Synthesis and pharmacology of a novel κ opioid receptor (KOR) agonist with a 1,3,5-trioxazatriquinane skeleton

Article abstract of DOI:10.1021/ml5000542

We designed and synthesized the 1,3,5-trioxazatriquinane derivatives with m-hydroxyphenyl groups. These compounds include the phenethylamine structure within them, which is a common structure observed in morphinan derivatives like morphine. Among the synthesized compounds, (-)-8c with two m-hydroxyphenyl groups selectively bound and exerted full agonist activity toward the κ opioid receptor (KOR). Subcutaneously administered (-)-8c exhibited significant antinociceptive effects via the KOR in a dose-dependent manner. These results suggest the emergence of a novel class of KOR agonist.

Full text of DOI:10.1021/ml5000542

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Pharmacology of a Novel κ Opioid Receptor (KOR)
Agonist with a 1,3,5-Trioxazatriquinane Skeleton
Shigeto Hirayama, Naohisa Wada, Toru Nemoto, Takashi Iwai, Hideaki Fujii, and Hiroshi Nagase*,†
School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
S
* Supporting Information
ABSTRACT: We designed and synthesized the 1,3,5-trioxazatriquinane
derivatives with m-hydroxyphenyl groups. These compounds include the
phenethylamine structure within them, which is a common structure
observed in morphinan derivatives like morphine. Among the synthesized
compounds, (−)-8c with two m-hydroxyphenyl groups selectively bound
and exerted full agonist activity toward the κ opioid receptor (KOR).
Subcutaneously administered (−)-8c exhibited significant antinociceptive
effects via the KOR in a dose-dependent manner. These results suggest
the emergence of a novel class of KOR agonist.
KEYWORDS: Opioid, analgesics, phenethylamine structure
win drugs consisting of two pharmacophore units in a
single molecule have been described in numerous
(Figure 1) was a selective MOR agonist.12 It is interesting that
SYK-385 exhibited the highest selectivity for the MOR over the
KOR among the reported MOR selective nonpeptide ligands.12
Although these results predicted that the 1,3,5-trioxazatriqui-
nane derivatives would exert novel pharmacological profiles, all
the above-mentioned derivatives contained large morphinan
units.
We next focused on the 1,3,5-trioxazatriquinane derivatives
with simple moieties. The derivatives 1 with aryl groups
included the phenethylamine structure, which is a common
structure observed not only in endogenous neuropeptides such
as enkephalins, dopamine, and adrenaline but also in
morphinan skeletons like morphine and naltrexone (Figure
2). Interestingly, when the 3D-alignment of a partial structure
of compound 1 (R = m-OH) was superimposed onto that of
naltrexone, the nitrogen atom, phenyl ring, and phenolic
hydroxy group in both compounds were located in very similar
positions to each other (Figure 3). This three point
pharmacophore is well documented for ligands of opioid
receptors.13,14
T
domains of medicinal chemistry. Symmetrical twin drugs with
flexible linkers can simultaneously fit into symmetrical binding
sites of a protein complex to afford increased activity or to
increase selectivity. In contrast, nonsymmetrical twin drugs may
bind to individual relevant binding sites to provide dual
actions.1 To obtain potential drugs with enhanced efficacy or
improved safety, drug discovery campaigns have focused on a
strategy designing multitarget drugs (DMLs)2,3 from the
viewpoint of polypharmacology.4 In the opioid field, many
twin drugs have been reported.5 For example, Portoghese et al.
designed nonsymmetrical twin drugs possessing selective
agonists and/or antagonists for the opioid receptor type to
investigate the heterodimer of the opioid receptor,6,7 whereas
Neumeyer et al. synthesized symmetrical and nonsymmetrical
twin drugs for the purpose of seeking potent analgesics with
fewer side effects.8 Recently, we developed a novel synthetic
method for attaching three identical or nonidentical pharma-
cophore units to a single scaffold, the 1,3,5-trioxazatriquinane
skeleton, i.e., a “triplet”.9 These triplets exerted interesting
pharmacological profiles. Subcutaneous administration of
symmetrical triplet KNT-93 with three oxymorphone units or
symmetrical twin drug KNT-123 with two oxymorphone units
(Figure 1) showed profound antinociceptive effects mediated
by the μ opioid receptor (MOR) in a dose-dependent manner.
