Synthesis and pharmacological evaluation of 6-naltrexamine analogs for alcohol cessation

Article abstract of DOI:10.1016/j.bmc.2009.07.069

A series of substituted aryl amide derivatives of 6-naltrexamine, 3 designed to be metabolically stable were synthesized and used to characterize the structural requirements for their potency to binding and functional activity of human mu (μ), delta (δ) a

Full text of DOI:10.1016/j.bmc.2009.07.069

Bioorganic & Medicinal Chemistry 17 (2009) 6671–6681
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological evaluation of 6-naltrexamine analogs
for alcohol cessation
Senait Ghirmai a, Marc R. Azar b,c, John R. Cashman a,
*
a Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA
b Behavioral Pharma Inc., 505 Coast Blvd. South, Suite 210, La Jolla, CA 92037, USA
c Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted aryl amide derivatives of 6-naltrexamine, 3 designed to be metabolically stable were
Received 5 June 2009
Revised 23 July 2009
Accepted 26 July 2009
Available online 6 August 2009
synthesized and used to characterize the structural requirements for their potency to binding and func-
tional activity of human mu (
assays showed that 4–10 had subnanomolar Ki values for
stimulation of [35S]GTP
S binding showed that several compounds acted as partial or inverse agonists
and antagonists of the and d, opioid or NOP receptors. The compounds showed considerable stability
l
), delta (d) and kappa (
j) opioid and nociceptin (NOP) receptors. Binding
l
and opioid receptors. Functional assays for
j
c
l
j
Keywords:
in the presence of rat, mouse or human liver preparations and NADPH. The inhibitory activity on the func-
tional activity of human cytochrome P450s was examined to determine any potential inhibition by 4–9.
Only modest inhibition of CYP3A4, CYP2C9 and CYP2C19 was observed for a few of the analogs. As a rep-
resentative example, radiolabeled 6 was examined in vivo and showed reasonable brain penetration. The
inhibition of ethanol self-administration in rats trained to self-administer a 10% (w/v) ethanol solution, uti-
Beta naltrexamides
Alcohol cessation agents
Metabolism
In vitro–in vivo studies
lizing operant techniques showed 5–8 to have very potent efficacy (ED50 values 19–50 lg/kg).
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
sesses superior pharmaceutical properties compared with 1a4 but
1b also suffers from hepatotoxic side effects.
Worldwide, dependence on alcohol is a serious public health
disorder with significant social and economic consequences. In
1994, naltrexone (compound 1a, Fig. 1) was approved by the Uni-
ted States FDA for treatment of alcoholism. Compound 1a, along
with acamprosate (C10H20CaN2O8S2) and disulfiram (C10H20N2S4)
are the only agents currently available to treat alcohol dependence.
Compound 1a has shown benefit for treating alcoholism in heavy
drinkers1 and in a number of clinical studies linked to moderate
to severe alcoholism.1,2 However, 1a is not successful in treating
all alcoholics and adverse effects including intolerable nausea3
and hepatotoxicity4 confound treatment of patients with liver dis-
ease. It may be that metabolic bioactivation of 1a to a reactive met-
abolic intermediate contributes to the hepatotoxicity observed.
Diminished effect over time, relatively low bioavailability2 and
Studies using rodent animal models have shown that 1a de-
creases alcohol self-administration,8,9 suggesting that these types
of agents may prevent the reinforcing effects of alcohol consump-
tion.1 However, some opioid receptor antagonists decrease both
ethanol and sucrose intake in rats.10 Certain opioid receptor ago-
nists stimulate food consumption in preclinical animal models of
obesity and opioid receptor antagonists inhibit energy-rich food
consumption.11 It may be that opioid receptor antagonists prevent
central reward mechanisms that share common neural substrates
responsible for the development of alcohol dependence.12
Opioid receptors are well-characterized receptors and numer-
ous studies suggest that alcohol interacts with endogenous opioid
systems13,14 (e.g., 1a is a pure opioid
l receptor antagonist with no
agonist activity and no abuse potential). Antagonizing opioid
receptors decrease the effects of alcohol-mediated pleasure-induc-
ing endogenous opioids. By attenuating the positive reinforcing ef-
fects of alcohol consumption, opioid receptor antagonists have
direct effects on alcohol-seeking behavior.10 A decrease in alcohol
consumption by antagonism of opioid receptors suggests direct ef-
fects on this reinforcement system and animal studies have shown
possibly relatively low affinity for d and
j
opioid receptors5 or ge-
netic variability of the opioid receptors6 may explain the less than
consistent efficacy of 1a.7 Nalmefene (compound 1b, Fig. 1), pos-
Abbreviations: BOP, benzotriazol-1-yl-oxy-tris-(dimethylamino) phosphonium
hexafluorophosphate; ESI, electrospray ionization; DTPA, diethylenetriaminepenta-
acetic acid; GTP, guanosine 50-triphosphate; TLC, thin layer chromatography; HPLC,
high-performance liquid chromatography.
* Corresponding author. Tel.: +1 858 458 9305; fax: +1 858 458 9311.
E-mail address: jcashman@hbri.org (J.R. Cashman).
that
l-, d- and j-opioid receptors contribute to alcohol-induced
reinforcement.15,16
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.069

6672
S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
2. Results
HO
HO
O
The chemical synthesis of a series of substituted aryl amide
derivatives of 6-b-naltrexamine 4–10 was efficiently accomplished
(Scheme 1) and used to characterize the structural requirements
for binding to and functional activity of human
O
O
N
N
OH
OH
l-, d-, j-opioid
H2C
and nociceptin receptors. Compound 1a was converted to its oxime
2 in a quantitative yield using hydroxylamine hydrochloride in the
presence of sodium acetate in refluxing ethanol.19 Reduction of the
oxime 2 to the corresponding amine 3 was accomplished by heat-
ing 2 with borane-tetrahydrofuran complex for 2 days. Following
1b
1a
Figure 1. Chemical structures of 1a and 1b.
Previously, 6-alpha and 6-beta-N-heterocyclic17 naltrexamines
an aqueous workup, amine 3 was obtained as a 1:9 (a/b) mixture
of diastereomers. The diastereomers were separated by chroma-
tography on silica gel and the stereochemistry at the C-6 position
was determined on the basis of the size of the NMR coupling con-
stant, J5,6. The amine 3 (only the beta diastereomer was used in this
work because previous work18 showed little stereoselectivity in
were synthesized and displayed considerable antagonist potency
and selectivity for the
amide naltrexamine18 derivatives were synthesized and showed
considerable potency and selectivity for -opioid receptors. At
the outset, we hypothesized that -opioid receptor antagonists
l-opioid receptor. In a separate study, N-aryl
j
j
opioid binding for b versus
a diastereomers) was coupled either
would show alcohol consumption cessation in animal models.
Most electron-rich N-aryl amide naltrexamines18 were not potent
at alcohol consumption cessation in animal models. However,
one electron-deficient aryl amide of naltrexamine (i.e., 17-cyclo-
with a carboxylic acid in the presence of benzotriazol-1-yl-oxy-
tris-(dimethylamino) phosphonium hexa-fluorophosphate (BOP)
and diisopropylethylamine or alternatively, with an acid chloride
in triethylamine. The product was treated with potassium carbon-
ate in methanol to remove the byproduct resulting from esterifica-
tion of the 3-position hydroxyl group, giving amides 4–10 in
moderate to high yields (60–97%). While the BOP coupling proce-
dure resulted in less esterification at the 3-position compared with
the acid chloride method, some esterification at the 3-position
could not be avoided. Thus, it was found to be more convenient
to run the reaction with an excess of the acid chloride, form the
intermediate amide ester and then purify the amide ester before
base hydrolysis to afford the desired amide.
propylmethyl-3,14b-dihydroxy-4,5a
-epoxy-6b-[(40-chloro) benz-
amido]morphinan) showed considerable efficacy as an in vivo
alcohol cessation agent.18 We thus investigated highly electron-
deficient aryl amides of naltrexamine as alcohol cessation agents.
Herein, we report on the rationale for and the design of a class
of metabolically stable compounds that have mixed potency and
efficacy as
l-, d- and j-opioid and NOP-receptor partial agonists,
inverse-agonists and/or antagonists as alcohol self-administration
cessation agents. Partial agonist agents are anticipated to show a
dual action by inhibiting alcohol reinforcement and stimulating
dopamine release to decrease craving. The rationale for the work
described herein was to develop long-lived, metabolically stable
analogues of 1a or 1b by replacing the metabolically labile 6-keto
or 6-methylene groups, respectively, with an amide moiety, thus
leading to agents with more sustained pharmacological activity
and potentially less hepatotoxicity.
The binding of compounds 1a, 1b, 4–10 to the l-, d- and j-opioid
receptors wasdeterminedina competitivebinding assay with the fol-
lowing radioligands: [3H][D-ala,2 N-MePhe,4 Gly-ol]enkephalin, 11
([3H]DAMGO, -opioidreceptoragonist),[3H][D-ala,2 D-leu5]enkeph-
l
alin, 12 ([3H]DADLE, d-opioid receptor agonist) and [3H] (5a,7a,8b)-
(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)-benze-
N
N
OH
OH
1. BH3, THF, reflux, 2 d
NH2OH-HCl, NaOAc
EtOH:H2O (80:5)
reflux, 2.5 h
2. KOH, reflux, 2h
3. 10% HCl, reflux, 2 h
O
O
HO
O
HO
N OH
2
1a
N
N
OH
OH
RCO2H, BOP, DIEA
CH2Cl2, rt, 90 min
O
R
O
O
HO
HN
HO
NH2
3
4-10
4: R = p-Methylphenyl
5:
R =p-Trif luoromethylphenyl
6: R = p-Bromophenyl
7: R = p-Iodophenyl
8:
9:
R = p-t-Butylphenyl
R = 3,4-Dichlorophenyl
10: R = p-Chlorophenyl
Scheme 1. Chemical synthesis of compounds 2–10.

