Synthesis and pharmacological evaluation of 6,7-indolo/thiazolo-morphinans - Further SAR of levorphanol

Article abstract of DOI:10.1021/jm0701674

To further extend the structure-activity relationships of levorphanol, two series of novel morphinans were prepared by incorporation of an indole or aminothiazole fragment to the hexyl ring (ring C) in levorphanol. Such morphinans differed from previously reported ligands in that such indole- or aminothiazole-containing morphinans displayed enhanced binding affinity to the δ opioid receptor, while the affinity to κ and μ receptors was slightly reduced.

Full text of DOI:10.1021/jm0701674

J. Med. Chem. 2007, 50, 2747-2751
2747
Synthesis and Pharmacological Evaluation of 6,7-Indolo/Thiazolo-MorphinanssFurther SAR of
Levorphanol
Ao Zhang,*,† Fuying Li,† Chunyong Ding,†,‡ Qizhen Yao,‡ Brian I. Knapp,§ Jean M. Bidlack,§ and John L. Neumeyer*,|
Bioorganic and Medicinal Chemistry Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
China, Department of Pharmacology and Physiology, School of Medicine and Dentistry, UniVersity of Rochester, Rochester, New York 14642,
Medicinal Chemistry Laboratory, McLean Hospital, HarVard Medical School, Belmont, Massachusetts 02478, School of Pharmacy, China
Pharmaceutical UniVersity, Nanjing 210009, China
ReceiVed February 13, 2007
To further extend the structure-activity relationships of levorphanol, two series of novel morphinans were
prepared by incorporation of an indole or aminothiazole fragment to the hexyl ring (ring C) in levorphanol.
Such morphinans differed from previously reported ligands in that such indole- or aminothiazole-containing
morphinans displayed enhanced binding affinity to the δ opioid receptor, while the affinity to κ and µ
receptors was slightly reduced.
Introduction
method, 4-OH-morphinan 13 was also obtained as a side
product. A modified Sawa’s Ullmann coupling (Cu/Cs2CO3/
Py) was used to convert the 4-OH of 12 to its phenyl ether
14.10-13 After protection of the 6-keto group, ketal 15 was
subjected to Na/NH3 (liquid) reduction (Birch reduction) at
-78 °C to yield 16, which was hydrolyzed in 1 N HCl to yield
the key intermediate 6-oxo-morphinan 1714 (Scheme 1). N-
Demethylation of 17 with ethyl chloroformate15 and the
subsequent realkylation with cyclopropylmethyl bromide (CP-
MBr) or cyclobutylmethyl bromide (CBMBr) afforded the
corresponding N-substituted 6-oxo-morphinans 18 and 19.
The Fisher indole cyclization strategy16 was used to prepare
the 6,7-indolo-morphinans 10a-c. Thus, treating the ketones
17-19 with PhNHNH2‚HCl in refluxing EtOH followed by
O-demethylation with BBr3 afforded the target compounds
10a-c in 40-60% yield (Scheme 2). Bromination17 of the
ketones 17-19 followed by treatment of the intermediates with
thiourea yielded aminothiazoles 21a-c. Similarly, O-demethy-
lation of 21a-c with BBr3 afforded 6,7-aminothiazolo-morphi-
nans 22a-c in 30-60% yield.
Results and Discussion. All new compounds including
indoles 10a-c, the 3-methoxy-6,7-indolomorphinans 20a-c,
and aminothiazolomorphinans 22a-c were evaluated for their
binding affinity at all three opioid receptors (µ, δ, and κ; Table
1) using a previously reported procedure.5,6 For comparison
purposes, opioid binding affinity data for compounds 46
(levorphanol), 56 (cyclorphan), 66 (ATPM), and indoles 7-98
were also included.
From the data shown in Table 1, the incorporation of an indole
moiety into the morphinans (4 and 5) dramatically changed the
opioid receptor selectivity. Compared to the κ and/or µ
selectivity profiles of levorphanol (4) and cyclorphan (5),4 all
these compounds 20a-c and 10a-c displayed high affinity and
selectivity at the δ receptor, although moderate to good affinities
at κ and µ receptors were also observed. Compared to the 3-OH
compounds, the corresponding 3-OCH3 morphinans generally
had reduced, but not abolished affinity at all three opioid
receptors. All 3-OH indoles 10a-c had similar affinity at the δ
receptor, with Ki values of 0.17-0.35 nM. N-Methyl indolo-
morphinan 10a displayed the highest selectivity for δ over κ
(40-fold) and µ (63-fold) receptors. Compounds 10b and 10c
also had good affinity at κ and µ, only slightly lower than that
of δ receptors.
