Synthesis and evaluation of aryl-naloxamide opiate analgesics targeting truncated exon 11-associated μ opioid receptor (MOR-1) splice variants

Article abstract of DOI:10.1021/jm300305c

3-Iodobenzoylnaltrexamide 1 (IBNtxA) is a potent analgesic acting through a novel receptor target that lack many side-effects of traditional opiates composed, in part, of exon 11-associated truncated six transmembrane domain MOR-1 (6TM/E11) splice variant

Full text of DOI:10.1021/jm300305c

Subscriber access provided by Columbia Univ Libraries
Article
Synthesis and evaluation of aryl-naloxamide opiate analgesics targeting
truncated exon 11-associated mu opioid receptor (MOR-1) splice variants
Susruta Majumdar, Joan Subrath, Valerie Le Rouzic, Lisa Polikar, Maxim Burgman, Kuni Nagakura, Julie
Ocampo, Nathan Haselton, Anna R. Pasternak, Steven Grinnel, Ying-Xian Pan, and Gavril W. Pasternak
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Jun 2012
Downloaded from http://pubs.acs.org on June 27, 2012
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 45
Journal of Medicinal Chemistry
1
2
3
4
Synthesis and evaluation of aryl-naloxamide opiate analgesics targeting
truncated exon 11-associated mu opioid receptor (MOR-1) splice variants
5
6
7
8
9
Susruta Majumdar, Joan Subrath, Valerie Le Rouzic, Lisa Polikar, Maxim Burgman,
Kuni Nagakura, Julie Ocampo, Nathan Haselton, Anna R. Pasternak, Steven Grinnell,
Ying-Xian Pan and Gavril W. Pasternak*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Molecular Pharmacology and Chemistry Program
and
Department of Neurology
Memorial Sloan-Kettering Cancer Center,
New York, NY 10065.
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 45
1
2
ABSTRACT
3
4
5
6
7
8
9
3-Iodobenzoylnaltrexamide 1 (IBNtxA) is a potent analgesic acting through a novel
receptor target that lack many side-effects of traditional opiates composed, in part, of exon 11-
associated truncated six transmembrane domain MOR-1 (6TM/E11) splice variants. To better
understand the SAR of this drug target, a number of 4,5-epoxymorphinan analogs were
synthesized. Results show the importance of a free 3-phenolic group, a phenyl ring at the 6
position, an iodine at the 3’or 4’ position of the phenyl ring and an N-allyl or c-propylmethyl
group to maintain high 6TM/E11 affinity and activity. 3-Iodobenzoylnaloxamide 15 (IBNalA)
with a N-allyl group displayed lower delta opioid receptor affinity than its naltrexamine analog,
was 10-fold more potent an analgesic than morphine, elicited no respiratory depression or
physical dependence and only limited inhibition of gastrointestinal transit. Thus, the aryl-
naloxamide scaffold can generate a potent analgesic acting through the 6TM/E11 sites with
advantageous side-effect profile and greater selectivity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
INTRODUCTION
9
Opiates have been the subject of intense research since the isolation of morphine in the
early 1800’s.1 Despite their efficacy and utility, side-effects greatly limit the usefulness of
virtually all the clinically available opiates, including respiratory depression, constipation and
physical dependence, reward and addiction. Attempts to separate these unwanted actions
from analgesia have largely been unsuccessful. A number of studies exploring the
pharmacology of naloxonazine dissociated morphine analgesia from respiratory depression,2, 3
inhibition of gastrointestinal transit,4, 5 and physical dependence.6 Yet, dissociation of these
actions functionally failed to yield selective analgesics lacking these side-effects. The
identification of delta and kappa1 receptors led to extensive efforts to generate alternative
analgesics acting through different receptors to achieve a better pharmacological profile.
Although highly selective agents were developed, they have yet to fill the clinical need. The
kappa1 receptor drugs that were tested clinically revealed unacceptable psychotomimetic
effects, as well a profound diuresis, while the utility of delta-selective agents have not yet been
fully validated. Thus, there still remains a need for potent analgesics with safer side-effect
profiles.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Clinicians have long appreciated differences among mu opioids, with some patients
responding better to one agent than another.7-9 These clinical observations, along with
preclinical pharmacological and binding studies, led to the initial suggestion of multiple mu
receptors,10 a concept that has been confirmed with the cloning of a vast array of splice
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 45
1
2
3
4
5
6
7
8
variants of the mu opioid receptor (MOR-1) gene Oprm1 in mice, rats and humans.11-20
Included in these variants is a set of truncated six transmembrane domain splice variants
generated by an alternative promoter associated with exon 11 of the OPRM1 gene of mice,16
rats21 and humans.22 Although it was suggested that these variants might be active alone,23, 24
the poor affinity of the expressed variants for opiates and the very high doses of drug necessary
to activate the receptors seems to make this unlikely.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Recently, we reported the synthesis25 and pharmacological characterization26 of a β-
naltrexamine analog, 3’-iodobenzoylnaltrexamide 1 (IBNtxA, fig 1). Pharmacological studies
revealed an analgesic with a potency 10-fold greater than morphine with no observable
respiratory depression, physical dependence, cross tolerance to morphine or rewarding
behavior in the conditioned place preference assay and limited inhibition of gastrointestinal
transit. This compound displayed high affinity for a binding site in brain that was distinct from
traditional mu, delta or kappa1 receptors, most clearly shown by its persistent analgesia and
binding in a triple knockout mouse lacking the traditional mu, delta and kappa1 receptors. The
loss of both its analgesia and its binding site in an exon 11 knockout mouse, further established
a unique mechanism of action and implicated exon 11-associated 6 transmembrane domain
splice variants (6TM/E11). Additional studies suggest that these truncated variants do not act
alone. Rather, 1 appears to label with high affinity a heterodimer of a 6TM/E11 variant with an
additional G-protein coupled receptor, although the partner is still unknown. We now describe
the structure-activity relationships for this new drug target by synthesizing several analogs of 1
with substitutions on the 6, 17 and 3 positions of the 4,5-epoxymorphinan scaffold. All
synthesized molecules were evaluated for their relative binding affinity for cloned mu (MOR-1),
4
ACS Paragon Plus Environment

Page 5 of 45
Journal of Medicinal Chemistry
1
2
kappa1 (KOR-1) and delta (DOR-1) sites in cell lines and 6TM/E11 sites in mouse brain and for
3
4
5
their analgesic activity in vivo with the radiant heat tail flick assay.
6
7
8
9
RESULTS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chemistry: The ketone at the 6-position of naloxone 4 and 3-methoxynaloxone 11 were
transformed to an amine (Opiate-NH2) by reductive amination using sodium cyanoborohydride
(NaBH3CN) and ammonium acetate (NH4OAc) to yield a mixture of beta and alpha isomers.27
The beta and alpha isomers were then purified by column chromatography. In a parallel
synthesis, substituted carboxylic acids 5-7 were converted to their respective N-succinimidyl
esters 8-10 by reacting them with N-hydroxysuccinimide (NHS) in the presence of DCC and THF.
The corresponding activated esters were then reacted with the beta or alpha isomer of the
opiate-NH2 in the presence of diisopropylethylamine (DIEA) and DCM. The corresponding aryl,
alkyl and carbocyclic amido derivatives of opiates were then purified by column
chromatography. Alternatively, the substituted carboxylic acids were directly coupled to the
opiate-NH2 using benzotriazol-1yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) and DIEA in DCM to give 3, 6-disubstituted derivatives. The 3,6-disubstituted opiate
derivatives were then subjected to basic hydrolysis with potassium carbonate to yield 6- alkyl,
aryl and carbocyclic derivatives of naloxamide and 3-methoxynaloxamide (Scheme 1; table 1).
1, 14-15 were synthesized as previously reported.25 The pharmacological characterization of 14
and 15 and all newly synthesized compounds (16-38) is summarized in table 1.
Pharmacology: 1 is a potent analgesic lacking many of the problematic side-effects
associated with traditional opiates. Our objective is to establish the criteria important for both
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 45
1
2
3
4
potency and selectivity for the 6TM/E11 sites by exploring the structure-activity relationship
(SAR).
5
6
7
8
9
The N-substituent is important for defining both affinity and analgesic activity. Receptor
binding studies reveal that an N-methyl substituent dramatically lowers the affinity of the
compound for the 6TM/E11 site, as shown with oxymorphone 2 (Ki >1 µM, compared to the c-
propylmethyl group in naltrexone 3 (Ki 32 nM) or the allyl group in naloxone 4 (Ki 53 nM) (fig 1;
table 1).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The increased affinity of 1 (Ki = 0.16 nM) illustrates the importance of substituents at the
6-position of β-naloxamine. To further explore the role of the 3’-iodophenyl moiety at the 6-
position of the 4,5-epoxymorphinan, we synthesized 14 and 15 which incorporated a 3’-
iodophenyl amido moiety into the oxymorphone and naloxone scaffold, respectively. 14 has a
Ki of 41 nM for 6TM/E11 sites. Although the addition of the 3’-iodophenyl amido moiety
increased the affinity by at least 50-fold, its affinity remained over 100-fold poorer for the
6TM/E11 site than either the naloxone analog (3-Iodobenzoylnaloxamide 15 (IBNalA)), Ki = 0.25
nM, fig 1) or the naltrexone analog (1, Ki 0.16 nM). The 3’-iodophenyl amido moiety also
influenced the pharmacology of the compounds. Oxymorphone 2 is a potent analgesic. Yet,
its analog, 14, showed no analgesia at doses as high as 10 mg/kg, s.c. despite a reasonable
affinity for mu receptors. Conversely, the 3’-iodophenylamido substituent at the 6 position
converted the naltrexone and naloxone analogs into potent analgesics, contrasting markedly
with the antagonist character of naloxone and naltrexone themselves. It is important to note
that 15 retained a similar affinity for the 6TM/E11 binding site and analgesic potency as 1 (fig
2a). Compared to 1, 15 had a 10-fold lower affinity for delta receptors and about 2 fold lower
6
ACS Paragon Plus Environment