S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
6673
neacetamide, 13 ([3H]U69593,
j
-opioid receptor agonist),
opioid and NOP-receptors, and compared to the standard selective
full agonists, 11, [D-pen2, D-pen5]-enkephalin, 14 (DPDPE), 13 and
nociceptin, 15, respectively. Table 2 summarizes the EC50 and Emax
values for compounds 4–16 in the presence of cloned human cell
14 [D-Pen2,5]-enkephalin (DPDPE), 15, naltrindole, 16 (ꢀ)-trans-
(1S,2S)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]
benzene acetamide hydrochloride ((ꢀ) U50,488), and 17, norbinal-
torphimine. Results of binding to the individual receptors, and the
membranes containing the l, d- or j-opioid or NOP receptors.
ratios of d- and
j
-binding relative to
l
- were summarized and
Para-alkyl substituted 4 and 8 were either very weak agonists or
completely not functional suggesting that electron donating groups
might be detrimental to functional activity. The 3,4-dichlorophenyl
listed in Table 1. The amides 4–10 were between 4 and 10-fold
more potent at the d-opioid receptors than 1a and 1b (Ki <4 nM
for 4–10 compared to 16.3 and 13.3 nM for 1a and 1b, respec-
tively). The 3,4-dichloro phenyl amide and the bulky t-butyl and
iodo phenyl amide analogs were the most potent with Ki values
around 1 nM. Compared to 1a and 1b, binding was also improved
with regard to the
afforded a Ki of <0.4 nM compared to 0.8 nM for 1a and 1.03 nM for
1b. The p-methyl phenyl analog 4 bound to the receptor with the
greatest affinity (Ki = 0.11 nM) suggesting that a smaller group at
the para position was favored for receptor binding. Finally, add-
ing an aryl amide at the 6-position on the naltrexamine core (i.e.,
4–10) did not significantly change the affinity for binding to the
derivative 9 was found to stimulate GTP
-, d- and
-opioid receptors (Emax ꢁ80–85%), with an EC50 value in
the low nanomolar range (EC50 = 2.3, 1.4, 0.9 for -, d-, -receptors,
respectively). Compounds 5–7 and 10 were partial agonists
(Emax values between 28% and 63%) at -, d-, and -opioid receptors
(Table 2). Compounds 4–10 had very low affinity for the NOP recep-
tor and did not stimulate agonist-induced GTP S binding.
cS binding as a full agonist at
l
j
l
j
j
-opioid receptor. The phenyl amide derivatives
l
j
j
c
In a second functional assay, compounds 4–10 were evaluated
as inverse-agonists. Compounds 4 and 8 were found to be partial
inverse-agonists at the l- and j-receptors. Compounds 4–10 were
found to potently decrease basal binding at NOP and compounds 7
and 9 were found to have high affinity as inverse-agonists. Com-
pound 8 was also observed to be a potent inverse-agonist at d-
j
l-
receptor compared to 1a and 1b, (i.e., Ki values between 0.3 and
1.09 nM were observed). All the compounds examined had at least
2–3-fold greater potency for the
d-receptor. Compound 5 was about sevenfold more selective for
the -receptor compared with the d-receptor.
j
-receptor compared to the
l
- or
and
tor (Table 3). Compound 4 was found to be a potent inverse-ago-
nist at -and -receptors with decreased potency (albeit with
j-receptors, with less potent inverse-agonism at the l-recep-
j
l
j
A functional assay was also run in order to evaluate the opioid
high efficacy) at the NOP receptor. Compound 6 was also observed
receptor-mediated activation of its associated G protein. Com-
to display inverse-agonism at the d-receptor albeit at higher con-
centrations (i.e., 10 nM–10 lM), in addition to potent agonism at
pounds 4–10 were evaluated using the [35S]GTP
c
S assay.20 In this
assay, a compound’s potency or affinity for the receptor was asso-
lower concentrations (i.e., 10 pM–10 nM).
ciated with its EC50 value for stimulating [35S]GTP
cS binding. Ago-
High affinity compounds that showed low or partial agonist
nist activity of each compound was determined at the
l
-, d-,
j
-
activity in the GTP
tion of agonist-induced GTP
4 produced strong inhibition at d- and
bition at -receptors, but not at the NOP-receptor (Table 4). Com-
pound 5 produced potent inhibition at both - and NOP-receptors,
but not at - or d-receptors. Compound 6 produced very potent
inhibition at the -receptor but no detectable inhibition at -, d-
or NOP receptors. Compounds 7 and 8 did not produce any detect-
able inhibition at any opioid receptor examined. Compounds 4, 5
and 6 appear to possess mixed activity as either agonists, in-
c
S binding experiments were tested for inhibi-
S binding at each receptor. Compound
-receptors and potent inhi-
c
Table 1
j
Inhibition values and selectivity for
l, d and j opioid binding
l
Compda
Ki (nM) SEM
Selectivity
d/ j/l
j
l
l
d
j
l
j
l
1ab
0.30
0
16.31 1.10
13.26 0.75
3.6 0.3
2.5 0.3
2.2 0.3
1.4 0.2
1.4 0.1
1.0 0.1
2.6 0.3
0.81 0.02
1.03 0.19
0.11 0.02
0.34 0.05
0.29 0.04
0.37 0.05
0.37 0.06
0.34 0.04
0.23 0.03
49
15
11
5.3
2.5
1.7
1.3
2.1
4.3
2.5
1.1
1bb
0.91 0.10
0.34 0.05
0.47 0.05
0.88 0.10
0.82 0.10
1.09 0.20
0.48 0.07
0.61 0.09
0.9
4
5
6
7
8
9
10
11
0.32
0.72
0.33
0.45
0.34
0.71
0.38
verse-agonists or antagonists for each of the
l-, d- and j-opioid
and NOP-receptors. As described below, further kinetic analysis
was done to characterize the pharmacological properties of these
latter compounds.
17
0.8
Salvonorin A
0.8
3. Structure–activity studies
Ki values were expressed as the mean SEM of two determinations.
a
The SAR of the aromatic amide portion of the opioid derivatives
was examined. Despite the limited number of compounds studied,
Compounds 4–10 were salts as described in the Methods.
b
Data was taken from Ref. 18.
Table 2
Stimulation of [35S]GTP
cS binding at opioid receptors by compounds 4–10 and the opioid agonists, 11, 14, 16 and 15
Compda
l
d
j
NOP
EC50
>10
16 2.1 nM
4.5 0.5 nM
8.8 1.7 nM
Emax
EC50
Emax
EC50
>10
9.9 1.7 nM
0.1 0.1 nM
29 3.4 nM
Emax
EC50
Emax
4
5
6
7
8
l
M
0
0.14 0.1 nM
5.1 0.2 nM
0.2 0.1 nM
5.1 2.9 nM
10 9.6
28 6.5
46 3.7
45 6.2
0
85 35
22 1.7
—
l
M
0
>10
>10
>10
>10
>10
>10
>10
—
l
M
M
M
M
M
M
M
0
0
0
0
0
0
0
0
63 14
38 4.3
53 2.8
0
85 7.4
46 3.8
124 7.9
—
36 6.4
42 3.9
28 3.1
0
80 7.4
31 2.6
—
l
l
l
l
l
l
>10
l
M
>10
l
M
>10
l
M
9
2.3 1.4 nM
6.8 1.7 nM
8.2 1.4 nM
—
—
—
1.4 1.4 nM
42 3.1 nM
—
15 2.6 nM
—
—
0.9 0.1 nM
7.1 2.7 nM
—
—
10
11
14
15
16
76 4.8
—
—
—
—
—
0
—
—
—
3.9 0.5 nM
—
109 11
0
0.4 0.2 nM
54 14
a
Compounds 4–10 were salts as described in the Methods. Emax values are expressed as mean SEM percentage of basal [35S]GTP
cS binding stimulation.

6674
S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
Table 3
Inhibition of basal [35S]GTP S binding at opioid receptors by compounds 4–10a
c
Compd
l
d
j
NOP
EC50
8.9 0.2 nM
Emax
EC50
Emax
EC50
Emax
EC50
Emax
4
5
6
7
8
33 2.4
0
0
0
46 1.6
0
0
>10
>10
l
l
M
M
0
0
2.4 0.4 nM
20 1.2
0
0
0
60 2.6
0
0
135 31 nM
20 11 nM
94 33 nM
3.6 1.5 nM
15 2.6 nM
0.1 0.4 nM
88 19 nM
138 4.8
104 4.4
92 7.4
173 6.3
92 2.9
87 6.9
320 6.8
>10
>10
>10
lM
lM
lM
>10
>10
>10
l
l
l
M
M
M
66 1.8 nM
>10
0.3 0.1 nM
35 4.8
0
28 9.1
0
0
l
M
4.0 0.1
l
M
0.4 1.2 nM
9
10
>10
>10
l
l
M
M
>10
>10
l
l
M
M
>10
>10
l
l
M
M
a
Compounds 4–10 were salts as described in the Methods. Emax values are expressed as mean SEM percentage of basal [35S]GTP
cS binding stimulation.
Table 4
cally stable in the presence of liver preparations from all three
species examined (i.e., rat, mouse, human) with the exception that
6 and 8 possessed similar metabolic stability as 1b in the presence
of rat liver microsomes (Table 5). Compounds 4 and 5 remained
unchanged in the mouse and human liver microsomes for the
length of the experiment. Compounds 6–9 were also very stable
in the mouse and human liver microsomes with a half life greater
than 112 min. Similarly, 4–9 were stable in mouse liver micro-
somes and compounds that were metabolically stable in the pres-
ence of mouse or human liver microsomal preparations did not
afford evidence of significant amounts of metabolite formation
based on inspection of the HPLC profiles (data not shown). In the
presence of rat liver microsomes, overall, the compounds were
somewhat less metabolically stable, but the half life values ob-
served did not preclude evaluation of the compounds in vivo.
The lack of metabolic instability, however, may have been the re-
sult of inhibition of CYP-dependent metabolism. To examine this
point more carefully, the effect of 4–9 on inhibition of selected
CYPs was examined.
