Transformation of naltrexone into mesembrane and investigation of the binding properties of its intermediate derivatives to opioid receptors

Article abstract of DOI:10.1016/j.bmc.2014.12.032

We transformed naltrexone (5) with the morphinan skeleton into mesembrane (4) belonging to the Sceletium alkaloids via key intermediate 6, characterized by a cis-fused hydroindole skeleton with a suspended phenyl ring fixed by an epoxy bridge. We then investigated the binding affinities of 4 and the key intermediate 6 derivatives to the opioid receptors. Among the tested compounds, 15′, with a cis-fused hydroindole core, bound to the three opioid receptor types with strong to moderate affinities. The observed differences of binding affinities among the tested compounds were reasonably explained by the conformational analyses of the compounds. The structure-activity relationship (SAR) of the tested compounds like 15′ with the hydroindole structure was completely different from the reported SAR of morphinan derivatives with the hydroisoquinoline skeleton. Compound 15′ with a structure that differs from the morphinans represents a useful fundamental skeleton with a novel chemotype that may contribute to the development of new opioid ligands.

Full text of DOI:10.1016/j.bmc.2014.12.032

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Transformation of naltrexone into mesembrane and investigation
of the binding properties of its intermediate derivatives to opioid
receptors
a
b
a
c
Kazuya Konoura a, Hideaki Fujii a, , Satomi Imaide , Hiroaki Gouda , Shigeto Hirayama , Shuichi Hirono ,
⇑
Hiroshi Nagase a,d
a Department of Medicinal Chemistry, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
b School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
c Department of Physical Chemistry for Drug Design, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
d International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We transformed naltrexone (5) with the morphinan skeleton into mesembrane (4) belonging to the
Sceletium alkaloids via key intermediate 6, characterized by a cis-fused hydroindole skeleton with a
suspended phenyl ring fixed by an epoxy bridge. We then investigated the binding affinities of 4 and
the key intermediate 6 derivatives to the opioid receptors. Among the tested compounds, 150, with a
cis-fused hydroindole core, bound to the three opioid receptor types with strong to moderate affinities.
The observed differences of binding affinities among the tested compounds were reasonably explained
by the conformational analyses of the compounds. The structure–activity relationship (SAR) of the tested
compounds like 150 with the hydroindole structure was completely different from the reported SAR of
morphinan derivatives with the hydroisoquinoline skeleton. Compound 150 with a structure that differs
from the morphinans represents a useful fundamental skeleton with a novel chemotype that may
contribute to the development of new opioid ligands.
Received 18 November 2014
Revised 15 December 2014
Accepted 15 December 2014
Available online xxxx
Keywords:
Mesembrane
Opioid
Sceletium alkaloids
Conformational analysis
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
nous agonists exert pharmacological responses via various effec-
tors, whereas biased agonists selectively induced cell responses
G protein-coupled receptors (GPCRs) are among the most
valuable drug targets and more than 25% of the FDA-approved
drugs interact with them.1 Originally, a substrate was thought to
interact with a GPCR one for one, and that an agonist exerted phar-
macological responses via inherent pathways. However, recent
investigations have revealed that the actual phenomena are more
complicated. GPCRs exist not only as monomeric receptors but also
as homo- or hetero-dimers and the dimeric receptors are the func-
tional unit in a living organism.2 Concerning the opioid receptor,
heterodimerization is known to occur between opioid receptor
through an effector.4–6 Moreover, GPCR splice variants, which are
mutated receptors produced from a gene with more than one exon,
have also been investigated. A large series of splice variants of the
MOR have already been identified.7,8 These phenomena, that is,
receptor dimerization, biased agonism, and splice variants have
been the subjects of investigations involving molecular biology,
molecular pharmacology, and the development of specific ligands.
For example, 60-guanidinonaltrindole (60-GNTI) and N-naphthoyl-
b-naltrexamine (NNTA) were reported to be DOR-KOR9 and
MOR-KOR10 agonists, respectively. Herkinorin and TRV-130 were
reported to be G protein-biased MOR agonists.11,12 Amidino-TAPA
produced antinociceptive effects via the MOR splice variants with
which the representative MOR agonist, morphine, did not inter-
act.13 Although the aforementioned specific agonists have been
reported, the development of novel and unique ligands is required
for the further investigation. Moreover, there have been almost no
structural guidelines for the design of such ligands. However, the
structures of the ligands might play an important role in
discrimination of receptor dimers, effectors engaging with GPCRs
(ligand bias), and/or receptor splice variants. Indeed, it has been
types such as d opioid receptor (DOR)—j opioid receptor (KOR),
l
opioid receptor (MOR)—DOR, and MOR—KOR as well as heterodi-
merization between opioid receptors and non-opioid receptors
such as MOR—cannabinoid CB1 receptor, DOR—chemokine CXCR2
receptor, and KOR—b2 adrenergic receptor.3 On the other hand,
GPCRs have been recently reported to engage effectors other than
G proteins such as b-arrestins. Traditional agonists like endoge-
⇑
Corresponding author. Tel.: +81 3 5791 6372; fax: +81 3 3442 5707.
E-mail address: fujiih@pharm.kitasato-u.ac.jp (H. Fujii).
http://dx.doi.org/10.1016/j.bmc.2014.12.032
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Konoura, K.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.032

2
K. Konoura et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Me
N
Me
N
Me
N
alkaloids (Fig. 1) had these binding determinants although the phe-
H
H
H
O
OH
O
nolic hydroxy group was masked by the methyl group. Moreover, it
is interesting that both the Sceletium alkaloids and a representative
opioid ligand like naltrexone (5) have a common structure, the
phenethylamine motif (indicated by red line, Fig. 1). However, nal-
trexone (5) has a trans-fused hydroisoquinoline structure with a
suspended phenyl ring fixed by both epoxy and methylene bridges,
whereas the Sceletium alkaloids possess a cis-fused hydroindole
skeleton with a freely rotatable phenyl ring. Therefore, we pro-
posed to transform naltrexone (5) into Sceletium alkaloids like
mesembrane (4) via a key intermediate 6 (Scheme 1), which has
cis-fused hydroindole skeleton with a suspended phenyl ring fixed
by an epoxy bridge,20 and to evaluate the binding affinities of
mesembrane (4) and key intermediate 6 derivatives for the opioid
receptors. We chose mesembrane (4) as a target compound
because among the Sceletium alkaloids 1–4, mesembrane (4) was
the structurally simplest compound lacking the oxo or hydroxy
groups which were potential substituents to interact with the opi-
oid receptors. Herein, we report the transformation from naltrex-
one (5) into mesembrane (4) and the binding abilities of 4 and
key intermediate 6 derivatives to the opioid receptors. We also
examine the structure–activity relationships (SARs) based on con-
formational analyses of the tested compounds.
OMe
OMe
OMe
OMe
OMe
OMe
mesembrenone (1)
mesembrenol (2)
mesembrine (3)
Me
N
H
OH
N
O
O
OMe
OH
naltrexone (5)
OMe
mesembrane (4)
Figure 1. Structures of Sceletium alkaloids 1–4 and naltrexone (5). The pheneth-
ylamine moieties are indicated by red lines.
reported that ligand bias would stem from a distinct receptor
conformation which is stabilized by each agonist. Therefore,
development of ligands with various chemotypes would be impor-
tant to investigate receptor dimers, ligand bias, and/or receptor
splice variants.
2. Results and discussion
Sceletium tortuosum ZembrinÒ, which is a mixture of three Scel-
etium alkaloids14 (mesembrenone (1) + mesembrenol (2) >70%,
mesembrine (3) <20%, Fig. 1), was recently reported to bind to
the opioid receptors with very weak affinities (10–30% of the con-
trol bindings remained).15 Mesembrane (4)16 is also a member of
the Sceletium alkaloids (Fig. 1). The basic nitrogen, phenyl ring,
and phenolic hydroxy group are known to be essential pharmaco-
phores to interact with the opioid receptors.17–19 The Sceletium
2.1. Transformation from naltrexone (5) into mesembrane (4)
The key intermediate 6 was prepared from naltrexone (5)
(Scheme 2). Naltrexone methyl ether, obtained by methylation of
the phenolic hydroxy group in naltrexone (5), was converted into
olefin 7 under Hofmann elimination reaction conditions. The oxo
and hydroxy groups in the obtained olefin 7 were sequentially
protected, followed by treatment with 2,2,2-trichloroethyl
Me
H
N
Me
N
OH
H
N
O
O
O
O
OMe
OH
naltrexone (5)
OMe
key intermediate 6
OMe
mesembrane (4)
Scheme 1. Strategy of transformation from naltrexone (5) into mesembrane (4) via key intermediate 6.