Page 7 of 45
Journal of Medicinal Chemistry
1
2
3
4
affinity for mu and kappa receptors. In view of this increased selectivity, we decided to pursue
the naloxone series of analogs.
5
6
7
8
9
The stereochemistry of the 6-position is important. The alpha isomer, 16, showed a 33-
fold decreased binding affinity for the 6TM/E11 site, a 23, 150 and 3 fold lower affinity for mu,
kappa and delta receptors and a 10-fold higher ED50 in analgesic assays (Table 1). Thereafter,
we focused upon β-naloxamine compounds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Moving the position of the iodine on the aryl ring influences activity. Although an iodine
at position 4’, 18, retains a very similar profile compared to the 3’ position, 15, placing it at
position 2’, 17, lowered its binding affinity for the 6TM/E11 sites by 100-fold with
approximately 10-fold lower affinities for mu, kappa and delta receptors and eliminated its
analgesic activity at doses as high as 10 mg/kg.
We next examined a series of halogens at the 3’ position of the aryl ring (19-21, Table 1),
and compared it to hydrogen in 22. We chose the 3’ position based on the higher affinity of the
3’-iodo-analog, 15 when compared to the 4’-iodo-analog, 18. 22 illustrates the importance of
the iodine when compared to a hydrogen. Removal of the iodine at either the 3’ or the 4’-
position lowered the affinity at the 6TM/E11 site by over 20-fold while the affinity across mu,
kappa and delta receptors also decreased by about 20-, 180- and 25-fold, respectively. The
6TM/E11 affinity increased as the size of the halogen increased and electron withdrawing
nature of the halogen atom decreased while no observable trends in affinity were seen with the
traditional mu, kappa and delta receptors. The analgesic activity correlated with the 6TM/E11
site binding, increasing from F to I. The existence of halogen bonding in protein-ligand
complexes has been shown for adenosine kinase inhibitors,28 HIV-RT inhibitors29, 30 and PD5
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 45
1
2
3
4
5
6
7
inhibitors.31 This raises the possibility that the ability of iodine to form weak halogen bonds32
in the protein pocket, rather than the electron-withdrawing nature of the halogen atom, maybe
responsible for its higher affinity for the 6TM/E11 sites.
8
9
To further evaluate the role of electron releasing or electron withdrawing groups on the
aryl ring on 6TM/E11 affinity we synthesized compounds with both electron releasing groups
(CH3, OCH3, OH and NMe2) and electron withdrawing groups (NO2, OCF3 and CF3; 23-30, table
1). No clear trend was seen with either series, with these substituents lowering the affinities for
the 6TM/E11 site and the traditional opioid receptors when compared to 15. However, the
analgesic activity of 24 with a CF3 at the 3’position and 25 with OMe at the 3’position of the aryl
ring stand out from the others with regards to their potent analgesic actions. 24 is 42, 4 and 10
fold more selective for kappa1 sites than 6TM/E11, mu or delta receptors. Similarly, 25 is 9, 27
and 100 fold more mu selective than 6TM/E11, kappa and delta receptors respectively, raising
the question whether the strong analgesic actions of 24 and 25 might be due to traditional
opioid receptors. If true, this would be unusual since the placement of an allyl group on the
nitrogen in the 17-position typically yield antagonists for the traditional receptors.
An aryl ring may be sufficient to instill high affinity for the 6TM/E11 site in view of the
similar affinity of analogs with either electron withdrawing or releasing groups. More complex
aryl rings (31-32, table 1) maintained a modest binding affinity for the 6TM/E11 site. 31, with
its naphthalene ring also shows modest analgesic activity. Despite its similar binding affinity for
the 6TM/E11 site, 32 was not analgesic. Both 31 and 32 have higher affinity for the mu receptor
over 6TM/E11 sites and offer no additional advantage in terms of 6TM/E11 selectivity over the
traditional opioid receptors.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
ACS Paragon Plus Environment

Page 9 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Lipophilicity alone appears to have little role in defining binding affinity for the 6TM/E11
sites or activity. Methyl, hexyl, dodecyl, cyclohexyl and adamantyl groups at the 6 position (33-
37, table 1) all showed poor binding affinity for the 6TM/E11 sites and moderate to poor
affinities across the traditional opioid receptors and no analgesic activity at doses as high as 10
mg/kg, s.c. This contrasts markedly with the aryl substituent’s. 22, with a simple phenyl ring (Ki
5nM; ED50 5 mg/kg, s.c.) is far superior to any of the above analogs, raising the question of a pi-
pi stacking interaction between the aromatic ring at the 6-position and the 6TM/E11 protein
binding site.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Finally, we examined the need for a free 3-OH group. It has long been known that
traditional mu opiates of the 4,5-epoxymorphinan series require a free hydroxyl group at this
position. Although codeine and oxycodone, which have a 3-methoxy group, are effective
analgesics, many believe that the activity results from demethylation to morphine and
oxymorphone, respectively. Like other opiates, the presence of a methyl group in 38 eliminates
binding across the 6TM/E11 sites and the traditional opioid receptors and analgesia.
To see if these compounds are acting in a manner similar to 1, we extended the
pharmacological assessment of 15, the naloxone analog. The N-allyl analog had an advantage
in that its affinity for delta receptors is approximately 10-fold lower than the CPM compound, 1.
Despite its potent analgesic activity, 15 displayed no respiratory depression at doses
approximately 4-fold greater than its analgesic activity, showed no evidence of physical
dependence after repeated administration based upon the absence of withdrawal signs
following the administration of naloxone or levallorphan and far less inhibition of GI transit
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 45
1
2
than morphine (Fig. 2). Thus, the overall pharmacology of 15 mimics that of 1, consistent with
3
4
5
activity through the 6TM/E11 target.
6
7
8
9
DISCUSSION
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Our earlier work with 1 illustrated the ability to develop opioids capable of acting
through a novel target, which is comprised, in part, by exon 11-associated splice variants of
MOR-1.26 The overall profile of this class of analgesic was quite unusual in that the agents had
no respiratory depression, physical dependence, or cross tolerance to morphine and only
limited effects on gastrointestinal transit. Furthermore, 1 retained full analgesic activity in a
MOR-1/DOR-1/KOR-1 triple knockout mouse lacking traditional mu, delta and kappa1 receptors
while it was without activity in an E11 knockout mouse with a disruption of the exon 11-
associated variants.26 Our current study explores a series of analogs. The pharmacological
profile of the analogous naloxone derivative, 15, was quite similar. Again, it was a potent
analgesic with no evidence of respiratory depression or physical dependence and limited
inhibition of gastrointestinal transit.
The structure-activity relationships of these analogs are interesting. The N-substituent is
important, as the analgesic activity is limited to the N-allyl, 15 or N-methylcyclopropyl 6β-
naltrexamine derivatives, 1. No analgesia could be detected for the N-methyl derivative, 14.
This was opposite to what might be expected since analgesia is seen only with oxymorphone
and not naloxone and naltrexone. This might reflect a reversal of the effect of the N-substituent
on the traditional agonist/antagonist character of the molecules, but the N-methyl group also
was associated with a marked decrease in affinity for the 6TM/E11 sites. It is interesting to
10
ACS Paragon Plus Environment