The antinociception induced by KNT-93 and KNT-123 was
54- and 5-fold more potent, respectively, than that of
morphine.10,11 4,5-Epoxymorphinan derivative SYK-134 (Fig-
ure 1) with the 1,3,5-trioxazatriquinane moiety, which was
synthesized as an analog of KNT-93 and -123, showed agonistic
activity selective to the KOR, while another derivative SYK-385
From these observations, we expected the compound 1 (R =
m-OH) (Figure 2) to interact with the opioid receptor with
adequate binding affinity. Herein, we report the synthesis of
1,3,5-trioxazatriquinane derivatives with one to three m-
hydroxyphenyl groups (1a-c (R = m-OH), 8a-d, and 11a,b).
The pharmacological properties of the synthesized derivatives
were also evaluated.
Received: February 3, 2014
Accepted: June 26, 2014
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
Figure 3. Superimposition of the 3D-alignment of naltrexone (yellow)
on that of a 1,3,5-trioxaxatriquinane derivative with a m-hydroxyphenyl
(green) group. The red circle indicates a nitrogen atom, the orange
circle indicates a phenyl ring, and the blue circle indicates a phenolic
hydroxy group. Two m-hydroxyphenyl groups were omitted from the
1,3,5-trioxazatriquinane derivative 1 (R = m-OH) for clarity.
Figure 1. Structures of KNT-93, KNT-123, SYK-134, and SYK-385.
11a,b, respectively. The treatment of 7c with 130 mol %
potassium 1-propanethiolate gave 8c, 12a, and 12b (Scheme
4). The relative configurations of all synthesized compounds
were determined by 2D-NMR experiments or X-ray crystallog-
raphy (see the Supporting Information for details).
The binding affinities of the prepared 1,3,5-trioxazatriqui-
nanes for the opioid receptors were evaluated with competitive
binding assays (Table 1). The assays were performed by a
previously reported procedure.15 Although most of the tested
compounds did not bind to the opioid receptors, 1b, 1c, and 8a
exhibited submicromolar binding affinities to the MOR or to
both the MOR and the KOR.
Surprisingly, compound 8c strongly bound to the KOR with
a Ki value of 6.09 nM. The affinity and selectivity of 8c were
almost the same as those of U-69,593 and U-50,488, known
standard KOR agonists. (−)-8c16 showed stronger binding
affinity for the KOR with a Ki value of 4.63 nM, whereas its
(+)-enantiomer 8c hardly bound to the KOR. Very recently,
Schmidhammer et al. reported phenethylamine derivatives with
the m-hydroxy group as agonists selective for the KOR.17
Taken together, this earlier report and our present results
suggest that the phenethylamine moiety with the m-
hydroxyphenyl group would be an important pharmacophore
to interact with the opioid receptors. Compounds 1b and 1c,
which included the structure of 8c, hardly bound to the KOR.
This outcome may result from the steric hindrance of the third
aryl moiety. Although compounds 11a and 11b with a m-
hydroxyphenyl group corresponded to partial structures of 8c,
these compounds exhibited no binding affinities, suggesting
that both aryl moieties of 8c would play an indispensable role in
binding to the KOR. Both compounds 8b and 8c had two aryl
groups with similar stereochemistry: one is in an endo-position,
and the other is in an exo-position. However, compound 8c
strongly bound to the KOR, while 8b did not. These
observation indicated that the relationships between the spatial
positions of these two aryl groups were important for binding
to the KOR. Compound 7c, which had the m-methoxyphenyl
instead of m-hydroxyphenyl groups, did not bind to the KOR,
suggesting that the hydroxy groups would be important
structural determinants to achieve binding. Compound 12a
indicated a sufficient binding affinity for the KOR, but
Figure 2. Structures of compounds containing the phenethylamine
unit.