OH
OH
OAc
N
O
Troc
d) - f)
g), h)
a) - c)
O
N
O
N
O
O
O
O
Me
Me
OH
OMe
OMe
naltrexone (5)
7
8
Me
N
OH
H
OHC
O
O
O
O
Troc
Troc
N
N
Me
OHC
Me
i), j)
k)
O
O
O
O
l), m)
O
HO
O
HO
6
OMe
OMe
OMe
9
10
11
Scheme 2. Synthesis of the key intermediate 6. Reagents and conditions: (a) MeI, K2CO3, DMF, (b) MeI, DMF, then concentrated, (c) 1 M NaOH (aq), 1,4-dioxane, (d) ethylene
glycol, TMSOTf, CHCl3, (e) Ac2O, (f) Troc-Cl, TEA, CH2Cl2, 72% from 5, (g) O3, CH2Cl2, then Me2S, (h) TEA, MeOH, 73% from 8, (i) mCPBA, CH2Cl2, (j) TEA, MeOH, 36% from 9, (k)
Zn, AcOH, filtered, then NaBH3CN, 92%, (l) Tf2NPh, NaH, DME, then PdCl2(MeCN)2 (10 mol %), dppp (25 mol %), TEA, HCO2H, (m) 1 M HCl, 79% from 11.
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K. Konoura et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
OH
OH
OH
R1
N
R1
N
N
Wolff-Kishner reduction24,25
Wolff-Kishner reduction26-28
6
6
OH
O
5
R1 = CPM, allyl
R1 = CPM
O
O
R2 = H, Me
R2 = H
R3 = TBS, Me
OH
OR2
OR3
Scheme 3. Wolff–Kishner reduction of naltrexone derivatives. CPM: cyclopropylmethyl.
chloroformate (Troc-Cl) to afford compound 8. In the reaction of
(cyclopropylmethy)methylamine 7 with Troc-Cl, the cyclopro-
pylmethyl group was selectively dealkylated.21 After ozonolysis
of 8, the acetyl group was removed by methanolysis. The obtained
dialdehyde 9 was oxidized by m-chloroperbenzoic acid (mCPBA),
followed by methanolysis of the obtained formate groups to give
ketone 10. Deprotection of the Troc group in 10 and subsequent
treatment with sodium cyanoborohydride (NaBH3CN) provided
compound 11. The phenolic hydroxy group in 11 was removed
by triflation and subsequent palladium catalyzed reduction, fol-
lowed by deacetalization afforded the key intermediate 6. The
key intermediate 6 was determined to have cis-fused hydroindole
structure by ROESY specta (Fig. S1).
In morphinan derivatives, the presence or absence of the epoxy
bridge is known to critically affect the pharmacological properties
of the compounds.22,23 Therefore, from the viewpoint of the
structural diversity, we attempted to prepare both hydroindole
derivatives with and without the epoxy bridge. Although Wolff–
Kishner reduction is the general method for converting an oxo
group into a methylene moiety, the Wolff–Kishner reduction of
4,5-epoxymorphinans has been demonstrated to provide two
kinds of products (Scheme 3).24–29 In hope that both compounds
with and without the ether bond would be obtained, we attempted
the Wolff–Kishner reduction of the key intermediate 6. As we
expected,29 compounds 12 and 13 were prepared in 73% and 25%
yield, respectively (Scheme 4).
Mesembrane (4) was prepared from compound 12 (Scheme 4).
Catalytic hydrogenation of 12 and subsequent treatment with
N-phenylbis(trifluoromethanesulfonimide) (Tf2NPh) afforded triflate
14. Palladium-catalyzed reduction of 14 unexpectedly gave two
products, 15 and epi-15. The configurations of these compounds
were determined by NOE or ROESY spectra (Fig. 2). We are pres-
ently investigating why the epimerization occurred during the pal-
ladium-catalyzed reduction of 14. Selective iodination of 15 using
trifluoroacetic acid (TFA) as a solvent successfully proceeded to
afford 16. The position of the iodo group was determined by HMBC
spectroscopy (Fig. S2). After the halogen-lithium exchange reaction
of 16 with n-butyl lithium, treatment with triisopropyl borate
(B(Oi-Pr)3) followed by treatment with sodium perborate tetrahy-
drate (NaBO3ꢀ4H2O) yielded phenol 17 (39%) concomitantly with
15 (47%). The objective mesembrane (4) was obtained by methyl-
ation of the phenolic hydroxy group in 17 with trimethylsilyldia-
zomethane (TMSCHN2). The observed spectral data of prepared 4
were identical to the literature data.30–32 For the binding assays,
O-methyl groups in 13, 15, and epi-15 were deprotected to give
the corresponding 130, 150, and epi-150 (Scheme 4).
2.2. Binding affinities for the opioid receptors
The binding abilities of mesembrane (4), 13, 130, 15, 150, epi-15,
and epi-150 for the opioid receptors were evaluated by competitive
binding assays (Table 1 and S1). Although 13 displayed no binding
Me
N
Me
N
Me
H
H
H
N
a)
+
O
O
OH
O
OMe
OMe
OR
13 (R = Me)
b)
12
6
13' (R = H)
Me
N
Me
N
Me
N
H
H
H
e)
c), d)
12
+
OTf
OMe
OR
15 (R = Me)
OR
epi-15 (R = Me)
14
b)
b)
15' (R = H)
-15' (R = H)
epi
Me
N
Me
N
Me
N
H
H
H
f)
g)
h)
15
OMe
OMe
mesembrane (4)
OMe
OMe
I
OH
17
16
Scheme 4. Synthesis of mesembrane (4) from 6. Reagents and conditions: (a) H2NNH2ꢀH2O, KOH, ethylene glycol, 12: 73%, 13: 25%, (b) BBr3, CH2Cl2, 130: 23%, 150: 62%, epi-
150: 87%, (c) H2, Pd/C, AcOH, (d) Tf2NPh, NaH, DME, 83% from 12, (e) Pd(MeCN)2Cl2 (10 mol %), dppp (25 mol %), HCO2H, TEA, DME, 15: 40%, epi-15: 24%, (f) NIS, TFA, 85%, (g) n-
BuLi, THF, then B(Oi-Pr)3, NaBO3ꢀ4H2O, 17: 39%, 15: 47%, (h) TMSCHN2, MeOH, 81%.
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K. Konoura et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
H
H
H
R
N
R1
R1
Me
N
Me
N
H
H
N
N
H
H
OH
OH
O
O
H
H
H
H
NOE
OH
OR2
OR2
(-)-18 (R = Me)
(-)-19 (R = allyl)
morphine (20)
(R1 = Me, R2 = H)
nalorphine (21)
(R1 = allyl, R2 = H)
codeine (22)
23 (R1 = Me, R2 = H)
24 (R1 = R2 = Me)
OMe
OR
ROESY
15
epi-15
Figure 2. Observed NOE or ROESY spectra of 15 and epi-15.
(R1 = R2 = Me)
Table 1
Binding affinities for the opioid receptorsa
Figure 3. Structures of compounds (ꢁ)-18–24.
Compound
Ki (nM)
MORb
DORc
KORd
of morphinan derivatives with a hydroisoquinoline fundamental
skeleton. The binding affinities of mesembrane (4) were extremely
low as were those for the compounds 13, 15, epi-15 with the meth-
oxy group. The extremely low affinities may result from the
absence of a phenolic hydroxy group in 4. The lack of values for
Ki in the report by Harvey et al. makes it difficult to compare the
binding affinities of 4 with those of Sceletium tortuosum Zem-
brinÒ.15 Concerning compound 4, the non-displaced bindings of
the labeled compounds were 82.7%, 92.3%, and 93.0% for the
Mesembrane (4)
Naltrexone (5)
13
130
15
150
epi-15
epi-150
9102
0.27
16,230
12.3
5800
0.70
NDe
NDe
NDe
2151
5533
90.0
10,650
494.7
2363
19,130
1149
42,380
6627
769.8
4775
163.2
48,940
584.7
a
Binding assays were carried out in duplicate using human MOR, DOR, or KOR
recombinant cell (CHO) membranes.
MOR, DOR, and KOR, respectively (Table S3) at 10 lM of 4, which
was the highest concentration of the tested compounds in our
[3H] DAMGO was used.
b
[3H] DPDPE was used.
c
[3H] U-69,593 was used.
d
e
binding assays. On the other hand, the affinities of Sceletium tortuo-
sum ZembrinÒ were evaluated at a concentration of 750
lg/mL,
Not determined. The highest concentration of the tested compound was 10 lM.
which was calculated to be about 2.6 mM. Under these assessed
conditions, the non-displaced bindings of the labeled compounds
were about 10–30% (see the Supplementary data for more detailed
discussion).15
affinities for any of the receptors, the compounds 130, 150, and epi-
150 with a phenolic hydroxy group bound to the opioid receptors
with sufficient binding affinities in the micromolar range, and 150
and epi-150 both showed the same rank order of the binding abili-
ties to the individual opioid receptor types: MOR > KOR > DOR.