Page 11 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
note that an aryl ring at the 6-position enhances affinity for the 6TM/E11 sites. All analogs
lacking the aryl ring had poorer affinity with Ki >10 nM. Placement of an iodine on the aryl ring
at the 3’ or 4’ position further enhances the affinity for the 6TM/E11 sites and as well as
analgesic activity.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Structurally analogous aryl naltrexamides have been described by Ghirmai et al., as
alcohol cessation agents33, 34 and by Zhang and co- workers as mu antagonists.35, 36 However,
none of these molecules were noted to have analgesic activity. Similarly, 6-arylamido
morphines have been reported as M6G analogues by McDougal et al.37 More recently,
Portoghese described a naphthoyl analog of 6β-naltrexamine (NNTA) analogous to the
naloxone version synthesized in our study 31 (Table 1) with analgesic activity and lacking
physical dependence and rewarding behavior in the place preference assay that he ascribed to
activation of a mu/kappa1 opioid receptor dimer.38 As with the 6β-naltrexamine, the 6β-
naloxamine analog was active in the mouse radiant heat tailflick assay. However, it is unclear
whether this compound is acting through the same target as 1 and 15. Compared to 15, the
napthoyl derivative, 31, showed over a 26-fold lower binding affinity for the 6TM/E11 site while
its analgesic activity was only 1.5-fold lower. The loss of NNTA analgesia as reported in their
MOR-1 knockout mouse38 does not help since the disruption of exons 2 and 3 in their mouse
leads to the loss of both the exon 11-associated 6TM variants and the full length MOR-1 ones
and therefore would be expected to abolish the actions of compounds acting through any of
the 6TM or 7 TM MOR-1 variants.
There is, in general, a reasonably good correlation between binding affinity for the
6TM/E11 sites and analgesic potency for most of the compounds, consistent with activity
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 45
1
2
3
4
5
6
7
8
through the 6TM/E11 sites. However, there are some exceptions. For example, the 3-OCH3
analog ,25, shows an analgesic potency over 3-fold greater than 15, despite an affinity for the
6TM/E11 site that is 10-fold poorer. A similar dissociation of analgesic activity and binding
affinity for the 6TM/E11 site is seen with 24 (4-CF3) and 30 (4-OCF3), raising questions about
their targets as well. Thus, interpretation of the analgesic activity and correlating it with
binding affinities must be done cautiously since a wide range of targets can all elicit the same
pharmacological response. Finally, both 1 and 15 show a remarkable side-effect profile,
confirming that it is possible to develop superior analgesics. However, it is premature to
extrapolate these observations to the other compounds that may, or may not, have a similar
side-effect profiles.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
A series of C-6-substituted 4,5-epoxymorphinans (i.e. aryl, alkyl, and carbocycle
naloxamides) were synthesized as analogs of 1 to probe the structure-activity relationships of
both affinity and selectivity of ligands for the 6TM/E11 sites. These compounds were prepared
by the reaction of 6β-naloxamine with alkyl, aryl and carbocyclic carboxylic acids or their
corresponding NHS activated esters. This is the first report of the pharmacological properties of
aryl naloxamides and their ability to demonstrate potent agonist activity, although structurally
analogous aryl naltrexamides have been reported before. To our knowledge, this is the first
study demonstrating aryl-naloxamide analgesics. Although many of the derivatives displayed
high affinity for other opiate receptor targets, particularly mu and kappa1, there were clear
predictors for the 6TM/E11 target. The most important was the aryl ring at the C-6 position of
12
ACS Paragon Plus Environment

Page 13 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
the 4,5-epoxymorphinan scaffold. All derivatives lacking a phenyl ring were completely inactive
in analgesia assays and had lower affinity in 6TM/E11 binding assays. The presence of an iodine
at either the 3’ or 4’position, but not the 2’ position, of the phenyl ring also increased the
6TM/E11 affinity and activity. Replacing the N-CPMof 1 with an N-allyl substituent, 15,
produced an analog with similar affinity for the 6TM/E11 site and a similar pharmacological
profile, as well as increased selectivity over the delta opioid receptor. The superior
pharmacological profile of 15 compared to traditional mu analgesics suggested that the
6TM/E11 site may provide a useful target in developing useful analgesics.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 45
1
2
3
4
5
6
7
8
9
EXPERIMENTAL PROCEDURES
Materials :
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Naltrexone, oxymorphone, levallorphan, morphine, U50,488H, [D-Pen2,D-
Pen5]enkephalin, [D-Ala2,MePhe4,Gly(ol)5]enkephalin and naloxone were a generous gift from
the Research Technology Branch of the National Institute of Drug Abuse (Rockville, MD) and
other chemicals were purchased from Fischer Scientific (Pittsburgh, PA, USA) and Sigma (St
Louis, MO, USA). Na125I was bought from Perkin-Elmer (Waltham, MA, USA).
Synthesis of compounds (15-38)
General Procedures: All reactions were carried out under positive nitrogen atmosphere
with a magnetic stirrer at ambient temperatures using oven-dried glassware. 1H-NMR were
taken on a 500 MHz Bruker Advance III instrument using CDCl3 as solvent and calibrated using
an internal reference. Chemical shifts are expressed in parts per million (ppm) and coupling
constants (J) in hertz . High resolution mass spectra were obtained with a Waters LCT premiere
spectrometer while a Waters SQD mass spectrometer instrument operating in the ESI mode
was also used. A reversed-phase HPLC using a Perkin Elmer LC pump series 200 and a 785A
uv/vis detector (214 nM) was used. A Varian microsorb MV 100-5 reversed-phase column (5 μm
× 4.6 mm × 250 mm) with the mobile phases being 0.1% TFA in water and 0.1% TFA in ACN with
a gradient elution at a flow rate of 1 mL/min was used. Silica gel (230-400 mesh) was used in
column chromatography. C18 sep paks were purchased from Waters (Milford, MA).
Radioiodinated samples were counted on a Wizard 1470 automatic gamma counter from Perkin
14
ACS Paragon Plus Environment