According to the previously reported method,11,12 we
prepared compounds 1a−c (R = m-OH), 8a−d, and 11a,b.
The synthesis of compounds 1a−c (R = m-OH) commenced
with m-methoxyacetophenone (2). α-Hydroxyaldehyde 3 and
its hemiacetal dimer 4 derived from 2 were treated with
ammonium chloride and sodium acetate to give oxazoline 5.
Oxazoline 5 reacted with the mixture of 3 and 4 in the presence
of camphorsulfonic acid (CSA) to provide 6a−c (Scheme 1).
Compounds 7a−d were prepared by the acidic condensation of
5 with glycolaldehyde dimer (Scheme 2). The key intermediate
of the synthesis of 10a,b was oxazoline 9 prepared from
glycolaldehyde dimer and the mixture of 3 and 4 (Scheme 3).
The O-methyl groups of thus prepared 6a−c, 7a−d, and 10a,b
were deprotected with boron tribromide or potassium thiolates
to afford the corresponding compounds 1a−c, 8a−d, and
B
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a
Scheme 1
a
Reagents and conditions: (a) TosMIC, K2CO3, MeOH, rt; (b) 2 M HCl aq, THF, rt; (c) NH4Cl, AcONa, MeOH, reflux; (d) 3, 4, CSA, CHCl3,
reflux; (e) BBr3, CH2Cl2, 0 °C to rt.
a
Scheme 2
a
Reagents and conditions: (a) glycolaldehyde dimer, CSA, CHCl3, reflux; (b) 1-dodecanethiol, t-BuOK, DMF, 130 °C; (c) 1-propanethiol, t-BuOK,
DMF, 130 °C.
a
Scheme 3
a
Reagents and conditions: (a) glycolaldehyde dimer, NH4Cl, AcONa, MeOH, rt; (b) glycolaldehyde dimer, CSA, CHCl3, reflux; (c) 1-
dodecanethiol, t-BuOK, DMF, 130 °C.
a
Scheme 4
a
Reagents and conditions: (a) 1-propanethiol, t-BuOK, DMF, 130 °C.
C
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ACS Medicinal Chemistry Letters
Letter
Table 1. Binding Affinities of 1,3,5-Trioxazatriquinane
a
Derivatives for Opioid Receptors
Ki (nM)
b
c
d
MOR
DOR
KOR
U-69,593
U-50,488
1a
>1000
>1000
>1000
652
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
651
1.37
1.67
>1000
1b
667
>1000
793
1c
928
8a
735
8b
>1000
>1000
>1000
293
>1000
8c
6.09
4.63
>1000
(−)-8c
(+)-8c
8d
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
11a
11b
7c
12a
52.8
>1000
12b
a
Binding assays were carried out in duplicate (KOR: cerebellum of
guinea pig; MOR and DOR: whole brain without cerebellum of
b
c
d
mouse). [3H] DAMGO was used. [3H] DPDPE was used. [3H] U-
69,593 was used.