Compound 150 most strongly bound to the opioid receptors and
its binding affinity for the MOR was highest (Ki = 90.0 nM) among
the three opioid receptor types. Compounds 13, 15, and epi-15
with a methoxy group exhibited lower binding abilities than did
the corresponding compounds 130, 150, epi-150 with the phenolic
hydroxy group. It is well-known that the phenolic hydroxy group
is an important determinant for binding to the opioid receptor17–
2.3. Conformational analysis
To examine the differences in the binding affinities among the
compounds 130, 150, and epi-150, we carried out conformational
analyses of these protonated compounds using Conformational
Analyzer with Molecular Dynamics And Sampling (CAMDAS) 2.1
program (Fig. 4, see the Supplementary data for details).37 We con-
sidered two different stereoisomers with R- or S-configuration at
the protonated nitrogen atom for each compound. Interestingly,
the most stable conformations of 130, 150, and epi-150 were found
to possess S-, S-, and R-configurations, respectively, at the proton-
ated nitrogen atom. As a result, we could more frequently see an
isomer with S-configuration for 130 and 150 and one of R-configura-
tion for epi-150 among the lower-energy conformers, but both con-
figurations for each compound were observed within 10 kcal/mol
of the most stable conformer (Table S4). The axial orientation of
the N–H bond in the protonated compounds was proposed to pro-
mote effective binding to the opioid receptor via the directional
enforced ionic bond which was an ionic bond reinforced by the
directional hydrogen bond.38 Therefore, we paid attention to the
conformers with S-configuration at the nitrogen atom, in which
the N–H bond was directed axially. Figure 4 includes superimposi-
tions of the low-energy conformers with S-configuration for 130,
150, and epi-150, in which the phenol rings were superimposed.
Although the spatial disposition of the nitrogen atom was tightly
restricted for 130, those of 150 and epi-150 were widely spread.
The bound conformation of b-funaltrexamine (b-FNA, an irrevers-
ible MOR antagonist, Fig. 4G) in complex with the MOR39 is also
shown in Figure 4H and I. A comparison of Figure 4B and H
indicated that the nitrogen atoms of many low-energy conformers
of 150 adopted a conformation which resembled that of the basic
nitrogen of the bound conformer of b-FNA. The basic nitrogen is
well-known to provide a pharmacophoric interaction with the
19
and that morphinan derivatives with the phenolic hydroxy
group like naltrexone (5) are stronger binders than the correspond-
ing derivatives with the methoxy group. These results suggested
that the tested compounds would bind to the opioid receptors with
the same binding mode as observed for the morphinan derivatives.
The binding affinities of 150 were sufficient but lower than those of
naltrexone (5), which was sometimes used as a message structure
to provide novel opioid ligands. The message structure is the moi-
ety responsible for exerting opioid activities according to the mes-
sage-address concept,33,34 which is a useful guideline for the
design of ligands selective for the opioid receptor types. Therefore,
we predicted that compound 150 would be a novel message
structure with a skeleton distinct from morphinans to provide
characteristic opioid ligands. However, both the introduction of
the epoxy bridge (compound 130) and trans-fusion of the hydroin-
dole ring system (compound epi-150) decreased the binding affini-
ties. The binding affinities of (ꢁ)-18 and (ꢁ)-19 with a freely
rotatable phenyl ring have been reported to be lower or compara-
ble to morphine (20) and nalorphine (21) (Fig. 3).35 Although no
binding affinities have been reported, the analgesic effects by 20
and codeine (22) with a trans-fused hydroisoquinoline structure
were stronger than those of 23 and 24 possessing a cis-fused ring
system (Fig. 3).36 Interestingly, the SAR of the tested compounds
with the hydroindole structure was completely different from that
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K. Konoura et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 4. Results of conformational analyses of the protonated 130, 150, and epi-150, and the structure of b-FNA. Structures within 10 kcal/mol of the most stable conformer
were collected for the protonated forms of 130, 150, and epi-150. In each figure, the green circles indicated the area around the position relative to the basic nitrogen adjacent to
the phenol ring in b-FNA. (A) Top view of conformers of protonated 130; (B) top view of conformers of protonated 150; (C) top view of conformers of protonated epi-150; (D)
side view of conformers of protonated 130; (E) side view of conformers of protonated 150; (F) side view of conformers of protonated epi-150; (G) structure of b-FNA. The
structure of b-FNA was drawn using the coordinates obtained in the X-ray crystallographic analyses of the b-FNA-MOR complex,39 in which b-FNA bound to the MOR through
a covalent bond provided by Michael addition of Lys233 in the MOR to the
a,b-unsaturated ester moiety in b-FNA. (H) Top view of conformer of b-FNA; (I) side view of
conformer of b-FNA; (J) top view of superimpositions of 130, 150, epi-150, and b-FNA. The indicated conformer of each compound was selected based on the location of the
nitrogen atom that most closely resembled that of b-FNA. Compounds 130, 150, and epi-150 are indicated by orange, green, and light purple, respectively; (K) side view of
superimpositions of 130, 150, epi-150, and b-FNA.
opioid receptor.17–19 These results suggested that 150 could bind to
the opioid receptors with a binding mode similar to that of the
morphinans, thereby accounting for it having the strongest binding
affinities among these three compounds. The compound epi-150
also had low-energy conformers with the basic nitrogen located
in a favorable region (green circle), but as indicated by the much
less congested model (Fig. 4C), the population of the adequate
low-energy conformers was much lower than that of 150. These
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K. Konoura et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
observations reasonably suggested compound epi-150 possessed
poorer binding affinities than 150. Taken together, both observations
on the most stable conformers of protonated 150 and epi-150
(Table S4) and populations of the adequate low-energy conformers
of 150 and epi-150 sufficiently accounted for the difference in the
binding affinities of these compounds. Furthermore, the most
stable conformer of compound 130 had S-configuration at the
protonated nitrogen atom, but it had almost no conformers having
the basic nitrogen located in the proper space. Figure 4J, which
pictures superimpositions of 130, 150, epi-150, and b-FNA, clearly
supported these observations. These phenomena resulted in the
weakest binding affinities of 130 to the opioid receptors.
4.2. Synthesis
4.2.1. (3aR,3a1S,9aR)-5-Methoxy-3a1-(2-(methyl((2,2,2-trichloro
ethoxy)carbonyl)amino)ethyl)-1,2,3a,3a1-tetrahydro-9aH-spiro
[phenanthro[4,5-bcd]furan-3,20-[1,3]dioxolan]-9a-yl acetate (8)
Under an Ar atmosphere, to a solution of naltrexone (5) hydro-
chloride (32.3 g, 85.5 mmol) in DMF (323 mL) were added K2CO3
(29.6 g, 214 mmol) and MeI (6.9 mL, 104 mmol), and the mixture
was stirred at room temperature for 3 h. The reaction mixture
was poured into distilled water and extracted with Et2O. The com-
bined organic layers were washed with brine and concentrated
under reduced pressure to give a solid material (32.82 g). The
obtained material was used in the next reaction without
purification.
3. Conclusion
Under an Ar atmosphere, to a solution of the obtained material
(32.82 g) in DMF (324 mL) was added MeI (58 mL, 874 mmol) with
stirring at 130 °C for 7 h. After removing solvent under reduced
pressure, to the residue were added 1,4-dioxane (320 mL) and
1 M aqueous solution of NaOH (325 mL) with stirring at 80 °C for
4 h. The cooled reaction mixture was poured into distilled water
and extracted with CHCl3. The combined organic layers were con-
centrated under reduced pressure to give crude product of 7
(31.97 g) as an oil. The obtained crude product was used in the
next reaction without purification.
Under an Ar atmosphere, to a solution of the crude 7 (31.97 g) in
CHCl3 (636 mL) were added ethylene glycol (25.5 mL, 457 mmol)
and TMSOTf (17.0 mL, 93.9 mmol) with stirring at 50 °C for 4 h.
The cooled reaction mixture was poured into a saturated aqueous
solution of NaHCO3 and extracted with CHCl3. The combined
organic layers were concentrated under reduced pressure to give
an oil (39.33 g). The obtained oil was used in the next reaction
without purification.
