Design and Synthesis of Potent and Highly Selective Orexin 1 Receptor Antagonists with a Morphinan Skeleton and Their Pharmacologies

Article abstract of DOI:10.1021/acs.jmedchem.6b01418

Nalfurafine, a κ-selective opioid receptor agonist, unexpectedly showed a selective antagonist activity toward the orexin 1 receptor (OX₁R) (K_i = 250 nM). Modification of the 17-amino side chain of the opioid ligand to an arylsulfonyl group and the 6-furan acrylamide chain to 2-pyridyl acrylamide led to compound 71 with improvement of the antagonist activity (OX₁R, K_i = 1.36 nM; OX₂R, not active) without any detectable affinity for the opioid receptor. The dihydrosulfate salt of 71, freely soluble in water, attenuated the physical dependence of morphine. Furthermore, all of the active nalfurafine derivatives in this study had almost no activity for OX₂R, which led to high OX₁R selectivity. These results suggest that nalfurafine derivatives could be a useful series of lead compounds to develop highly selective OX₁R antagonists.

Full text of DOI:10.1021/acs.jmedchem.6b01418

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Article
Design and Synthesis of Potent and Highly Selective Orexin 1 Receptor
Antagonists with a Morphinan Skeleton and their Pharmacologies
Hiroshi Nagase, Naoshi Yamamoto, Masahiro Yata, Sayaka Ohrui, Takahiro Okada, Tsuyoshi Saitoh,
Noriki Kutsumura, Yasuyuki Nagumo, Yoko Irukayama-Tomobe, Yukiko Ishikawa, Yasuhiro Ogawa,
Shigeto Hirayama, Daisuke Kuroda, Yurie Watanabe, Hiroaki Gouda, and Masashi Yanagisawa
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01418 • Publication Date (Web): 04 Jan 2017
Downloaded from http://pubs.acs.org on January 4, 2017
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Design and Synthesis of Potent and Highly Selective Orexin 1 Receptor Antagonists with a
Morphinan Skeleton and their Pharmacologies
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*
†‡
†
‡
†
‡
Hiroshi Nagase, Naoshi Yamamoto, Masahiro Yata, Sayaka Ohrui, Takahiro Okada,
†
†
†
†
Tsuyoshi Saitoh, Noriki Kutsumura, Yasuyuki Nagumo, Yoko IrukayamaꢀTomobe, Yukiko
†
†
ǁ
§
§
Ishikawa, Yasuhiro Ogawa, Shigeto Hirayama, Daisuke Kuroda, Yurie Watanabe, Hiroaki
§
†
Gouda, Masashi Yanagisawa
†
International Institute for Integrative Sleep Medicine (WPIꢀIIIS), University of Tsukuba, 1ꢀ1ꢀ1
Tennodai, Tsukuba, Ibaraki 305ꢀ8575, Japan
‡
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1ꢀ1ꢀ1 Tennodai,
Tsukuba, Ibaraki 305ꢀ8571, Japan
ǁ
Laboratory of Medicinal Chemistry, School of Pharmacy, Kitasato University, 5ꢀ9ꢀ1 Shirokane,
Minatoꢀku, Tokyo 108ꢀ8641, Japan
§
School of Pharmacy, Showa University, 1ꢀ5ꢀ8 Hatanodai, Shinagawaꢀku, Tokyo 142ꢀ8555,
Japan
KEYWORDS
Orexin, Opioid, OX R antagonist, Morphinan skeleton
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ABSTRACT
Nalfurafine, a κꢀselective opioid receptor agonist, unexpectedly showed a selective antagonist
activity toward the orexin 1 receptor (OX R) (K = 250 nM). Modification of the 17ꢀamino side
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chain of the opioid ligand to an arylsulfonyl group and the 6ꢀfuran acrylamide chain to 2ꢀpyridyl
acryl amide led to compound 71 with improvement of the antagonist activity (OX R: K = 1.36
1
i
nM, OX R: Not active) without any detectable affinity for the opioid receptor. The
2
dihydrosulfate salt of 71, freely soluble in water, attenuated the physical dependence of
morphine. Furthermore, all of the active nalfurafine derivatives in this study had almost no
activity for OX R, which led to high OX R selectivity. These results suggest that nalfurafine
2
1
derivatives could be a useful series of lead compounds to develop highly selective OX R
1
antagonists.