Inhibition of agonist-stimulated [35S]GTP
pounds 4–8a compared to 1a, 17 and 18
c
S binding at opioid receptors by com-
Compd
Ki
l
d
j
NOP
>10
4.2 0.3 nM
4
5
6
7
8
1a
17
18
6.2 1.9 nM
0.1 0.02 nM
15 1.4 pM
637 10 pM
0.3 0.2 pM
>10
>10
lM
>10
>10
>10
>10
lM
lM
lM
lM
>10
>10
>10
>10
lM
lM
lM
lM
>10
>10
>10
>10
—
lM
lM
lM
lM
l
l
M
M
3.6 0.2 nM
—
—
66.8 12.6 nM
0.3 0.1 nM
—
42 4.0 pM
—
4.8 2.3 pM
—
a
Compounds 4–8 were salts as described in the Methods. Values are expressed
M), 14 (200 nM, 16 (2 M) and 15 (NOP;
S binding stimulation was performed at -, d-, -opioid and
nociceptin (NOP) receptors, respectively.
as mean ( SEM) Ki for inhibition of 11 (1
M) basal [35S]GTP
l
l
1
l
c
l
j
a few conclusions could be reached. In general, electron withdraw-
ing para-monosubstituted or meta, para-disubstituted aromatic
groups showed the greatest potency and efficacy for the
(Table 2). Thus, compound 10 (the 4-chloro-substituted aromatic
amide) showed significant affinity for the -receptor and had
l-receptor
5. CYP Inhibition
l
It is known that cyclopropyl methyl-containing amines can in-
hibit CYP.21,22 To understand the metabolic stability data described
above and to examine the possible extent and selectivity of CYP
inhibition, selected compounds (i.e., 4–9) were examined along
with 1b for their ability to inhibit selective functional activities
of human CYP enzymes. The observed percent inhibition for selec-
tive functional inhibition of CYP-3A4, -2B6, -2C9, -2C19 and -2D6
were reported in Table 6. The enzyme assays were done using stan-
dard conditions as previously described.23 Compounds 4–9 were
weaker inhibitors than 1b for the CYPs studied except in the case
of CYP2C19 that appeared to be more sensitive than 1b to inhibi-
tion by 5, 8, and 9. In general, the enzymes mainly involved in inhi-
bition by 4–9 were CYP3A4 and 2C19. In addition, compound 6
inhibited CYP2B6 with greater potency than 1b. Replacement of
the C-6 exo methylene group of 1b with an aryl amide group in this
EC50 values in the low nM range (Tables 1 and 2). Electron-rich
aryl-substituted compounds 4 and 8 showed no detectable stimu-
lation of [35S]GTP
c
S binding. Compounds 4 and 6 possessed the
greatest potency against the d receptor but aside from compound
9, the compounds tested did not markedly stimulate [35S]GTP
cS
binding. With the exception of the electron-rich aryl-substituted
compounds 4 and 8, all of the compounds examined had relatively
good potency for the
j-receptor. The efficacy of 4–10 for the
j
receptor largely paralleled that observed for the
l
receptor. No
detectable potency for the NOP receptor was observed for com-
pounds 4–10 (Table 2). In summary, the opioid receptors appear
to favor binding of compounds with highly electron-deficient and
lipophilic substituents at the meta and para position of C-6 substi-
tuted aromatic amides of naltrexamine. The electronic effect of the
aromatic substituent on the in vivo ED50 value was more pro-
nounced (see in vivo analysis, below).
Table 5
Metabolic stability of 4–9a in the presence of liver preparations
4. Metabolic stability
Compound
Half Life (mins)
Mouse
Rat
Human
As a prelude to studying the test compounds in vivo, TLC- and
HPLC-based analytical methods and biochemical assays were used
to assess the metabolic stability of selected compounds in the pres-
ence of rat, mouse and human liver preparations and the appropri-
ate NADPH generating system. These studies were done to
ascertain the stability of the compounds toward oxidative metab-
olism in advance of more detailed studies with highly purified hu-
man CYPs and FMO3 as well as to determine if the compounds
possessed sufficient metabolic stability for in vivo studies. Com-
pared to 1b, the candidate compounds 4–9 were quite metaboli-
1bb
4
5
6
7
100
373
379
97
164
94
20
20
NCc
NCc
273
480
NCc
NCc
NCc
NCc
NCc
112
555
301
8
9
135
a
Compounds 4–9 were salts as described in the Methods.
Data taken from Ref. 18.
b
c
NC, no change.

S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
6675
Table 6
iv (100 lg/kg) route of administration. After oral administration,
Percent inhibition of CYP3A4, CYP2B6, CYP2C9, CYP2C19 and CYP2D6 by selected
naltrexamides
the Tmax was 57 min and the apparent T1/2 was 2.5 h. After iv
administration, the Tmax was 22 min and the T1/2 was 45 min. A
separate group of three male Wistar rats was administered the
oxalate salt of radiolabeled 6 via the oral route of administration
and killed after 1.5 h. Brain tissue and blood was immediately pro-
cured and chilled on ice and prepared for analysis as described in
the Methods section. The amount of radiolabeled 6 oxalate present
in each animal at 1.5 h was determined by examining an aliquot of
brain homogenate and plasma by scintillation counting. The
Compda
Percent inhibitionb
CYP2C9
CYP3A4
CYP2B6
CYP2C19
CYP2D6
1b
4
5
6
7
60
36
29
36
41
13
11
5
6
9
18
6
9
35
12
9
8
7
17
7
31
NDc
12
52
19
17
49
39
12
ND
ND
10
8
9
5
7
amount of radiolabeled
6 in brain tissue and plasma was
5
6.5 0.8 ng/gm and 2.8 0.3 ng/mL, respectively. The brain tis-
sue/plasma ratio of 2.3 at the time of measurement suggested that
adequate brain concentrations of 6 was present to proceed with
in vivo alcohol self-administration cessation studies.
a
Compounds 4–9 were salts as described in the Methods.
Percent inhibition in the presence of 10 lM test compound. The test compound
b
was preincubated for 2–5 min with the enzyme and cofactor and then the appro-
priate substrate was added and the rate of product was monitored and compared
with the complete system without the test compound present. Values are the
average of 2–3 determinations. The range of the values never exceeded 10–15%.
7. In vivo alcohol self-administration studies
c
ND, no detectable inhibition was observed at the concentration of the test
compound examined.
In vivo studies were intended to test the effects of compounds
4–10 on baseline ethanol (EtOH) intake in rats trained to self-
administer a 10% (w/v) ethanol solution, utilizing an operant tech-
nique model. This model is commonly used to examine the effects
of novel compounds on reinforcing effects of ethanol.25–30 Control
groups consisting of rats trained to orally self-administer a 0.025%
saccharin (SACC) solution were used to examine non-specific ef-
fects of the experimental compounds. 1b hydrochloride was used
as a positive control. Initially, dose range studies were conducted
and if compounds appeared biologically active, more detailed stud-
ies were conducted. Preliminary determinations, showed that 4–8
and 10 possessed ED50 values of 0.25, 0.019, 0.042, 0.038, 0.05 and
0.5 mg/kg, respectively. Because compound 10 showed inhibition
of alcohol self-administration with an ED50 of approximately
0.5 mg/kg and was considerably less potent than the other com-
pounds examined, it was not studied further. Additionally, after
sc administration of 0.025 mg/kg of 9, a potent decrease in alcohol
consumption was observed (i.e., 77%), but 9 also caused profound
analgesia and consequently further studies were not pursued with
this compound. Compounds 5–8 were then administered sc in a
separate drug-naive cohort of rats using a within-subjects Latin
Square dose design. Results from testing compounds 5–8 at doses
ranging from 0.00625 to 0.05 mg/kg showed significant effects in
the alcohol self-administration model (Table 7). For 1b [F = 13.1,
P <0.0001], 5 [F = 5.3, P <0.006], and 6 [F = 7.3, P <0.001], treatment
with opioid 30 min prior to testing had an overall effect on operant
self-administration of 10% ethanol. Compared with vehicle, post
hoc analysis of 1b, 5 and 6 showed that doses of 0.0125, 0.025
and 0.05 mg/kg significantly inhibited operant self-administration
of 10% ethanol. For compounds 7 [F = 5.7, P <0.004] and 8 [F = 4.9, P
<0.008], treatment had an overall effect on operant self-adminis-
tration of 10% ethanol. Compared with vehicle, post hoc analysis
series attenuated the inhibitory potency toward CYP. This suggests
a significant contribution of the C-6 moiety in the interaction of 1b
with CYP and for the C-6 substituted amides examined herein, it
suggests a decreased interaction with CYP. Because CYP3A4 and
CYP2D6 often make significant contributions to opioid metabo-
lism, adverse drug–drug interactions, metabolic bioactivation and
therefore possible side-effects, this new synthetic class of opioid
analog is attractive. Decreased interaction with CYP in part may ex-
plain some of the metabolic stability observed for the compounds
in this and related series.24 On the basis of the data from the
in vitro metabolism studies, we judged the compounds to be suffi-
ciently stable and of low CYP inhibitory potency to study them
in vivo in an animal model of ethanol self-administration. Com-
pound 6 was selected to examine the putative metabolism in
greater detail.
6. In vitro and in vivo studies with compound 6
A radiometric assay and an HPLC assay were set up to examine
the possible metabolism of radiolabeled 6. Compound 6 was cho-
sen as a representative compound to study because its radiosyn-
thesis was very efficient. To confirm the results from the
radiometric studies, we developed an HPLC method to analyze
N-oxygenated and amide hydrolysis of compound 6. In the
presence of rat liver microsomes and after extractive work-up
and HPLC analysis, compound 6 hydrolysis was linearly dependent
on time (0–15 min) and protein concentration (i.e., 0–0.4 mg of
protein). However, the rate of hydrolysis was quite low and ranged
between 0.7 and 0.9 nmol/min/mg of protein. No significant
amount of the N-oxide of 6 was detected. In the presence of human
liver microsomes, compound 6 hydrolysis was linearly dependent
on time (0–30 min) and protein concentration (i.e., 0–0.5 mg of
protein). The rate of hydrolysis in human liver microsomes was
lower than for rat liver microsomes and ranged between 0.2 and
0.5 nmol/min/mg of protein. In contrast to rat liver microsomes,
in the presence of human liver microsomes a significant amount
of 6 N-oxide was formed (i.e., 10–23 pmol/min/mg of protein). For-
mation of the N-oxide of 6 was dependent on pH; the rate doubled
upon going from pH 7.4 to pH 10. Highly purified human FMO3
catalyzed the formation of 6 N-oxide (i.e., 0.9–1.1 nmol/min/mg
of protein) but this rate was quite low. In summary, overall, the
metabolism of 6 was quite low and the data agreed with the rela-
tive metabolic stability described above (Table 5).