We transformed naltrexone (5) with the morphinan skeleton
into mesembrane (4) belonging to the Sceletium alkaloids via key
intermediate 6 to investigate the binding affinities of 4 and deriv-
atives of the key intermediate 6 to the opioid receptors. Compound
150, which had the cis-fused hydroindole skeleton, exhibited strong
to moderate binding affinities for the MOR and KOR. Although
compound epi-150, which was a trans-fused congener of 150, also
displayed moderate binding to the MOR and KOR, the affinities of
epi-150 were lower than those of 150. In contrast to compounds
150 and epi-150, compound 130 showed greatly reduced binding.
The SARs of the tested compounds 130, 150, and epi-150 with
hydroindole structures were completely different from that of
morphinan derivatives with the hydroisoquinoline fundamental
skeleton. The observed differences of binding affinities among
the tested compounds were reasonably explained by the confor-
mational analyses and by the comparison of the relative relation-
ship of the spacial locations between the protonated nitrogen
and phenol ring among the tested compounds and b-FNA. Com-
pound 150 with a structure that differed from the morphinans like
naltrexone (5) is expected to be a useful fundamental structure
with a novel chemotype to develop characteristic opioid ligands,
which might provide useful ligands to investigate receptor dimers,
ligand bias, and/or receptor splice variants. Synthesizing deriva-
tives of 150 and evaluating their binding affinities and functional
activities for the opioid receptors, including receptor dimers and
ligand bias, are in progress.
Under an Ar atmosphere,
a solution of the obtained oil
(39.33 g) in Ac2O (354 mL) was stirred at 80 °C for 4 h. The solvent
was removed under reduced pressure and then further removed
under azeotropic conditions with toluene. The solution of the res-
idue in CHCl3 was washed with saturated aqueous solution of
NaHCO3 and concentrated under reduced pressure to give an oil
(42.24 g). The obtained oil was used in the next reaction without
purification.
Under an Ar atmosphere, to a solution of the obtained oil
(42.24 g) in CH2Cl2 (800 mL) were added Troc-Cl (23.6 mL,
171 mmol) and Et3N (35.7 mL, 256 mmol) at 0 °C, and the mixture
was stirred at the same temperature for 5 h. The reaction mixture
was poured into a saturated aqueous solution of NaHCO3 and
extracted with CHCl3. The combined organic layers were concen-
trated under reduced pressure. The residue was purified by a silica
4. Experimental
4.1. General information
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification. Melting points
were determined on a Yanako MP-500P melting point apparatus
and were uncorrected. Infrared (IR) spectra were recorded on a
JASCO FT/IR-460Plus. Nuclear magnetic resonance (NMR) spectra
were recorded on an Agilent Technologies VXR-400NMR for 1H
NMR and 13C NMR. Chemical shifts were reported as d values
(ppm) referenced to the deuterated solvent (CHCl3 or pyridine).
Mass spectra (MS) were obtained on a JMS-AX505HA, JMS-700
MStation, or JMS-100LP instrument by applying an electrospray
ionization (ESI) method. Elemental analyses were determined with
a Yanako MT-5 and JM10 for carbon, hydrogen, and nitrogen. The
progress of the reaction was determined on Merck Silica Gel Art.
5715 (TLC) visualized by exposure to ultraviolet light or with
sodium phosphomolybdate-ethanol, anisaldehyde-sulfuric acid-
ethanol, or ninhydrin-citric acid buffer-butanol stain. Column
chromatographies were carried out using Kanto Silica Gel 60 N
gel column chromatography (hexane/AcOEt = 2:1) to give
8
(35.44 g, 61.4 mmol, 72% from 5 hydrochloride) as a colorless
amorphous material.
MS (ESI): [M+Na]+ m/z = 598. HR-MS (ESI): [M+Na]+ calcd for
C
25H28Cl3NNaO8: 598.07486, found: 598.07782. IR (film): 1720,
1226, 1167, 1040 cmꢁ1 1H NMR (CDCl3, 400 MHz): d 1.39–1.50
.
(m, 1H), 1.55–1.67 (m, 2H), 1.87–1.98 (m, 0.35H), 1.98–2.24 (m,
1.65H), 2.09 (s, 1.95H), 2.13 (s, 1.05H), 2.78–2.91 (m, 1.7H), 2.79
(s, 1.05H), 2.82 (s, 1.95H), 3.13–3.27 (m, 0.65H), 3.53 (ddd,
J = 4.5, 13.0, 13.4 Hz, 0.65H), 3.79–3.94 (m, 2H), 3.88 (s, 3H), 4.04
(dd, J = 6.6, 13.0 Hz, 1H). 4.17–4.27 (m, 1H), 4.50 (d, J = 12.0 Hz,
0.65H), 4.69 (d, J = 12.0 Hz, 0.35H), 4.70 (d, J = 12.0 Hz, 0.35H),
4.72–4.82 (m, 1.65H), 6.31 (d, J = 9.8 Hz, 1H), 6.42 (d, J = 9.8 Hz,
0.65H), 6.46 (d, J = 9.8 Hz, 0.35H), 6.58–6.74 (m, 2H). 13C NMR
(CDCl3, 100 MHz): d 22.4, 26.9, 27.2, 27.4, 32.1, 32.5, 33.9, 34.9,
45.6, 46.2, 51.2, 56.2, 65.1, 66.56, 66.59, 74.86, 74.90, 86.5, 86.9,
95.0, 95.3, 95.5, 95.7, 107.8, 113.03, 113.08, 118.2, 118.3, 122.1,
122.2, 124.4, 124.5, 127.0, 127.5, 128.7, 128.9, 144.16, 144.19,
146.02, 146.05, 154.05, 154.10, 169.9, 170.2.
(60–230
l
m), Fuji Silysia CHROMATOREXÒ PSQ 60B (60
m).
lm), or
Fuji Silysia CHROMATOREXÒ NH-DM2035 (60
l
Please cite this article in press as: Konoura, K.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.032

K. Konoura et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
4.2.2. 2,2,2-Trichloroethyl (2-((1S,4aR,9bS)-1,9-diformyl-1-hydroxy-
6-methoxy-2,3,4a,9b-tetrahydro-1H-spiro[dibenzo[b,d] furan-
4,20-[1,3]dioxolane]-9b-yl)ethyl)(methyl)carbamate (9)
Ozone was bubbled into a solution of 8 (10.23 g, 17.7 mmol) in
CH2Cl2 (1000 mL) at ꢁ78 °C for 1 h. After vigorous bubbling with
N2 gas for 30 min, Me2S (25 mL, 338 mmol) was added to the reac-
tion mixture at the same temperature, and then the reaction was
stirred at room temperature for 12 h. After removing the solvent
under reduced pressure, to a solution of the residue in MeOH
(100 mL) was added Et3N (100 mL) with stirring for 4 h. The resi-
due obtained by removing the solvent under reduced pressure
was purified by silica gel column chromatography (CHCl3/
AcOEt = 5:1) to give 9 (7.31 g, 12.9 mmol, 73%) as a colorless amor-
phous material.
obtained by concentration of the combined organic layers under
reduced pressure was purified by silica gel (Fuji Silysia
CHROMATOREXÒ NH-DM2035) column chromatography (toluene/
CHCl3 = 2:1) to give 11 (6.42 g, 19.3 mmol, 92%) as a colorless
amorphous material.
MS (ESI): [M+H]+ m/z = 334. HR-MS (ESI): [M+H]+ calcd for
C
18H24NO5: 334.16545, found: 334.16811. IR (film): 2953, 2897.
2830, 1495, 1267, 1205, 1031 cmꢁ1 1H NMR (CDCl3, 400 MHz): d
.
1.56–1.80 (m, 2H), 1.80–1.88 (m, 2H), 2.17–2.27 (m, 1H), 2.41 (s,
3H), 2.44–2.55 (m, 1H), 2.64–2.74 (m, 1H), 3.15–3.24 (m, 2H),
3.77 (s, 3H), 3.91–4.13 (m, 4H), 4.34 (s, 1H), 6.31 (d, J = 8.7 Hz,
1H), 6.61 (d, J = 8.7 Hz, 1H). 13C NMR (CDCl3, 100 MHz): d 15.2,
28.4, 35.6, 36.0, 50.2, 55.3, 56.6, 65.3, 65.8, 66.9, 88.6, 107.3,
108.7, 113.2, 123.4, 137.5, 146.9, 148.1. Mp: 151 °C (dec.).
MS (ESI): [M+Na]+ m/z = 588. HR-MS (ESI): [M+Na]+ calcd for
C
23H26Cl3NNaO9: 588.05708, found: 588.05482. IR (film): 2952,
4.2.5. (3aS,6aR,11bR)-8-Methoxy-3-methyl-1,2,3,3a,4,5-hexahy-
drobenzofuro[3,2-d]indol-6(6aH)-one (6)
1715, 1605, 1287, 757 cmꢁ1 1H NMR (CDCl3, 400 MHz): d 1.75–
.