Analogs of opioids, such as morphine (1), codeine (2),
thebaine (3), and levorphanol (4), are of current interest due to
their utilities as analgesics and as building blocks for potential
therapeutic agents for the treatment of drug abuse.1-3 With
levorphanol (4) as the structural prototype, a number of
morphinanoids have been developed (Figure 1)4-6 by substitu-
tion of the N-methyl group in 4 resulting in 5 (cyclorphan) with
high affinity at both κ and µ opioid receptors, while further
changes at the 3-position of this compound with the aminothia-
zole moiety produced highly κ-selective morphinans, such as 6
(ATPM, Ki ) 0.049 nM).6 However, no structure-activity
relationship (SAR) study has been conducted investigating the
modifications of the hexyl ring (ring C) of levorphanol (4).
Extensive SAR studies on ring C of other opioids have been
conducted resulting in numerous structures possessing various
pharmacological properties. For example, starting from naltr-
exone, Portoghese et al.7 developed the 6,7-indole-fused mor-
phinoid 7 (naltrindole, NTI), a widely used δ opioid receptor
antagonist. Similarly, Coop and co-workers8 reported several
series of 4-hydroxy-6,7-indole morphinoids with a 14-OH group
(8) or lacking a 14-OH group (9) by opening the 4,5-furanyl
ring. These compounds also displayed interesting pharmacologi-
cal properties at opioid receptors. Thus, it would be of interest
to develop indole analogs without the 4-OH, 14-OH, and 4,5-
furanyl groups (Figure 1, compound 10). These new compounds
can be viewed as direct ring C-modified indole analogs of
levorphanol (4). We report herein the synthesis and pharma-
cological investigation of the indole morphinans 10a-c, as well
as the aminothiazole morphinans 22a-c.
Chemistry. The synthesis was initiated from codeine (2),
which was treated with n-BuLi followed by hydrogenation to
afford the dihydrothebainone 129 (Scheme 1). This compound
can also be prepared from dihydrocodone 11 by treating with
zinc powder in concd HCl and HOAc.8 However, using this
* To whom correspondence should be addressed. Tel.: 86-21-50806035
(A.Z.); 1-617-855-3388 (J.L.N.). Fax: 86-21-50806035 (A.Z.); 1-617-855-
2519 (J.L.N.). E-mail: aozhang@mail.shcnc.ac.cn (A.Z.); neumeyer@
mclean.harvard.edu (J.L.N.).
† Shanghai Institute of Materia Medica.
‡ China Pharmaceutical University.
§ University of Rochester.
| Harvard Medical School.
10.1021/jm0701674 CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/09/2007

2748 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Brief Articles
Figure 1. Representative opioids.
Scheme 1a
a Reagents and conditions: (i) BuLi, THF, 0 °C-rt; (ii) Pd/C, H2, AcOH; (iii) Zn, HCl, AcOH; (iv) PhBr, Cu, Cs2CO3, Py, 110 °C; (v) ethylene glycol,
TsOH, benzene, reflux: (vi) Na, NH3 (liquid), -78 °C-rt; (vii) HCl (1 N); (viii) ClCOOEt, K2CO3, CHCl3, reflux, then HCl, AcOH, reflux; (ix) CPMBr or
CBMBr, NaHCO3, EtOH, reflux.
Scheme 2a
a Reagents and conditions: (i) PhNHNH2‚HCl, TsOH, EtOH, reflux; (ii) BBr3, CH2Cl2, -78 °C; (iii) Br2, AcOH, 60 °C; (iv) NH2CSNH2, 110 °C.
The aminothiazolomorphinans 22a-c all displayed good
affinity at all three opioid receptors, with the selectivity
remaining at the κ or µ receptors, which was similar to the
prototypes 4 and 5. N-Methyl aminothizaole 22a had a Ki of
0.49 nM at the µ receptor and selectivity of 4- and 8-fold for κ
and δ receptors, respectively. Compound 22b was equipotent

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2749
Table 1. Ki Values Inhibition of δ, κ, and µ Opioid Binding to CHO Membranes by Novel Compoundsa
Ki (nM) ( SE
selectivity
cmpd
[3H]DAMGO (µ)
[3H]naltrindole (δ)
[3H]U69,593 (κ)
µ/κ
δ/κ
4 (levorphanol)b
0.21 ( 0.02
0.062 ( 0.003
1.5 ( 0.2
4.2 ( 2.3
1.9 ( 0.1
29 ( 2
2.3 ( 0.3
0.034 ( 0.002
0.049 ( 0.005
30.4 ( 3.6
3160 ( 210
7900 ( 890
390 ( 16
0.1
2
31
0.9
0.6
2
0.9
1.6
4
2
4
4
0.2
2
2
5 (cyclorphan)b
56
590
0.007
0.007
0.001
0.1
0.03
1
0.3
0.1
0.2
2
6 (ATPM)b
7 (NTI)c
8c
27 ( 1
0.22 ( 0.13
22 ( 7
1850 ( 380
13300 ( 700
340 ( 12
9c
20a
10a
20b
10b
20c
10c
22a
7 ( 1
35 ( 5
22 ( 4
0.35 ( 0.05
1.5 ( 0.1
0.19 ( 0.02
8.7 ( 0.5
0.17 ( 0.03
4.2 ( 1.1
0.23 ( 0.01
1.1 ( 0.2
14 ( 1
5.9 ( 0.6
1.4 ( 0.2
1.1 ( 0.1
0.64 ( 0.01
89 ( 3
330 ( 15
3.9 ( 0.7
1.0 ( 0.08
2.2 ( 0.3
0.49 ( 0.03
0.12 ( 0.007
0.37 ( 0.03
22b
22c
0.07 ( 0.004
0.14 ( 0.008
3
8
3
a CHO membranes, 0.5 mg of protein/sample, were incubated with 12 different concentrations of the compounds in the presence of receptor-specific
radioligands at 25 °C, in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5. Nonspecific binding was determined using 10 uM naloxone. Data are the mean
values ( SEM from three experiments, performed in triplicate. b Data for compounds 4 (levorphanol), 5 (cyclorphan), and 6 (ATPM) were obtained from
reference 6. c Data for compounds 7 (naltrindole, NTI), 8, and 9 were from the reference 8.
