Structure-Activity Relationship Studies of 6α- and 6β-Indolylacetamidonaltrexamine Derivatives as Bitopic Mu Opioid Receptor Modulators and Elaboration of the message-Address Concept to Comprehend Their Functional Conversion

Article abstract of DOI:10.1021/acschemneuro.8b00349

Structure-activity relationship (SAR) studies of numerous opioid ligands have shown that introduction of a methyl or ethyl group on the tertiary amino group at position 17 of the epoxymorphinan skeleton generally results in a mu opioid receptor (MOR) agonist while introduction of a cyclopropylmethyl group typically leads to an antagonist. Furthermore, it has been shown that introduction of heterocyclic ring systems at position 6 can favor antagonism. However, it was reported that 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(2′-indolyl)acetamido]morphinan (INTA), which bears a cyclopropylmethyl group at position 17 and an indole ring at position 6, acted as a MOR agonist. We herein report a SAR study on INTA with a series of its complementary derivatives to understand how introduction of an indole moiety with α or β linkage at position 6 of the epoxymorphinan skeleton may influence ligand function. Interestingly, one of INTA derivatives, compound 15 (NAN) was identified as a MOR antagonist both in vitro and in vivo. Molecular modeling studies revealed that INTA and NAN may interact with different domains of the MOR allosteric binding site. In addition, INTA may interact with W293 and N150 residues found in the orthosteric site to stabilize MOR activation conformation while NAN does not. These results suggest that INTA and NAN may be bitopic ligands and the type of allosteric interactions with the MOR influence their functional activity. These insights along with our enriched comprehension of the message-address concept will to benefit future ligand design.

Full text of DOI:10.1021/acschemneuro.8b00349

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Article
Structure activity relationship studies of 6#- and 6#-
indolylacetamidonaltrexamine derivatives as bitopic mu
opioid receptor modulators and elaboration of the ‘message-
address concept’ to comprehend their functional conversion
Samuel Obeng, Huiqun Wang, Abdulmajeed Jali, David L Stevens,
Hamid I Akbarali, William L Dewey, Dana E Selley, and Yan Zhang
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00349 • Publication Date (Web): 29 Aug 2018
Downloaded from http://pubs.acs.org on September 4, 2018
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Structure activity relationship studies of 6αꢀ and 6βꢀ
indolylacetamidonaltrexamine derivatives as bitopic
mu opioid receptor modulators and elaboration of
the ‘messageꢀaddress concept’ to comprehend their
functional conversion
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†
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Samuel Obeng, Huiqun Wang, Abdulmajeed Jali, David L. Stevens, Hamid I. Akbarali,
‡
‡
†,*
William L. Dewey, Dana E. Selley, Yan Zhang
†
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Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University,
00 E Leigh Street, Richmond, Virginia 23298, United States
‡Department of Pharmacology and Toxicology, Virginia Commonwealth University, 410 North
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2th Street, Richmond, Virginia 23298, United States
*
Corresponding author: Tel: +1 (804) 828ꢀ0021. Eꢀmail address: yzhang2@vcu.edu (Y. Zhang).
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ABSTRACT
Structureꢀactivity relationship (SAR) studies of numerous opioid ligands have shown that
introduction of a methyl or ethyl group on the tertiary amino group at position 17 of the
epoxymorphinan skeleton generally results in a mu opioid receptor (MOR) agonist while
introduction of a cyclopropylmethyl group typically leads to an antagonist. Furthermore, it has
been shown that introduction of heterocyclic ring systems at position 6 can favor antagonism.
However, it was reported that 17ꢀcyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ[(2′ꢀ
indolyl)acetamido]morphinan (INTA), which bears a cyclopropylmethyl group at position 17
and an indole ring at position 6, acted as a MOR agonist. We herein report a SAR study on INTA
with a series of its complementary derivatives to understand how introduction of an indole
moiety with α or β linkage at position 6 of the epoxymorphinan skeleton may influence ligand
function. Interestingly, one of INTA derivatives, compound 15 (NAN) was identified as a MOR
antagonist both in vitro and in vivo. Molecular modeling studies revealed that INTA and NAN
may interact with different domains of the MOR allosteric binding site. In addition, INTA may
interact with W293 and N150 residues found in the orthosteric site to stabilize MOR activation
conformation while NAN does not. These results suggest that INTA and NAN may be bitopic
ligands and the type of allosteric interactions with the MOR influence their functional activity.
These insights along with our enriched comprehension of the “messageꢀaddress” concept will to
benefit future ligand design.
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Keywords: opioids, SAR, bitopic, allosteric, functional conversion, messageꢀaddress concept
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INTRODUCTION
Since opioid binding sites were first proposed in the early 1950s and 1960s, and discovered in
mammalian brain tissue in 1973, extensive pharmacological studies have identified several types
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of opioid receptors. To date, four opioid receptors have been cloned, the mu opioid receptor
(MOR), kappa opioid receptor (KOR), delta opioid receptor (DOR), and the opioid receptorꢀlike
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orphan receptor (ORL) or nociceptin/orphanin FQ receptor (NOP). Opioid receptors share
high sequence homology and belong to the superfamily of seven transmembraneꢀspanning G
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proteinꢀcoupled receptors (GPCRs). GPCRs play important roles in mediating the actions of
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many known neurotransmitters and hormones.
Effects of opioids are mainly inhibitory
resulting in significant inhibition of nerve firing and reduction in neurotransmitter release. The
rewarding, dependenceꢀproducing, and analgesic effects of opioids result from activation of
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MORs in several brain regions.
KOR agonists have been shown to produce dysphoric and
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psychotomimetic effects, while DOR activation results in inhibition of anxiety and stress.
As a result, some opioid agonists and antagonists have been applied in the treatment of a number
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of diseases. In general, a compound is characterized as an opioid agonist only if its effect is
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competitively inhibited by an opioid antagonist.
Structureꢀactivity relationship (SAR) studies of opioids have revealed that introduction of a
methyl or ethyl group on the 17ꢀamino group of the epoxymorphinan skeleton generally results
in a MOR agonist while introduction of an allyl or cyclopropylmethyl group at the same position
typically leads to an opioid antagonist. Also, a hydroxyl group at position 14 may strengthen the
23–25
binding to the opioid receptors, like naltrexone.
In addition, some compounds with
heterocyclic rings at position 6 of the epoxymorphinan group such as 17ꢀcyclopropylmethylꢀ
3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6α (isoquinolineꢀ3ꢀcarboxamido)morphinan (NAQ) and 17ꢀ
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cyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ(4′ꢀpyridylcarboxamido)morphinan (NAP)
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have been shown to be MOR selective antagonists (Figure 1).
However, introduction of an
allyl or cyclopropylmethyl group on the 17ꢀamino group of the epoxymorphinan skeleton may
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also lead to an opioid agonist.
For example, Le Naour et al. reported that 17ꢀ
cyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ[(2’ꢀindolyl)acetamido]morphinan (INTA),
which is similar to NAQ and NAP structureꢀwise, actually acted as an opioid agonist and
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activated KORꢀDOR and KORꢀMOR heterodimers in HEK293 cells (Figure 1). It was shown
that INTA produced potent antinociception and was devoid of tolerance and dependence while it
showed no aversive effects in the conditioned place preference assay. Similar to NAQ and NAP,
INTA carries a heterocyclic ring at position 6 and a cyclopropylmethyl group at position 17, thus
it was intriguing how INTA acted as an agonist while NAQ and NAP acted as antagonists.