Table 7
Effect of 5–8a on the number of ethanol self-administrations in rats
Compd
N
Vehicle
Dose (lg/kg)
6.25
12.5
25
50
1b
5
6
7
8
10
10
10
10
10
39.6 3.2
30.6 3.9
41.1 6.0
33.3 5.5
39.2 5.4
NDc
26.1 3.8b
20.5 3.2b
29.7 3.9b
25.0 2.7
35.6 5.7
22.2 3.4b
12.8 1.6b
25.6 3.5b
24.7 3.7
28.0 3.1
17.1. 1.5b
NDc
26.4 3.6
NDc
19.3 2.6b
13.3 1.5b
19.9 4.1b
NDc
NDc
a
Compounds 5–8 were salts as described in the Methods.
Statistically significant compared to vehicle-treated rats (P <0.05).
b
c
ND, no data collected at this dose based on preliminary screening in a separate
g/kg dose) or total sup-
cohort of rats showing no efficacy at this dose (for 6.25
pression of saccharin controls (for 50 g/kg dose).
l
The oxalate salt of radiolabeled 6 was administered to two
groups of three male Wistar rats via oral gavage (400
lg/kg) and
l

6676
S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
as pain medication candidates.24 It was noted that glucuronides
were less potent than glucose derivatives but certain substituted
Table 8
Effect of 5–8a on the number of saccharin self-administrations in 1 h in rats
aryl amides showed significant selectivity for the
j
-receptor.33
Compd
N
Vehicle
Dose (
l
g/kg)
Recently published17 related studies have shown that 6b-N-hetero-
6.25
12.5
25
50
cyclic substituted naltrexamides were
lopioid receptor selective. In
1b
5
6
7
8
6
6
6
6
6
21.0 10.6
33.3 7.2
31.5 9.1
16.8 7.0
14.0 7.1
NDc
17.5 6.0
24.8 8.0
11.3 3.6
6.0 1.7
7.3 1.9
13.7. 5.6
NDc
a previous study18 we initially examined glucose derivatives as alco-
hol cessation agents but the compounds did not possess significant
23.8 6.8
10.0 3.1b
13.2 2.3
6.2 3.0
NDc
10.5 5.2b
4.8 1.6
7.2 3.5
NDc
potency in vitro against the d- and j-receptors and were not effica-
NDc
16.2 8.8
6.2 2.3
cious in vivo to warrant further investigation. Next, we synthesized
analogs of saccharides where C-6 aryl amide substituents mimicked
the polar functionalities of a saccharide and the compounds retained
significant potency. In good agreement with that described previ-
ously,34 carboxyl-containing C-6 aryl amides possessed poor affinity
a
Compounds 5–8 were salts as described in the Methods.
Statistically significant compared to vehicle-treated rats (P <0.05).
ND, no data collected at this dose based on preliminary screening in separate
g/kg dose) or total sup-
b
c
cohort of rats showing no efficacy at this dose (for 6.25
pression of saccharin controls (for 50 g/kg dose).
l
l
for the l- and j
-opioid receptors.18 On that basis, we made a number
of analogs to explore SAR and in brief, electron-deficient aryl amides
appeared to show the greatest promise in terms of potency and
opioid receptor antagonism. As shown in Table 2, not all of the
showed that only a dose of 0.05 mg/kg significantly inhibited oper-
ant self-administration of 10% ethanol. To test whether the effect of
the compounds were selective for ethanol, the effect of 1b and 5–8
on self-administration of saccharin (0.025%) (Table 8) was exam-
ined. Treatment with 1b [F = 1.0, P = 0.4135] and 8 [F = 0.68.7,
P = 0.578] did not have an overall effect on the operant self-admin-
compounds tested stimulated full [32S]GTP
c
S binding at the
-receptors. Compounds 4 and 8 showed no functional agon-
ism of the -receptor. Addition of a meta-chloro substituent to 10
l-,
d- and
j
l
provided a compound (i.e., 9) that elicited considerable potency to
all three receptors. Thus, introduction of small substituent changes
can result in dramatic biological effects in this series. Surprisingly,
a Hammett relationship was not observed for 4–10 on the basis of
in vitro data but a strong Hammett relationship was observed for
C-6 aryl amide substituents and alcohol cessation ED50 values
in vivo. The finding from in vivo studies that 5–8 were much more
potent as ethanol cessation agents compared with 1a suggests that
the partial agonist activity at all three opioid receptors might be nec-
essary for full ethanol cessation activity. We surmise that the in vivo
istration of saccharin compared with vehicle. Compound
5
[F = 6.06, P = 0.0065], compound 6 [F = 4.52, P = 0.019], and com-
pound 7 [F = 3.7, P = 0.037] did have an overall effect on saccharin
self-administration. In light of these non-specific effects, post-hoc
analysis of a dose of 0.025 mg/kg for 5 and a dose of 0.05 mg/kg
for 6 showed that these doses were the only doses examined that
significantly inhibited self-administration of saccharin, compared
with vehicle. The ED50 value for ethanol self-administration ob-
served for hydrochlorides of 1a and 1b in similar experiments31
was approximately 0.5 and 0.04 mg/kg, respectively. The efficacy
for inhibition of ethanol self-administration by 5–8 compared very
favorably to that of 1b, and in some cases, (i.e., compounds 5, 6 and
7) were apparently more efficacious.
efficacy of 5–8 is dependent on interaction with all three
l-, d-, and
j-opioid receptors because a compound such as 4 that is highly
selectiveforthed-opioidreceptorisnotoptimalforalcoholcessation
in vivo. The profile of opioid receptor binding coupled with the met-
abolic stability of 5–8 contributes to the optimal functional activity
as alcohol self-administration cessation agents in vivo.
Hepatotoxicity of 1a and 1b is a concern because, generally, the
liver of individuals that abuse ethanol is severely compromised.
Because a relationship between metabolic bioactivation and hepa-
totoxicity has been established,35 the design of the compounds
described herein may afford a decrease in the metabolic lability
of 1b analogs by providing compounds with increased bioavailabil-
ity as a result of modification of the C-6 position. As shown in
Tables 4 and 5, we identified compounds that possessed greater
metabolic stability and generally less propensity to interact with
hepatic CYP than 1b. It may be that decreasing the affinity of opioid
derivatives described herein for metabolic enzymes and increasing
the metabolic stability results in a class of compounds with less
potential for hepatotoxicity.
On the basis of the results of the binding and functional assay
and metabolic stability studies shown herein, selected compounds
were evaluated for their effects in an animal model of ethanol self-
administration. One objective was to try to determine which opioid
receptor contributed to ethanol self-administration cessation.
Based on limited data presented above, it appears that individual
8. In vivo SAR
The effect of the C-6 meta- or para-aryl amide substituent of the
opioid on the relative efficacy of compounds 5–9 to inhibit ethanol
self-administration in vivo was examined with regression correla-
tion analysis using various physical organic parameters.32 A plot of
the log ED50 value versus the electronic substituent sigma values
provided a linear correlation with a slope of rho (q) value of 1.55
and an R2 value of 0.925. A plot of the log ED50 value versus the
hydrophobicity substituent pi values provided a less linear correla-
tion with a slope of 1.35 and an R2 value of 0.59. Likewise, an exam-
ination of steric effects with a plot of the log ED50 value versus the
steric substituent values (Fs) provided a non-linear correlation with
a slope of ꢀ0.793 and an R2 value of 0.563. On thebasis of the R2 value
and the goodness of fit the suggestion is that the in vivo ED50 values
for alcohol cessation can be explained to a great extent by the C-6
meta-orpara-arylamideelectronicsubstituenteffectsandtoa much
less extent on the basis of hydrophobicity or steric effects.
antagonism of
anol self-administration cessation. It may be that potent
l
-, d- or
j
-receptors alone is not sufficient for eth-
9. Conclusion
j
-antago-
nism can compensate for weak d-antagonism but what is more
likely is that ethanol self-administration cessation efficacy requires
partial agonism of all three opioid receptors but requires strong j-
opioid antagonism. Such agents (i.e., 5–7) showed efficacious
in vivo activity in an animal model of ethanol self-administration
in rats trained to self-administer a 10% (w/v) ethanol solution. This
is in agreement with a recent study that showed that an opioid
A series of C-6 aryl-substituted amide derivatives of 6-b-naltrex-
amine where the amide was appended with substituted aromatic
groups was prepared by the reaction of 6-b-naltrexamine,
compound 3b, with aryl acid chlorides or carboxylic acids. The com-
pounds were evaluated as ligands for the human l-, d-, j- and noci-
ceptin opioid receptors with the goal of identifying a potent and
selective alcohol self-administration cessation agent. Originally,
metabolically stable saccharide derivatives of opioids were studied
with strong
j-receptor antagonism (i.e., 1b) was more effective
at ethanol self administration cessation than an opioid with broad

S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
6677
receptor antagonism (i.e., 1a).31 Consequently, compounds such as
5–7 and related agents may represent an exciting lead for develop-
ing the next generation of opioid compounds useful in the treat-
ment of alcohol abuse.
0.05 M tetrabutylammonium hydroxide and 80% methanol using
isocratic elution at a flow rate of 1 mL/min. Elemental analysis was
done by NuMega Resonance labs inc. San Diego, CA.