Table 2. Emax/EC50 and Imax/IC50 Values for the Stimulation and Inhibition of [35S]GTPγS Binding by Novel Compoundsa
µ (mean ( S.E)
κ (mean ( S.E)
δ (mean ( S.E)
cmpd alone EC50
with DAMGO
cmpd alone EC50
with U50,488
cmpd alone EC50
with SNC-80
cmpds
(nM)/Emax(%)
IC50(nM)/Imax(%)
(nM)/Emax(%)
IC50(nM)/Imax(%)
(nM)/Emax(%)
IC50 (nM)/Imax(%)
10a
20b
10b
20c
10c
22a
22b
22c
NT/NT
NT/NT
440 ( 160/43 ( 3
12 ( 6/84 ( 3
NT/NT
NA/23 ( 5
NA/45 ( 7
3 ( 1/64 ( 4
26 ( 9/68 ( 1
NT/NT
24 ( 8/68 ( 7
61 ( 7/67 ( 5
NT/NT
190 ( 22/87 ( 7
21 ( 3/44 ( 2
0.34 ( 0.08/37 ( 2
2.6 ( 0.2/40 ( 5
NT/NT
NI/NI
17 ( 6/49 ( 1
0.47 ( 0.09/23 ( 2
1.5 ( 0.5/56 ( 1
0.16 ( 0.06/22 ( 2
23 ( 9/54 ( 6
0.8 ( 0.4/37 ( 4
8.4 ( 1.0/67 ( 2
0.17 ( 0.04/49 ( 1
2.5 ( 0.9/51 ( 3
1.5 ( 0.7/45 ( 8
NI/NI
1.7 ( 2.5/72 ( 6
92 ( 10/47 ( 3
NT/NT
110 ( 10/68 ( 2
140 ( 3/42 ( 6
0.4 ( 0.02/34 ( 2
2.6 ( 0.3/33 ( 4
1.2 ( 0.4/73 ( 4
NT/NT
NI/NI
NI/NI
NI/NI
NI/NI
0.6 ( 0.2/55 ( 2
NI/NI
NI/NI
NI/NI
93 ( 17/44 ( 4
a CHO membranes, expressing either the κ or µ receptor, were incubated with varying concentrations of the novel compounds in the presence of 0.8 nM
[35S]GTPγS. Data are the mean values ( SE from three experiments, performed in triplicate. NA ) not available, no value could be determined; NI ) no
inhibition; NT ) not tested, no experiments were done to determine value.
at all three receptors, with Ki values of 0.07, 0.1, and 0.2 nM at
κ, µ, and δ receptors, respectively.
receptors, with Ki values of 0.07, 0.1, and 0.2 nM at κ, µ, and
δ receptors, respectively. These results further confirm Por-
toghese’s and Coop’s findings that the introduction of an indole
function serves to reduce κ and µ receptor affinity and enhances
the affinity at the δ receptor. However, the reduction in affinity
at κ and µ receptors of compounds 10a-c and 22a-c was not
significant. In comparison to the indole moiety, the aminothia-
zole component also improves δ receptor affinity, but the affinity
at κ and µ receptors is not significantly affected.
The stimulation and inhibition of [35S]GTPγS binding studies
were shown in Table 2. The indolomorphinans 10b and 10c
displayed both agonist and antagonist activities at µ and δ
receptors. At κ receptors, 10b was a partial agonist/antagonist,
whereas 10c was a full agonist (Emax ) 87%) without inhibition
to the κ agonist, U50,488. It was of interest to note that
10b was a κ and δ partial agonist, but its 3-methoxy analog
20b was a κ and δ agonist. Similarly, 10c was a δ partial ago-
nist, but its 3-methoxy analog 20c was a δ agonist. All
aminothiazolomorphinans 22a-c showed both agonist and
antagonist actions at µ but only pure agonist properties at the δ
receptor. At κ receptor, both 22a and 22b displayed pure agonist
activity, but 22c had both agonist and antagonist properties
(Table 2).