1.92 (m, 2H), 2.00–2.18 (m, 1H), 2.23–2.49 (m, 2H), 2.74–3.16
(m, 5H), 3.28–3.43 (m, 1H), 3.91 (s, 3H), 4.04–4.22 (m, 4H), 4.42–
4.76 (m, 3H), 4.93 (s, 0.4H), 4.95 (s, 0.6H), 6.90 (d, J = 8.5 Hz, 1H),
7.43 (d, J = 8.5 Hz, 0.6H), 7.44 (d, J = 8.5 Hz, 0.4H), 9.316 (s, 0.6H),
9.324 (s, 0.4H), 9.85 (s, 0.6H), 9.87 (s, 0.4H). 13C NMR (CDCl3,
100 MHz): d 26.1, 26.7, 27.9, 28.0, 28.6, 28.7, 34.1, 34.8, 45.6,
46.3, 56.0, 56.2, 65.17, 65.21, 65.5, 74.8, 75.0, 78.8, 78.9, 85.8,
87.0, 95.7, 106.16, 106.23, 111.57, 111.63, 127.1, 127.3, 128.9,
129.0, 129.6, 129.67, 129.73, 148.2, 148.4, 150.7, 153.9, 154.1,
191.2, 191.6.
Under an Ar atmosphere, to a solution of 11 (1.92 g, 5.76 mmol)
in DME (58 mL) was added NaH (55% in oil, 379 mg, 8.69 mmol) at
0 °C and the reaction mixture was stirred at the same temperature
for 30 min. Tf2NPh (3.12 g, 8.73 mmol) was added to the reaction
mixture with stirring at room temperature for 3 h. To the reaction
mixture were added Et3N (9.7 mL, 69.6 mmol) and HCO2H
(1.75 mL, 46.4 mmol) at 0 °C, subsequently added dppp (600 mg,
1.45 mmol) and Pd(MeCN)2Cl2 (150 mg, 0.578 mmol) at room tem-
perature, and the mixture was stirred at 80 °C for 7 h. To the reac-
tion mixture were added Et3N (6.0 mL, 43.0 mmol) and HCO2H
(1.0 mL, 26.5 mmol) at room temperature with stirring at 80 °C
for 10 h. Further HCO2H (1.0 mL, 26.5 mmol) was added to the
reaction mixture at room temperature with stirring at 80 °C for
4 h. The cooled reaction mixture was poured into distilled water
and extracted with AcOEt. The combined organic layers were con-
centrated under reduced pressure. A solution of the residue in
AcOEt was filtered through a short amine silica gel column (CHRO-
MATOREXÒ NH-DM2035). The solvent was removed under
reduced pressure to give an oil (1.80 g). The obtained oil was used
in the next reaction without purification.
4.2.3. 2,2,2-Trichloroethyl (2-((4aR,9bS)-9-hydroxy-6-methoxy-
1-oxo-2,3,4a,9b-tetrahydro-1H-spiro[dibenzo[b,d]furan-4,20-
[1,3]dioxolane]-9b-yl)ethyl)(methyl)carbamate (10)
Under an Ar atmosphere, to a solution of 9 (32.96 g, 58.3 mmol)
in CH2Cl2 (662 mL) was added 77% mCPBA (27.4 g, 122 mmol) and
the mixture was refluxed with stirring for 4 h. To the cooled reac-
tion mixture were added saturated aqueous solutions of Na2S2O3
and NaHCO3, and the mixture was extracted with CHCl3. The
combined organic layers were concentrated under reduced
pressure and to a solution of the residue in MeOH (322 mL) was
added Et3N (326 mL) with stirring at room temperature for 6 h.
The residue obtained by removing the solvent under reduced pres-
sure was purified by silica gel column chromatography (hexane/
AcOEt = 2:3) to give 10 (10.96 g, 20.9 mmol, 36%) as a colorless oil.
MS (ESI): [M+Na]+ m/z = 546. HR-MS (ESI): [M+Na]+ calcd for
A solution of the obtained oil (1.80 g) in 1 M HCl (10 mL) was
stirred at 100 °C for 5 h. After cooling, the reaction mixture was
basified (pH 9) with a saturated aqueous solution of NaHCO3 and
extracted with CHCl3. After concentration, the residue was purified
by silica gel column chromatography (hexane/AcOEt = 2:1) to give
6 (1.24 g, 4.54 mmol, 79% from 11) as a colorless amorphous
material.
C
21H24Cl3NNaO8: 546.04652, found: 546.04505. IR (film): 3303,
2952, 1715, 1505, 1163, 1033 cmꢁ1 1H NMR (CDCl3, 400 MHz): d
.
MS (ESI): [M+H]+ m/z = 274. HR-MS (ESI): [M+H]+ calcd for
1.97–2.07 (m, 1H), 2.08–2.38 (m, 3H), 2.66–2.82 (m, 2H), 2.87–
3.08 (m, 4H), 3.36–3.49 (m, 1H), 3.79 (s, 3H), 3.86–4.16 (m, 3H),
4.20–4.31 (m, 1H), 4.58–4.88 (m, 3H), 6.40 (d, J = 8.8 Hz, 1H),
6.73 (d, J = 8.8 Hz, 1H), 7.70 (s, 0.45H), 7.82 (s, 0.55H). 13C NMR
(CDCl3, 100 MHz): d 26.3, 33.8, 34.4, 34.7, 35.5, 35.7, 44.9, 45.5,
56.67, 56.72, 61.7, 61.8, 65.7, 65.9, 66.2, 66.4, 75.1, 87.0, 87.8,
95.6, 106.3, 109.7, 109.8, 112.5, 112.9, 115.18, 115.24, 138.3,
138.4, 147.8, 148.1, 153.9, 154.3, 213.5, 213.6.
C
16H20NO3: 274.14432, found: 274.14220. IR (film): 1719, 1491,
1459, 1292, 1201, 1126 cmꢁ1 1H NMR (pyridine-d5, 600 MHz): d
.
1.74–1.86 (m, 2H), 1.92 (ddd, J = 7.8, 8.9, 13.3 Hz, 1H), 2.10 (s,
3H), 2.15–2.18 (m, 1H), 2.21 (ddd, J = 3.1, 9.8, 13.3 Hz, 1H), 2.27
(ddd, J = 7.8, 9.1, 9.8 Hz, 1H), 2.44 (ddd, J = 1.9, 7.1, 17.5 Hz, 1H),
2.53 (ddd, J = 8.2, 11.4, 17.5 Hz, 1H), 3.04 (ddd, J = 3.1, 8.9, 9.1 Hz,
1H), 3.82 (s, 3H), 4.64 (s, 1H), 6.86 (d, J = 7.5 Hz, 1H), 6.95 (d,
J = 8.0 Hz, 1H), 7.05 (dd, J = 7.5, 8.0 Hz, 1H). 13C NMR (pyridine-d5,
150 MHz): d 20.0, 32.0, 36.2, 38.7, 54.6, 56.3, 60.4, 69.1, 93.8,
113.5, 115.9, 123.1, 132.9, 145.6, 148.6, 206.0. Mp: 122–124 °C.
4.2.4. (3aS,6aR,11bR)-8-Methoxy-3-methyl-1,2,3,3a,4,5-
hexahydro-6aH-spiro[benzo-furo[3,2-d]indole-6,20-[1,3]
dioxolan]-11-ol (11)
4.2.6. 2-Methoxy-6-((3aR,7aS)-1-methyl-1,2,3,6,7,7a-hexahydro-
3aH-indol-3a-yl)phenol (12) and (3aS,6aS,11bR)-8-Methoxy-3-
methyl-1,2,3,3a,4,5,6,6a-octahydrobenzofuro[3,2-d]indole (13)
Under an Ar atmosphere, to a solution of 6 (1.61 g, 5.90 mmol)
in ethylene glycol (58 mL) was added H2NNH2ꢀH2O (1.45 mL,
29.9 mmol), and the mixture was stirred at 80 °C for 2 h. KOH
(2.33 g, 41.5 mmol) was added to the reaction mixture at room
temperature with stirring at 130 °C for 23 h. The cooled reaction
mixture was poured into a saturated aqueous solution of NH4Cl
(2.37 g, 44.3 mmol) and extracted with CHCl3. After concentration,
Under an Ar atmosphere, to
a solution of 10 (10.96 g,
20.9 mmol) in AcOH (218 mL) was added zinc powder (6.90 g,
105 mmol) with stirring at room temperature for 2 h. The reaction
mixture was filtered through a Celite pad and to the obtained fil-
trate was added NaBH3CN (3.95 g, 62.9 mmol) at 0 °C, and then
the mixture was stirred at the same temperature for 17 h under
an Ar atmosphere. After removing the solvent under reduced pres-
sure, a solution of the residue in CHCl3 was poured in a saturated
aqueous solution of NaHCO3 and extracted with CHCl3. The residue
Please cite this article in press as: Konoura, K.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.032

8
K. Konoura et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
the residue was purified by silica gel column chromatography
(CHCl3/MeOH = 40:1 ? 5:1 ? 2:1) to give 12 (1.12 g, 4.32 mmol,
73%) as a colorless oil and 13 (379 mg, 1.46 mmol, 25%) as a color-
less crystal.