Microsomes from rat, mouse and human liver expressing func-
tional human cytochrome P-450s were purchased from BD Gentest
(Woburn, MA) or made in house and microsomes from baculovirus-
infected cells co-expressing cytochrome P-450s (3A4, 2B6, 2C9,
2C19and2D6),NADPH-cytochromeP-450reductaseandcytochrome
b5 (BACULOSOMESÒ) were purchased from PanVera LLC (Madison,
WI). Human flavin-containing monooxygenase form 3 (FMO3) was
prepared in house according to the method described before.36 The
receptor binding studies were conducted by the National Institute
of Mental Health Psychoactive Drug Screening Program (Chapel Hill,
NC) following a previously described procedure.37
10. Methods
10.1. Chemicals
Hydrochloride salts of 1a and 1b were obtained from Tyco Mallinc-
rodt (St. Louis, MO). Compounds 9 and 10 and their HCl salts were
prepared as previously described.18 NADP+, glucose-6-phosphate, glu-
cose-6-phosphate dehydrogenase, diethylenetriaminepentaacetic acid
(DTPA), MgCl2, 11 [D-Ala,2 N-MePhe,4 Gly5-ol]-enkephalin (DAMGO),
14 [D-Pen2,5]-enkephalin (DPDPE), 15, naltrindole 16 (ꢀ)-trans-
(1S,2S)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] ben-
zene acetamide hydrochloride ((ꢀ) U50,488), 17, norbinaltorphimine
10.3. Naltrexone oxime (2)
18, guanosine 50-[
c-thio]triphosphate tetralithium salt (GTPcS),
Compound 1a (500 mg, 1.46 mmol, 1 equiv), NH2OH-HCl
(147 mg, 2.12 mmol, 1.5 equiv) and NaOAc (294 mg, 3.58 mmol,
2.5 equiv)weredissolvedinabsoluteethanol(8 mL)andthemixture
was heated at reflux for 2.5 h and then concentrated to dryness.
Water (20 mL) was added and the mixture was made basic with
K2CO3 and extracted with CHCl3. The CHCl3 extract was washed with
brine, dried over Na2SO4, filtered and concentrated to give a white
solid (463 mg, 89%): ESI-MS m/z 357 (MH+); 1H NMR (CDCl3) d 6.75
(d, J = 8.2 Hz, 1H), 6.61 (d, J = 8.2 Hz, 1H), 5.0 (s, 1H), 3.15 (m, 2H),
2.65–1.30 (m, 10H), 0.86 (m, 1H), 0.56 (m, 2H), 0.20 (m, 2H).
guanosine 50-diphosphate sodium salt (GDP), 2-hydroxy-ethylpiper-
azine-N-2-ethane sulfonic acid (HEPES), DL-dithiothreitol, tricine, eth-
ylenediaminetetraacetic acid (EDTA), ethanol and saponin were all
obtained from Sigma–Aldrich Chemical Company (St. Louis, MO) and
were used as received. All of the solvents and buffers were obtained
in the highest grade commercially available from VWR (San Diego,
CA). [35S]-Guanosine 50-( -thio)triphosphate ([35S]-GTP
c cS) (250 lCi;
9.25 MBq) was supplied from Perkin–Elmer (Boston, USA). [14C]Bromo-
benzoic acid was purchased from American Radiolabeled Chemicals,
Inc., (St. Louis, MO). Sodium chloride was purchased from Fisher Scien-
TM
tific (Fairlawn, USA). Zeocin 100 mg/mL and Geneticin 50 mg/mL were
TM
10.4.
a6–Naltrexamine (3a) and b6–naltrexamine (3b)
both purchased from Invitrogen (Carlsbad, CA). Wheatgerm Agglutinin
SPA Beads were purchased from Amersham Biosciences (Little Chalfont,
England), Protein Assay Reagent was purchased from BIORAD (Hercu-
les, USA) and the mammalian protease inhibitor tablet was purchased
from Roche Diagnostics, (Indianapolis, IN).
Compound 2 (5.83 g, 16.3 mmol, 1 equiv) was dissolved in THF
(40 mL) and transferred by cannula over 10 min to a solution of
BH3.THF (300 mL, 300 mmol, 1 M solution in THF, 18 equiv) held
at 10 °C. A white precipitate formed and then slowly dissolved as
the reaction was heated at reflux for 48 h. The solution was cooled
to room temperature and water (10 mL) and 1 N KOH (200 mL)
was added slowly. The solution was then reheated at reflux for
2 h. The pH was decreased to 2.5 with 10% HCl (225 mL) and the
solution was heated at reflux for an additional 2 h. The THF was re-
moved under vacuum and the aqueous solution was made basic
(pH 8–9) with K2CO3. The mixture was extracted with CHCl3
(4 ꢂ 150 mL) and the extract was dried over Na2SO4, filtered and
concentrated. The resulting oil was purified by chromatography
on SiO2 (26 ꢂ 60 cm, elution with CH3CN/MeOH/NH4OH, 25/5/1,
v/v/v) providing 3b (b-diastereomer) (2.14 g, 38%) as a white-yel-
low solid: Rf = 0.2; 1H NMR (CDCl3 with two drops of CD3OD) d
6.61 (d, J = 8.1 Hz, 1H), 6.49 (d, J = 8.1 Hz, 1H), 4.17 (d, J = 7.5 Hz,
1H), 3.39–0.45 (20 H); MS m/z 343 (MH+). An additional 0.64 g
10.2. General procedures
Synthetic chemical reactions were run under a positive pressure
of nitrogen with magnetic stirring at ambient temperature using
oven-dried glassware unless otherwise indicated. Air- and mois-
ture-sensitive liquids were transferred via syringe through rubber
septa. Silica gel (230–400 mesh) was used for column chromatogra-
phy. DMF was dried by filtration through a column of neutral alu-
mina and stored over activated 4 Å molecular sieves under
nitrogenprior to use. All other solventsand reagents wereused asre-
ceived. 1H NMR and 13C NMR were recorded at 300.0 and 75.4 MHz,
respectively, on a Varian Mercury 300 instrument. Chemical shifts
were reported in ppm (d) relative to CDCl3 at 7.26 ppm and
77 ppm, respectively. NMR spectra were recorded in CDCl3 unless
stated otherwise. Melting points were reported uncorrected. High
resolution mass spectra were obtained with a VG 7070 spectrometer
with an Opus V3.1 and DEC 3000 Alpha Station data system at the
University of California, Riverside or a Waters LCT Premier instru-
ment operating in the ESI mode at the University of California, Irvine.
Analytical purities were determined by straight and reversed-phase
HPLC using a Hitachi D2500 Hitachi Chromato-integrator, an L-6000
Hitachi pump and an L-4200 UV–vis Hitachi detector (285 nM).
(12%) of material consisting of a mixture of
was also isolated. Repeated chromatography gave an analytically
pure sample of the
-diastereomer, compound 3a: Rf = 0.16; 1H
a- and b-diastereomers
a
NMR d 6.65 (d, J = 8.1 Hz, 1H), 6.46 (d, J = 8.1 Hz, 1H), 4.50 (d,
J = 3.0 Hz, 1H), 3.34 (dt, J = 3.9, 12.6 Hz, 1H), 3.04 (t, J = 6.6 Hz,
1H), 2.95 (s, 1H), 2.63–0.29 (17H); ESI-MS m/z 343 (MH+).
10.5. General procedure for amidation of 6-b-naltrexamine
with an acid chloride
When an AXXI-chrom normal phase column (5
l
m i.d. ꢂ 4.6 mm
6-b-Naltrexamine (104 mg, 0.3 mmol, 1 equiv) was dissolved in
CH2Cl2 (4 mL) and NEt3 (0.13 mL, 0.93 mmol, 3.1 equiv) and a
substituted benzoyl chloride (0.73 mmol, 2.4 equiv) were added.
The solution was stirred for 2 h at room temperature and concen-
trated to dryness. The residue was filtered through a column of
SiO2 (CH2Cl2/MeOH, 20/1, v/v). The resulting solid was dissolved
in anhydrous methanol (3 mL) and K2CO3 (300 mg, 2.2 mmol,
o.d. ꢂ 250 mm) was used (condition A), the mobile phase was
MeOH/2-propanol/HClO4 (55:45:0.01, v:v) at a flow rate of 1 mL/
min. When a reversed-phase system was used (condition B) HPLC
was done with an L-7100 Hitachi pump, an L-7400 UV–vis Hitachi
detector (285 nM), an L-7200 Hitachi autosampler and a D-7000 Hit-
achi chromato-integrator employing a Supleco reversed-phase col-
umn (5
lm ꢂ 4.6 mm ꢂ 250 mm). The mobile phase was 20%

6678
S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
7.3 equiv) was added. The mixture was stirred at room tempera-
ture for 12 h, concentrated and purified by SiO2 chromatography.
C30H31F3N2O8+2H2O: C, 56.25; H, 5.51; N, 4.37. Found: C, 56.79;
H, 5.93; N, 4.38. HPLC (condition A) tR = 4.80 min (98%).
10.6. General procedure for the amidation of 6-b-naltrexamine
10.6.5. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6b-
with a carboxylic acid
[(4’-bromo)benzamido]morphinan (6)
Compund 6 was synthesized according to the general procedure
described above; 6-b-naltrexamine (70 mg, 0.2 mmol, 1 equiv), p-
bromobenzoic acid (62 mg, 0.31 mmol, 1.5 equiv), BOP (137 mg,
0.31 mmol, 1.5 equiv) and N,N-diisopropylethylamine (0.11 mL,
0.61 mmol, 3 equiv) were combined in dichloromethane (2 mL)
followed by basic hydrolysis with K2CO3 to give the title compound
as a white foam (101 mg, 94%). Rf = 0.02 (CH2Cl2/MeOH, 20/1, v/v);
ESI/MS m/z = 525 (MH+); 1H NMR d 7.71 (d, J = 8.1 Hz, 2H), 7.51 (d,
J = 8.1 Hz, 2H), 6.73 (d, J = 7.8 Hz,, 2H), 6.52 (d, J = 7.8 Hz, 1H), 4.56
(d, J = 6.0 Hz, 1H), 4.16–4.13 (m, 1H), 3.12–1.46 (m, 11H), 0.52 (m,
2H), 0.12 (m, 2H); 13C NMR d 166.1, 143.2, 139.9, 133.4, 132.5,
131.7, 130.5, 129.1, 128.2, 125.4, 124.1, 121.8, 119.4, 118.1, 92.6,
70.2, 62.4, 61.9, 59.3, 47.1, 37.6, 36.8, 36.7, 35.9, 9.3, 3.9; HRMS
calcd for C27H30BrN2O4 525.1389, found 525.1382.