Page 15 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Elmer (Waltham, MA, USA). All chemicals and were either purchased from Fischer Scientific
(Pittsburgh, PA, USA) or Sigma (St Louis, MO). All synthesized compounds were characterized
and purity determined by 1H-NMR, 13C-NMR, HPLC, MS and HRMS. Analytical data confirmed
the purity of the products ≥95%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of β-naloxamine 12 and β-3-methoxynaloxamine 13: Reductive amination of
naloxone 4 and 3-methoxynaloxone 11 was carried out using a literature protocol published by
Jiang et al.27 Typically, 10 g of opiate (30 mmol), was stirred with NH4OAc (22g, 0.3 mol, 10 eqv)
in 40 mL dry methanol for 10 minutes at room temperature. NaBH3CN (1.31 g, 21 mmol, 0.7
eqv) in 5 mL dry methanol was then added to the reaction mixture and contents stirred
overnight. The reaction was quenched by addition of 10 mL 1N NaOH, the solvents were
evaporated on a rotavapor at 40°C. The residue was then extracted with 30 mL DCM three
times; the organic extracts were combined and washed with 25 mL water. The organic extracts
were dried over Na2SO4 and concentrated to a white solid, which was purified by silica gel
column chromatography. The reaction gave a mixture of alpha and beta isomers. The respective
isomers were isolated by column chromatography using 87:10:3 of EtOAc: MeOH: NH4OH as the
eluent. The beta isomer had a higher Rf than the alpha isomer on a TLC plate and eluted first
when the mixture was subjected to column chromatography. Yields for beta isomer were about
2.5-3 g (25-30%). The stereochemistry of the diastereomers at the C-6 position were
determined on the basis of the size of the NMR coupling constant, J 5, 6. For the α-isomer, the
chemical shift of the C-5 H is δ 4.58 with 3.5 Hz as the J 5,6 coupling constant, compared to the
literature value 27 of δ 4.55 and 4.0 Hz; for the β-isomer, the chemical shift of the C-5 H is δ 4.24
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 45
1
2
3
4
with 7.1 Hz as the J 5,6 coupling constant, comparing to the literature value25, 27 of δ 4.18 and 7.4
Hz.
5
6
7
8
9
Synthesis of N-hydroxysuccinimide (NHS) esters of carboxylic acids 8-10: Substituted
carboxylic acid (5-7, 7.8 mmol), NHS (1g, 8.6 mmol, 1.1 eqv), DCC (1.79 g, 8.6 mmol, 1.1 eqv) in
20 mL dry THF were stirred overnight. The white suspension was filtered and the clear filterate
was evaporated on a rotavapor at 40°C. The white solid seen was purified by column
chromatography using EtOAc/hexanes as eluents. A singlet at δ2.9 integrating to 4 protons in
1H-NMR and corresponding to four protons of succinimide was seen in all NHS esters of
substituted carboxylic acids. Crude yields were about 80-100%. The crude activated ester was
used in the next step without any purification.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of aryl-α-naloxamide, aryl-β-naloxamides and β-3-methoxynaloxamides 15-
38: Coupling of α-naloxamine, β-naloxamine 12 or β-3-methoxynaloxamine 13 (Opiate-NH2) to
the corresponding carboxylic acid was carried out by either reacting the amine to the NHS-
activated carboxylic acid ester or by direct coupling of the amine to the carboxylic acid in
presence of BOP.
Procedure I: Opiate-NH2 (12-13, 200 mg, 0.6 mmol) was reacted with DIEA (116 ul, 0.66
mmol, 1.1 eqv) and NHS esters of substituted carboxylic acids (8-10, 0.66 mmol, 1.1 eqv) in dry
DCM (5 mL) for 2 h. The reaction was diluted to 20 mL with DCM and washed with 5 mL water.
The organic extracts were dried over Na2SO4 and then concentrated to a white solid, which was
purified by silica gel flash column chromatography using a gradient run of 1-5% MeOH:DCM as
eluents.
16
ACS Paragon Plus Environment

Page 17 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Alternate procedure II: Opiate-NH2 (12-13, 200 mg, 0.6 mmol) was reacted with BOP
(271 mg, 1.2 mmol, 2 eqv), DIEA (313 ul, 1.8 mmol, 3 eqv) and substituted carboxylic acid (1.2
mmol, 2 eqv) in dry DCM (5 mL) for 2 h. The reaction mixture poured into a small silica gel
column and eluted with 100 mL EtOAc. The ethyl acetate fraction was evaporated and a white
solid was obtained. The solid obtained was hydrolyzed in K2CO3 and MeOH. Briefly, the
contents, usually a white suspension were stirred with K2CO3 (622 mg, 4.22 mmol, 7 eqv) and
MeOH for 3h.The white suspension seen was filtered and the filterate concentrated to a
yellowish oil or a white solid. The oily residue or white solid obtained was then purified by flash
column chromatography using a gradient run of 1-5% MeOH: DCM as the eluent.
Synthesis of individual embodiments:
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-iodo)benzamido]morphinan (15).
Compound 15 was synthesized according to the general procedure (I) described above using β-
naloxamine, NHS ester of 3-iodobenzoic acid 8 and DIEA in DCM. A white solid was obtained.
Yield: 255 mg (75%); mp 203-207 oC dec; [α]20D -161.2 (c 0.1, CHCl3); 1H-NMR (500 MHz, CDCl3)
δ: 8.16 (s, 1H), 7.8 (d, J=8.9 Hz, 1H), 7.76 (d, J=8.9 Hz, 1H), 7.15-7.11 (m, 1H), 6.69 (d, J=10.6 Hz,
1H), 6.57 (d, J=10.6 Hz, 1H), 5.8 (m, 1H), 5.23-5.16 (m, 2H), 4.57 (d, J=8.85 Hz, 1H), 4.13 (m, 1H),
3.14-1.2 (m, 14H). 13C NMR (150 MHz, CDCl3) δ: 165.4, 142.9, 140.3, 139.2, 136.4, 136.2, 135.2,
130.6, 130.1, 126.1, 124.7, 119.3, 118.1, 117.6, 94.3, 92.9, 70.2, 62.4, 57.8, 50.5, 47.3, 43.6,
31.5, 29.0, 23.2, 22.7 ppm. ESI-MS m/z: 559.1 (MH+). HRMS calcd for C26H28N2O4I (MH+),
559.1094; found, 559.1099. HPLC purity: 96.5%.
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3′-iodo)benzamido]morphinan (16).
Compound 16 was synthesized according to the general procedure (I) described above using α-
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 45
1
2
3
4
5
6
7
8
naloxamine, NHS ester of 3-iodobenzoic acid 8 and DIEA in DCM. A white solid was obtained.
Yield: 248 mg (73%); mp 134-136 oC; [α]20D -225.7 (c 0.1, CHCl3); 1H-NMR (500 MHz, CDCl3) δ:
8.01 (s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 6.70 (d, J =
8.1 Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H ), 6.37 (d, J = 8.2 Hz, 1H), 5.80 (m, 1H), 5.18 (d, J = 18.5 Hz,
1H,), 5.15 (d, J = 10.9 Hz, 1H), 4.74 (m, 2H), 3.50-1.00 (m, 15 H) ppm. 13C NMR (150 MHz, CDCl3)
δ: 165.5, 145.1, 140.3, 137.2, 136.6, 136.0, 135.2, 130.8, 130.1, 126.3, 125.9, 119.4, 118.0,
117.3, 94.2, 90.1, 69.7, 62.3, 58.1, 47.2, 46.7, 42.9, 33.3, 28.9, 23.0, 21.0 ppm. MS (ESI) m/z (%)
559 (MH+). HRMS calcd for C26H28N2O4 I (MH+), 559.1094; found, 559.1107. HPLC purity: 97.5%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′-iodo)benzamido]morphinan (17).
Compound 17 was synthesized according to the general procedure (II) described above using β-
naloxamine, 2-iodobenzoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white
solid was obtained. Yield: 204 mg (60%); mp 208-211 oC dec; [α]20D -180.0 (c 0.1, CHCl3); 1H-
NMR (500 MHz, CDCl3) δ: 7.87 (d, J=8.35 Hz, 1H), 7.42 (d, J=8.35, 1H), 7.38-7.36 (m, 1H), 7.11-
7.08 (m, 1H), 6.75 (d, J=8.35, 1H), 6.6 (d, J=8.35, 1H), 6.41 (m, 1H), 5.78 (m, 1H), 5.14 (m, 2H),
4.51 (d, J=8.35, 1H), 4.17 (m, 1H), 3.49-1.26 (m, 14H). 13C NMR (150 MHz, CDCl3) δ: 169.2,
142.9, 142.2, 139.9, 139.6, 135.2, 131.1, 130.8, 128.3, 128.2, 124.8, 119.3, 118.0, 117.6, 93.2,
92.4, 70.2, 62.4, 57.7, 50.8, 47.5, 43.6, 31.0, 29.5, 23.5, 22.7 ppm. MS(ESI) m/z (%) 559 (MH+).
HRMS calcd for C26H28N2O4 I (MH+), 559.1094; found, 559.1115. HPLC purity: 96.5%.
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4′-iodo)benzamido]morphinan (18).
Compound 18 was synthesized according to the general procedure (II) described above using β-
naloxamine, 4-iodobenzoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white
18
ACS Paragon Plus Environment