Therefore, a series of compounds were prepared as INTA complementary analogues in order to
understand how introduction of an indole moiety with an α or β linkage at position 6 of the
epoxymorphinan skeleton would influence ligand function.
Interestingly, we identified 17ꢀcyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6αꢀ(indoleꢀ7ꢀ
carboxamido)morphinan (NAN) as a potent MOR antagonist. Per the ‘messageꢀaddress concept’,
ligands that bind opioid receptors have distinct pharmacophores responsible for efficacy
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(message) or selectivity (address).
Both NAN and INTA bear an identical epoxymorphinan
message portion with subtle differences in the linker and address regions of the molecule. Thus,
it would be expected that they carry similar efficacies at the opioid receptors; however, the
opposite was observed.
Studies have shown that allosteric ligands of the MOR show varying abilities to modulate the
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orthosteric ligand’s affinity, efficacy, or both.
Allosteric ligands bind to a site on the receptor,
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termed as allosteric binding site, that is distinct from the site that endogenous orthosteric ligands
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bind (orthosteric binding site).
An allosteric ligand can act as a positive allosteric modulator
(PAM) which enhances the affinity or efficacy of an orthosteric ligand, a negative allosteric
modulator (NAM) which inhibits the intrinsic affinity or efficacy, or a silent allosteric modulator
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(SAM) which occupies the allosteric binding site and has no effect on the affinity or efficacy. In
addition, bitopic (dual steric) ligands which bind to both the orthosteric and allosteric binding
sites of a single receptor monomer have been shown to have higher affinity, increase or decrease
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intrinsic efficacy, improve offꢀrates and induce functional selectivity.
Thus, bitopic
compounds may combine the advantages of orthosteric and allosteric ligands and carry unique
pharmacological properties, e.g. higher selectivity.
We herein describe our effort to understand the SAR of INTA analogues as well as to investigate
the molecular mechanisms of how structurally similar MOR ligands, NAN and INTA, result in
the opposite functional effects on the MOR by application of classic “messageꢀaddress” concept
in addition to allosteric modulation of GPCRs.
Figure 1. Structural similarities between INTA and some known MOR ligands.
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RESULTS AND DISCUSSION
CHEMISTRY
Scheme 1. Synthetic route for INTA analogues.
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The INTA analogues were synthesized in a similar way as described previously.
Briefly,
stereoselective reductive amination of naltrexone with benzyl amine followed by catalytic
hydrogenation under acidic conditions produced 6αꢀnaltrexamine (6αꢀNTA) dihydrochloride salt
in a total yield of 67%, while reductive amination of naltrexone with dibenzyl amine followed by
catalytic hydrogenation under acidic conditions led to 6βꢀnaltrexamine (6βꢀNTA)
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dihydrochloride salt in a total yield of 59%. A variety of commercially available indole
carboxylic acids were coupled with 6αꢀNTA and 6βꢀNTA using the EDCI/HOBt coupling
method. The reaction mixture was then treated with K CO to obtain the 6ꢀmonosubstituted NTA
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derivatives with yields ranging from 23 to 81% which were then converted to HCl salts.
BIOLOGICAL RESULTS
The binding affinity and selectivity of these naltrexamine derivatives in three opioid receptors
(mouse MOR, KOR and DOR) were determined using monocloned opioid receptorꢀexpressed
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27,28,31
Chinese hamster ovary (CHO) cells as described previously
through radioligand binding
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assay. The [ S]ꢀGTPγS functional assay using mouse MOR was applied to determine whether
the synthesized compounds acted as agonist, partial agonist, or antagonist of the MOR by
determining their efficacy for Gꢀprotein activation relative to a full agonist, DAMGO. An in vivo
study was further conducted in mice using the tail immersion assay to determine whether the
compounds had antinociceptive effects or antagonized the antinociceptive effects of morphine.
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IN VITRO RADIOLIGAND BINDING AND [ S]ꢀGTPγS FUNCTIONAL ASSAYS
The binding affinities of the synthesized compounds for the MOR, KOR and DOR together with
3
their efficacy at the mouse MOR (mMOR) were compared with those of INTA. [ H]Naloxone
3
was used to label the mMOR while [ H]diprenorphine was used to label both the mKOR and
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mDOR. The [ S]ꢀGTPγS functional assay was used to determine the potency and efficacy of
these derivatives at the mMOR and the results were interpreted as potency (EC ) and efficacy
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(
%Emax relative to DAMGO). The binding, selectivity, potency and efficacy data for the 6βꢀ
INTA analogues are summarized in Table 1 and the results for the 6αꢀINTA analogues are
summarized in Table 2.
As shown in Table 1, compounds 1ꢀ9 showed subnanomolar affinity for the MOR and KOR
while having a lower affinity at the DOR except for compound 3 that bound to KOR with
nanomolar affinity. Particularly, INTA showed similar affinity for the MOR and KOR and was
about 5ꢀfold selective for MOR over DOR and 9ꢀfold selective for KOR over DOR. These
results corroborated the findings from Le Naour et. al, where they reported that INTA produced
3
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similar stimulation in the MOR and KOR and the least stimulation in the DOR. INTA showed
the lowest selectivity for the MOR over the DOR compared to other compounds.
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Among the 6αꢀINTA analogues (compounds 10ꢀ18), it was observed that all the compounds
bound to the MOR with subnanomolar affinity while having relatively lower affinity at the KOR,
and even lower at the DOR. The reason for the greater binding affinity of these ligands for the
MOR and KOR compared to other opioid antagonist with different heterocyclic rings attached to
position 6 (examples include NAP and NAQ) could be due to additional hydrogen bonding
interactions between the hydrogen atom at position 1 of the indole ring and an acceptor group in
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the binding pocket. It has been shown that methylation of the indole 1ꢀnitrogen atom led to
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reduction in activation of singly or coexpressed opioid receptors. Even though MOR and DOR
have a relatively higher percent sequence identity (62%) than that of MOR and KOR (57%), the
percent sequence identities for TM3 and TM7 where the address region is located is relatively
greater for the MOR and KOR (TM3 = 100%, TM7 = 86%) than those of the MOR and DOR
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(
TM3 = 90%, TM7 = 78%). This could account for the overall similar binding affinities at the
MOR and KOR over the DOR.
It was also noticed that the position of substitution on the indole ring (compounds 1ꢀ6 and 10ꢀ15)
did not result in a significant change in the MOR binding affinity except for compound 13. Also,
increasing the alkyl chain length of the indole analogues (compounds 7ꢀ9 and 16ꢀ18) at position
3
did not result in significant change in their MOR binding affinity, except a slightly lower
affinity of compound 16. This suggests that the binding pocket of the address site of the MOR
may be spacious enough to allow the rotation and extension of the indole group.
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Table 1. Opioid receptor binding affinity and MOR [ S]ꢀGTPγS functional assay results for 6βꢀ
INTA analogues.
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K (nM)
i
Selectivity
MOR [ S]ꢀGTPγS
assay
Compd.