4.2.8. (3aS,7aS)-3a-(3-Methoxyphenyl)-1-methyloctahydro-1H-
indole (15) and (3aS,7aR)-3a-(3-methoxyphenyl)-1-methylocta-
hydro-1H-indole (epi-15)
Under an Ar atmosphere, a solution of Pd(MeCN)2Cl2 (8.6 mg,
Compound 12: MS (ESI): [M+H]+ m/z = 260. HR-MS (ESI):
[M+H]+ calcd for C16H22NO2: 260.16505, found: 260.16360. IR
33.1
stirred at room temperature for 1 h. To the reaction mixture were
added a solution of 14 (111.4 mg, 308.4 mol) in DME (1.8 mL),
lmol) and dppp (32.0 mg, 77.6 lmol) in DME (0.5 mL) was
(film): 3521, 2935, 1469, 1257 cmꢁ1
.
1H NMR (CDCl3, 400 MHz):
l
d 1.50–1.62 (m, 1H), 1.63–1.72 (m, 1H), 1.80–1.98 (m, 2H), 2.07–
2.18 (m, 1H), 2.46 (s, 3H), 2.57–2.63 (m, 1H), 2.99 (dd, J = 7.1,
7.3 Hz, 2H), 3.16 (dd, J = 4.2, 9.0 Hz, 1H), 3.87 (s, 3H), 5.89–5.97
(m, 2H), 6.67 (dd, J = 8.0, 8.0 Hz, 1H), 6.76 (dd, J = 1.5, 8.0 Hz, 1H),
6.86 (dd, J = 1.5, 8.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz): d 19.8,
20.7, 36.8, 38.0, 48.4, 52.5, 55.8, 67.3, 109.3, 117.1, 119.7, 125.3,
133.97, 134.05, 145.7, 148.6.
Et3N (0.4 mL, 2.87 mmol), and HCO2H (0.07 mL, 1.85 mmol), and
the mixture was stirred at 100 °C for 37 h in a sealed tube. To
the reaction mixture were added Et3N (0.4 mL, 2.87 mmol) and
HCO2H (0.07 mL, 1.85 mmol) with further stirring at 100 °C for
8 days. The cooled reaction mixture was poured into 2 M aqueous
solution of NaOH and extracted with AcOEt. After removing the
solvent, a solution of the residue in CHCl3 was filtered through a
short amine silica gel column (CHROMATOREXÒ NH-DM2035).
The filtrate was concentrated under reduced pressure. The residue
was purified by preparative TLC (AcOEt) to give 15 (30.2 mg,
Compound 13: MS (ESI): [M+H]+ m/z = 260. HR-MS (ESI):
[M+H]+ calcd for C16H22NO2: 260.16505, found: 260.16545. IR
(film): 2936, 2778, 1493, 1459, 1292, 1201, 1027 cmꢁ1 1H NMR
.
(pyridine-d5, 400 MHz): d 1.26–1.40 (m, 2H), 1.48–1.72 (m, 3H),
1.86 (ddd, J = 3.9, 9.6, 13.3 Hz, 1H), 1.92–2.04 (m, 2H), 2.18 (s,
3H), 2.35 (ddd, J = 7.7, 9.0, 9.6 Hz, 1H), 2.40–2.45 (m, 1H), 3.02
(ddd, J = 3.9, 8.8, 9.0 Hz, 1H), 3.80 (s, 3H), 4.57 (dd, J = 5.0,
6.9 Hz, 1H), 6.90–6.94 (m, 2H), 6.99 (dd, J = 6.6, 8.6 Hz, 1H). 13C
NMR (pyridine-d5, 100 MHz): d 16.0, 22.1, 27.0, 37.8, 39.7, 54.2,
54.8, 56.2, 68.6, 89.7, 113.0, 116.0, 121.9, 136.6, 145.7, 148.2.
Mp: 72–73 °C.
0.123 mmol, 40%) as a colorless oil and epi-15 (17.9 mg,
0.073 mmol, 24%) as a colorless oil.
Compound 15: MS (ESI): [M+H]+ m/z = 246. HR-MS (ESI):
[M+H]+ calcd for C16H24NO: 246.18579, found: 246.18405. IR
(film): 2933, 1607, 1581, 1448, 1246, 1053, 701 cmꢁ1 1H NMR
.
(CDCl3, 400 MHz): d 1.09–1.22 (m, 1H), 1.33–1.40 (m, 1H), 1.44–
1.68 (m, 3H), 1.77–2.02 (m, 5H), 2.27–2.35 (m, 1H), 2.34 (s, 3H),
2.61–2.66 (m, 1H), 3.28 (ddd, J = 4.5, 9.8, 9.8 Hz, 1H), 3.81 (s, 3H),
6,73 (ddd, J = 0.7, 2.5, 8.0 Hz, 1H), 6.94 (dd, J = 2.1, 2.5 Hz, 1H),
6.97 (ddd, J = 0.7, 2.1, 8.3 Hz, 1H), 7.24 (dd, J = 8.0, 8.3 Hz, 1H).
13C NMR (CDCl3, 100 MHz): d 20.3, 22.8, 23.6, 35.9, 40.6, 40.8,
47.8, 54.3, 55.0, 68.5, 109.8, 113.7, 119.3, 128.9, 149.4, 159.3.
Compound
13
hydrochloride:
Anal.
Calcd
for
C
16H21NO2ꢀHClꢀ0.2H2O: C, 60.54; H, 7.75; N, 4.41, found: C,
60.34; H, 7.57; N, 4.29. Mp: 201 °C (dec.).
Compound
15
hydrochloride:
Anal.
Calcd
for
4.2.7. 2-Methoxy-6-((3aS,7aS)-1-methyloctahydro-3aH-indol-
3a-yl)phenyl trifluoromethanesulfonate (14)
Under an Ar atmosphere, to a solution of 12 (111.6 mg,
430.9 lmol) in AcOH (2.3 mL) was added 10% Pd on carbon
C
16H23NOꢀHClꢀ0.15H2O: C, 67.54; H, 8.61; N, 4.92, found: C,
67.44; H, 8.57; N, 4.91. Mp: 224 °C (dec.).
epi-15: MS (ESI): [M+H]+ m/z = 246. HR-MS (ESI): [M+H]+ calcd
for C16H24NO: 246.18579, found: 246.18459. IR (film): 2935, 1578,
(33.7 mg), and after exchange of Ar into H2, the reaction was stir-
red at 80 °C for 21 h. The cooled reaction mixture was filtered
through a Celite pad and washed with MeOH. After concentration
of the filtrate, the residue was dissolved with CHCl3. The obtained
solution was poured into a saturated aqueous solution of NaHCO3
and extracted with CHCl3. The combined organic layers were con-
centrated under reduced pressure. A solution of the residue in hex-
ane/AcOEt (1:3) was filtered through a short amine silica gel
column (CHROMATOREXÒ NH-DM2035). The solvent was removed
under reduced pressure to give a dark brown solid (108.8 mg). The
obtained solid was used in the next reaction without purification.
Under an Ar atmosphere, to a solution of the obtained solid
(108.8 mg) in DME (2.0 mL) was added NaH (55% in oil, 56.0 mg,
1.28 mmol) at 0 °C, and the mixture was stirred at the same tem-
perature for 20 min. To the reaction mixture was added Tf2NPh
(300 mg, 0.839 mmol) at 0 °C with stirring at the same tempera-
ture for 25 min, followed by addition of Tf2NPh (150 mg,
0.420 mmol) with stirring at room temperature for 2 h. The reac-
tion mixture was poured into 2 M aqueous solution of NaOH and
extracted with AcOEt. After concentration, the residue was purified
by preparative TLC (AcOEt) to give 14 (140.4 mg, 0.357 mmol, 83%
from 12) as a colorless crystal.
1455, 1240, 1051, 709 cmꢁ1 1H NMR (CDCl3, 600 MHz): d 1.12
.