Page 19 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
solid was obtained. Yield: 146 mg (43%); mp 210-213 oC dec; [α]20D -163.2 (c 0.1, CHCl3); 1H-
NMR (500 MHz, CDCl3) δ: 7.78 (d, J=9.8 Hz, 2H), 7.53 (d, J=9.8 Hz, 2H), 6.7 (d, J=9.8 Hz, 1H), 6.57
(d, J=9.8 Hz, 1H), 5.82 (m, 1H), 5.23-5.2 (m, 2H), 4.51 (d, J=8.2 Hz, 1H), 4.23 (m, 1H), 3.19-1.5 (m,
14H). 13C NMR (150 MHz, CDCl3) δ 166.1, 143.2, 139.0, 137.7, 135.2, 133.9, 130.5, 128.7, 124.9,
119.3, 118.1, 117.5, 98.4, 92.7, 70.2, 62.4, 57.8, 49.9, 47.1, 43.5, 31.9, 28.7, 22.9, 22.7 ppm.
MS(ESI) m/z (%) 559 (MH+). HRMS calcd for C26H28N2O4 I (MH+), 559.1094; found, 559.1099.
HPLC purity: 90.1%, α-isomer: 8.6%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-fluoro)benzamido]morphinan (19).
Compound 19 was synthesized according to the general procedure (II) described above using β-
naloxamine, 3-fluorobenzoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white
solid was obtained. Yield: 192 mg (70%); mp 193-196 oC dec; [α]20D -135.5 (c 0.1, CHCl3); 1H-
NMR (500 MHz, CDCl3) δ: 7.59 (d, J=9.2 Hz, 1H), 7.55 (d, J=9.2Hz, 1H), 7.41-7.36 (m, 2H), 7.21-
7.17 (m, 1H), 6.73 (d, J=9.2 Hz, 1H), 6.59 (d, J=10Hz, 1H), 5.81 (m, 1H), 5.23-5.16 (m, 2H), 4.51
(d, J=9.2 Hz, 1H), 4.25 (m, 1H), 3.14-1.28 (m, 14H). 13C NMR (150 MHz, CDCl3) δ 166.6, 143.0,
140.1, 136.4, 130.3, 130.2, 122.8, 119.2, 118.8, 118.7, 118.6, 114.6, 114.4, 93.1, 70.6, 62.4,
57.7, 50.8, 47.4, 43.7, 31.2, 29.3, 23.9, 22.7 ppm. MS(ESI) m/z (%) 451 (MH+). HRMS calcd for
C26H28N2O4F(MH+), 451.2033; found, 451.2031. HPLC purity: 94.3%.
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-chloro)benzamido]morphinan (20).
Compound 20 was synthesized according to the general procedure (II) described above using β-
naloxamine, 3-chlorobenzoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white
solid was obtained. Yield: 205 mg (72%); mp 207-211 oC dec; [α]20D -181.7 (c 0.1, CHCl3); 1H-
NMR (500 MHz, CDCl3) δ: 7.82 (s, 1H), 7.69 (d, J=7.85 Hz, 1H), 7.47 (d, J=7.85Hz, 1H), 7.39-7.35
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 45
1
2
3
4
5
6
7
8
(m, 1H), 6.73 (d, J=8.05 Hz, 1H), 6.59 (d, J=8.05 Hz, 1H), 5.82-5.81 (m, 1H), 5.2-5.17 (m, 2H),
4.51-4.5 (d, J=5 Hz, 1H), 4.25 (m, 1H), 3.14-1.28 (m, 14H). 13C NMR (150 MHz, CDCl3) δ 165.7,
142.9, 139.2, 136.1, 135.2, 134.6, 131.5, 130.5, 129.8, 127.5, 125.1, 124.7, 119.3, 118.1, 117.6,
92.7, 70.3, 62.4, 57.8, 50.5, 47.2, 43.6, 31.6, 29.0, 23.2, 22.7 ppm. MS(ESI) m/z (%) 467 (MH+).
HRMS calcd for C26H28N2O4Cl(MH+), 467.1738; found, 467.1737. HPLC purity: 97.4%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-bromo)benzamido]morphinan (21).
Compound 21 was synthesized according to the general procedure (II) described above using β-
naloxamine, 3-bromobenzoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white
solid was obtained. Yield: 218 mg (70%); mp 152-154 oC; [α]20D -193.1 (c 0.1, CHCl3); 1H-NMR
(500 MHz, CDCl3) δ: 7.96 (s, 1H), 7.72 (d, J=8.75 Hz, 1H), 7.61 (d, J=8.75 Hz, 1H), 7.31-7.28 (m,
1H), 7.24-7.22 (m, 1H), 6.72 (d, J=8.75 Hz, 1H), 6.58 (d, J=8.75 Hz, 1H), 5.8 (m, 1H), 5.23-5.16 (m,
2H), 4.52 (d, J=8.75 Hz, 1H), 4.18 (m, 1H), 3.14-1.5 (m, 14H). 13C NMR (150 MHz, CDCl3) δ 165.6,
143.2, 139.4, 136.4, 135.4, 135.2, 134.5, 130.4, 130.1, 125.6, 124.5, 122.7, 119.3, 118.1, 117.8,
92.5, 70.2, 62.4, 57.8, 50.2, 47.1, 43.5, 31.9, 28.7, 23.0, 22.7 ppm. MS (ESI) m/z (%) 511 (MH+).
HRMS calcd for C26H28N2O4Br (MH+), 511.1232; found, 511.1250. HPLC purity: 94.4%.
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-(benzamido)morphinan (22). Compound 22
was synthesized according to the general procedure (II) described above using β-naloxamine,
benzoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white solid was obtained.
Yield: 84 mg (32%); mp 177-181 oC; [α]20D -58.4 (c 0.1, CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 7.82
(d, J=9.2 Hz, 2H), 7.51-7.42 (m, 3H), 7.20 (m, 1H), 6.74 (d, J=9.2Hz, 1H), 6.59 (d, J=9.2 Hz, 1H),
5.82 (m, 1H), 5.23-5.17 (m, 2H), 4.5 (d, J=7.65 Hz, 1H), 4.26 (m, 1H), 3.13-1.25 (m, 14H). 13C
NMR (150 MHz, CDCl3) δ 166.9, 143.3, 139.2, 135.2, 134.5, 131.5, 130.7, 128.6, 127.0, 125.0,
20
ACS Paragon Plus Environment