R
µ
κ
δ
κ/µ
δ/µ
EC (nM) % Emax
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of
DAMGO
1
(INTA)
2
0.29 ± 0.04
0.19 ± 0.02
0.18 ± 0.00
0.16 ± 0.01
1.54 ± 0.47
7.17 ± 1.23
0.6
0.8
5.3
0.21 ± 0.01
0.22 ± 0.07
92.42 ± 2.81
37.7
79.48 ± 7.41
3
4
0.24 ± 0.03
0.26 ± 0.04
1.94 ± 0.30
0.51 ± 0.04
49.84 ± 12.69
17.67 ± 4.78
8.1
2.0
207.6
68.0
8.16 ± 4.72
2.98 ± 1.25
16.22 ± 1.40
22.32 ± 3.50
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6
0.25 ± 0.04
0.19 ± 0.01
0.17 ± 0.01
0.52 ± 0.09
6.23 ± 0.83
0.7
2.7
24.9
0.19 ± 0.02
1.96 ± 1.01
71.79 ± 4.37
37.94 ± 5.85
30.45 ± 9.49
160.3
7
8
0.24 ± 0.03
0.17 ± 0.01
0.95 ± 0.11
0.39 ± 0.05
37.02 ± 1.21
77.10 ± 28.29
4.0
2.3
154.3
453.5
1.44 ± 0.75
1.73 ± 1.10
48.27 ± 4.45
45.08 ± 5.18
9
0.20 ± 0.03
0.18 ± 0.04
25.30 ± 4.34
0.9
126.5
1.04 ± 0.17
36.29 ± 4.11
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Table 2. Opioid receptor binding affinity and MOR [ S]ꢀGTPγS functional assay results for 6αꢀ
INTA analogues.
0
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K (nM)
i
Selectivity
MOR [ S]ꢀGTPγS
assay
Compd.
R
µ
κ
δ
κ/µ
δ/µ
EC (nM) % Emax of
50
DAMGO
10
0.36 ± 0.03
0.29 ± 0.04
0.93 ± 0.13
0.98 ± 0.13
14.24 ± 2.78
10.54 ± 2.89
2.6
3.4
39.5
36.3
6.75 ± 3.20
1.00 ± 0.13
36.91 ± 2.40
1
1
33.34 ± 3.51
28.90 ± 0.95
32.24 ± 1.26
12
0.26 ± 0.04
0.76 ± 0.11
1.49 ± 0.35
3.45 ± 0.99
9.26 ± 2.83
5.7
4.5
35.6
35.2
4.99 ± 2.19
1.55 ± 0.08
1
1
3
4
26.72 ± 7.72
0.43 ± 0.05
0.23 ± 0.02
1.63 ± 0.28
1.69 ± 0.35
12.78 ± 3.36
13.69 ± 1.39
3.8
7.3
29.7
59.5
6.06 ± 1.25
3.85 ± 2.32
26.62 ± 0.66
19.11 ± 3.31
15 (NAN)
1
6
0.84 ± 0.12
0.44 ± 0.04
3.14 ± 0.45
1.36 ± 0.18
9.17 ± 2.67
3.7
3.1
10.9
57.0
4.75 ± 0.31
2.09 ± 0.19
34.14 ± 0.97
41.55 ± 6.14
17
25.07 ± 6.83
1
8
0.29 ± 0.04
0.15 ± 0.03
6.73 ± 0.40
0.5
23.2
1.89 ± 1.04
54.89 ± 6.35
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Not surprisingly, INTA was identified as a MOR full agonist with subnanomolar potency (Table
3
5
1
) in the [ S]ꢀGTPγS functional assay, while compound 2 and 5 also showed high MOR
efficacies relative to DAMGO. In addition to INTA, compounds 2 and 4 have previously been
3
4
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0
synthesized by Le Naour et al. and the results we obtained were similar to theirs. For other
compounds, even though they carried the same substitution (indole moiety) as the side chain,
most of them showed moderate to low efficacy at the MOR. Particularly, compound 3 and
compound 15 (NAN) acted as MOR antagonists since both had efficacies lower than 20%. Based
on our previous studies, we have observed that compounds with less than 20% relative efficacies
26,28,31
for Gꢀprotein activation in MORꢀexpressing CHO cells tend to be antagonists in vivo.
Furthermore, INTA and NAN together with their respective diastereomers (compound 10 and
compound 6) were compared to understand how the stereochemistry at position 6 of the
epoxymorphinan skeleton would affect binding affinity and efficacy. It was observed that the
configuration of the linker at position 6 did not lead to a significant difference in their MOR
binding affinity but influenced their potency and efficacy on the MOR more evidently. For
instance, changing the linker of INTA from βꢀconfiguration to αꢀconfiguration (compound 10)
did not result in a significant change in MOR binding affinity (INTA K = 0.29 ± 0.04 nM,
i
compound 10 K = 0.36 ± 0.03 nM) but produced a 32ꢀfold and 2.5ꢀfold reduction in potency and
i
efficacy, respectively (INTA EC = 0.21 ± 0.01 nM, INTA E = 92.42 ± 2.81%, compound 10
5
0
max
EC50 = 6.75 ± 3.20 nM, compound 10 Emax = 36.91 ± 2.40%). Meanwhile, changing the
configuration of the linker in NAN from α to β (compound 6) did not result in a significant
change in MOR binding affinity either (NAN K = 0.23 ± 0.02 nM, compound 6 K = 0.19 ± 0.01
i
i
nM), but resulted in an increase in efficacy (NAN Emax = 19.11 ± 3.31%, compound 6 Emax
=
3
7.94 ± 5.85%). In all, it seemed that changing the linker from the αꢀconfiguration to the βꢀ
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configuration at position 6 resulted in an increase in efficacy on the MOR without affecting
binding affinity on the receptor significantly.
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WARM WATER IMMERSION ASSAY
35
To corroborate the findings of the [ S]ꢀGTPγS functional assay, an in vivo assay was conducted
using the warmꢀwater immersion assay to evaluate whether the compounds synthesized produced
acute antinociceptive effects (opioid agonists) or antagonized morphine’s antinociceptive effect
27
(opioid antagonists). The warmꢀwater immersion assay was performed as described previously.
The test was conducted 20 minutes after injection of the compounds because morphine’s effect
starts to peak 20 minutes after s.c. administration. The results were interpreted as the percentage
of maximum possible effect (% MPE). A higher % MPE indicates a stronger antinociception
effect by the ligand. Figure 2A and 2B showed the antinociceptive effects of these INTA
analogues at a single dose of 10 mg/kg. Similar to the report from Le Naour et. al, among all the
3
5
ligands tested, INTA, identified as the most potent and efficacious MOR agonists in the [ S]ꢀ
GTPγS functional assay, also acted as an opioid agonist with the highest antinociception in the
warmꢀwater immersion assay. The % MPE values of the remaining INTA analogues, except for
compounds 6 and 7, were less than 20%, which corresponded to their lower efficacy observed in
35
the [ S]ꢀGTPγS functional assay. The low % MPE values for compounds 2 and 5, which showed
3
5
high potency and efficacy in the [ S]ꢀGTPγS binding assay, could be due to their poor
pharmacokinetic properties such as low CNS permeability, since the tail immersion response is
5
5
mainly mediated at the level of the spinal cord in the CNS.