INTRODUCTION
1
2
Orexin (orexinꢀA and ꢀB, also known as hypocretinꢀ1 and ꢀ2 ) are a pair of lateral hypothalamic
neuropeptides originally identified as the endogenous ligands for two previously orphan G
proteinꢀcoupled receptors, orexin 1 receptor (OX R) and orexin 2 receptor (OX R). An essential
1
2
role of the orexin system in regulation of sleep and wakefulness was initially demonstrated by
3
a
3b
the discoveries that OX Rꢀdeficient dogs and preproꢀorexin knockout mice both exhibited
2
symptoms highly similar to the sleep disorder narcolepsy/cataplexy. Whereas OX R/OX R
1
2
double null mice exhibit a severe narcoleptic phenotype indistinguishable from that seen in
preproꢀorexin knockout mice, OX Rꢀnull mice show a somewhat milder narcolepsy phenotype.
2
Moreover, OX Rꢀnull mice exhibit no appreciable sleep/wakefulnessꢀrelated phenotype,
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suggesting that OX R, rather than OX R, plays a predominant role in sleep/wake regulation, and
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1
an intact OX Rꢀmediated signaling is sufficient to prevent the symptoms of
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narcolepsy/cataplexy.
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Many researchers have attempted to develop nonꢀpeptide orexin antagonists to evaluate the role
5
of orexin receptors (OXRs), especially focused on sleep indications. Many selective OX R
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6
7
7
8
antagonists (1ꢀSORAs); 4 (SBꢀ334867), 5 (SBꢀ408124), 6 (SBꢀ674042), 7 (GSKꢀ1059865),
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and selective OX R antagonists (2ꢀSORAs); 8 (TCSꢀOX2ꢀ29), 9 (MKꢀ3697) , as well as dual
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1
12
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OX R/OX R antagonists (DORAs); suvorexant (1) , almorexant (2) , 3 (SBꢀ649868) , have
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2
been reported (Figure 1).
Quite recently, Merck released suvorexant (1) (DORA) in Japan and the United States for
1
1
treatment of insomnia. However, no orexin agonist had been reported until recently. In 2015,
we reported the design and synthesis of the first nonꢀpeptide OX R agonists; one of the agonists,
2
compound 10, showed potent and selective agonistic activity for OX R (OX R: EC = 2,750
2
1
50
1
4
nM; OX R: EC = 28 nM, E = 94%) (Figure 1). Central injection of dihydrochloride of
max
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50
compound 10 (260 nmol) in mice increased wake time to a similar degree achieved with 3 nmol
orexinꢀA.
Orexins have been reported to be also involved in regulation of a wide range of behaviors other
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5,16
than sleep/wake, e.g., hedonic feeding behavior and reward seeking.
Especially, OX R was
1
reported to contribute to regulating rewardꢀrelated behaviors. An increasing body of work shows
17a,18
that orexin neurons play a part in the behavioral presentation of addiction to morphine,
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7b,17c
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cocaine,
amphetamine, heroin, nicotine, ethanol, and cannabinoids. Generally,
orexin seems to be involved in the modulation of highly motivated reward seeking, especially
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when the seeking is triggered by external cues. 1ꢀSORAs, 4 and 7 were reported to attenuate
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the expression of conditioned place preference induced by cocaine and amphetamine in rats.
Orexin might also be involved in opioid addiction; for example, orexin knockout mice, as well as
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wild type mice treated with the 1ꢀSORA, 4, reduced morphine withdrawal.
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Figure 1. The structures of the dual orexin receptor antagonists 1–3, selective OX R antagonists
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4–7, selective OX R antagonists 8–9, and selective OX R agonist 10.
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We have been interested in the interaction of the OX R with opioid receptors on the basis of our
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long history in the opioid research field, and also intrigued by the biological relevance for the
coexpression of excitatory orexin and inhibitory dynorphin (11, endogenous κ opioid agonist)
27
and the coexistence of their opposing effects within the same vesicle in the orexin neuron. To
obtain specific ligands for clarifying the potential role for their coexistence, we attempted to
design and synthesize OX R antagonists derived from κ opioid receptor (KOR) ligands.