6-b-Naltrexamine (100 mg, 0.29 mmol, 1 equiv), the substituted
benzoic acid (0.58 mmol, 2 equiv) and BOP (258 mg, 0.58 mmol,
2 equiv) were dissolved in CH2Cl2 (3 mL). To this solution, Pr2EtN
(0.15 mL, 0.88 mmol, 3 equiv) was added and the mixture was stir-
red at room temperature for 2 h. The solution was concentrated
and filtered through a short column of SiO2 (eluted with EtOAc)
providing a white material. This product was dissolved in MeOH
(3 mL) and K2CO3 (300 mg, 2.2 mmol, 7.5 equiv) was then added.
The mixture was stirred at room temperature for 3 h and concen-
trated to dryness. The residue was purified by SiO2 chromatogra-
phy (CH2Cl2/MeOH, 20/1, v/v) to provide the target compound.
10.6.1. 17-Cyclopropylmethyl-3, 14b-dihydroxy-4, 5
a-epoxy-6
b-[(40-methyl)benzamido]morphinan (4)
Compound 4 was synthesized according to the general proce-
dure described above; 6-b-naltrexamine (100 mg, 0.29 mmol,
1 equiv), p-toluoyl chloride (0.09 mL, 0.7 mmol, 2.4 equiv) and tri-
ethylamine (0.13 mL, 0.91 mmol, 3.1 equiv) were combined in
dichloromethane followed by basic hydrolysis with K2CO3 to give
the title compound as a white solid (107 mg, 79%). Rf = 0.04
(CHCl3/MeOH, 20/1, v/v); mp = 207.6 °C; ESI/MS m/z = 461 (MH+);
1H NMR (CDCl3/CD3OD, 9/1, v/v) d 7.68 (d, J = 8.1 Hz, 2H), 7.23 (d,
J = 8.1 Hz, 2H), 6.67 (d, J = 8.1 Hz,, 2H), 6.51 (d, J = 8.1 Hz, 1H),
4.40 (d, J = 6.6 Hz, 1H), 4.15–4.05 (m, 1H), 3.09–2.96 (m, 2H),
2.60 (m, 2H), 2.34 (s, 3H), 2.12–1.40 (m, 6H) 0.50 (m, 2H), 0.09
(m, 2H); 13C NMR (CDCl3/CD3OD, 9/1, v/v) d 168.1, 142.8, 142.1,
139.9, 131.0, 130.4, 128.9, 128.1, 127.3, 126.7, 123.7, 118.6, 93.0,
70.4, 62.3, 61.9, 59.0, 49.6, 48.7, 47.3, 22.6, 9.3, 3.9, 3.6; HRMS calcd
for C28H33N2O4 461.2440, found 461.2440.
10.6.6. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6b-
[(40-bromo) benzamido]morphinan-oxalate (6-oxalate salt)
The amide product was converted to its oxalate salt using
1 equiv of oxalic acid dihydrate in methanol. Anal. Calcd for
C27H29BrN2O4+2C2H2O4+H2O: C, 51.46; H, 4.88; N, 3.87. Found: C,
51.66; H, 5.16; N, 3.24. HPLC (condition A) tR = 5.15 min (98%).
10.6.7. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6b-
[(40-iodo) benzamido]morphinan (7)
Compound 7 was synthesized according to the general proce-
dure described above; b-naltrexamine (50 mg, 0.20 mmol,
1 equiv), p-iodobenzoic acid (55 mg, 0.22 mmol, 1.1 equiv), BOP
(97 mg, 0.22 mmol, 1.1 equiv) and N,N-diisopropylethylamine
(0.08 mL, 0.44 mmol, 2.2 equiv) were combined in dichlorometh-
ane (2 mL) followed by basic hydrolysis with K2CO3 to give the title
compound as a white foam (83 mg, 97%). Rf = 0.05 (CH2Cl2/MeOH,
10.6.2. 17-Cyclopropylmethyl-3,14b-dihydroxy-4, 5
a
-epoxy-6 b-
20/1, v/v); ESI/MS m/z = 572.9 (MH+); 1H NMR
d 7.68 (d,
[(40-methyl)benzamido]morphinan-oxalate (4-oxalate salt)
Compound 4 (50 mg, 0.11 mmol, 1 equiv) was converted to its
oxalate salt using 1 equiv of oxalic acid in methanol (3 mL). Anal.
Calcd for C30H34N2O8+3H2O: C, 59.59; H, 6.67; N, 4.63. Found: C,
60.35; H, 6.81; N, 4.59. HPLC (Condition A) tR = 5.14 min (99%).
J = 8.1 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 6.72 (d, J = 8.1 Hz, 2H),
6.51 (d, J = 8.1 Hz, 1H), 4.56 (d, J = 6 Hz, 1H), 4.11–4.08 (m, 1H),
3.1–1.44 (m, 11H), 0.51 (m, 2H), 0.11 (m, 2H); 13C NMR d 166.4,
143.2, 140, 137.8, 137.1, 133.9, 130.5, 129.1, 128.2, 124, 119.4,
118.1, 92.3, 70.2, 62.4, 62.0, 59.2, 47.2, 37.6, 36.8, 36.7, 35.9, 9.3,
3.9; HRMS calcd for C27H30IN2O4 573.1250, found 573.1237.
10.6.3. 17-Cyclopropylmethyl-3,14b-dihydroxy-4, 5a-epoxy-6b-
[(40-trimethylfluoro)benzamido]morphinan (5)
10.6.8. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6b-
Compound 5 was synthesized according to the general proce-
dure described above; 6-b-naltrexamine (100 mg, 0.29 mmol,
1 equiv), 4-(trifluoromethyl)benzoyl chloride (0.12 mL, 0.73 mmol,
2.5 equiv) and triethylamine (0.12 mL, 0.88 mmol, 3 equiv) were
combined in dichloromethane followed by basic hydrolysis with
K2CO3 to give the title compound as a white solid (117 mg, 78%
yield). Rf = 0.11 (CHCl3/MeOH, 20/1, v/v); mp = 157.5 °C; ESI/MS
m/z = 515 (MH+); 1H NMR d 7.92 (d, J = 8.4 Hz, 2H), 7.63 (d,
J = 8.4 Hz, 2H), 6.67 (d, J = 7.8 Hz,, 2H), 6.53 (d, J = 7.8 Hz, 1H),
4.60 (d, J = 5.4 Hz, 1H), 4.16–4.13 (m, 1H), 3.15–1.44 (m, 11H),
0.54 (m, 2H), 0.13 (m, 2H); 13C NMR d 166.2, 142.6, 139.4, 137.4,
130.5, 128.1, 127.3, 125.4, 125.1, 124.3 121.8, 119.4, 118.1, 92.6,
70.4, 62.3, 61.9, 59.2, 51.3, 50.8, 47.2, 22.6, 9.3, 3.9; HRMS calcd
for C28H30F3N2O4 515.2158, found 515.2137.
[(40-iodo) benzamido]morphinan-oxalate (7-oxalate salt)
The amide product was converted to its oxalate salt using
1 equiv of oxalic acid dihydrate in methanol. Anal. Calcd for
C29H31IN2O8+2H2O: C, 49.87; H, 5.05; N, 4.01. Found: C, 49.44; H,
5.23; N, 5.74. HPLC (condition A) tR = 5.20 min (98%).
10.6.9. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5
a-epoxy-6b-
[(40-t-butyl) benzamido]morphinan (8)
Compound 8 was synthesized according to the general proce-
dure described above; b-naltrexamine (50 mg, 0.15 mmol,
1 equiv), 4-t-butylbenzoyl chloride (0.14 mL, 0.70 mmol, 4.7 equiv)
and NEt3 (0.07 mL, 0.88 mmol, 5.9 equiv) were combined in dichlo-
romethane (2 mL) followed by basic hydrolysis with K2CO3 to give
the title compound as a white solid (47 mg, 64%). Rf = 0.09 (CH2Cl2/
MeOH, 20/1, v/v); mp = 151.1 °C; ESI/MS m/z = 503 (MH+), 501
(MHꢀ); 1H NMR d 7.75 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H),
6.72 (d, J = 7.8 Hz, 2H), 6.55 (d, J = 7.8 Hz, 1H), 4.53 (d, J = 5.7 Hz,
1H), 4.21–4.18 (m, 1H), 3.14–1.44 (m, 11H), 0.54 (m, 2H), 0.13
(m, 2H); 13C NMR d 167.2, 143.1, 139.5, 137.4, 131.4, 130.6,
127.3, 126.6, 125.5, 124.5, 119.4, 118.0, 93.2, 70.1, 62.4, 62.0,
10.6.4. 17-Cyclopropylmethyl-3,14b-dihydroxy-4, 5a-epoxy-6b-
[(40-trimethylfluoro) benzamido]morphinan-oxalate (5-oxalate
salt)
The amide product was converted to its oxalate salt using
1 equiv of oxalic acid dihydrate in methanol. Anal. Calcd for

S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
6679
59.3, 49.6, 47.2, 34.2, 31.3, 31.0, 9.3, 3.9; HRMS calcd for
10.9. General procedure for [35S]GTP
cS binding assay
C31H39N2O4 503.2910, found 503.2893.