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A
B
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D
C
Figure 2. Warmꢀwater tail immersion assay in mice (n=5) at 56 ± 0.1 °C. All tested compounds were administered
subcutaneously (s.c.). Antinociceptive effects of (A) INTA analogues based on 6βꢀnaltrexamine, and (B) INTA
analogues based on 6αꢀnaltrexamine. Compounds (10 mg/kg) were injected at time 0. Twenty minutes after
injection, tail withdrawal response was assessed with warm water. Antagonism of the antinociceptive effect of
morphine by (C) INTA analogues based on 6βꢀnaltrexamine, and (D) INTA analogues based on 6αꢀnaltrexamine.
Tested compounds (10 mg/kg) were injected at time 0. Five minutes later, morphine (10 mg/kg) was administered.
Twenty minutes after morphine injection, tail flick was tested using warm water. * Indicates P < 0.05 compared to
vehicle, ** indicates P < 0.01 compared to vehicle while *** indicates P < 0.001. ns indicates P > 0.05. In Fig. 2A
and B, all compounds except 1 had significantly less antinociception than morphine. While in Fig. 2B and C, only
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compounds 6, 7, 15 and naltrexone significantly antagonized morphine’s antinociception compared to distilled water
treated mice.
Figures 2C and 2D shows the inhibition of morphine’s antinociceptive effect at 10 mg/kg in
the presence of each INTA indole analogue at a single dose of 10 mg/kg. As expected, NAN,
0
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which showed a high potency and low efficacy in the [ S]ꢀGTPγS functional assay, was
identified as the most potent opioid antagonist and its AD value was determined as 2.07 (0.40ꢀ
5
0
1
0.74) mg/kg (95% CL) from a following doseꢀresponse study (Figure 3). We also observed that
3
5
compound 3 which had a low efficacy in the [ S]ꢀGTPγS functional assay did not significantly
inhibit morphine’s antinociceptive effect. This could be due to its poor pharmacokinetic
properties preventing it from getting into the CNS.
Figure 3. Dose dependent studies on NAN for opioid antagonist effect. NAN had an AD50 value of 2.07 (0.40ꢀ
10.74) mg/kg (95% CL).
MOLECULAR MODELING STUDY
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SAR studies of numerous opioids have revealed that a cyclopropylmethyl group on the tertiary
5
6
amino group at position 17 typically leads to opioid antagonism as observed with naltrexone as
well as NAN. On the contrary, INTA, which also carries a cyclopropylmethyl group at position
1
1
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0
1
7, acted as an MOR agonist. Hence, docking studies were conducted to understand how these
two structurally similar compounds, INTA and NAN (Figure 4), with the identical ‘message’
structure but slightly different ‘address’ portions produced almost opposite functional effects.
The ligands INTA and NAN were both sketched using SybylX2.1, and then docked into the
57
58
agonistꢀbound (PDB ID: 5C1M) and antagonistꢀbound (PDB ID: 4DKL)
MORs using
5
9
GOLD 5.4, respectively. The highest scored (CHEMꢀPLP) docking solutions were chosen as
active
inactive
the optimal binding poses, named INTA_MOR
and NAN_MOR
complexes.
Figure 4. The chemical structures of INTA (A) and NAN (B) together with their atom notation derived from the
complexes obtained from docking studies. The ‘message’ and ‘address’ portions of the molecules are marked out in
different colors.
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Figure 5. Binding poses of (A) INTA in the agonistꢀbound (5C1M) and (B) NAN in the antagonistꢀbound (4DKL)
MOR crystal structures from docking results. Protein shown as cartoon model in lightꢀgreen (A) and lightꢀpink (B);
INTA, NAN, and key amino acid residues shown as stick model. Carbon atoms: INTA (cyan), NAN (magenta),
amino acid residues in (A) (green) and in (B) (orange); oxygen atoms (red); nitrogen atoms (blue). 2D
representations of binding modes of (A) INTA and (B) NAN in 5C1M and 4DKL, respectively, are also shown.
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From the docking results, it was observed that the epoxymorphinan moiety (‘message’ portion)
of both INTA and NAN bound to the MOR orthosteric binding site in a similar fashion as
2
6,60
reported for other known MOR ligands.
The ‘message’ portions formed ionic interactions
1
1
1
1
1
1
1
1
1
1
2
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0
with D147, hydrogen bonding interactions with Y148, and hydrophobic interactions with
residues M151, W293, and H297. Meanwhile, the indole ring (‘address’ portion) of INTA
seemed to be able to form hydrophobic interactions with H54 (Figure 5A), while this interaction
inactive
did not exist in the NAN_MOR
complex. Moreover, the ‘address’ portions of both INTA
and NAN could form πꢀπ stacking interactions with W318 located at the allosteric binding site,
active
but the interaction in the INTA_MOR
complex was stronger than that in the
inactive
NAN_MOR
complex based on the distances between the ligand and the residue (Table 3).
Another observation from the docking study of INTA was that in order to accommodate the βꢀ
configuration of the indole side chain in the protein, the cyclohexyl ring that the side chain
attached to had to adopt a twisted boat conformation. In the case of NAN, the cyclohexyl ring
actually was in a twisted chair conformation to retain the αꢀconfiguration of the indole side
chain.
Many studies have proved that molecular dynamics (MD) simulation is an effective method to
6
1–64
explore the binding modes between proteins and their ligands.
Therefore, an MD simulation
active
inactive
study was further conducted on the INTA_MOR
and NAN_MOR
complexes using
NAMD 2.8 to investigate the potential allosteric modulation mechanisms of the ‘address’
portions of INTA and NAN to the binding of their ‘message’ portions with the orthosteric
65
binding site of the MOR. Prior to conducting the MD simulations, the two ligandꢀreceptor
6
6
complexes were first embedded in a lipid (POPC) bilayer membrane using VMD 1.9.3. Next,
each complex was solvated by TIP3 water layers. After that, any water molecules and POPC
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lipid molecules at a distance of 0.75 Å or less from the ligandꢀreceptor complex were removed.
Finally, sodium and chloride ions were added to the solvated system to make the ion
concentration approximately 0.1 nM. A representative ligandꢀreceptor complex embedded in a
POPC bilayer membrane and solvated by a water box is displayed in the supporting information
0
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(Figure S1, A).
active
inactive
A 10 ns MD simulations were then conducted for the INTA_MOR
and NAN_MOR
complexes. After the simulation, the rootꢀmeanꢀsquare deviation (rmsd) values of all the protein
backbone atoms relative to the respective starting structures were calculated to evaluate the
active
stability of the two systems (Figure S1, B). The average rmsd values of the INTA_MOR
and
inactive
NAN_MOR
complexes were determined as 1.45 Å and 1.31 Å, respectively.