Experimental Section
General. Melting points were determined on a Thomas-Hoover
1
capillary tube apparatus and are reported uncorrected. H and 13C
NMR spectra were recorded on a Brucker AC300 spectrometer
using tetramethylsilane as an internal reference. Element analyses,
performed by Atlantic Microlabs, Atlanta, GA, were within (0.4%
of theoretical values. Analytical thin-layer chromatography (TLC)
was carried out on 0.2 mm Kieselgel 60F 254 silica gel plastic
sheets (EM Science, Newark). Flash chromatography was used for
the routine purification of reaction products. The column output
was monitored with TLC.
General Procedure for Indolic Morphinans 20a-c and 10a-
c: A solution of 17-19 (0.63 mmol), phenylhydrazine‚HCl (100
mg, 0.69 mmol), and TsOH‚H2O (120 mg, 0.63 mmol) in EtOH
(15 mL) was heated at reflux for 2 days. The cooled reaction
mixture was evaporated, and the residue was diluted with water
(20 mL). The solution was basified (pH 9) with NH4OH and
extracted with CH2Cl2. The extracts were combined, washed with
brine, and dried over anhydrous Na2SO4. After removal of the
solvent, the crude product was purified by column chromatography
(EtOAc/Et3N ) 10/1), giving 20a-c as a yellow solid.
In summary, we have extended the SARs of levorphanol (4)
by incorporation of an indole or aminothiazole fragment to the
ring C. Two series of novel morphinans 10a-c and 22a-c were
prepared and their pharmacological properties at opioid receptors
were evaluated. Compared to the high κ and/or µ and low δ
affinity profiles of morphinans described in the previous
reports,4-6 these compounds were potent at the δ receptor. All
3-OH indoles 10a-c had similar Ki values (0.17-0.35 nM) at
the δ receptor. Compound 10a displayed the highest selectivity
for δ over κ (40-fold) and µ (63-fold) receptors. The aminothia-
zolomorphinan 22a had a Ki of 0.49 nM at µ receptor and
selectivities of 4- and 8-fold for κ and δ receptors, respectively.
Compound 22b can be considered as equipotent at all three

2750 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Brief Articles
The 3-methoxy indolic morphinans 20a-c (0.3 mmol) were then
dissolved in 5 mL of CH2Cl2, cooled to -78 °C, and dropped slowly
into a solution of BBr3 (1 M in CH2Cl2, 4 mL) in 4 mL of CH2Cl2.
The mixture was stirred at -78 °C for 4 h and then at rt overnight.
The solution was cooled to -78 °C again, MeOH (10 mL) was
added dropwise, and the mixture was stirred at -78 °C to rt
overnight. The solvent was evaporated and the residue was
recrystallized from MeOH to give 10a-c as HBr salts.
anhyd MeOH and then evaporated. The residue was dried in vacuo
and recrystallized from the mixture of MeOH and Et2O to give the
3-hydroxy aminothiazolic morphinans 22a-c.
1
21a: yellow oil (66.7%). MS m/z (%) 342 (MH+); H NMR
(CDCl3, 300 MHz) δ 7.00 (d, J ) 8.4 Hz, 1H), 6.84 (d, J ) 2.1
Hz, 2H), 6.66 (dd, J ) 2.1, 8.4 Hz, 1H), 5.11 (s, 2H), 3.70 (s, 3H),
3.56 (m, 1H), 3.38 (m, 2H), 3.05 (m, 2H), 2.45-1.26 (m, 10H).
22a: This compound was prepared as a free base and purified
by column chromatography (EtOAc/ MeOH/Et3N ) 10/4/2) to give
a yellow solid (43.5%). Mp > 250 °C; MS m/z (%) 328 (MH+);
1H NMR (CDCl3, 300 MHz) δ 6.96 (d, J ) 8.4 Hz, 1H), 6.73 (d,
J ) 2.1 Hz, 2H), 6.68 (dd, J ) 2.1, 8.4 Hz, 1H), 4.36 (s, 2H), 3.76
(m, 1H), 3.35 (m, 3H), 2.52 (m, 6H), 1.98 (m, 1H), 1.80 (m, 1H),
1.62 (m, 3H). The free base was converted to its acetic acid salt.
Anal. (C18H21N3OS‚CH3COOH) C, H, N.
20a: yellow solid (57.4%). Mp 97-100 °C (dec); MS (EI) 358
(M+); 1H NMR (CDCl3) δ 7.97 (s, 1H), 7.29 (d, J ) 7.5 Hz, 1H),
7.23 (m, 1H), 7.01 (m, 3H), 6.80 (d, J ) 2.4 Hz, 1H), 6.60 (dd, J
) 2.7, 8.7 Hz, 1H), 3.66 (s, 3H), 3.48 (d, J ) 16.5 Hz, 1H), 3.19
(m, 1H), 2.88 (m, 4H), 2.40 (s+m, 6H), 2.19 (m, 1H), 2.03 (m,
2H), 1.62 (m, 1H); Anal. (C24H26N2O‚0.5H2O) C, H, N.