Triplicate assays were done in 96-well plates on ice with each
reaction containing [35S]GTP
S (50 pM), cell membrane (10
protein), GDP (5 M), and SPA beads (0.5 mg) with assay buffer
10.6.10. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5
a
-epoxy-
c
lg
6b-[(40-t-butyl) benzamido]morphinan-oxalate (8-oxalate salt)
The amide product was converted to its oxalate salt using
1 equiv of oxalic acid dihydrate in methanol. Anal. Calcd for
C33H40N2O8+2H2O: C, 63.04; H, 7.05; N, 4.46. Found: C, 62.58; H,
7.23; N, 4.49. HPLC (condition A) tR = 5.32 min (98%).
l
(pH 7.5; 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 10 mg/mL
saponin) and the opioid ligands as before.18 Non-specific binding
was determined in the presence of GTP
c
S (10
l
M). Single drug
dose–response curves (0.1 nM–10
l
M) of [35S]GTP
cS stimulated
binding were done at each opioid receptor with each compound
and compared to the standard opioid agonist compounds 11, 14,
10.6.11. Radiosynthesis of [14C]-17-Cyclopropylmethyl-3,14b-
dihydroxy-4,5
a
-epoxy-6b-[(40-bromo) benzamido]morphinan
16 and 15 for the
Inhibition of opioid agonist-stimulated [35S]-GTP
selective opioid agonists 11 (1
M), 14 (200 nM), (ꢀ) 16 (2
and 15 (1 M) for the -, d-, - and nociceptin receptors, respec-
tively, were done in the presence of varying concentrations
(10 pM–10 M) of each compound. Membranes and GDP were
incubated with the antagonists for 30 min, before the opioid ago-
nists, [35S]GTP
S and SPA beads were added. Assay plates were
l
-, d-,
j
- and nociceptin receptors, respectively.
S binding of
M)
([14C] (6a)
c
In a dry screw cap culture tube was placed 6-b-naltrexamine
l
l
(10 mg, 29.9
1 equiv), 100
BOP (4.4 mg, 9.95
l
l
mol, 3 equiv), 4-bromobenzoic acid (2 mg, 9.9
Ci of carboxyl-labeled [14C]-bromobenzoic acid and
mol, 1 equiv). The mixture was dissolved in
dichloromethane (0.2 mL). The mixture was placed under an atmo-
sphere of argon and i-Pr2EtN (5.1 L, 29.9 mol, 3 equiv) was
l
mol,
l
l
j
l
l
l
l
c
added and stirred at room temperature for 4 h. The mixture was
evaporated to dryness and purified by PTLC (CH2Cl2/MeOH, 8/1,
v/v) Rf = 0.54, to give 4.9 mg, 91% product yield and 70% radioactiv-
ity yield. The product gave an ESI/MS m/z = 527 (MH+) and 529
(MH++14C). Conversion of a portion of the product to an oxalate salt
gave radiolabeled 6a oxalate salt.
shaken for 45 min at 25 °C, and then centrifuged (1500 rpm,
5 min, 25 °C) before [35S]GTP
cS-stimulated binding was assessed
using the NXT TOPCOUNTER.
10.10. Rat and mouse liver microsome and human liver S-9
stability assays
10.6.12. 17-Cyclopropylmethyl-3,14b-dihydroxy-4,5
a-epoxy-6b-
A typical assay mixture contained rat or mouse liver microsomes
[(40-bromo) benzamido]morphinan N-oxide
or human liver S-9 (0.4–0.5 mg of protein), 100
lM potassium phos-
A solution of compound 6 (10 mg, 29.9
romethane (0.3 mL) was combined with meta-chloroperbenzoic
acid (4 mg, 23 mol, 0.8 equiv) and allowed to stir at room temper-
ature for 5 h. The mixture was evaporated to dryness and purified
with PTLC (EtOAc/MeOH, 20/1, v/v) to give 6.1 mg of the desired N-
oxide (60% yield). Rf = 0.06; ESI/MS m/z = 541 (MH+).
l
mol, 1 equiv) in dichlo-
phate buffer (pH 7.4), 40 M test compound, an NADPH-generating
l
system consisting of 0.5 mM NADP+, 0.5 mM glucose-6-phosphate,
5 IU/mL glucose-6-phosphate dehydrogenase, 1 mg/mL DTPA and
7 mM MgCl2 for a final incubation volume of 0.1 mL.23 Incubations
were run for 0, 10, 25, 40 and 60 min in air with shaking at 37 °C in
a water bath and were terminated by the addition of 1 mL CH2Cl2/
2-propanol (3:1, v:v). After centrifugation at 13,000 rpm for 5 min,
the organic fraction was collected and the solvent was removed with
a stream of argon. The residue was reconstituted in methanol
l
10.7. General procedures for cell culture
HEK293 cells stably transfected with FLAG-tagged mouse
l
-
(200
was analyzed by high-performance liquid chromatography with an
Axxi-chrom normal-phase column (4.6 mm ꢂ 250 mm, 5 m) or
with a Supelco reversed-phase HS F5 pentafluorophenyl column
m) as described above. Standard conditions
utilized an isocratic, ternary-solvent system consisting of solvents A
(methanol), B (isopropanol) and C (aqueous 70% HClO4) set at a flow
rate of 1.5 mL/min (straight-phase), or A, D (water) and E (HCO2H)
set at a flow rate of 1.0 mL/min (reversed-phase), k = 254 nm with
retention times (tR) evaluated in min.
lL), centrifuged at 13,000 rpm for 5 min and the supernatant
and -opioid and human nociceptin receptors or hemaglutinin-
j
tagged mouse d-opioid receptors were confirmed with Fluores-
cence Activated Cell Sorter (FACS) analysis and confocal micro-
scopic visualization of cells on coverslips stained with fluorescent
antibodies (SF: M1 & Alexa IgG2b; HA: HA11 & Alexa IgG1). Cells
were cultured under 7% CO2 in Dulbecco’s modified Eagle’s med-
ium containing 10% fetal bovine serum in the presence of 0.4 mg/
l
(4.6 mm ꢂ 250 mm, 5
l
mL Zeocin (for
l-and d-receptor cells), 0.5 mg/mL of Geneticin
(for -receptor cells), or 0.2 mg/mL hygromycin (for NOP-receptor
j
cells) to select for the presence of the transfected plasmid
(pcDNA3.1Zeo and pcDNA3.1) that codes for both the opioid recep-
tor and antibiotic resistance.
10.11. CYP inhibition assays
To measure CYP3A4 activity, testosterone 6-hydroxylation, was
determined as previously described.23 To measure CYP2C9, diclofe-
nac hydroxylase activity was measured.38 For determination of
CYP2B6, CYP2C19 and CYP2D6 activity, isozyme specific Vivid Blue
substrate O-dealkylation was determined via a modified Panvera
Vivid Assay Protocol as previously described.23
10.8. General procedure for membrane preparation
HEK293 cells expressing the
l-, d-, j- and nociceptin (NOP)
receptors were grown in 10 cm dishes. When the cells were nearly
100% confluent, cells were washed twice with ice-cold phosphate
buffered saline and scraped from the dishes with a HME lysis buf-
fer (pH 7.5; 100 mM HEPES, 8 mM MgCl2, 4 mM EDTA, 10 mg/mL
saponin and one mammalian protease inhibitor tablet). The mate-
rial was pelleted (14,000 rpm, 15 min, 4 °C) and resuspended in
HME buffer. Following a rapid freeze (N2)/thaw cycle, the material
was sonicated at 4 °C, repelleted and resuspended in HME buffer
and stored at ꢀ80 °C until used. Protein concentrations of mem-
brane samples were determined by visible spectophotometry
(595 nm) using the BIORAD protein assay reagent and found to
10.12. Metabolism studies of compound 6
As a representative example, metabolic incubations were done
with 6 in the presence of human or rat liver microsomes or highly
purified human FMO3. The incubation mixture contained the
NADPH-generating system as described above, 1 mg/mL DTPA
and 7 mM MgCl2, 0.4 mg of microsomes or 10
FMO3 in a total volume of 0.25 mL combined and mixed at 4 °C.
The incubation was initiated by the addition of 6 (30 M) and
lg of human
be 5.8
lg/lL (l
), 7.3
lg/lL (d), 8.6
lg/lL (j) and 3.5
l
g/l
L (NOP).
l

6680
S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
placed in a 37 °C shaking incubator. At the appropriate time, the
incubation was stopped by the addition of 2 volumes of ice cold
acetonitrile (for the TLC assay) and an aliquot was directly placed
on an LK5DF preabsorbent TLC plate (Whatman, Maidstone, UK)
using an eluant of EtAOc/MeOH/NH4OH, 20/5/0.2, v/v) that sepa-
rated compound 6, 6-N-oxide and bromobenzoic acid with Rf val-
[ cS-stimulated binding expressed as a percentage of the
35S]GTP
basal stimulation:% basal stimulation = (ligand count ꢀ no treat-
ment)/basal stimulation ꢂ 100%, where basal stimulation = no
treatment ꢀ non-specific binding. Calculated EC50 values represent
the concentration of the compound required to produce 50% max-
imal stimulation of [35S]GTP
cS binding. The apparent functional Ki
ues of 0.58, 0.28 and 0.11, respectively. For analysis, 50
l
g of 6, 6-
values were determined using the Cheng and Prusoff equation
(Ki = IC50/(1 + [L]/ED50),40 where IC50 is the concentration that pro-
duces 50% inhibition of opioid selective agonist-stimulated
N-oxide and bromobenzoic acid was used as TLC standards and the
UV–vis bands corresponding to these regions were scraped and
placed in scintillation vials for counting and quantification. For
the HPLC assay, the incubation was stopped by the addition of iso-
propanol/CH2Cl2 (3/1, v/v), mixed thoroughly and the organic layer
was separated by centrifugation. The organic extracts were evapo-
rated to dryness, taken up in MeOH and the products were sepa-
[
35S]GTP
c
S binding, [L] is the concentration of agonist and ED50
is the concentration of agonist that produces 50% maximal stimu-
lation of [35S]GTP
S binding as determined from agonist-response
c
curves. All statistics, regression analyses and determination of
EC50, IC50 and Ki values were done using GraphPad PrismÒ (version
4.02, GraphPad, San Diego, CA). Data from in vitro binding or func-
tional assays were analyzed by non-linear regression using a sig-
moidal curve with variable slope.
rated by HPLC (i.e., Supleco column (4.6 mm ꢂ 25 cm, 5
lm) with
a mobile phase of CH3CN/potassium phosphate buffer, 1/1, v/v,
pH 3) that separated 3b, 6-N-oxide and 6, with retention volumes
of 4.1, 8.4 and 9.2 mL, respectively) at 235 nm. The analytes were
quantified on the basis of HPLC peak height.