Table 3. The measured shortest distances between atoms on critical amino acid residues and
active
inactive
atoms on the ligands from INTAꢀMOR
MD simulations.
and NANꢀMOR
complexes before and after
Complex
Atom of
ligand
Atom of
Distance from docking
study (Å)
Distance from MD
(Å)
residue
ND1@H54
OD2@D147
OH@Y148
CB@N150
CE@M151
CZ3@W293
CE1@H297
NZ@K303
CH2@W318
OD2@D147
active
INTA_MOR
N37@INTA
N21@INTA
O15@INTA
C37@INTA
C5@INTA
C4@INTA
C6@INTA
C63@INTA
C58@INTA
N21@NAN
3.0
4.0
3.4
5.5
3.8
3.8
3.3
3.1
3.2
3.0
4.3
3.1
4.0
4.5
4.7
4.1
4.0
4.3
3.9
3.1
inactive
NAN_MOR
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O15@NAN
C37@NAN
C26@NAN
C34@NAN
O54@NAN
C52@NAN
C4@NAN
C6@NAN
C63@NAN
C63@NAN
OH@Y148
3.0
6.5
3.6
8.6
5.2
5.3
5.4
4.5
3.6
3.7
3.5
6.8
4.2
4.8
4.5
3.9
5.7
5.4
6.9
5.4
CG@N150
CE@M151
CD2@L232
NZ@K233
CE@K233
CZ3@W293
CE1@H297
NZ@K303
CZ2@W318
1
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0
Previous studies have shown that when a system’s rmsd value is less than 3 Å, it indicates that
6
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the system has achieved dynamic equilibrium.
In addition, it seemed that the rmsd values of
both systems remained stable after 5 ns MD simulations (Figure S1, B). Therefore, it can be
concluded that both systems achieved stability after 10 ns MD simulations and snapshots from
the last 4 ns of MD simulations were collected to analyze the interactions between the ligand and
the receptor.
As shown in Figures 6A and 6B, owing to the hydrophobic interactions with W318 and H54, the
‘
address’ portion of INTA still interacted with the same domain (K303 and W318) of the
allosteric binding site in the active MOR after 10 ns MD simulations (Table 3, Figure S2). On
the other hand, the ‘address’ portion of NAN formed new hydrophobic interactions with L232
and K233 and its binding domain seemed to shift from K303 and W318 to L232 and K233 (a
5
8
residue that formed a covalent bond with βꢀFNA in the MOR inactive crystal structure) after 10
ns MD simulations (Table 3). As a consequence, the cyclopropylmethyl group of INTA was
buried deeply into the orthosteric binding site, and much closer to N150 than that of NAN (Table
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) to induce a hydrophobic interaction. This hydrophobic interaction could stabilize the
conformation of the side chain of N150, which may further help secure the hydrogen bonding
interaction between the side chain of N150 and the backbone carbonyl of I146 in the
active
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INTA_MOR
complex. Such a postulation was based on the fact that the distances between
the nitrogen atom at the side chain of N150 and the backbone carbonyl of I146 in the
active
INTA_MOR
complex was 3.2 Å, (Figure 6A) very close to the same measurement in the
5
7
BU72ꢀbound active MOR crystal structure (2.8 Å) respectively.
Another observation from the binding of the ‘address’ portion of both INTA and NAN with the
allosteric binding site was that the distance between the aromatic moiety of epoxymorphinan
skeleton in INTA and W293 (4.1 Å) was 1.6 Å shorter than that of NAN to the same residue (5.7
Å) (Table 3, Figures 6A and 6B). As previous studies reported, the hydrophobic interaction
between an agonist and W293 could stabilize the conformation of the side chain of W293 in its
5
7
active state. Therefore, this may also provide another possible explanation on INTA acting as
an agonist.
Bitopic ligands typically carry two distinct parts that interact with the orthosteric binding site and
allosteric binding site and are connected by a linker. Since the indole side chain (the ‘address’
portion of the two molecules) attached to the ‘message’ portion of the epoxymorphinan skeleton
through opposite configurations, we further looked into how different linkers would influence
their function on the MOR. As INTA has a βꢀconfiguration linker while NAN has an α one, this
led to opposite conformations of the cyclohexyl ring in the skeletons in order to accommodate
the ‘address’ portion of the molecules when the ‘message’ portions of NAN and INTA bound to
the orthosteric binding site of the MOR. This may further make the ‘message’ portion and
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cyclopropylmethyl group of INTA reach deeply into the orthosteric binding site and form
interactions with W293 and N150, respectively, to stabilize their active conformations.
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active
inactive
Figure 6. The binding modes of INTA_MOR
(A) and NAN_MOR
(B) complexes after MD simulations.
active
inactive
Protein shown as cartoon model in lightꢀgreen (INTA_MOR
complex) and lightꢀpink (NAN_MOR
complex); INTA, NAN, and key amino acid residues shown as stick model. Carbon atoms: INTA (cyan); NAN
active
inactive
(
magenta); key amino acid residues in INTA_MOR
complex (green) and in NAN_MOR
complex (orange).
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Oxygen atoms (red); nitrogen atoms (blue). The red dash lines represent the critical distances discussed. 2D
active
inactive
representations of binding modes of (A) INTA and (B) NAN in INTA_MOR
complexes, respectively are also shown.
and in NAN_MOR
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CONCLUSION
In summary, a series of 6αꢀ and 6βꢀindolylacetamidonaltrexamine derivatives were prepared to
understand the SAR of INTA. NAN, as one of INTA analogues, was identified as an MOR
antagonist from in vitro radioligand competition binding and functional assays. Further in vivo
studies showed that NAN antagonized morphine’s antinociceptive effects while INTA produced
potent antinociception. Molecular modeling studies were conducted to further understand how
two structurally similar compounds, NAN and INTA, showed almost opposite functional
activities. The hydrophobic interactions between the ‘address’ portion of INTA and residues
W318 and H54 at the allosteric binding site may cause the ‘message’ portion and
cyclopropylmethyl group of INTA to reach deeply into the orthosteric binding site and form
favorable interactions with W293 and N150 to stabilize the MOR in its active state
conformation. On the other hand, the ‘address’ portion of NAN formed hydrophobic interactions
with residues L232 and K233 at another allosteric binding site, which prevented the message
portion of NAN to reach deep enough into the orthosteric binding site. In other words, the indole
moiety of INTA may be acting as a PAM while that of NAN may be acting as a NAM at the
MOR. Since NAN and INTA have the same ‘message’ portion that bind to the orthosteric
binding site and distinct ‘address’ portions that may interact with the allosteric binding site,
NAN and INTA may be called bitopic ligands. These results further suggested that even though
two ligands may carry identical ‘message’ portion, they could “deliver” totally opposite function
if the ‘address’ portion (even with some minor structural differences) of the compounds do not
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interact with the same domain of the allosteric binding site of MOR. It is believed that such an
elaboration of the “messageꢀaddress” concept may enhance our comprehension of structureꢀ
activity relationship of opioid ligands and provide insight for future rational molecule design.
1
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EXPERIMENTAL SECTION
Chemical Synthesis. General methods. Chemical reagents were purchased from either Sigmaꢀ
Aldrich or Alfa Aesar. Reactions were monitored using Analtech Uniplate F254 TLC plates.
Compounds were purified using silica gel columns (230−400 mesh, Merck). The IR spectra were
obtained using a Thermo Scientific smart iTR instrument. Proton (400 MHz) and carbonꢀ13 (100
MHz) nuclear magnetic resonance (NMR) spectra were acquired at ambient temperature with
tetramethylsilane as the internal standard on a Bruker Ultrashield 400 Plus spectrometer. MS
analysis was performed using the Perkin Elmer AxION TOF mass spectrometer. HPLC analysis
was done with a Varian ProStar 210 system on MicrosorbꢀMV 100ꢀ5 C8/C18 column (250 mm ×
4
.6 mm) at 254 nm, eluting with acetonitrile (0.1% TFA)/water (40/60) at 1 mL/min over 30
min. All above analytical methods were used to determine purity of the newly synthesized
compounds, and their purity is confirmed as ≥ 95%.