Table 2. Agonist Activities of 1,3,5-Trioxazatriquinane
a
Derivatives for Opioid Receptors
EC50 (Emax)
Figure 4. Analgesic effects induced by (−)-8c in the mouse acetic acid
writhing test. Each mouse was injected intraperitoneally (i.p.) with
0.6% acetic acid at a dose of 10 mL/kg 15 min after (−)-8c
administration (s.c.). After a 10 min delay, the animals were observed
for an additional 10 min, during which the number of abdominal
constrictions was counted. (a) Dose response effect; (b) Effects of
pretreatment with MOR antagonist β-FNA, DOR antagonist NTI, or
KOR antagonist nor-BNI on s.c. (−)-8c-induced antinociception in
mice. Groups of mice were pretreated with β-FNA21−25 (40 mg/kg),
NTI (3 mg/kg), or nor-BNI17,26−28 (20 mg/kg) at 24 h, 30 min, or 24
h, respectively, before s.c. administration of (−)-8c (7 mg/kg). Each
group was comprised of 8 mice. Data are presented as group means
SEM. Statistical significance of differences was assessed by repeated
one-way ANOVA followed by the Bonferroni test. ***p < 0.001 vs veh
or veh/veh.
MOR
DOR
KOR
U-69,593
8c
N.T.
N.D.
N.D.
N.T.
N.D.
12.0 nM (100%)
97.5 nM (98.2%)
50.3 nM (96.8%)
(−)-8c
83.7 nM (23.4%)
a
[35S] GTPγS binding assays were carried out in duplicate using
human MOR, DOR, or KOR expressed in CHO cells. DAMGO,
DPDPE, or U-69,593 was used as the standard MOR, DOR, or KOR
agonist, respectively. N.T.: not tested; N.D.: not detected.
compound 12b did not. The comparison of the binding
affinities between 12a and 12b indicated the importance of the
spatial location required for the m-hydroxyphenyl group.
According to our initial working hypothesis (Figure 3), the
phenolic hydroxy group on the endo-aryl group may play a
crucial role in binding to the opioid receptor. However, it was
not 12b with the hydroxyl group but rather 12a with the
methoxy group in the endo-aryl group that showed sufficient
binding to the KOR. This outcome indicated we should
restructure our binding model of 1,3,5-trioxazatriquinane
derivatives toward the opioid receptors. We are now
investigating the binding mode of (−)-8c with the KOR
using our three-dimensional pharmacophore model applicable
to some KOR agonists.18−20
The functional activities of 8c and its eutomer (−)-8c were
assessed by the [35S]GTPγS binding assays in human receptor
transfected CHO cells. Procedures similar to those previously
reported27 were used. Both compounds showed selective full
agonist activities for the KOR (Table 2). We next evaluated the
analgesic effects of (−)-8c by the acetic acid writhing test in
mice. Subcutaneous administration of (−)-8c dose-dependently
exhibited significant antinociception (ED50: 3.5 mg/kg, s.c.,
Figure 4a). The antinociceptive effects induced by (−)-8c were
significantly reversed by the selective KOR antagonist nor-BNI
but not by the selective MOR antagonist β-FNA or the
selective DOR antagonist NTI (Figure 4b).
In summary, we have designed and synthesized the 1,3,5-
trioxazatriquinane derivatives possessing m-hydroxyphenyl
groups. Among the synthesized compounds, (−)-8c selectively
bound and exerted full agonist activity for the KOR.
Subcutaneous administration of (−)-8c exhibited significant
antinociceptive effects via the KOR in a dose-dependent
manner. These results suggest the emergence of a novel class of
KOR agonist. We are now investigating the binding mode of
(−)-8c with the KOR.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for the synthesis and characterization
of the compounds, the in vitro activity assay, the in vivo mouse
acetic acid writhing assay, and the spectral data of the reported
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
D
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ACS Medicinal Chemistry Letters
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(12) Wada, N.; Fujii, H.; Koyano, K.; Hirayama, S.; Iwai, T.; Nemoto,
T.; Nagase, H. Synthesis of novel triplet drugs with 1,3,5-
trioxazatriquinane skeletons and their pharmacologies. 3: Synthesis
of novel triplet drugs with the bis(epoxymethano) or bis-
(dimethylepoxymethano) structure (double-capped triplet). Bioorg.
Med. Chem. Lett. 2012, 22, 7551−7554.
(13) Beckett, A. H.; Casy, A. F. Synthetic analgesics: stereochemical
considerations. J. Pharm. Pharmacol. 1954, 6, 986−1001.