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Quite recently, the heterodimerization of the OX R and KOR has been reported, shedding
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further light on the potential function for the colocalization of the corresponding two peptides
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with opposite effects,
which were shown in not only the endogenous peptide, but also with
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exogenous alkaloids. For example, as we mentioned above, OX R antagonist 4 attenuated drug
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7b,21b,30
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addiction
and most KOR agonists represented by 16 (Uꢀ50488H) induced severe
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aversion (like the psychotomimetic effect) and dysphoria, which was ample reason to eliminate
the derivatives of 16 at the early stage of the clinical trials. Intriguingly, only nalfurafine (12)
among all of the KOR agonists showed neither addiction nor drug aversion and was released as
26a‒e
an antipruritic agent for kidney dialysis patients in Japan in 2009.
Even now, many
researchers have been interested in and are investigating why only nalfurafine (12) caused no
aversion. We postulated that nalfurafine (12) might bind with the OX RꢀKOR dimer to prevent
1
aversion, but 16 binds with pure KOR to afford an intrinsic aversive effect. Furthermore, we
expected that nalfurafine (12) would also bind with OX R in addition to binding with KOR, if
1
the nalfurafine could show affinity for the receptor heterodimer. Based on these considerations,
we evaluated nalfurafine (12) with a calcium transient assay in CHO cells expressing human
OX R or OX R, to examine the possible activity of nalfurafine for orexin receptors. As we
1
2
expected, nalfurafine (12) showed antagonistic activity for OX R (OX R: K = 250 nM, OX R:
1
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i
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Not active) (Figure 2).
Interestingly, only nalfurafine (12) showed activity for the OX R, but no effect was noted with
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the µ opioid receptor (MOR) antagonist βꢀfunaltrexamine (βꢀFNA), the δ opioid receptor
(DOR)
selective
agonist
(6R,6aS,14aR)ꢀ17ꢀmethylꢀ5,6,7,14ꢀtetrahydroꢀ6aHꢀ6,14aꢀ
35
(epiminoethano)naphtho[2,1ꢀb]acridineꢀ2,6aꢀdiol (KNTꢀ127),
the antagonist naltrindole
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(
NTI), and the KOR selective antagonist norꢀbinaltorphimine (norꢀBNI). Furthermore, the
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Upjohnꢀtype KOR agonist 16 showed no activity for OX R. The structure of nalfurafine (12)
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contains a tyrosine moiety as the Nꢀterminal structure of dynorphin (11), while 16 does not. The
fact that only nalfurafine (12), with the partial structure of dynorphin (11), showed activity for
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OX R in our opioid chemical library, might be a clue for clarifying the role of the
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aforementioned coexistence of dynorphin and orexin.
HꢀTyrꢀGlyꢀGlyꢀPheꢀLeuꢀArgꢀArgꢀX
Dynorphin A: X = IleꢀArgꢀProꢀLysꢀLeuꢀLysꢀTrpꢀAspꢀAsnꢀGlnꢀOH
Dynorphin B: X = GlnꢀPheꢀLysꢀValꢀValꢀThrꢀOH
11 (Dynorphin)
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17
N
N
OH
O
OH
Me
O
O
6
N
6
N
N
O
Me
Me
Cl
O
O
O
N
3
R
3
Cl
OH
15
16 (Uꢀ50488H)
R = OH; 12 (Nalfurafine) OX R: K = 250 ± 37.1 nM, OX R: Not active
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i
2
R = OMe; 13
R = H; 14
OX R: K = 22.5 ± 2.56 nM, OX R: Not active
1 i 2
OX R: K = 1245 ± 132 nM, OX R: Not active
1
i
2
Figure 2. The structures of dynorphin (11), nalfurafine (12), nalfurafine derivatives 13‒15, and
16.
The above described antagonists in Figure 1 have rather simple flexible structures which consist
of aromatic or alicyclic rings connected to each other with amide or urea bonds. In contrast,
nalfurafine (12) has a characteristic rigid morphinan skeleton with a tyrosine moiety which is a
partial structure of dynorphin (11) (Figure 2). So, we expected that the antagonistic activity and
selectivity of nalfurafine for OX R could be easily improved by using our modification
1
techniques developed over the long course of our opioid research.