General procedure for amide coupling reaction. The carboxylic acid derivatives (3 eq.),
hydrobenzotriazole (HOBt) (3 eq.), 4 Å molecular sieves, triethylamine (9 eq.), Nꢀ(3ꢀ
dimethylaminopropyl)ꢀN′ꢀethylcarbodiimide
hydrochloride
(EDCI)
(3
eq.),
and
dimethylformamide (DMF) 2 mL were added into a threeꢀnecked flask on an iceꢀwater bath and
stirred for 15 minutes. Naltrexamine (1 eq.) suspended in 2 mL DMF was added dropwise and
then stirred on the water bath for 24 hours. When the reaction was completed, the reaction
mixture was filtered with celite and the filtrate was concentrated to remove DMF. MeOH (7 mL)
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and K CO (3 eq.) were added to the concentrate and stirred at ambient temperature. When
2
3
hydrolysis of the ester at position 3 was complete, the mixture was filtered through celite again
and concentrated to remove MeOH. The mixture was then purified using column
2
7
0
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chromatography with dichloromethane: MeOH (20:1) and 1% NH OH to give the free base.
4
1
13
Upon confirmation by H NMR and C NMR, the free base was then transformed into
hydrochloride salt by dissolving in MeOH (0.1 mL) and dichloromethane (2 mL), adding HCl
methanol solution (1.25 M, 4 equiv) on an iceꢀwater bath, and stirring for 5 min. Diethyl ether
(10 mL) was then added. Two hours later, the precipitate was collected by filtration and dried in
1
vacuum to give the target compound as a hydrochloride salt, which was characterized by H
1
3
NMR, C NMR, IR, MS and the purity determined using HPLC (For spectra see Supporting
Information).
17ꢀCyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ(indoleꢀ2ꢀcarboxamido)morphinan
(
1)
The title compound was prepared by following the general procedure in 32% yield. H NMR
(400 MHz, DMSOꢀd6) 11.60 (s, 1H, exchangeable), 9.33 (s, 1H, exchangeable) 8.88 (s, 1H,
exchangeable), 8.73 (d, J = 7.92 Hz, 1H, exchangeable), 7.62 (d, J = 7.96 Hz, 1H), 7.42 (d, J =
.24 Hz, 1H), 7.18 (m, 2H), 7.04 (t, J = 7.52 Hz, 1H), 6.73 (d, J = 8.08 Hz, 1H), 6.66 (d, J = 7.96
Hz, 1H), 6.24 (s, 1H, exchangeable), 4.84 (d, J = 7.72 Hz, 1H), 3.89 (s, 1H), 3.71 (m, 1H), 3.33
m, 2H), 3.11ꢀ3.06 (m, 2H), 2.88 (m, 1H), 2.50 (m, 2H), 1.91 (q, J = 11.84 Hz, 1H), 1.80 (d, J =
3.56 Hz, 1H) 1.63 (m, 1H), 1.49ꢀ1.40 (m, 2H), 1.09 (m, 1H), 0.68 (m, 1H), 0.60 (m, 1H), 0.52
1
δ
8
(
1
1
3
(
m, 1H), 0.42 (m, 1H). C NMR (100 MHz, DMSOꢀd6)
δ 160.73, 142.02, 141.09, 136.23,
1
31.30, 129.62, 126.97, 125.26, 123.46, 121.52, 120.64, 119.83, 119.39, 117.87, 112.23, 102.63,
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9.85, 69.62, 61.67, 56.69, 50.66, 46.37, 45.61, 29.37, 23.78, 22.91, 5.62, 5.07, 2.55. M.p. 235ꢀ
+
+
37 °C. HRMS (m/z) [M] calculated for C H N O 485.2315, found [MH] 486.2411. IR
2
9
31
3
4
ꢀ1
(ATR, cm ) ν
3212, 2167, 1975, 1617, 1551, 1506, 1315, 1239,1125, 1034, 815, 747, 668.
max
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
4
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0
1
2
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7
8
9
0
1
2
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9
0
1
2
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3
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9
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1
2
3
4
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7
8
9
0
1
7ꢀCyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ(indoleꢀ3ꢀcarboxamido)morphinan
2)
The title compound was prepared by following the general procedure in 27% yield. H NMR
400 MHz, DMSOꢀd6) 11.64 (s, 1H, exchangeable), 9.34 (s, 1H, exchangeable), 8.89 (s, 1H,
(
1
(
δ
exchangeable), 8.13ꢀ8.10 (m, 3H, 1 proton exchangeable), 7.42 (d, J = 7.88 Hz, 1H), 7.16ꢀ7.06
(m, 2H), 6.73 (d, J = 8.08 Hz, 1H), 6.65 (d, J = 8.12 Hz, 1H), 6.24 (s, 1H, exchangeable), 4.82
(d, J = 7.76 Hz, 1H), 3.90 (d, J = 4.28 Hz, 1H), 3.70 (m, 1H), 3.36 (m, 1H), 3.12ꢀ3.05 (m, 3H),
2
.88 (m, 2H), 1.89 (q, J = 12.88 Hz, 1H), 1.78 (d, J = 13.2 Hz, 1H), 1.63 (m, 1H), 1.48ꢀ1.41 (m,
13
2
H), 1.09 (m, 1H), 0.68 (m, 1H), 0.50 (m, 1H), 0.52 (m, 1H), 0.42 (m, 1H). C NMR (100 MHz,
164.46, 142.18, 141.03, 135.90, 129.70, 127.60, 125.86, 121.98, 120.89, 120.63,
20.43, 119.29, 117.89, 111.79, 110.30, 90.17, 69.69, 61.70, 56.70, 50.21, 46.38, 45.60, 29.40,
DMSOꢀd6)
δ
1
2
+
7.30, 23.99, 22.93, 5.61, 5.12, 2.53. M.p. 272ꢀ274 °C. HRMS (m/z) [M] calculated for
+
ꢀ1
C H N O 485.2315, found [MH] 486.2411. IR (ATR, cm ) ν 3267, 1671, 1621, 1539,
max
29
31
3
4
1
505, 1455, 1313, 1176, 1119.
7ꢀCyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ(indoleꢀ4ꢀcarboxamido)morphinan
1
(3)
The title compound was prepared by following the general procedure in 31% yield. H NMR
400 MHz, DMSOꢀd6) 11.31 (s, 1H, exchangeable), 9.34 (s, 1H, exchangeable), 8.87 (s, 1H,
exchangeable), 8.39 (d, J = 8.12 Hz, 1H, exchangeable), 7.55 (d, J = 8.04 Hz, 1H), 7.48 (d, J =
1
(
δ
7
.16 Hz, 1H), 7.43 (m, 1H), 7.15 (t, J = 7.72 Hz, 1H), 6.88 (s, 1H), 6.73 (d, J = 8.12 Hz, 1H),
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.66 (d, J = 8.16 Hz, 1H), 6.19 (s, 1H, exchangeable), 4.89 (d, J = 7.76 Hz, 1H), 3.88 (d, J = 4.92
Hz, 1H), 3.77ꢀ3.72 (m, 1H), 3.32 (m, 1H), 3.13ꢀ3.04 (m, 2H), 2.87 (m, 1H), 1.93 (q, J = 12.08
Hz, 1H), 1.78 (d, J = 13.68 Hz, 1H), 1.65 (m, 1H), 1.46 (m, 2H), 1.09 (m, 2H), 0.70ꢀ0.68 (m,
13
0
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9
0
1
H), 0.60 (m, 1H), 0.53ꢀ0.50 (m, 1H), 0.43ꢀ0.41 (m, 1H). C NMR (100 MHz, DMSOꢀd6)
δ
1
67.77, 142.15, 141.02, 136.35, 129.67, 126.30, 126.09, 125.75, 120.64, 120.19, 119.34, 118.50,
117.90, 114.31, 101.78, 89.92, 69.68, 61.68, 56.68, 50.89, 46.37, 45.61, 29.43, 27.29, 23.66,
+
2
4
2.90, 5.58, 5.11, 2.50. M.p. 259ꢀ261 °C. HRMS (m/z) [M] calculated for C H N O
2
9
31
3
4
+
ꢀ1
85.2315, found [MH] 486.2434. IR (ATR, cm ) ν
3207, 2166, 1639, 1503, 1455, 1319,
max
1237, 1195, 1123, 1037, 917.
7ꢀCyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ(indoleꢀ5ꢀcarboxamido)morphinan
4)
The title compound was prepared by following the general procedure in 61% yield. H NMR
400 MHz, DMSOꢀd6) 11.36 (s, 1H, exchangeable), 9.32 (s, 1H, exchangeable) 8.88 (s, 1H,
exchangeable), 8.48 (d, J = 8.08 Hz, 1H, exchangeable), 8.18 (s, 1H), 7.66 (dd, J =1.56 Hz, J =
1
(
1
(
δ
1
2
8
2
4
1
.56 Hz, 1H), 7.42 (m, 2H), 6.73 (d, J = 8.12 Hz, 1H), 6.65 (d, J = 8.16 Hz, 1H), 6.53 (t, J =
1
.04 Hz, J = 2.00 Hz, 1H), 6.20 (s, 1H, exchangeable), 4.89 (d, J =7.80 Hz, 1H), 3.88 (d, J =
2
.96 Hz, 1H), 3.71 (m, 1H), 3.35 (m, 2H), 3.11ꢀ3.04 (m, 2H), 2.87 (m, 1H), 1.90 (q, J = 12.6 Hz,
H), 1.78 (d, J = 13.76 Hz, 1H), 1.62 (m, 1H), 1.49ꢀ1.39 (m, 2H), 1.09 (m, 1H), 0.68 (m, 1H),
1
3
0.59 (m, 1H), 0.52 (m, 1H), 0.42 (m, 1H). C NMR (100 MHz, DMSOꢀd )
δ
166.77, 142.30,
41.30, 137.41, 129.77, 126.99, 126.60, 125.29, 120.56, 120.49, 119.91, 119.16, 117.92, 110.80,
02.05, 90.06, 69.80, 61.81, 56.74, 51.11, 46.52, 45.62, 29.44, 27.41, 23.93, 23.06, 5.72, 5.09,
6
1
1
+
+
2.65. M.p. 290ꢀ292 °C. HRMS (m/z) [M] calculated for C H N O 485.2315, found [MH]
2
9
31
3
4
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ꢀ1
4
86.2434. IR (ATR, cm ) ν
3125, 2162, 1980, 1622, 1595, 1547, 1360, 1311, 1249, 1124,
max
1
037, 919, 760, 672.
17ꢀCyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ(indoleꢀ6ꢀcarboxamido)morphinan
5)
The title compound was prepared by following the general procedure in 29% yield. H NMR
(400 MHz, DMSOꢀd6) 11.43 (s, 1H, exchangeable), 9.33 (s, 1H, exchangeable), 8.87 (s, 1H,
exchangeable), 8.54 (d, J = 7.4 Hz, 1H, exchangeable), 7.99 (s, 1H), 7.58 (s, 2H), 7.50 (s, 1H),
.73 (d, J = 6.36 Hz, 1H), 6.66 (d, J = 7.68 Hz, 1H), 6.49 (s, 1H), 6.20 (s, 1H, exchangeable),
4.90 (d, J = 5.92 Hz, 1H), 3.88 (s, 1H), 3.71 (m, 1H), 3.17ꢀ3.06 (m, 2H), 2.89 (m, 2H), 2.5 (s,
1
1
1
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2
2
2
2
2
2
2
3
3
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3
3
3
3
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3
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4
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0
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1
δ
6
1
0
H), 1.90 (m, 1H), 1.78 (m, 1H), 1.62 (m, 1H), 1.49ꢀ1.39 (m, 2H), 1.09 (m, 2H), 0.68 (m, 1H),
13
.60 (m, 1H), 0.52 (m, 1H), 0.42 (m, 1H). C NMR (100 MHz, DMSOꢀd )
δ 166.85, 142.16,
6
140.23, 135.05, 129.76, 127.74, 127.20, 119.34, 118.39, 117.95, 116.98, 111.16, 101.20, 90.74,
6
2
9.62, 61.76, 58.35, 51.50, 49.99, 39.96, 39.76, 30.60, 29.99, 24.65, 22.25, 3.72, 3.46. M.p. 254ꢀ
+
+
56 °C. HRMS (m/z) [M] calculated for C H N O 485.2315, found [MH] 486.2396. IR
2
9
31
3
4
ꢀ1
(
ATR, cm ) ν 3223, 2152, 1970, 1608, 1551, 1312, 1035, 920, 820, 740.
max
1
7ꢀCyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ(indoleꢀ7ꢀcarboxamido)morphinan
6)
The title compound was prepared by following the general procedure in 56% yield. H NMR
(400 MHz, DMSOꢀd6) 11.07 (s, 1H, exchangeable), 9.37 (s, 1H, exchangeable), 8.90 (s, 1H,
exchangeable), 8.75 (d, J = 8.04 Hz, 1H, exchangeable), 7.78ꢀ7.75 (m, 2H), 7.35 (m, 1H), 7.10
t, J = 7.60 Hz, 1H), 6.74 (d, J = 8.12 Hz, 1H), 6.67 (d, J = 8.16 Hz, 1H), 6.49 (m, 1H), 6.26 (s,
1H, exchangeable), 4.93 (d, J = 7.8 Hz, 1H), 3.91 (d, J = 4.84 Hz, 1H), 3.82ꢀ3.78 (m, 2H), 3.18ꢀ
(
1
δ
(
3
.06 (m, 3H), 2.91ꢀ2.86 (m, 1H), 2.02ꢀ1.93 (q, J = 11.56 Hz, 1H), 1.82 (d, J = 13.64 Hz, 1H),
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1
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1
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2
2
2
2
2
2
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3
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3
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3
3
3
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4
4
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.65 (m, 1H), 1.56ꢀ1.41 (m, 2H), 1.10 (t, J = 7.00 Hz, 2H), 0.71ꢀ0.68 (m, 1H), 0.62ꢀ0.60 (m,
1
3
H), 0.55ꢀ0.52 (m, 1H), 0.44ꢀ0.42 (m, 1H). C NMR (100 MHz, DMSOꢀd )
δ 164.50, 139.51,
6
138.46, 131.48, 127.15, 126.67, 123.95, 121.72, 118.20, 117.28, 117.02, 115.79, 115.40, 113.77,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
3
8.68, 87.35, 67.21, 62.45, 59.20, 54.25, 48.23, 43.86, 43.16, 26.96, 24.75, 21.14, 20.39, 12.43,
+
.07, 2.65. M.p. 246ꢀ248 °C. HRMS (m/z) [M] calculated for C H N O 485.2315, found
2
9
31
3
4
+
ꢀ1
[MH] 486.2411. IR (ATR, cm ) ν
3090, 2162, 1979, 1633, 1540, 1327, 1248, 1040.