(14) Beckett, A. H. Analgesics and their antagonists: some steric and
chemical considerations. I. The dissociation constants of some tertiary
amines and synthetic analgesics, the conformations of methadone-type
compounds. Pharm. Pharmacol. 1956, 8, 848−859.
(15) Ida, Y.; Nemoto, T.; Hirayama, S.; Fujii, H.; Osa, Y.; Imai, M.;
Nakamura, T.; Kanemasa, T.; Kato, A.; Nagase, H. Synthesis of
quinolinomorphinan-4-ol derivatives as δ opioid receptor agonists.
Bioorg. Med. Chem. 2012, 20, 949−961.
(16) The optical resolution of racemic 8c and the determination of
the absolute configuration of each enantiomer are described in detail in
the Supporting Information.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: nagase.hiroshi.gt@u.tsukuba.ac.jp.
Present Address
†H.N.: International Institute for Integrative Sleep Medicine,
University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-
8577, Japan.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
CHO, Chinese hamster ovary; CSA, camphorsulfonic acid;
DAMGO, [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin; DOR, δ
opioid receptor; DPDPE, [D-Pen2, D-Pen5]-enkephalin; β-
FNA, β-funaltrexamine; KOR, κ opioid receptor; MOR, μ
opioid receptor; nor-BNI, nor-binaltorphimine; NTI, naltrin-
dole; TosMIC, p-toluenesulfonylmethyl isocyanide
(17) Spetea, M.; Berzetei-Gurske, I. P.; Guerrieri, E.; Schmidhammer,
H. Discovery and pharmacological evaluation of a diphenethylamine
derivative (HS665), a highly potent and selective κ opioid receptor
agonist. J. Med. Chem. 2012, 55, 10302−10306.
(18) Yamaotsu, N.; Fujii, H.; Nagase, H.; Hirono, S. Identification of
the three-dimensional pharmacophore of κ-opioid receptor agonists.
Bioorg. Med. Chem. 2010, 18, 4446−4452.
(19) Yamaotsu, N.; Hirono, S. 3D-pharmacophore identification for
κ-opioid agonists using ligand-based drug-design techniques. Top.
Curr. Chem. 2011, 299, 277−307.
(20) According to our three-dimensional pharmacophore model of
KOR agonists (see refs 18 and 19), the binding modes were classified
into four types. (−)-8c may belong to the binding mode type IV,
which differs from type II as indicated in Figure 3 and includes both
aromatic and hydrogen-bonding accepting and/or donating inter-
actions.
(21) We determined the dosage and administration time of β-FNA in
accordance with refs 22−25.
(22) Takemori, A. E.; Larson, D. L.; Portoghese, P. S. The
irreversible narcotic antagonistic and reversible agonistic properties
of the fumaramate methyl ester derivative of naltrexone. Eur. J.
Pharmacol. 1981, 70, 445−451.
REFERENCES
■
(1) Contreras, J. M.; Sippl, W. Homo and Heterodimer Ligands: the
Twin Drug Approach. In The Practice of Medicinal Chemistry, 3rd ed.;
Wermuth, C. G., Ed.; Academic Press: London, 2008; Chapter 18.
(2) Morphy, R.; Rankovic, Z. Multi-target Drugs: Strategies and
Challenges for Medicinal Chemistry. In The Practice of Medicinal
Chemistry, 3rd ed.; Wermuth, C. G., Ed.; Academic Press: London,
2008; Chapter 27.
(3) Designing Multi-Target Drugs; Morphy, J. R., Harris, C. J., Eds.;
The Royal Society of Chemistry: Cambridge, 2012.
(4) Polypharmacology in Drug Discovery; Peters, J. U., Ed.; John Wiley
& Sons, Inc.: Hoboken, NJ, 2012.
(5) Fujii, H. Twin and triplet drugs in opioid research. Top. Curr.