To the best of our knowledge, no OXR ligand with a morphinan skeleton has been reported. First
results of testing nalfurafine derivatives for OX R antagonism showed that the 6ꢀαꢀamide isomer
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5 of nalfurafine had almost no activity for OXRs. Therefore, we focused our modification on
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the 6ꢀβꢀamide isomers. Herein, we report the results of the modification of nalfurafine (12) and
the pharmacological effects of the resulting selective and potent antagonists of OX R.
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RESULTS AND DISCUSSIONS
We started with the methylation of 3ꢀOH in nalfurafine (12) to afford 3ꢀmethyl ether 13 which
showed 11 times more potent antagonistic activity for OX R than nalfurafine (Figure 2). We also
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synthesized 3ꢀdehydroxynalfurafine 14 which showed very weak activity for OXRs. These
facts led us to use the 3ꢀmethyl ether as a lead compound for succeeding modifications of the
nalfurafine derivatives.
2
6b,41
Next the 3ꢀfurylacryl group of the 6ꢀamide side chain in nalfurafine methyl ether 13
was
converted to benzyl, phenylacryl and 2ꢀfurylacryl amide groups. These derivatives 17–19 gave
no improved activities (Table 1). Then we decided to hold the 6ꢀamide side chain on the 3ꢀ
furylacryloyl group in the following modification.
Table 1. Assay results of the 6ꢀsubstituted nalfurafine derivatives for orexin receptor antagonism
K (nM)
i
Compound
R
–
OX R
OX R
1
2
a
5
18.9 ± 0.688
250 ± 37.1
2070 ± 482
a
b
nalfurafine (12)
–
b
b
a
–
–
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389 ± 88.8
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K values represented the mean ± SEM. These values were calculated using IC50 values of
i
nalfurafine derivatives at least three independent calcium assays performed in triplicate (Chengꢀ
a
b
Prusoff equation). Its HCl salt was assayed. K value was not calculated, IC value was over
i
50
than 10,000 nM (cut off value) or was not obtain from concentrationꢀresponse curve.
We then replaced the 17ꢀcyclopropylmethyl (CPM) group in nalfurafine methyl ether 13 by
various acyl substituents to improve the activity for OX R (Scheme 1). The phenolic hydroxyl
1
group in naltrexone hydrochloride (20) was methylated and the 6ꢀketone group was protected as
1
,3ꢀdioxolane, followed by acetylation of the 14ꢀOH under refluxing in Ac O. The CPM group
2
of the resulting acetate was exchanged for a Troc group at 140 °C with an excess amount of
TrocCl to afford carbamate 21. The carbamate and the acetate group in 21 were hydrolyzed with
aqueous KOH at 110 °C. After removing the 1,3ꢀdioxolane group in amine 22, the secondary
amine was protected with Boc to give ketone 23 in good yield. The imine formation of 23 with
Nꢀbenzylmethylamine, followed by reduction with NaBH CN afforded βꢀamine 24.
3
Hydrogenation of 24 and amidation of the resulting amine with (E)ꢀ3ꢀ(furanꢀ3ꢀyl)acryloyl
4
2
chloride afforded 17ꢀBoc nalfurafine derivative 26. Deprotection of the Boc group in
compound 26 gave key intermediate 27, which was converted to 17ꢀamide nalfurafine
derivatives 28–31. The antagonistic activities for OXRs of the obtained 17ꢀBoc and amide
2
+
derivatives were estimated in the Ca assays.
Scheme 1. Synthesis of 17ꢀcarbonyloxy compounds 28–31 from naltrexone hydrochloride (20).