10a: gray solid (57.6%). Mp > 250 °C; MS (EI) 344 (M+
-
1
1
21b: yellow oil (58.2%). MS m/z (%) 382 (MH+); H NMR
HBr); H NMR (CD3OD) δ 7.20 (m, 2H), 6.99 (m, 2H), 6.88 (m,
1H), 6.82 (m, 1H), 6.61 (m, 1H), 3.93 (s, 1H), 3.63 (d, J ) 16.8
Hz, 1H), 3.30 (m, 2H), 3.16 (m, 2H), 2.92 (s, 3H), 2.82 (m, 2H),
2.57 (m, 1H), 2.34 (m, 1H), 2.15 (m, 1H), 1.83 (m, 1H), 1.17 (m,
1H); Anal. (C23H25N2O‚HBr‚1.7H2O) C, H, N.
(CDCl3, 300 MHz) δ 6.85 (d, J ) 8.4 Hz, 1H), 6.71 (d, J ) 2.7
Hz, 2H), 6.53 (dd, J ) 2.4, 8.4 Hz, 1H), 4.77 (s, 2H), 3.57 (s, 3H),
3.27 (d, J ) 16.8 Hz, 1H), 3.23 (m, 1H), 2.66 (d, J ) 6.6 Hz, 1H),
2.61 (m, 1H), 2.41(m, 6H), 1.89 (m, 3H), 1.20 (m, 2H), 0.77 (m,
2H), 0.39 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 165.1, 158.2,
143.6, 140.3, 129.0, 128.5, 116.8, 111.7, 110.3, 59.8, 55.1, 54.6,
44.9, 41.3, 41.2, 37.4, 37.2, 24.5, 23.9, 9.3, 3.8, 3.7.
20b: yellow foam (42.4%). Mp 147-150 °C (dec); MS (EI) 398
(M+); 1H NMR (CDCl3) δ 7.97 (s, 1H), 7.29 (d, J ) 7.5 Hz, 1H),
7.23 (m, 1H), 7.01 (m, 3H), 6.80 (d, J ) 2.4 Hz, 1H), 6.60 (dd, J
) 2.7, 8.7 Hz, 1H), 3.66 (s, 3H), 3.48 (d, J ) 16.5 Hz, 1H), 3.19
(m, 1H), 2.88 (m, 4H), 2.69 (m, 1H), 2.47 (m, 4H), 2.13 (m, 3H),
1.63 (m, 1H), 0.95 (m, 1H), 0.57 (m, 2H), 0.18 (m, 2H).
10b: this compound was prepared as its HCl salt (68.7%). MS
(EI) 384 (M+); 1H NMR (CDCl3) δ 8.47 (s, 1H), 7.73 (d, J ) 6.6
Hz, 1H), 7.63 (d, J ) 7.2 Hz, 1H), 7.46 (m, 2H), 7.35 (d, J ) 8.7
Hz, 1H), 7.25 (m, 1H), 7.00 (d, J ) 8.7 Hz, 1H), 3.97 (s, 1H),
3.77 (d, J ) 15.9 Hz, 1H), 3.11 (m, 8H), 2.63 (t, J ) 11.4 Hz,
1H), 2.41 (m, 1H), 1.96 (m, J ) 11.4 Hz, 1H), 1.74 (m, 2H), 1.36
(m, 1H), 0.99 (d, J ) 7.2 Hz, 2H), 0.60 (m, 2H); Anal. (C26H29-
ClN2O‚1.3H2O) C, H, N.
22b: yellow solid (61.7%). Mp > 270 °C; MS m/z (%) 368 (M+
- HBr); 1H NMR (CD3OD, 300 MHz) δ 7.10 (d, J ) 8.4 Hz, 1H),
6.78 (d, J ) 2.7 Hz, 2H), 6.71 (dd, J ) 2.4, 8.4 Hz, 1H), 4.26 (s,
2H), 3.40 (m, 9H), 2.80 (m, 4H), 2.29 (m, 2H), 1.91 (d, J ) 14.4
Hz, 1H), 1.17 (m, 1H), 0.79 (m, 2H), 0.52 (m, 2H); 13C NMR (CD3-
OD, 75 MHz) δ 171.1, 158.2, 137.5, 132.4, 131.0, 124.9, 116.2,
114.9, 111.8; Anal. (C21H25N3OS‚2.1HBr‚2H2O) C, H, N.