(dddd, J = 3.8, 3.9, 13.3, 13.4 Hz, 1H), 1.25–1.35 (m, 1H), 1.37
(ddd, J = 3.5, 13.2, 13.3 Hz, 1H), 1.43–.48 (m, 1H), 1.64–1.81 (m,
5H), 2.16–2.30 (m, 2H), 2.37 (s, 3H), 2.60–2.65 (m, 1H), 3.04 (dd,
J = 8.4, 17.9 Hz, 1H), 3.79 (s, 3H), 6.70 (dd, J = 2.6, 8.1 Hz, 1H),
7.21 (dd, J = 7.9, 8.1 Hz, 1H), 7.37 (br d, J = 7.9 Hz, 1H), 7.52 (br s,
1H). 13C NMR (CDCl3, 150 MHz): d 22.3, 24.1, 25.7, 38.1, 39.8,
41.6, 49.3, 53.2, 55.1, 76.8, 109.6, 115.8, 121.2, 128.7, 147.4, 159.3.
epi-15 hydrochloride: Anal. Calcd for C16H23NOꢀHClꢀ1.2H2O: C,
63.33; H, 8.77; N, 4.62, found: C, 63.25; H, 8.68; N, 4.38. Mp was
not able to be measured due to high hygroscopicity.
4.2.9. (3aS,7aS)-3a-(4-Iodo-3-methoxyphenyl)-1-methyloctahydro-
1H-indole (16)
Under an Ar atmosphere, to a solution of 15 (125.1 mg,
510.6 lmol) in TFA (2.5 mL) was added NIS (114.7 mg, 509.8 lmol)
at 0 °C and the reaction temperature was gradually raised to room
temperature with stirring for 24 h. After removing the solvent
under reduced pressure, to the residue was added saturated aque-
ous solution of Na2S2O3 (1 mL) and basified (pH 9) with 1 M aque-
ous solution of NaOH, and then the mixture was extracted with
CHCl3. After concentration, the residue was purified by preparative
MS (ESI): [M+H]+ m/z = 394. HR-MS (ESI): [M+H]+ calcd for C17-
H23F3NO4S: 394.12999, found: 394.12743. IR (film): 2939, 1411,
TLC (chloroform/methanol = 20:1) to give 16 (160.6 mg, 433 lmol,
1203, 1125, 883, 748 cmꢁ1
.
1H NMR (CDCl3, 400 MHz): d 0.82–
85%) as a dark brown oil.
0.95 (m, 1H), 1.35–1.60 (m, 3H), 1.75–1.99 (m, 4H), 2.11–2.24
(m, 2H), 2.29 (s, 3H), 2.32–2.46 (m, 1H), 2.49–2.53 (m, 1H), 3.23
(ddd, J = 3.3, 9.2, 9.2 Hz, 1H), 3.86 (s, 3H), 6.91 (dd, J = 1.4, 8.2 Hz,
1H), 7.06 (dd, J = 1.4, 8.2 Hz, 1H), 7.23 (dd, J = 8.2, 8.2 Hz, 1H). 13C
NMR (CDCl3, 100 MHz): d 20.6, 23.1, 24.0, 35.5, 38.0, 40.4, 46.7,
54.7, 55.6, 67.9, 110.7, 119.1 (q, J = 322.4 Hz), 120.8, 127.3, 138.6,
141.7, 150.9. Mp: 88–90 °C.
MS (ESI): [M+H]+ m/z = 372. HR-MS (ESI): [M+H]+ calcd for C16-
H23INO: 372.08243, found: 372.08208. IR (film): 2933, 1461, 1393,
1045 cmꢁ1 1H NMR (CDCl3, 400 MHz): d 1.06–1.19 (m, 1H), 1.33–
.
1.44 (m, 1H), 1.45–1.66 (m, 3H), 1.75–2.02 (m, 5H), 2.28–.37 (m,
1H), 2.35 (s, 3H), 2.60–2.64 (m, 1H), 3.30 (ddd, J = 4.7, 9.2, 9.2 Hz,
1H), 3.88 (s, 3H), 6.74 (dd, J = 2.1, 8.2 Hz, 1H), 6.83 (d, J = 2.1 Hz,
1H), 7.67 (d, J = 8.2 Hz, 1H). 13C NMR (CDCl3, 100 MHz): d 20.2,
Please cite this article in press as: Konoura, K.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.032

K. Konoura et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
22.8, 23.6, 36.0, 40.5, 40.7, 47.9, 54.2, 56.2, 68.4, 82.3, 110.2, 121.3,
138.7, 149.9, 157.8.
4.2.13. (3aS,6aS,11bR)-3-Methyl-1,2,3,3a,4,5,6,6a-octahydroben-
zofuro[3,2-d]indol-8-ol (130)
A colorless crystal. 23% yield.
4.2.10. 2-methoxy-4-((3aS,7aS)-1-methyloctahydro-3aH-indol-
3a-yl)phenol (17)
MS (ESI): [M+H]+ m/z = 246. HR-MS (ESI): [M+H]+ calcd for
C
15H20NO2: 246.14940, found: 246.14767. IR (film): 2937, 1611,
Under an Ar atmosphere, to a solution of 16 (160.6 mg,
1465, 1232, 1176 cmꢁ1 1H NMR (pyridine-d5, 400 MHz): d 1.26–
.
433
l
mol) in THF (4.5 mL) was added a 2.65 M solution of n-BuLi
mol) at ꢁ78 °C and the mixture was stir-
1.43 (m, 2H), 1.50–1.72 (m, 3H), 1.89 (ddd, J = 3.9, 9.2, 12.3 Hz,
1H), 1.94–2.03 (m, 2H), 2.18 (s, 3H), 2.35 (ddd, J = 7.7, 8.8, 9.2 Hz,
1H), 2.45 (dd, J = 3.5, 3.5 Hz, 1H), 3.03 (ddd, J = 3.9, 8.8, 8.8 Hz,
1H), 4.56 (dd, J = 4.9, 7.0 Hz, 1H), 6.85 (dd, J = 1.2, 7.4 Hz, 1H), 7.00
(dd, J = 7.4, 7.9 Hz, 1H), 7.17 (dd, J = 1.2, 7.9 Hz, 1H). 13C NMR (pyr-
idine-d5, 100 MHz): d 16.1, 22.1, 27.0, 37.9, 39.8, 54.4, 54.9, 68.6,
89.4, 114.1, 117.0, 122.2, 136.5, 143.8, 147.5. Mp: 155 °C.
Compound 130 hydrochloride: Anal. Calcd for C15H19NO2ꢀ
HClꢀ0.2H2O: C, 63.13; H, 7.20; N, 4.91, found: C, 62.99; H, 7.03;
N, 4.93. Mp: 248 °C (dec.).
in hexane (0.2 mL, 530
l
red at the same temperature for 50 min. To the reaction mixture
was added B(Oi-Pr)3 (0.3 mL, 1.31 mmol) at ꢁ78 °C and the reac-
tion temperature was gradually raised to room temperature with
stirring for 19 h. To the reaction mixture were added 1 M aqueous
solution of NaOH (2 mL) and NaBO3ꢀ4H2O (200 mg, 1.30 mmol).
After stirring for 1 h, to the reaction mixture were added distilled
water (2 mL), MeOH (0.5 mL), and NaBO3ꢀ4H2O (200 mg,
1.30 mmol), and then the mixture was stirred for 2 h. The reaction
mixture was poured into AcOEt and adjusted to pH 8 with NH4Cl,
and then extracted with AcOEt. After removing the solvent under
reduced pressure, the residue was purified by preparative TLC
4.2.14. 3-((3aS,7aS)-1-Methyloctahydro-3aH-indol-3a-yl)phenol
(150)
(CHCl3/MeOH = 10:1) to give 17 (44.4 mg, 170
l
mol, 39%) as a col-
A colorless crystal. 62% yield.
orless crystal and concomitantly with 15 (49.7 mg, 203
lmol, 47%).
MS (ESI): [M+H]+ m/z = 232. HR-MS (ESI): [M+H]+ calcd for
MS (ESI): [M+H]+ m/z = 262. HR-MS (ESI): [M+H]+ calcd for
C
15H22NO: 232.17014, found: 232.17026. IR (film): 2933, 1583,
C16H24NO2: 262.18070, found: 262.18043. IR (film): 2934, 1519,
1449, 1241, 779, 701 cmꢁ1 1H NMR (CDCl3, 400 MHz): d 1.09–
.
1284, 1211, 1032 cmꢁ1
.