max
1
7ꢀCyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ[2ꢀ(indolꢀ3ꢀyl)acetamido)morphinan
7)
The title compound was prepared by following the general procedure in 49% yield. H NMR
400 MHz, DMSOꢀd6) 10.89 (s, 1H, exchangeable), 9.32 (s, 1H, exchangeable), 8.82 (s, 1H,
(
1
(
δ
exchangeable), 8.20 (d, J = 7.92 Hz, 1H, exchangeable), 7.54 (d, J = 7.76 Hz, 1H), 7.34 (d, J =
8.04 Hz, 1H), 7.18 (d, J = 2.08 Hz, 1H), 7.06 (t, J = 7.08 Hz, 1H), 6.98 (t, J = 7.24 Hz, 1H), 6.70
(
d, J = 8.12 Hz, 1H), 6.62 (d, J = 8.16 Hz, 1H), 6.14 (s, 1H, exchangeable), 4.60 (d, J = 7.84 Hz,
H), 3.82 (d, J = 5.2 Hz, 1H), 3.50 (q, J = 14.96 Hz, 2H), 3.40 (m, 1H), 3.32 (m, 1H), 3.28 (m,
H), 3.07ꢀ3.01 (dd, J = 5.96 Hz, J = 19.24 Hz, 2H), 2.84 (m, 1H), 2.43ꢀ2.39 (m, 2H), 1.68 (m,
1
1
2
0
1
2
H), 1.49ꢀ1.28 (m, 3H), 1.07 (m, 1H), 0.67ꢀ0.66 (m, 1H), 0.60ꢀ0.56 (m, 1H), 0.51ꢀ0.48 (m, 1H),
13
.41ꢀ0.39 (m, 1H). C NMR (100 MHz, DMSOꢀd )
δ 170.94, 170.86, 141.99, 140.99, 135.88,
6
129.55, 127.03, 123.57, 121.02, 120.58, 119.36, 118.53, 118.42, 117.83, 111.28, 108.49, 99.49,
6
2
9.56, 61.54, 56.62, 46.32, 45.49, 32.78, 29.17, 27.21, 23.45, 22.82, 5.55, 5.08, 2.46. M.p. 292ꢀ
+
+
94 °C. HRMS (m/z) [M] calculated for C H N O 499.2471, found [MH] 500.2539. IR
3
0
33
3
4
ꢀ1
(ATR, cm ) ν
3270, 1666, 1549, 1463, 1303,1274, 1232, 1174, 1124, 1030, 898.
max
17ꢀCyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ[3ꢀ(indolꢀ3ꢀyl)
propanamido)morphinan (8)
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The title compound was prepared by following the general procedure in 70% yield. H NMR
(
400 MHz, DMSOꢀd6) 10.77 (s, 1H, exchangeable), 9.34 (s, 1H, exchangeable), 8.83 (s, 1H,
δ
exchangeable), 8.14 (d, J = 7.8 Hz, 1H, exchangeable), 7.52 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.04
Hz, 1H), 7.10 (s, 1H), 7.06 (t, J = 7.96 Hz, 1H), 6.97 (t, J = 7.88 Hz, 1H), 6.72 (d, J = 8.12 Hz,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H), 6.63 (d, J = 8.16 Hz, 1H), 6.18 (s, 1H, exchangeable), 4.55 (d, J = 7.8 Hz, 1H), 3.85 (s, 1H),
3.45 (m, 1H), 3.34ꢀ3.29 (m, 2H), 3.09ꢀ3.00 (m, 2H), 2.92ꢀ2.83 (m, 3H), 2.45ꢀ2.42 (m, 4H), 1.71ꢀ
1
.68 (m, 2H), 1.53ꢀ1.50 (m, 1H), 1.42 (d, J = 9.76 Hz, 1H), 1.33 (t, J = 12.32 Hz, 1H), 1.08 (m,
1
3
1
H), 0.68ꢀ0.63 (m, 1H), 0.60ꢀ0.57 (m, 1H), 0.52ꢀ0.48 (m, 1H), 0.42ꢀ0.38 (m, 1H). C NMR (100
MHz, DMSOꢀd6)
δ
141.98, 140.93, 135.97, 129.53, 126.86, 121.96, 120.98, 120.64, 119.41,
1
2
18,26, 117.89, 113.57, 111.27, 89.91, 69.58, 61.58, 56.67, 50.47, 46.31, 45.49, 39.62, 29.21,
+
7.21, 23.43, 22.83, 20.85, 5.55, 5.11, 2.47, 0.012. M.p. 279ꢀ281 °C. HRMS (m/z) [M]
+
ꢀ1
calculated for C H N O 513.2628, found [MH] 514.2714. IR (ATR, cm ) ν 3187, 2160,
max
3
1
35
3
4
1
640, 1541, 1505, 1450, 1325, 1271, 1234, 1124, 1032, 854.
1
7ꢀCyclopropylmethylꢀ3,14βꢀdihydroxyꢀ4,5αꢀepoxyꢀ6βꢀ[4ꢀ(indolꢀ3ꢀyl)
butanamido)morphinan (9)
1
The title compound was prepared by following the general procedure in 39% yield. H NMR
(
400 MHz, DMSOꢀd6)
exchangeable), 8.09 (d, J = 7.88 Hz, 1H, exchangeable), 7.51 (d, J = 7.84 Hz, 1H), 7.33 (d, J =
.04 Hz, 1H), 7.12 (d, J = 2.08 Hz, 1H), 7.08 (t, J = 7.46 Hz, 1H), 6.97 (t, J = 7.42 Hz, 1H), 6.72
d, J = 8.12 Hz, 1H), 6.64 (d, J = 8.16 Hz, 1H), 6.18 (s, 1H, exchangeable), 4.56 (d, J = 7.84 Hz,
H), 3.85 (d, J = 4.84 Hz, 1H), 3.09ꢀ3.03 (dd, J = 5.88 Hz, J = 19.48 Hz, 2H), 2.88ꢀ2.84 (m,
δ 10.79 (s, 1H, exchangeable), 9.35 (s, 1H, exchangeable), 8.84 (s, 1H,
8
(
1
1
2
1H), 2.68 (t, J = 7.48 Hz, 2H), 2.44ꢀ2.41 (m, 2H), 2.16 (t, J = 7.32 Hz, 2H), 1.89ꢀ1.85 (m, 2H),
1
.75ꢀ1.70 (m, 2H), 1.56ꢀ1.53 (m, 1H), 1.43 (d, J = 9.68 Hz, 1H), 1.38ꢀ1.31 (m, 1H), 1.12ꢀ1.05
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