Page 21 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
119.2, 118.1, 117.5, 93.3, 70.2, 62.5, 57.8, 49.8, 47.2, 43.6, 31.7, 28.9, 23.2, 22.7 ppm. MS (ESI)
m/z (%) 433 (MH+). HRMS calcd for C26H29N2O4 (MH+), 433.2127; found, 433.2125. HPLC purity:
90.4%.
8
9
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-methyl)benzamido]morphinan (23).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound 23 was synthesized according to the general procedure (II) described above using β-
naloxamine, 3-toluic acid, BOP and DIEA in DCM followed by base hydrolysis. A white solid was
obtained. Yield: 133 mg (49%); mp 218-221 oC dec; [α]20D -163.4 (c 0.1, CHCl3); 1H-NMR (500
MHz, CDCl3) δ: 7.67 (m, 2H), 7.51 (s, 1H), 7.35 (d, J=8.1 Hz, 1H), 6.71 (d, J=8.1Hz, 1H), 6.61 (d,
J=8.1Hz, 1H), 5.82 (m, 1H), 5.23-5.17 (m, 2H), 4.55 (d, J=7.05 Hz, 1H), 4.06 (m, 1H), 3.36-1.5 (m,
16H). 13C NMR (150 MHz, CDCl3) δ 167.3, 143.1, 139.3, 138.4, 135.2, 134.4, 132.3, 130.7, 128.4,
127.8, 124.8, 123.9, 119.2, 118.1, 117.6, 93.3, 70.2, 62.5, 57.8, 50.2, 47.3, 43.6, 31.5, 29.1, 23.5,
22.7, 21.4 ppm. MS(ESI) m/z (%) 447 (MH+). HRMS calcd for C27H31N2O4 (MH+), 447.2284;
found, 447.2290. HPLC purity: 98.6%.
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-trifluoromethyl)benzamido]morphinan
(24). Compound 24 was synthesized according to the general procedure (II) described above
using β-naloxamine, 3-trifluorotoluic acid, BOP and DIEA in DCM followed by base hydrolysis. A
white solid was obtained. Yield: 210 mg (69%); mp 88-91 oC; [α]20D -126.7 (c 0.1, CHCl3); 1H-NMR
(500 MHz, CDCl3) δ: 8.01 (s, 3H), 8.0 (m, 1H), 7.89-7.88 (m, 1H), 7.65 (m, 1H), 7.45 (m, 1H),
6.62 (d, J=8.15 Hz, 1H), 6.5 (d, J=8.15 Hz, 1H), 5.78-5.74 (m, 1H), 5.2-5.13 (m, 2H), 4.67 (d,
J=6.15 Hz, 1H), 4.11-4.02 (m, 1H), 3.54-1.24 (m, 14H). 13C NMR (150 MHz, CDCl3) δ 165.5, 143.1,
139.2, 135.2, 130.5, 130.3, 129.1, 128.1, 124.8, 124.2, 119.3, 118.1, 117.5, 92.8, 70.2, 62.4,
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 45
1
2
3
4
57.8, 50.2, 47.2, 43.5, 31.8, 28.8, 23.0, 22.7 ppm. MS (ESI) m/z (%) 501 (MH+). HRMS calcd for
C27H28N2O4F3 (MH+), 501.2001; found, 501.2004. HPLC purity: 96.4%.
5
6
7
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-methoxy)benzamido]morphinan (25).
8
9
Compound 25 was synthesized according to the general procedure (II) described above using β-
naloxamine, 3-anisic acid, BOP and DIEA in DCM followed by base hydrolysis. A white solid was
obtained. Yield: 169 mg (60%); mp 113-115 oC; [α]20D -124.4 (c 0.1, CHCl3); 1H-NMR (500 MHz,
CDCl3) δ: 7.56 (d, J=9 Hz, 1H), 7.39-7.26 (m, 3H), 7.0 (m, 1H), 6.72 (d, J=8.1 Hz, 1H), 6.55 (d,
J=8.1 Hz, 1H), 5.78-5.74 (m, 1H), 5.24-5.17 (m, 2H), 4.52 (d, J=6.2 Hz, 1H), 4.12-4.11 (m, 1H),
3.78 (s, 3H), 3.72-1.25 (m, 14H). 13C NMR (150 MHz, CDCl3) δ 166.9, 159.8, 143.1, 139.3, 135.9,
134.9, 130.6, 129.5, 124.6, 119.2, 118.8, 117.8, 117.7, 112.4, 93.0, 70.2, 62.5, 57.8, 55.4, 50.2,
47.2, 43.7, 31.5, 29.0, 23.3, 22.7 ppm. MS (ESI) m/z (%) 463 (MH+). HRMS calcd for C27H31N2O5
(MH+), 463.2233; found, 463.2232. HPLC purity: 97.0%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-amino)benzamido]morphinan (26).
Compound 26 was synthesized according to the general procedure (II) described above using β-
naloxamine, 3-amino benzoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white
solid was obtained. Yield: 82 mg (30%); mp 191-193 oC dec; [α]20D -148.7 (c 0.1, CHCl3); 1H-NMR
(500 MHz, CDCl3) δ: 7.2-7.16 (m, 1H), 7.1 (d, J=7.95 Hz, 1H), 6.90 (d, J=7.95 Hz, 1H), 6.8 (d,
J=7.95 Hz, 1H), 6.75 (d, J=8.1 Hz, 1H), 6.69 (d, J=8.1 Hz, 1H), 5.81-5.8 (m, 1H), 5.19-5.16 (m, 2H),
4.46 (d, J=5.85 Hz, 1H), 4.21-4.19 (m, 1H), 3.48-1.22 (m, 16H). 13C NMR (150 MHz, CDCl3) δ
167.6, 146.6, 143.0, 139.6, 135.5, 135.3, 130.7, 129.3, 124.4, 119.1, 118.3, 118.0, 117.9,116.8,
114.5, 92.8, 70.4, 62.5, 57.7, 50.7, 47.3, 43.6, 31.2, 29.2, 23.7, 22.7 ppm. MS (ESI) m/z (%) 448
(MH+). HRMS calcd for C26H30N3O4 (MH+), 448.2236; found, 448.2230. HPLC purity: 95.4%.
22
ACS Paragon Plus Environment

Page 23 of 45
Journal of Medicinal Chemistry
1
2
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-dimethylamino)benzamido]morphinan
3
4
5
6
7
8
9
(27). Compound 27 was synthesized according to the general procedure (II) described above
using β-naloxamine, 3-dimethylamino benzoic acid, BOP and DIEA in DCM followed by base
hydrolysis. A white solid was obtained. Yield: 174 mg (60%); mp 170-173 oC; [α]20D -127.3 (c 0.1,
CHCl3); 1H-NMR (500 MHz, CDCl3) δ: 7.21 (s, 1H), 7.02 (d, J=10 Hz, 1H), 6.89-6.85 (m, 1H), 6.75
(d, J=10 Hz, 1H), 6.59 (m, 1H), 5.79 (m, 1H), 5.22-5.16 (m, 2H), 4.48 (d, J=10 Hz, 1H), 4.22 (m,
1H), 3.14-1.5 (m, 20H). 13C NMR (150 MHz, CDCl3) δ 167.8, 150.7, 143.2, 139.4, 135.3, 130.7,
129.1, 119.1, 118.0, 117.6, 115.3, 114.2, 111.6, 93.1, 70.2, 62.5, 57.8, 53.4, 50.2,47.2, 43.6,
40.5, 31.5, 29.1, 23.4, 22.7 ppm. MS (ESI) m/z (%) 476 (MH+). HRMS calcd for C28H34N3O4
(MH+), 476.2549; found, 476.2544. HPLC purity: 96.5%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-hydroxy)benzamido]morphinan (28).
Compound 28 was synthesized according to the general procedure (II) described above using β-
naloxamine, 3-hydroxy benzoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white
solid was obtained. Yield: 107 mg (39%); mp 201-204 oC dec; [α]20D -170.7 (c 0.1, CHCl3); 1H-
NMR (500 MHz, CDCl3) δ: 7.44 (m, 3H), 7.3-7.28 (m, 2H), 6.99 (d, J=7.75 Hz, 1H), 6.71 (d, J=7.75
Hz, 1H), 6.6 (d, J=7.75 Hz, 1H), 5.82-5.8 (m, 1H), 5.22-5.17 (m, 2H), 4.51 (d, J=7.75 Hz, 1H), 4.062
(m, 1H), 3.51-1.51 (m, 14H). 13C NMR (150 MHz, CDCl3) δ 168.0, 156.7, 142.7, 139.8, 135.1,
134.8, 130.6, 129.7, 124.1, 119.5, 119.2, 118.2, 118.1, 115.1, 93.3, 70.6, 62.4, 57.7, 51.3, 47.4,
43.7, 30.9, 29.3, 24.2, 22.7 ppm. MS (ESI) m/z (%) 449 (MH+). HRMS calcd for C26H29N2O5
(MH+), 449.2076; found, 449.2080. HPLC purity: 95.5%.
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(3′-nitro)benzamido]morphinan (29).
Compound 29 was synthesized according to the general procedure (II) described above using β-
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 45
1
2
3
4
5
6
7
8
naloxamine, 3-nitro benzoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white
solid was obtained. Yield: 172 mg (59%); mp 228-231 oC dec; [α]20D -128.8 (c 0.1, CHCl3); 1H-
NMR (500 MHz, CDCl3)δ: 8.68 (s, 1H), 8.36-8.34 (m, 1H), 8.22 (d, J=11.8 Hz, 1H), 7.67-7.63 (m,
2H), 6.69 (d, J=11.8 Hz, 1H), 6.58 (d, J=11.8 Hz, 1H), 5.81 (m, 1H), 5.2-5.17 (m, 2H), 4.59 (d, J=9.8
Hz, 1H), 4.27 (m, 1H), 3.14-1.25 (m, 14H). 13C NMR (150 MHz, CDCl3) δ 164.6, 148.1, 141.6,
139.4, 135.5, 135.2, 133.7, 130.6, 129.4, 125.8, 124.6, 121.9, 119.4, 118.1, 117.3, 92.1, 70.6,
62.4, 57.9, 51.9, 47.4, 43.4, 38.6, 31.0, 29.5, 23.7,22.7 ppm. MS(ESI) m/z (%) 478 (MH+). HRMS
calcd for C26H28N3O6 (MH+), 479.1978; found, 478.1967. HPLC purity: 97.0%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4′-trifluoromethoxy)benzamido]morphinan
(30). Compound 30 was synthesized according to the general procedure (II) described above
using β-naloxamine, 4-(trifluoromethoxy)benzoic acid, BOP and DIEA in DCM followed by base
hydrolysis. A white solid was obtained. Yield: 248 mg (79%); mp 138-140 oC; [α]20D -136.0 (c 0.1,
CHCl3); 1H-NMR (500 MHz, CDCl3)δ: 7.87 (d, J=11.75 Hz, 1H), 7.44 (d, J=11.75 Hz, 1H), 7.24 (m,
2H), 6.72 (d, J=11.75 Hz, 1H), 6.58 (d, J=11.75 Hz, 1H), 5.81 (m, 1H), 5.23-5.16 (m, 2H), 4.53 (d,
J=9.8 Hz, 1H), 4.24 (m, 1H), 3.33-1.28 (m, 14H). 13C NMR (150 MHz, CDCl3) δ 165.7, 151.4, 143.1,
139.1, 135.1, 132.8, 130.5, 129.0, 124.7, 120.5, 119.3, 118.2, 117.6, 92.6, 70.3, 62.4, 57.8, 50.2,
47.1, 43.5, 31.8, 28.8, 23.0, 22.6 ppm. MS (ESI) m/z (%) 517 (MH+). HRMS calcd for
C27H28N2O5F3 (MH+), 517.1950; found, 517.1956. HPLC purity: 99.9%.
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2 '-naphthyl)acetamido]morphinan (31).
Compound 31 was synthesized according to the general procedure (I) described above
using β-naloxamine, NHS ester of naphthalene-2-carboxylic acid 9, and DIEA in DCM. A white
solid was obtained. Yield: 261 mg (89%); mp 210-214 oC dec; [α]20D -260.2 (c 0.1, CHCl3); 1H-
24
ACS Paragon Plus Environment