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N
7
NTroc
NH
NBoc
OH
NBoc
OH
OH —HCl
OAc
OH
1
4
a–d
e
f–g
i
h
6
O
O
O
O
NMeBn
O
O
O
O
O
O
O
O
3
OH
OMe
OMe
22
OMe
23
OMe
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2
0 (Naltrexone hydrochloride)
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21
O
NBoc
OH
NBoc
NH
R
N
OH
OH
OH
O
O
j
l
k
NHMe
O
N
N
N
Me
Me
Me
O
O
O
O
O
O
OMe
OMe
OMe
OMe
2
5
26
28–31
27
Reagents and conditions: a) MeI, K CO , DMF, rt; b) ethylene glycol, pꢀTsOH·H O, toluene,
2
3
2
reflux; c) Ac O, reflux; d) TrocCl, K CO , 1,1,2,2ꢀtetrachloroethane, 140 °C, 82% (4 steps); e)
2
2
3
KOH aq., DMSO, 110 °C, 98%; f) HCl aq., 80 °C; g) (Boc) O, (iꢀPr) NEt, CH Cl , rt, 91%; h)
2
2
2
2
BnNHMe, PhCO H, pꢀTsOH·H O, PhH, reflux; evap.; NaBH CN, MS4A, EtOH, 0 °C to rt,
2
2
3
84%; i) H , Pd/C, MeOH, rt, quant; j) (E)ꢀ3ꢀ(furanꢀ3ꢀyl)acryloyl chloride, Et N, CH Cl , rt, 79%;
2
3
2
2
k) HCl–MeOH, rt, 82%; l) Ac O, pyridine, rt, 97% for 28; cyclopropanecarbonyl chloride,
2
pyridine, rt, 82% for 29; pꢀtoluoyl chloride, Et N, CH Cl , rt, 85% for 30; cinnamoyl chloride,
3
2
2
Et N, CH Cl , rt, 77% for 31.
3
2
2
Unexpectedly, the 17ꢀBoc nalfurafine derivative 26 showed the strongest antagonist activity
OX R: K = 4.15 nM) among the resulting amide derivatives shown in Table 2. However, the
(
1
i
OX R selectivity over OX R was not as high as those of the acetamide and cyclopropylamide
1
2
derivatives (28 and 29) (Table 2).
Table 2. Assay results of the 17ꢀBoc and amide derivatives for orexin receptor antagonism
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
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4
4
4
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5
5
5
5
5
5
5
5
5
5
6
O
R
N
OH
O
O
N
Me
O
OMe
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
K (nM)
i
Compound
R
OX R
OX R
1
2
a
2
2
6
8
4.15 ± 0.235
541 ± 59.9
–
a
–
a
2
9
12.0 ± 0.715
14.0 ± 1.98
–
30
725 ± 41.5
a
3
1
14.0 ± 2.20
–
K values represented the mean ± SEM. These values were calculated using IC50 values of
i
nalfurafine derivatives at least three independent calcium assays performed in triplicate (Chengꢀ
a
Prusoff equation). K value was not calculated, IC value was over than 10,000 nM (cut off
i
50
value) or was not obtain from concentrationꢀresponse curve.
The key functional group in the aforementioned first selective OX R agonists reported in 2015
2
1
4a
were sulfonamides, which led us to synthesize 17ꢀsulfonamide nalfurafine derivatives. The
synthetic route for the 17ꢀsulfonamide derivatives are shown in Scheme 2. The syntheses of the
alkylꢀ and arylsulfoamide derivatives 32–58 were attained by sulfoamidation of the amine 27.
Dimethylaminobenzenesulfonamide derivatives 59–61 were synthesized from the corresponding
nitrobenzenesulfonamide derivatives 50–52 by reduction of the nitro group and reductive
amination of the resulting amine. Deprotection of the Boc group in 24 and sulfonamidation with
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5
ꢀbromoꢀ2ꢀmethoxybenzensulfonyl chloride afforded sulfonamide 63. The benzyl group and
bromine atom were removed by hydrogenation, followed by amidation with (E)ꢀ3ꢀ(furanꢀ3ꢀ
yl)acyloyl chloride to give the oꢀMeO derivative 64 in good yield.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Synthesis of the 17ꢀsulfonamide derivatives
Reagents and Conditions: a) RSO Cl, Et N, CH Cl , rt, 66–99%; b) SnCl , HCl, CH Cl , 40
2
3
2
2
2
2
2
°
C; c) paraformaldehyde, NaBH CN, AcOH, 40 °C, 73–90% (2 steps); d) HCl–MeOH, rt, 99%;
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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
e) 5ꢀbromoꢀ2ꢀmethoxybenzensulfonyl chloride, Et N, CH Cl , rt, 84%; f) H , Pd/C, MeOH, rt; g)
3
2
2
2
(
E)ꢀ3ꢀ(furanꢀ3ꢀyl)acyloyl chloride, Et N, CH Cl , rt, 77% (2 steps).