1
21c: yellow oil (30.7%). MS m/z (%) 396 (MH+); H NMR
(CDCl3, 300 MHz) δ 6.93 (d, J ) 8.4 Hz, 1H), 6.75 (d, J ) 2.4
Hz, 1H), 6.59 (dd, J ) 2.4, 8.1 Hz, 1H), 4.92 (s, 2H), 3.62 (s, 3H),
3.33 (d, J ) 17.1 Hz, 1H), 2.95 (m, 2H), 2.50 (m, 5H), 1.98 (m,
3H), 1.79 (m, 5H), 1.44 (d, J ) 12.6 Hz, 1H), 1.24 (m, 2H), 0.82
(m, 2H); 13C NMR (CD3OD, 75 MHz) δ 165.0, 158.0, 143.5, 140.2,
135.0, 128.5, 116.8, 111.5, 110.2, 61.3, 55.0, 54.7, 45.0, 41.2, 41.1,
37.2, 37.0, 34.7, 27.8, 27.7, 24.5, 23.9, 18.8.
20c: yellow foam (51.2%). Mp 95 °C (soften); MS (EI) 412
1
(M+); H NMR (CDCl3, 300 MHz) δ 7.71 (s, 1H), 7.29 (d, J )
7.5 Hz, 1H), 7.25 (d, J ) 7.8 Hz, 1H), 7.02 (m, 3H), 6.78 (d, J )
2.7 Hz, 1H), 6.59 (dd, J ) 2.4, 8.1 Hz, 1H), 3.67 (s, 3H), 3.45 (d,
J ) 16.8 Hz, 1H), 3.18 (d, J ) 6.0 Hz, 1H), 3.06 (d, J ) 18.3 Hz,
1H), 2.85 (m, 2H), 2.60 (m, 5H), 2.3 7 (m, 2H), 2.17-1.56 (m,
7H); 13C NMR (CDCl3, 75 MHz) δ 157.9, 140.5, 135.6, 132.0,
129.7, 128.5, 127.6, 120.8, 119.0, 117.5, 110.8, 110.4, 110.3, 109.0,
61.5, 55.3, 55.0, 45.2, 41.7, 41.2, 37.7, 35.0, 34.1, 27.9, 27.8, 24.0,
22.5, 18.8; Anal. (C28H32N2O‚0.3H2O) C, H, N.
22c: yellow solid (42%). Mp > 270 °C; MS m/z (%) 381 (M+
- HBr); 1H NMR (CD3OD, 300 MHz) δ 7.11 (d, J ) 8.4 Hz, 1H),
6.77 (m, 1H), 6.72 (d, J ) 8.4 Hz, 1H), 3.97 (s, 2H), 3.47 (m, 2H),
3.30 (m, 5H), 2.77 (m, 5H), 2.25 (m, 4H), 1.96 (m, 5H); 13C NMR
(CD3OD, 75 MHz) δ 171.1, 158.2, 137.4, 132.4, 131.0, 124.9,
116.2, 114.9, 111.8, 60.3, 57.9, 39.4, 38.8, 36.4, 33.0, 32.3, 28.2,
28.0, 24.2, 23.9, 19.4; Anal. (C22H27N3OS‚2HBr‚2H2O) C, H, N.
General Procedure for Opioid Binding to the Human µ, δ,
and κ Opioid Receptors. Chinese hamster ovary (CHO) cells stably
transfected with the human κ opioid receptor (hKOR-CHO), δ
opioid receptor (hDOR-CHO), and the µ opioid receptor (hMOR-
CHO) were obtained from Drs. Larry Toll (SRI International, Palo
Alto, CA) and George Uhl (NIDA Intramural Program, Bethesda,
MD), respectively. The cells were grown in 100 mm dishes in
Dulbecco’s modified Eagle’s media (DMEM), supplemented with
10% fetal bovine serum (FBS) and penicillin-streptomycin (10 000
units/mL) at 37 °C in a 5% CO2 atmosphere. The affinity and
selectivity of the compounds for the multiple opioid receptors were
determined by incubating the membranes with radiolabeled ligands
and 12 different concentrations of the compounds at 25 °C in a
final volume of 1 mL of 50 mM Tris-HCl, pH 7.5. Incubation times
of 60 min were used for the µ-selective peptide [3H]DAMGO and
the κ-selective ligand [3H]U69,593. A 3 h incubation was used with
the δ-selective antagonist [3H]naltrindole.
10c: gray powder (43.1%). Mp > 270 °C; MS (EI) 398 (M+
-
1
HBr); H NMR (CD3OD) δ 7.44 (m, 1H), 7.28 (d, J ) 7.8 Hz,
1H), 7.23 (d, J ) 7.8 Hz, 1H), 6.99 (m, 2H), 6.80 (m, 1H), 6.62
(m, 1H), 3.84 (s, 1H), 3.58 (d, J ) 15.9 Hz, 1H), 3.36 (m, 2H),
3.21 (m, 3H), 3.07 (m, 1H), 2.93 (m, 1H), 2.73 (m, 2H), 2.27 (m,
3H), 1.94 (m, 7H); 13C NMR (CD3OD, 75 MHz) δ 156.7, 138.1,
136.2, 131.6, 129.4, 127.2, 123.3, 121.0, 118.9, 117.4, 114.9, 111.6,
110.9, 106.4, 59.4, 57.6, 46.9, 38.6, 36.2, 32.7, 31.2, 27.9, 27.5,
23.9, 22.3, 18.8; Anal. (C27H30N2O‚1.5HBr) C, H, N.