Page 25 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
NMR (500 MHz, CDCl3): δ 8.17 (s, 1H), 7.78-7.70 (m, 4H), 7.47 (t, J= 7.5 Hz, 1H), 7.40 (t, J = 7.5
Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.51 (d, J = 8.1 Hz, 1H), 5.79 (m, 1H),
5.16 (m, 2H), 4.80 (m, 1H), 4.73 (d, J = 4.3 Hz, 1H), 3.10-1.05 (m, 15 H) ppm. 13C NMR (150 MHz,
CDCl3) δ 167.3, 145.3, 137.4, 134.6, 132.5, 131.7, 128.9, 128.1, 127.6, 127.5, 127.4, 126.4,
123.9, 119.2, 117.4, 93.5, 90.2, 70.2, 69.7, 62.4, 58.1, 47.1, 46.6, 42.9, 33.3, 29.0, 22.9, 21.0
ppm. MS (ESI) m/z (%) 483 (MH+). HRMS calcd for C30H31N2O4 (MH+), 483.2284; found,
483.2293. HPLC purity: 97.3%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4′-phenyl)benzamido]morphinan (32).
Compound 32 was synthesized according to the general procedure (I) described above using β-
naloxamine, NHS ester of biphenyl-4-carboxylic acid 10, and DIEA in DCM. A white solid was
obtained. Yield: 263 mg (85%); mp 210-214 oC dec; [α]20D -145.8 (c 0.1, CHCl3); 1H-NMR (500
MHz, CDCl3) δ: 7.89 (d, J=8.15 Hz, 2H), 7.66-7.61 (m, 4H), 7.46 (m, 3H), 7.38 (m, 1H), 6.74 (d,
J=8.15 Hz, 1H), 6.61 (d, J=8.15 Hz, 1H), 5.82-5.79 (m, 1H), 5.23-5.17 (m, 2H), 4.53-4.52 (d, J=5.15
Hz, 1H), 4.31-4.29 (m, 1H), 3.15-1.25 (m, 14H). 13C NMR (150 MHz, CDCl3) δ 166.7, 144.2, 143.2,
140.0, 139.2, 135.2, 133.1, 130.6, 128.9, 128.0, 127.6, 127.2, 124.8, 119.2,118.1,117.6, 92.9,
70.2, 62.5, 57.8, 50.1, 47.2, 43.6, 31.7, 28.9, 23.2, 22.7 ppm. ESI-MS m/z: 509.09 (MH+). HPLC
purity: 95.9%.
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-(acetylamido)morphinan (33). Compound 33
was synthesized according to the general procedure (II) described above using β-naloxamine,
acetic acid, BOP and DIEA in DCM followed by base hydrolysis. A white solid was obtained.
Yield: 120 mg (53%); mp 204-207 oC dec; [α]20D -157.0 (c 0.1, CHCl3); 1H NMR (500 MHz, CDCl3):
δ 6.70 (d, J = 8.2 Hz, 1H,), 6.56 (d, J = 8.2 Hz, 1H), 5.96 (d, J = 9.2 Hz, 1H), 5.76 (m, 1H,), 5.18 (d, J
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 45
1
2
3
4
5
6
7
8
= 17.8 Hz, 1H,), 5.14 (d, J = 10.5 Hz, 1H,), 4.33 (d, J = 6.5 Hz, 1H), 3.89 (m, 1H), 3.15-0.80 (m, 18
H) ppm. 13C NMR (150 MHz, CDCl3) δ 170.6, 142.8, 139.8, 135.2, 130.8, 124.3, 119.1, 118.0,
117.8, 93.0, 70.4, 62.4, 57.7, 50.7, 47.4, 43.7, 30.9, 29.5, 23.9, 23.6, 22.7 ppm. MS(ESI) m/z (%)
371 (MH+). HRMS calcd for C21H27N2O4 (MH+), 371.1971; found, 371.1965. HPLC purity: 97.0%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-(hexylamido)morphinan (34). Compound 34
was synthesized according to the general procedure (II) described above using β-naloxamine,
hexanoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white solid was obtained.
Yield: 130 mg (50%); mp 113-116 oC; [α]20D -101.6 (c 0.1, CHCl3); 1H NMR (500 MHz, CDCl3): δ
6.71 (d, J = 8.2 Hz, 1H,), 6.55 (d, J = 8.2 Hz, 1H), 6.07 (d, J = 9.2 Hz, 1H), 5.77 (m, 1H), 5.18 (d, J =
17.4 Hz, 1H), 5.14 (d, J = 10.1 Hz, 1H,), 4.34 (d, J = 6.4 Hz, 1H), 3.91 (m, 1H), 3.15-0.80 (m, 26 H)
ppm. 13C NMR (150 MHz, CDCl3) δ 173.0, 142.9, 139.6, 135.3, 130.8, 124.7, 119.2, 118.0, 117.6,
93.7, 70.1, 62.5, 57.7, 50.1, 47.4, 43.6, 37.1, 31.4, 31.1, 29.5, 25.4, 23.9, 22.7, 22.4, 14.0 ppm.
MS(ESI) m/z (%) 427 (MH+). HRMS calcd for C26H35N2O4 (MH+), 427.2597; found, 427.2591.
HPLC purity: 96.0%.
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-(dodecylamido)morphinan (35). Compound
35 was synthesized according to the general procedure (II) described above using β-
naloxamine, dodecanoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white solid
was obtained. Yield: 202 mg (65%); mp 95-98 oC; [α]20D -99.9 (c 0.1, CHCl3); 1H NMR (500 MHz,
CDCl3): δ 6.71 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H,), 6.07 (d, J = 9.2 Hz, 1H,), 5.76 (m, 1H),
5.18 (d, J = 17.4 Hz, 1H, ), 5.14 (d, J = 10.1 Hz, 1H), 4.34 (d, J = 6.4 Hz, 1H), 3.91 (m, 1H), 3.10-
0.86 (m, 38 H) ppm. 13C NMR (150 MHz, CDCl3) δ 173.1, 142.9, 139.7, 130.7, 119.2, 117.7, 93.7,
70.1, 62.5, 57.7, 53.4, 50.1, 47.4, 43.7, 37.1, 31.9, 29.6, 29.5, 29.3, 29.2, 25.7, 23.9, 22.7, 14.1
26
ACS Paragon Plus Environment

