis, as this compound
exhibited the greatest binding affinity of the non-P-gp substrates
and would not be prone to demethoxylation in vivo. Tail flick,
phenylquinone, and hot plate assays were performed in mice
as previously described,28 and results were compared to those
previously determined for morphine.29
As shown in Table 2, compound 7 produces potent antinoci-
ception, exhibiting greater potency compared to morphine in
all assays performed. Compound 7 is approximately 10 times
more potent than morphine in the tail flick (0.2 vs 1.92 mg/kg)
and phenylquinone (0.03 vs 0.4 mg/kg) assays. These data
confirm previous studies30 that describe the high opioid potency
of 6-desoxy derivatives of morphine.
Opioid Binding. Binding assays were performed as described,27
using [3H]diprenorphine in membranes from C6 rat glioma cells
expressing recombinant µ or δ receptors and CHO cells expressing
the recombinant κ receptors.
Antinociception Studies. Antinociception was determined using
the tail flick (TF), p-phenylquinone (PPQ), and hot plate (HP) assays
in mice as described previously by the Drug Evaluation Committee
(DEC) of the College on Problems of Drug Dependence (CPDD).28
Acknowledgment. We thank Dr. Kellie Hom (NMR) and
Dr. Julie A. Ray (MS) for their assistance in compound
identification. This work was supported by a predoctoral
fellowship from the National Institute on Drug Abuse (NIDA,
DA 021049; C.W.C.) as well as a NIDA K-award (K02 DA
19834; A.C.). Opioid receptor binding and antinociception
evaluation was performed by the Drug Evaluation Committee
(DEC) of the College on Problems of Drug Dependence
(CPDD).
Conclusions
Results of in vitro ATPase activity assays show that P-gp
substrate activity in the morphine series can be attenuated by
masking or removing hydrogen bonding substitiuents at the 3-
and 6-positions. Substitution to the 3-phenol generally reduces
opioid receptor binding affinity; however, removal of the
6-hydroxyl moiety greatly increases binding to all three opioid
receptor subtypes. Additionally, 6-desoxymorphine analogs
exhibit diminished selectivity for binding µ receptors over δ
and κ. Antinociception studies performed on 6-desoxymorphine
(7) show an approximate 10-fold increase in antinociceptive
potency compared to morphine. These results indicate that
compound 7 is a µ opioid agonist with morphine-like potency
and no P-gp substrate activity and is, therefore, an ideal lead
compound to elucidate the effect of P-gp on the development
of analgesic tolerance of opioids.
Supporting Information Available: Routine experimental and
spectroscopic data; results from elemental analysis. This material
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Experimental Section
Chemistry. Compounds were hydrogenated using a Parr hydro-
genation apparatus, and HCl salts were lyophilized using a VirTis
Freezemobile lyophilizer where described. H NMR spectra were
1
obtained using a 500 MHz Varian NMR. Melting points were
determined in open capillary tubes using a Mel-Temp melting point
apparatus. TLC was performed on silica gel 60 GF plates (Analtech,
Inc., Newark, DE). Microanalysis was performed by Atlantic
Microlab, Inc. (Norcross, GA). Opioids used in this study were a
generous gift from Mallinckrodt, Inc. (St. Louis, MO). Reagents
were purchased from Sigma-Aldrich, Inc. Detailed syntheses of
known opioids 3-5 and 7-9 are available in Supporting Informa-
tion.
7,8-Dihydro-3-desoxyheterocodeine (6). Compound 319,20 (460
mg, 1.70 mmol) was 6-O-methylated using silica gel (2.0 g) and
TMS-CHN2 (1.94 g, 10 equiv) daily for 5 days. On days 2–5, 1–2
drops of water were added. On day 5, the reaction was quenched
with water, and the organic layer was separated. The aqueous layer
was extracted with CH2Cl2, and the combined organic layers were
dried (Na2SO4) and evaporated under reduced pressure. Column