3 2 2
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
Table 3. Assay results of the 17ꢀsulfonamide derivatives for orexin receptor antagonism
K (nM)
i
Compound
R
OX R
OX R
1
2
b
32
33
12.1 ± 1.14
–
b
5.94 ± 0.237
–
b
3
3
4
5
8.14 ± 0.606
2.07 ± 0.222
–
b
–
b
36
7.60 ± 0.570
–
b
37
4.05 ± 0.471
1.93 ± 0.138
–
b
38
–
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b
39
3.47 ± 0.274
–
b
b
4
4
0
1
5.13 ±1.02
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.10 ± 0.184
b
42
2.27 ± 0.204
–
b
b
4
4
3
4
6.21 ± 0.655
2.05 ± 0.209
–
–
b
45
6.19 ± 0.934
–
b
b
46
12.6 ± 0.790
1.96 ± 0.359
–
–
47
b
48
7.30 ± 0.748
–
b
b
49
13.9 ± 0.451
1.81 ± 0.142
–
–
50
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1
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1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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
b
51
5.05 ± 0.343
–
b
b
5
5
2
9
12.5 ± 1.26
–
–
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
a
a
a
1.92 ± 0.190
b
60
7.25 ± 0.557
–
b
b
6
6
1
4
12.9 ± 1.26
6.37 ±0.921
–
–
b
53
2.44 ± 0.388
–
b
b
54
55
8.99 ± 1.22
–
–
1.70 ± 0.194
b
5
5
6
7
2.98 ± 0.364
–
b
b
5.85 ± 0.794
4.16 ± 0.696
–
–
58
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9
K values represented the mean ± SEM. These values were calculated using IC50 values of
i
nalfurafine derivatives at least three independent calcium assays performed in triplicate (Chengꢀ
a
b
Prusoff equation). Its HCl salt was assayed. K value was not calculated, IC value was over
i
50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
than 10,000 nM (cut off value) or was not obtain from concentrationꢀresponse curve.
The activities of the sulfonamides are shown in Table 3. The activities of the two alkyl
sulfonamide derivatives 32 and 33 were lower than that of the 17ꢀBoc derivative 26. Almost all
the substituted sulfonamide derivatives showed high activities in the single digit nM range and
high selectivities for OX R, with only minor differences among the respective oꢀ, mꢀ, pꢀ
1
substitutions. We observed only small differences in activities and selectivities between the
substituted derivatives, independent of the electron donating (Me, NMe , except for OMe) or
2
electron withdrawing (CF , CN, NO ) character of the substitutents.
3
2
The observed tendency for the highest potency for the oꢀsubstituted derivatives suggests that the
oꢀsubstituent group might induce a rotation around the Ar–SO NR single bond by steric
2
2
hindrance with the oꢀsubstituents or by dipoleꢀdipole interaction between the F group and the
sulfonamide group forcing the phenyl ring into an adequate fitting position at the receptor site,
thus increasing the activity.
We carried out conformational analyses of 50 (oꢀnitrobenzenesulfonamide) and 52 (pꢀ
nitrobenzenesulfonamide) to compare their most stable conformations. As shown in Figure 3, we
found that the spatial dispositions of oꢀnitroꢀ and pꢀnitrobenzenesulfonamides were quite
different between the most stable conformers of 50 and 52. Further, from the superimpositions of
lower energy conformers of 50 and 52, we found that the oꢀnitrobenzenesulfonamide of 50 could
be widely spread out, but the pꢀnitrobenzenesulfonamide of 52 was spatially restricted. These
results again suggested the oꢀsubstituent group might facilitate the rotation around ArꢀSO NR .
2
2
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2
2
2
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2
2
2
2
2
2
3
3
3
3
3
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
Figure 3. The most stable conformations and the superimpositions of the lowꢀenergy conformers
of 50 and 52.