General Procedure for Aminothiazolic Morphinans 22a-c:
To a solution of ketone 17-19 (0.31 mmol) in a mixture of 1 mL
of AcOH and 2 drops of HBr (48%) was added Br2 (50 mg, 16
µL, 0.31 mmol) slowly. The solution was heated at 60 °C for 1 h.
Thiourea (48 mg, 0.63 mmol) was added and the mixture was
refluxed overnight. The reaction mixture was cooled to rt, poured
into ice/water, basified with NH4OH, and extracted with CHCl3.
The organic layer was washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo. The crude product was purified
by column chromatograph (EtOAc/Et3N ) 10/1 to 4/1) to give
21a-c as a yellow oil.
To a solution of 3-methoxy aminothiazole 21a-c (0.21 mmol)
in 5 mL of CH2Cl2, cooled to -78 °C, was dropped slowly a
solution of BBr3 (1 M in CH2Cl2, 5 mL) in 5 mL of CH2Cl2. The
mixture was stirred at -78 °C for 2 h and then at rt overnight.
After cooling to -78 °C again, a solution of MeOH (10 mL) was
dropped slowly. The mixture was stirred at rt for 2 h, and the
solvents were evaporated. The residue was dissolved in 10 mL of
General Procedure for [35S]GTPγS Binding Studies To
Measure Coupling to G Proteins. Membranes from CHO cells
stably expressing either the human κ or µ opioid receptor were
used in the experiments. Cells were scraped from tissue culture
plates and then centrifuged at 1000 g for 10 min at 4 °C. The cells
were resuspended in phosphate-buffered saline, pH 7.4, containing
0.04% EDTA. After centrifugation at 1000 g for 10 min at 4 °C,
the cell pellet was resuspended in membrane buffer, which consisted
of 50 mM Tris-HCl, 3 mM MgCl2, and 1 mM EGTA, pH 7.4. The

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2751
(3) Fries, D. S. Opioid Analgesics. In Foye’s Principles of Medicinal
Chemistry; Williams, D. A., Lemke, T. L., Eds.; Lippincott Williams
& Wilkins: PA, 2002; p 453.
(4) Neumeyer, J. L.; Zhang, A.; Xiong, W.; Gu, X.; Hilbert, J. E.; Knapp,
B. I.; Negus, S. S.; Mello, N. K.; and Bidlack, J. M. Design and
Synthesis of Novel Bivalent Morphinan Ligands for Opioid Recep-
tors. J. Med. Chem. 2003, 46, 5162-5170.
(5) Zhang, A.; Xiong, W.; Bidlack, J. M.; Hilbert, J. E.; Knapp, B. I.;
Wentland, M. P.; Neumeyer, J. L. 10-Ketomorphinan and 3-Substituted-
3-desoxymorphinan Analogues as Mixed κ and µ Opioid Ligands:
Synthesis and Biological Evaluation of Their Binding Affinity at
Opioid Receptors. J. Med. Chem. 2004, 47, 165-174.
(6) Zhang, A.; Xiong, W.; Hilbert, J. E.; DeVita, E. K.; Bidlack, J. M.;
Neumeyer, J. L. 2-Aminothiazole-Derived OpioidssBioisosteric
Replacement of Phenols. J. Med. Chem. 2004, 47, 1886-1888.
(7) Portoghese, P. S.; Sultana, M.; Takemori, A. E. Design of Peptido-
mimetic δ Opioid Receptor Antagonist Using The Message-Address
Concept. J. Med. Chem. 1990, 33, 1714-1720.
(8) Coop, A.; Rothman, R. B.; Dersch, C.; Partilla, J.; Porreca, F.; Devis,
P.; Jacobson, A. E.; Rice, K. C. δ Opioid Affinity and Selectivity of
4-Hydroxy-3-methoxyindolomorphinan Analogues Related to Nal-
trindole. J. Med. Chem. 1999, 42, 1673-1679.
(9) Coop, A.; Rice, K. Direct and Simple Conversion of Codeine to
Thebainone-A and Dihydrothebainone. Heterocycles 1999, 50, 39-
42.
(10) Sawa, Y. K.; Tsuji, N.; Maeda, S. Elimination of the 4-Hydroxyl
Group of the Alkaloids Related to Morphine. I. Tetrahedron 1961,
15, 144-153.