1H NMR (CDCl3, 400 MHz): d 1.09–1.23
1.23 (m, 1H), 1.33–1.51 (m, 2H), 1.53–1.72 (m, 2H), 1.82–2.00
(m, 5H), 2.29–2.39 (m, 1H), 2.36 (s, 3H), 2.66–2.71 (m, 1H), 3.29
(ddd, J = 4.9, 8.9, 9.3 Hz, 1H), 6.66 (br dd, J = 1.8, 8.0 Hz, 1H),
6.85–6.88 (m, 1H), 6.91 (br d, J = 7.9 Hz, 1H), 7.16 (dd, J = 7.9,
8.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz): d 20.4, 22.6, 23.5, 36.0,
40.6, 41.1, 47.8, 54.4, 68.9, 112.8, 114.4, 118.7, 129.1, 149.5,
156.1. Mp: 141 °C.
(m, 1H), 1.32–1.43 (m, 1H), 1.53–1.65 (m, 3H), 1.74–1.96 (m,
5H), 2.25–2.34 (m, 1H), 2.32 (s, 3H), 2.54–2.58 (m, 1H), 3.24
(ddd, J = 4.7, 9.1, 9.1 Hz, 1H), 3.89 (s, 3H), 5.37–5.60 (br, 1H),
6.83–6.90 (m, 3H). 13C NMR (CDCl3, 100 MHz): d 20.4, 22.9, 23.6,
36.1, 40.6, 41.1, 47.5, 54.3, 55.9, 68.7, 109.7, 113.7, 119.6, 139.7,
143.2, 146.1. Mp: 131 °C.
Compound 150 hydrochloride: Anal. Calcd for C15H21NOꢀ
HClꢀ0.15H2O: C, 66.60; H, 8.31; N, 5.18, found: C, 66.89; H, 8.27;
N, 4.90. Mp: 255 °C (dec.).
4.2.11. (3aS,7aS)-3a-(3,4-Dimethoxyphenyl)-1-methyloctahydro-
1H-indole (mesembrane) (4)
Under an Ar atmosphere, to
a solution of 17 (44.4 mg,
170 mol) in MeOH (1.35 mL) was added a 2 M solution of
l
4.2.15. 3-((3aS,7aR)-1-Methyloctahydro-3aH-indol-3a-yl)phenol
(epi-150)
TMSCHN2 in Et2O (0.5 mL, 1.0 mmol) at 0 °C and the mixture was
stirred at the same temperature for 30 min, and then further stir-
red at room temperature for 40 min. To the reaction mixture was
added AcOH/MeOH (1:1) at 0 °C until no foam was observed to
be formed. After concentration, a solution of the residue in CHCl3
was poured into a saturated aqueous solution of NaHCO3 and
extracted with CHCl3. After removing the solvent under reduced
pressure, the residue was purified by preparative TLC (CHCl3/
A colorless oil. 87% yield.
MS (ESI): [M+H]+ m/z = 232. HR-MS (ESI): [M+H]+ calcd for
C
15H22NO: 232.17014, found: 232.16793. IR (film): 2934, 1583,
1455, 1241, 756, 711 cmꢁ1 1H NMR (CDCl3, 400 MHz): d 1.11
.
(dddd, J = 3.9, 3.9, 13.0, 13.2 Hz, 1H), 1.24–1.36 (m, 1H), 1.37
(ddd, J = 3.4, 12.9, 13.0 Hz, 1H), 1.41–1.49 (m, 1H), 1.64–1.82 (m,
5H), 2.17–2.24 (m, 1H), 2.29 (ddd, J = 2.7, 10.3, 10.3 Hz, 1H), 2.38
(s, 3H), 2.57–2.63 (m, 1H), 3.11 (ddd, J = 8.2, 8.2, 10.2 Hz, 1H),
6.63 (ddd, J = 1.0, 2.6, 7.9 Hz, 1H), 7.15 (dd, J = 7.9, 8.0 Hz, 1H),
7.22 (dd, J = 1.9, 8.0 Hz, 1H), 7.46 (dd, J = 1.0, 1.9 Hz, 1H). 13C
NMR (CDCl3, 100 MHz): d 22.2, 24.0, 25.7, 38.4, 39.8, 42.2, 49.4,
53.3, 77.1, 112.3, 116.5, 120.3, 129.2, 146.9, 155.5.
MeOH = 18:1) to give 4 (37.8 mg, 138 lmol, 81%) as a yellow oil.
MS (ESI): [M+H]+ m/z = 276. HR-MS (ESI): [M+H]+ calcd for
C
17H26NO2: 276.19635, found: 276.19884. IR (film): 2931, 1520,
1463, 1254, 1147, 1029 cmꢁ1 1H NMR (CDCl3, 400 MHz): d 1.08–
.
1.22 (m, 1H), 1.32–1.42 (m, 1H), 1.43–1.51 (m, 1H), 1.51–1.66
(m, 2H), 1.75–2.00 (m, 5H), 2.27–2.36 (m, 1H), 2.34 (s, 3H), 2.60–
2.64 (m, 1H), 3.28 (ddd, J = 4.8, 9.1, 9.2 Hz, 1H), 3.85 (br s, 3H),
3.87 (br s, 3H), 6.80 (d, J = 8.3 Hz, 1H), 6.85–6.92 (m, 2H). 13C
NMR (CDCl3, 100 MHz): d 20.4, 22.8, 23.6, 35.9, 40.7, 40.9, 47.5,
54.3, 55.8, 55.9, 68.7, 110.6, 110.7, 118.8, 140.1, 146.8, 148.6.
epi-150 hydrochloride: Anal. Calcd for C15H21NOꢀHClꢀ0.15H2O: C,
66.60; H, 8.31; N, 5.18, found: C, 66.63; H, 8.24; N, 5.09. Mp: 227 °C
(dec.).
4.3. Binding assay
Compound
4
hydrochloride: Anal. Calcd for C17H25NO2ꢀ
HClꢀ0.9H2O: C, 62.24; H, 8.54; N, 4.27, found: C, 62.20; H, 8.42;
The competitive binding assays were performed using human
MOR, DOR, or KOR recombinant cell (CHO) membranes. [3H]DAM-
GO (2 nM), [3H]DPDPE (2 nM), and [3H]U69,593 (2 nM) were used
to label the MOR, DOR, and KOR, respectively. Nonspecific binding
N, 4.28. Mp: 195 °C (dec.).
4.2.12. Demethylation with BBr3
(General procedure) Under an Ar atmosphere, to a solution of
starting material in CH2Cl2 was added a 1 M solution of BBr3 in
CH2Cl2 (3 equiv) dropwise at ꢁ10 °C, and the mixture was stirred
at the same temperature for 1 h. To the reaction mixture was
added 30% aqueous solution of NH3 and the reaction temperature
was raised to room temperature, and then the mixture was
extracted with CHCl3. After removing the solvent under reduced
pressure, the residue was purified by preparative TLC.
was measured in the presence of 1.0 lM unlabeled DAMGO, DPDPE
or U-69,593. Ki values were calculated according to the Cheng–
Prusoff equation.40
4.4. Conformational analysis
SYBYL6.91 (Tripos, St Louis, MO, USA) was first employed to
construct initial structures for compounds 130, 150, and epi-150.
Please cite this article in press as: Konoura, K.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.032

10
K. Konoura et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
We protonated the nitrogens of compounds 130, 150, and epi-150 to
prepare two initial structures with different configurations at the
nitrogen for each compound (Fig. S3). Then, the following calcula-
tions were performed for each initial structure using the CAMDAS
2.1 program. Ten molecular dynamic (MD) calculations were
simultaneously performed using different conformers generated
randomly using the initial structure. Each of the MD calculations
was carried out for 1000 ps with an integral time step of 1 fs. The
lengths of covalent bonds were fixed with SHAKE algorithm
through the MD.41 The temperature of the system was maintained
at 1200 K in order to enhance the sampling efficiency. The Merck
Molecular Force Field (MMFF) was used to evaluate the potential
energy surface of the molecule.42 To mimic the shield effects of sol-
vent molecules on electrostatic interactions, the electrostatic
potential term was neglected. Conformers were sampled at 100
step intervals, thus producing 10,000 conformations for each MD
calculation. A total of 100,000 conformations were preclustered
with a dihedral angle deviation threshold of 30°. The dihedral
angles used to cluster similar conformations are indicated for each
compound by arrows in Fig. S3. Each of the conformers obtained
after preclustering was then minimized until the root mean square
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of
the
potential-energy
gradient
fell
below
0.05 kcal molꢁ1 Åꢁ1. The minimized conformers were reclustered
with a dihedral angle deviation threshold of 30°, furnishing a final
conformer set.
Supplementary data
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Supplementary data (supplementary data (Figs. S1–S3, Tables
S1–S4 and conformational analyses) and more discussion (compar-
ison of binding affinities between mesembrane and Sceletium tor-
tuosum ZembrinÒ)) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.12.032.
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