Page 27 of 45
Journal of Medicinal Chemistry
1
2
3
4
ppm. MS(ESI) m/z (%) 511 (MH+). HRMS calcd for C31H47N2O4 (MH+), 511.3536; found,
511.3550. HPLC purity: 95.3%.
5
6
7
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-(cyclohexylamido)morphinan (36). Compound
8
9
36 was synthesized according to the general procedure (II) described above using β-
naloxamine, cyclohexanoic acid, BOP and DIEA in DCM followed by base hydrolysis. A white
solid was obtained. Yield: 174 mg (65%); mp 236-239 oC dec; [α]20D -135.2 (c 0.1, CHCl3); 1H
NMR (500 MHz, CDCl3): δ 6.71 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H), 6.14 (d, J = 9.1 Hz, 1H),
5.77 (m, 1H), 5.18 (d, J = 17.4 Hz, 1H), 5.14 (d, J = 10.0 Hz, 1H), 4.33 (d, J = 6.1 Hz, 1H), 3.93 (m,
1H), 3.15-0.80 (m, 26 H) ppm. 13C NMR (150 MHz, CDCl3) δ 176.0, 143.1, 139.5, 135.3, 130.8,
124.7, 119.1, 118.0, 117.6, 93.7, 70.1, 62.5, 57.7, 49.7, 47.3, 45.7, 43.6, 31.3, 29.7, 29.6, 29.3,
25.8, 25.7, 23.6, 22.7ppm. MS (ESI) m/z (%) 439 (MH+). HRMS calcd for C26H35N2O4 (MH+),
439.2597; found, 439.2602. HPLC purity: 93.2%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17-Allyl-3,14β-dihydroxy-4,5α-epoxy-6β-[2-adamantylamido]morphinan (37).
Compound 37 was synthesized according to the general procedure (II) described above using β-
naloxamine, 1-Adamantyl carboxylic acid, BOP and DIEA in DCM followed by base hydrolysis. A
white solid was obtained. Yield: 227 mg (76%); mp 153-156 oC; [α]20D -129.0 (c 0.1, CHCl3); 1H
NMR (500 MHz, CDCl3) δ: 6.71 (d, J = 8.2 Hz, 1H,), 6.55 (d, J = 8.2 Hz, 1H), 6.22 (d, J = 9.5 Hz, 1H),
5.77 (m, 1H), 5.28 (s, 1H), 5.18 (d, J = 17.2 Hz, 1H), 5.14 (d, J = 10.2 Hz, 1H), 4.31 (d, J= 5.9 Hz,
1H), 3.97 (m, 1H), 3.15-0.76 (m, 29 H) ppm. 13C NMR (150 MHz, CDCl3) δ 178.1, 162.6, 143.2,
139.6, 135.3, 130.7, 124.4,119.0,118.0, 117.8, 93.0, 70.2, 62.4, 57.8, 49.6, 47.1, 43.6, 40.7, 39.1,
38.6, 36.5, 31.7, 31.5, 28.9, 28.1, 23.2, 22.6 ppm. MS (ESI) m/z (%) 491 (MH+). HRMS calcd for
C30H39N2O4 (MH+), 491.2910; found, 491.2912. HPLC purity: 91.9%.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 45
1
2
17-Allyl-4,5α-epoxy-14β-hydroxy-3β-methoxy-6β-[(3′-iodo)benzamido]morphinan
3
4
5
6
7
8
9
(38). Compound 38 was synthesized according to the general procedure (I) described above
using 3-OMe-β-naloxamine, NHS ester of 3-iodobenzoic acid 10 and DIEA in DCM. A white solid
was obtained. Yield: 296 mg (85%); mp 125-127 oC; [α]20D -133.7 (c 0.1, CHCl3); 1H-NMR δ: 8.19
(s, 1H), 7.8 (m, 1H), 7.42 (m, 1H), 7.16 (m, 1H), 6.75 (d, J=10 Hz, 1H), 6.66 (d, J=10 Hz, 1H), 5.85
(m, 1H), 5.18 (m, 2H), 4.61 (d, 1H), 4.08 (m, 1H), 3.85 (s, 2H), 3.15-0.1(m, 14H). 13C NMR (150
MHz, CDCl3) δ 165.2, 144.9, 144.3, 136.4, 130.8, 130.1, 127.3, 126.3, 124.4, 120.9, 119.4, 115.8,
94.2, 91.9, 72.7, 71.8, 71.7, 60.8, 57.0, 56.9, 50.0, 47.1, 30.9, 28.1, 26.7, 22.6 ppm. MS (ESI) m/z
(%) 573 (MH+). HRMS calcd for C27H30N2O4I (MH+), 573.1250; found, 573.1252. HPLC purity:
96.6%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Receptor-Binding Assays: Competition-binding assays in CHO cells stably expressing
MOR-1 (mu), DOR-1 (delta) or KOR-1 (kappa) were performed at 25°C in potassium phosphate
buffer (50 mM; pH 7.4), with the inclusion of MgSO4 (5 mM) in the MOR-1 assays. All
competition assays were carried out using 125I-BNtxA ( ) as described.39 Specific binding was
1
defined as the difference between total binding and nonspecific binding, determined in the
presence of levallorphan (8 μM). Protein concentrations were between 30-40 μg/mL and
incubation times were 90 minutes. 6TM/E11 competition binding assays using 125I-BNtxA (
1;
0.15 nM) were carried out in whole brain membrane homogenates (0.5 ml; 0.5 mg protein) at
25°C in potassium phosphate buffer (50 mM; pH 7.4) with magnesium sulfate (5 mM) for 90
minutes in the presence of CTAP, U50488H and DPDPE, all at 200nM,to block traditional opioid
binding sites. Specific binding was defined as the difference between total binding and
28
ACS Paragon Plus Environment

Page 29 of 45
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
nonspecific binding, determined in the presence of levallorphan (8 μM). Protein concentration
was determined as described by Lowry et al., 40 using bovine serum albumin as the standard.
Tail Flick Analgesia Assays: Male CD-1 mice (25-35 g; Charles River Breeding
8
9
Laboratories, Wilmington, MA) were maintained on a 12-hr light/dark cycle with Purina rodent
chow and water available ad libitum. Mice were housed in groups of five until testing. All
animal experiments were reviewed and approved by the Institutional Animal Care and Use
Committee of Memorial Sloan-Kettering Cancer Center. Analgesia was determined using the
radiant heat tail-flick technique41 using a machine from (Ugo Basile; model number 37360).
The intensity was set to achieve a baseline between 2-3 sec. The latency to withdraw the tail
from a focused light stimulus was measured electronically using a photocell. Baseline latencies
(2.0-3.0 sec) were determined before experimental treatments for all animals as the mean of
two trials. Post-treatment tail-flick latencies were determined as indicated for each experiment,
and a maximal latency of 10 sec for tail-flick was used to minimize tissue damage. Drugs were
given subcutaneously and cumulative dose-response experiments carried out with two
independent assays with each group (n=10). The combined results presented as the ED50 with
95% confidence limits (n=20) presented. Analgesia was defined quantally as a doubling, or
greater, of the baseline latency. Similar results were obtained analyzing the data in a graded
response manner. Analgesic ED50’s and confidence limits were determined using non-linear
regression analysis Graph Pad Prism (Graphpad Software, La Jolla, CA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Respiratory Depression assay: Respiratory rate was assessed in awake, freely- moving,
adult male mice using the MouseOx Pulse Oximeter system (Starr Life Sciences, Pittsburgh, PA).
Each animal was habituated to the device for 30 min and then tested. A five- second average
29
ACS Paragon Plus Environment