(11) Sawa, Y. K.; Tsuji, N.; Maeda, S. Elimination of the 4-Hydroxyl
Group of the Alkaloids Related to Morphine. II. Tetrahedron 1961,
15, 154-159.
(12) Sawa, Y. K.; Tsuji, N.; Okabe, K.; Miyamoto, T. Elimination of the
4-Hydroxyl Group of the Alkaloids Related to Morphine. VI.
Synthesis of (-)-3-Hydroxy-N-methyl-C-nor-morphinan derivatives.
Tetrahedron 1965, 21, 1121-1128.
(13) Sawa, Y. K.; Horiuchi, M. Elimination of the 4-Hydroxyl Group of
the Alkaloids Related to Morphine. Synthesis of (-)-3-Methoxy-N-
methylisomorphinan Derivatives. Tetrahedron 1965, 21, 1133-1139.
(14) Sawa, Y. K.; Maeda, S. Elimination of the 4-Hydroxyl Group of the
Alkaloids Related to Morphine. III. Tetrahedron 1964, 20, 2247-
2253.
(15) Olfoson, R. A.; Marts, J. T.; Seret, J. P.; Piteau, M.; Malfroot, T. A.
A New Reagent for the Selective, High Yield N-Dealkylation of
Tertiary Amines: Improved Synthesis of Naltrexone and Albuphine.
J. Org. Chem. 1984, 49, 2081-2082.
(16) Kubota, H.; Rothman, R. B.; Dersch, C.; McCullough, K.; Pinto, J.;
Rice, K. C. Synthesis and Biological Activity of 3-Substituted
3-Desoxynaltrindole Derivatives. Bioorg. Med. Chem. Lett. 1998, 8,
799-804.
membranes were homogenized by with a Dounce homogenizer,
followed by centrifugation at 40 000 g for 20 min at 4 °C. The
membrane pellet was resuspended in membrane buffer, and the
centrifugation step was repeated. The membranes were then
resuspended in assay buffer, which consisted of 50 mM Tris-HCl,
3 mM MgCl2, 100 mM NaCl, and 0.2 mM EGTA, pH 7.4. The
protein concentration was determined by the Bradford assay using
bovine serum albumin as the standard. The membranes were frozen
at -80 °C until use.
CHO cell membranes expressing either the human κ opioid
receptor (15 µg of protein per tube) or µ opioid receptor (7.5 µg of
protein per tube) were incubated with 12 different concentrations
of the agonist in assay buffer for 60 min at 30 °C in a final volume
of 0.5 mL. The reaction mixture contained 3 µM GDP and 80 pmol
of [35S]GTPγS. Basal activity was determined in the presence of 3
µM GDP and in the absence of an agonist, and nonspecific binding
was determined in the presence of 10 µM unlabeled GTPγS. Then
the membranes were filtered onto glass fiber filters by vacuum
filtration, followed by three washes with 3 mL of ice-cold 50 mM
Tris-HCl, pH 7.5. Samples were counted in 2 mL of Ecoscint A
scintillation fluid. Data represent the percent of agonist stimulation
[35S]GTPγS binding over the basal activity, defined as [(specific
binding/basal binding) × 100] - 100. All experiments were
repeated at least three times and were performed in triplicate. To
determine antagonist activity of a compound at the µ opioid
receptors, CHO membranes expressing the µ opioid receptor were
incubated with the compound in the presence of 200 nM of the
agonist DAMGO. To determine antagonist activity of a compound
at the κ opioid receptors, CHO membranes expressing the κ opioid
receptor were incubated with the compound in the presence of 100
nM of the κ agonist U50,488.
Acknowledgment. We thank financial support from the
NIDA Grants R01-DA14251 (to J.L.N.), K05-DA 00360 (to
J.M.B.), and from Chinese National Natural Science Foundation
(3067251) and Shanghai Commission of Science and Technol-
ogy (06ZR14102; to A.Z.).
Supporting Information Available: Procedure for the prepara-
tion of morphinans 17-19 and results from the elemental analysis
of the final compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(17) Katsuura, K.; Yamaguchi, K.; Sakai, S.-I.; Mitsuhashi, K. Thiazole
Analogs of Benzomorphans. II. Synthesis of Novel Thiazolo[5,4-f]-
morphans. Chem. Pharm. Bull. 1983, 31, 1518-1527.
(18) Davidson, M. R.; Gregg, B. T. Improved Synthesis of Dihydrothe-
bainone and its 14â-Isomer. Synth. Commun. 1998, 28, 547-558.
References
(1) Eguchi, M. Recent Advances in Selective Opioid Receptor Agonists
and Antagonists. Med. Res. ReV. 2004, 24, 182-212.
(2) Aldrich, J. V.; Vigil-Cruz, S. C. Narcotic Analgesics. In Burger’s
Medicinal Chemistry and Drug DiscoVery; Abraham, D., Ed.; John
Wiley & Sons: New York, 2003; p 329.
JM0701674