10.16. Ethanol self-administration analysis
Data were collected on-line simultaneously from multiple oper-
ant chambers. Results of the operant procedure are reported as
mean cumulative number of bar presses for ethanol or saccharin.
In general, tests for homogeneity of variance were first performed
on the data. If the scores did not violate the assumption of homo-
geneity of variance, appropriate analyses of variance (ANOVA)
were done. Data were analyzed using the StatView statistical pack-
age on a PC-compatible computer. Mixed-design ANOVAs were
used with drug treatments as a within-subjects factor (i.e., re-
peated measures design for drug treatment). A priori analysis
examining individual drug doses to vehicle control dose was con-
ducted using paired t-tests. Significant drug effects were defined
as having p <0.05 compared to vehicle-treated rats.
10.13. In vivo distribution studies with compound 6
Animal studies in male Wistar rats (275–310 g) with jugular
vein and femoral artery catheters were administered radiolabeled
6 oxalate salt (100 lg/kg iv and 400 lg/kg, oral). For plasma anal-
ysis, blood was obtained from the catheters at various time points
up to 8 h and centrifuged at 4 °C. An aliquot of plasma was counted
by scintillation counting. Brain distribution of radiolabeled 6 oxa-
late salt was also investigated in male Wistar rats administered
400 lg/kg by the oral route of administration. After 90 min post
dosing, animals were anesthetized by ip ketamine/xylazine and
blood samples were obtained by cardiac puncture. Brain tissues
were immediately removed, weighed, homogenized with a mortar
and pestle in borate buffer (pH 8.5)/acetonitrile, 1/1, v/v), centri-
fuged and an aliquot was measured by scintillation counting.
Acknowledgements
The authors thank Dr. James MacDougall, Mr. Luke Guo and Mr.
John Buza for their help with some of the synthetic and analytical
work described herein (supported in part by a grant from the David
Copley Foundation). The authors thank Ms. Meike Motika for help
with some of the animal work. Receptor binding data was gener-
ously provided by the National Institute of Mental Health’s Psycho-
active Drug Screening Program, Contract # NO1MH32004 (NIMH
PDSP). This work was financially supported by a grant from the Na-
tional Institutes of Health (Grant AA016029 to MA).
10.14. General procedure for oral ethanol and saccharin
operant self-administration training
Ethanol or saccharin (SACC) self-administration training was
conducted in standard operant cages (Coulbourn Instruments,
PA) located in sound-attenuated, ventilated cubicles. Two 35-ml
syringes dispensed either ethanol/SACC or water through plastic
tubing into two stainless steel drinking cups mounted 4 cm above
the grid floor and centered on the front panel of each chamber.
Each drinking cup held 2 reinforcer deliveries (0.1 ml fluid/rein-
forcer). Two retractable levers were located 4.5 cm to either side
of the drinking cups. Fluid delivery and recording of operant re-
sponses were controlled by a microcomputer.
Briefly, animals were trained to voluntarily self-administer 10%
(w/v) ethanol (n = 10) or saccharin (n = 6) by the oral route using
the saccharin fadeout method39 and were tested for their response
for ethanol or saccharin solution in a two-lever free choice situa-
tion. Once baseline ethanol and saccharin intake were achieved
(i.e., when responding across three consecutive days varied less
than 20% and response rates correspond to pharmacologically rel-
evant blood alcohol levels (BALs)), dose response testing for each
compound commenced. To allow for a complete dissipation of
any carry-over effects, a one week washout period, where rats
were re-baselined during daily 30 min operant sessions, occurred
between testing of each compound.
References and notes
1. Bouza, C.; Angeles, M.; Munoz, A.; Amate, J. M. Addiction 2004, 99, 811.
2. Anton, R. F.; Moak, D. H.; Waid, L. R.; Latham, P. K.; Malcolm, R. J.; Dias, J. K. Am.
J. Psychiatry 1999, 156, 1758.
3. Croop, R. S.; Faulkner, E. B.; Labriola, D. F. Arch. Gen. Psychiatry 1997, 54, 1130.
4. Mason, B. J.; Salvato, F. R.; Williams, L. D.; Ritvo, E. C.; Cutler, R. B. Arch. Gen.
Psychiatry 1999, 56, 719.
5. Tabakoff, B.; Hoffman, P. L. Life Sci. 1983, 32, 197.
6. Oslin, D. W.; Berrettini, W. H.; O’Brien, C. P. Addict. Biol. 2006, 11, 397.
7. Roozen, H. G.; de Waart, R.; van der Windt, D. A.; van den Brink, W.; de Jong, C.
A.; Kerkhof, A. J. Eur. Neuropsychopharmacol. 2006, 16, 311.
8. Benjamin, D.; Grant, E. R.; Pohorecky, L. A. Brain Res. 1993, 621, 137.
9. Stromberg, M. F.; Mackler, S. A.; Volpicelli, J. R.; O’Brien, C. P. Alcohol 2001, 23,
109.
10. Pastor, R.; Aragon, C. M. Neuropsychopharmacology 2006, 31, 1489.
11. Reid, L. D. Am. J. Clin. Nutr. 1985, 42, 1099.
12. Yeomans, M. R.; Gray, R. W. Neurosci. Biobehav. Rev. 2002, 26, 713.
13. Grisel, J. E.; Grahame, N. J.; Mogil, J. S.; Belknap, J. K.; Low, M. J. Soc. Neurosci.
Abstr. 1995, 21, 1699.
14. Gianoulakis, C.; Krishnan, B.; Thavundayil, J. Arch. Gen. Psychiatry 1996, 53, 250.
15. Herz, A. Psychopharmacology (Berl) 1997, 129, 99.
16. Ulm, R. R.; Volpicelli, J. R.; Volpicelli, L. A. J. Clin. Psychiatry 1995, 56, 5.
17. Li, G.; Aschenbach, L. C.; Chen, J.; Cassidy, M. P.; Stevens, D. L.; Gabra, B. H.;
Selley, D. E.; Dewey, W. L.; Westkaemper, R. B.; Zhang, Y. J. Med. Chem. 2009, 52,
1416.
10.15. Data analyses
Non-specific [35S]GTP
the presence of unlabeled GTP
c
S-stimulated binding was determined in
c
S (10 M) with ligand-induced
l

S. Ghirmai et al. / Bioorg. Med. Chem. 17 (2009) 6671–6681
6681
18. Ghirmai, S.; Azar, M. R.; Polgar, W. E.; Berzetei-Gurske, I.; Cashman, J. R. J. Med.
Chem. 2008, 51, 1913.
29. Roberts, A. J.; McDonald, J. S.; Heyser, C. J.; Kieffer, B. L.; Matthes, H. W.; Koob,
G. F.; Gold, L. H. J. Pharmacol. Exp. Ther. 2000, 293, 1002.
19. Mohamed, M. S.; Portoghese, P. S. J. Org. Chem. 1986, 51, 105.
20. Traynor, J. R.; Nahorski, S. R. Mol. Pharmacol. 1995, 47, 848.
21. Tullman, R. H.; Hanzlik, R. P. Drug Metab. Rev. 1984, 15, 1163.
22. Bondon, A.; Macdonald, T. L.; Harris, T. M.; Guengerich, F. P. J. Biol. Chem. 1989,
264, 1988.
23. Denton, T. T.; Zhang, X.; Cashman, J. R. Biochem. Pharmacol. 2004, 67, 751.
24. MacDougall, J. M.; Zhang, X. D.; Polgar, W. E.; Khroyan, T. V.; Toll, L.; Cashman,
J. R. J. Med. Chem. 2004, 47, 5809.
25. Heyser, C. J.; Schulteis, G.; Koob, G. F. Alcohol Clin. Exp. Res. 1997, 21, 784.
26. Heyser, C. J.; Roberts, A. J.; Schulteis, G.; Koob, G. F. Alcohol Clin. Exp. Res. 1999,
23, 1468.
27. Heyser, C. J.; Moc, K.; Koob, G. F. Neuropsychopharmacology 2003, 28, 1463.
28. June, H. L.; Grey, C.; Warren-Reese, C.; Durr, L. F.; Ricks-Cord, A.; Johnson, A.;
McCane, S.; Williams, L. S.; Mason, D.; Cummings, R.; Lawrence, A. Alcohol Clin.
Exp. Res. 1998, 22, 2174.
30. Holter, S. M.; Spanagel, R. Psychopharmacology (Berl) 1999, 145, 360.
31. Walker, B. M.; Koob, G. F. Neuropsychopharmacology 2008, 33, 643.
32. Lowry, T. H.; Richardson, K. S. Mechanism and Theory in Organic Chemistry, 3rd
ed.; Harper and Row: New York, 1987.
33. Cashman, J. R.; MacDougall, J. M. Curr. Top. Med. Chem. 2005, 5, 585.
34. Stevens, W. C., Jr.; Jones, R. M.; Subramanian, G.; Metzger, T. G.; Ferguson, D.
M.; Portoghese, P. S. J. Med. Chem. 2000, 43, 2759.
35. Doss, G. A.; Baillie, T. A. Drug Metab. Rev. 2006, 38, 641.
36. Brunelle, A.; Bi, Y. A.; Lin, J.; Russell, B.; Luy, L.; Berkman, C.; Cashman, J. Drug
Metab. Dispos. 1997, 25, 1001.
37. Roth, B. L.; Laskowski, M. B.; Coscia, C. J. J. Biol. Chem. 1981, 256, 10017.
38. Yano, J. K.; Denton, T. T.; Cerny, M. A.; Zhang, X.; Johnson, E. F.; Cashman, J. R. J.
Med. Chem. 2006, 49, 6987.
39. Rassnick, S.; Pulvirenti, L.; Koob, G. F. Alcohol 1993, 10, 127.
40. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.