Chem. 2011, 299, 239−275 and references cited therein..
(6) Bhushan, R. G.; Sharma, S. K.; Xie, Z.; Daniels, D. J.; Portoghese,
P. S. A bivalent ligand (KDN-21) reveals spinal δ and κ opioid
receptors are organized as heterodimers that give rise to δ1 and κ2
phenotypes. selective targeting of δ-κ heterodimers. J. Med. Chem.
2004, 47, 2969−2972.
(7) Daniels, D. J.; Lenard, N. R.; Etienne, C. L.; Law, P. Y.; Roerig, S.
C.; Portoghese, P. S. Opioid-induced tolerance and dependence in
mice is modulated by the distance between pharmacophores in a
bivalent ligand series. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 19208−
19213.
(8) Peng, X.; Knapp, B. I.; Bidlack, J. M.; Neumeyer, J. L. Synthesis
and preliminary in vitro investigation of bivalent ligands containing
homo- and heterodimeric pharmacophores at μ, δ, and κ opioid
receptors. J. Med. Chem. 2006, 49, 256−262.
(9) Nagase, H.; Watanabe, A.; Harada, M.; Nakajima, M.; Hasebe, K.;
Mochizuki, H.; Yoza, K.; Fujii, H. Novel synthesis of a 1,3,5-
trioxazatriquinane skeleton using a nitorogen clamp. Org. Lett. 2009,
11, 539−542.
(23) Ward, S. J.; Portoghese, P. S.; Takemori, A. E. Pharmacological
profiles of β-funaltrexamine (β-FNA) and β-chlornaltrexamine (β-
CAN) on the mouse vas deferens preparation. Eur. J. Pharmacol. 1982,
80, 377−384.
(24) Hayes, A. G.; Skingle, M.; Tyers, M. B. Effect of β-
funaltrexamine on opioid side-effects produced by morphine and U-
50,488H. J. Pharm. Pharmacol. 1985, 37, 841−843.
(25) Aceto, M. D.; Dewey, W. L.; Portoghese, P. S.; Takemori, A. E.
Effects of β-funaltrexamine (β-FNA) on morphine dependence in rats
and monkeys. Eur. J. Pharmacol. 1986, 123, 387−393.
(26) We determined dosage and administration time of nor-BNI in
accordance with refs 17 and 27.
(27) Endoh, T.; Matsuura, H.; Tanaka, C.; Nagase, H. Nor-
binaltorphimine: A potent and selective κ-opioid receptor antagonist
with long-lasting activity in vivo. Arch. Int. Pharmacodyn. Ther. 1992,
316, 30−42.
(28) Nemoto, T.; Fujii, H.; Narita, M.; Miyoshi, K.; Nakamura, A.;
Suzuki, T.; Nagase, H. Syntheses of 4,6′-epoxymorphinan derivatives
and their pharmacologies. Bioorg. Med. Chem. 2008, 16, 4304−4312.
(10) Nagase, H.; Watanabe, A.; Nemoto, T.; Nakajima, M.; Hasebe,
K.; Mochizuki, H.; Fujii, H. Synthesis of novel triplet drugs with 1,3,5-
trioxazatriquinane skeletons and their pharmacologies. 1: Synthesis of
triplet drugs with morphinan skeletons. Bioorg. Med. Chem. Lett. 2011,
21, 4023−4026.
(11) Nagase, H.; Koyano, K.; Wada, N.; Hirayama, S.; Watanabe, A.;
Nemoto, T.; Nakajima, M.; Nakao, K.; Mochizuki, H.; Fujii, H.
Synthesis of novel triplet drugs with 1,3,5-trioxazatriquinane skeletons
and their pharmacologies. 2: Synthesis of novel triplet drugs with the
epoxymethano structure (capped homotriplet). Bioorg. Med. Chem.
Lett. 2011, 21, 6198−6202.
E
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