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The activities of pꢀsubstituted sulfonamide derivatives with electron donating groups (including
+
halogen) were slightly higher than those of the electron deficient group (the NHMe2 group
resulted from protonation under physiological condition and would become an electron
withdrawing group), quite a different observation from oꢀsubstituted derivatives. As the pꢀ
substituted derivatives would provide neither steric nor dipoleꢀdipole interaction to the
sulfonamide group, the activity may directly be affected by the electron effect of the substituent
on the aromatic ring.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Demethylation of the 3ꢀOMe group in compound 61
Reagents and Conditions: a) BBr , CH Cl , –78 °C to rt, 51%.
3
2
2
Scheme 4. Synthesis of the compounds with pyridyl moiety
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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
O
O
O
S
S
N
O
NO2
NMe2
N
OH
OH
a
b, c
22
O
O
O
O
O
O
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
OMe
OMe
6
6
67
O
S
O
N
O
S
O
NMe2
OH
N
NMe2
OH
d
e
O
NMeR
O
O
OMe
OMe
6
8
69: R = Bn
70: R = H
f
O
S
O
N
NMe2
OH
g
O
N
Me
R
O
OMe
7
7
7
1: R = 2ꢀpyridyl
2: R = 3ꢀpyridyl
3: R = 4ꢀpyridyl
Reagents and Conditions: a) oꢀnitrobenzenesulfonyl chloride, Et N, CH Cl , rt, 99%; b) Fe,
3
2
2
NH Cl, EtOH, H O, 90 °C; c) (CH O) , NaBH CN, AcOH, 40 °C, 97% (2 steps); d) HCl aq.,
4
2
2
n
3
THF, 90 °C, 90%; e) BnNHMe, PhCO H, PhH, reflux; evap.; NaBH CN, MeOH, THF, 0 °C,
2
3
8
0%; f) H , Pd/C, MeOH, rt, 96%; g) RCH=CHCO H, HATU, (iꢀPr) NEt, DMF, rt, 95–97%.
2 2 2
Table 4. Assay results of the 65, 71–73 for orexin receptor antagonism
K (nM)
i
Compound
OX R
OX R
1
2
a
6
5
320 ± 72.1
–
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a
7
1
2
1.36 ± 0.174
42.3 ± 2.25
–
a
7
–
a
a
73
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
K values represented the mean ± SEM. These values were calculated using IC50 values of
i
nalfurafine derivatives at least three independent calcium assays performed in triplicate (Chengꢀ
a
Prusoff equation). K value was not calculated, IC value was over than 10,000 nM (cut off
i
50
value) or was not obtain from concentrationꢀresponse curve.
Table 5. Assay results for opioid receptors
K (nM)
i
ꢁ
δ
κ
Compound
3
3
3
([ H]DAMGO) ([ H]DPDPE) ([ H]Uꢀ69593)
5
>1,000
.99
>1,000
693
>1,000
5
0.238
a
nalfurafine (12)
(3.4–10.6)
>1,000
(223–2154)
(0.147–0.385)
184
2
6
>1,000
(130–261)
29
>1,000
>1,000
>1,000
>1,000
971
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
>1,000
200
31
34
61
65
b
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1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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
(
654–1441)
>1,000
K values with 95% confidential intervals were obtained from radioligandꢀbased competitive
(35–295)
c
7
1
>1,000
>1,000
i
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
a
b
c
receptor binding assay. Its HCl salt was assayed. Its MeSO H salt was assayed. Its 2H SO salt
3
2
4
was assayed.
The activity of 2,4,6ꢀtrimethylbenzenesulfonamide derivative 58 was almost equivalent with that
of oꢀMeꢀsubstituted derivative 55. These results also support the idea that steric hindrance on the
aromatic ring could induce a favorable rotation around the SO –Ar bond.
2
The 6ꢀNH carboxyamide derivatives of the oꢀdimethylaminosulfonamideꢀ6ꢀpyridylacrylamide
derivatives showed markedly lower activity (about 100 times lower) for OXRs than the
corresponding 6ꢀNMe amide derivative.
Intriguingly, the above obtained 17ꢀcarboxyamide and sulfonamide derivatives with a 3ꢀmethoxy
group showed extremely lower affinity for opioid receptors (MOR, DOR, and KOR: K = >1,000
i
nM) compared with that of nalfurafine (MOR: K = 5.99 nM, DOR: K = 693 nM, KOR: K =
i
i
i
0
.238 nM) (Table 5). Even the pꢀdimethylaminobenzenesulfonamide derivative with a 3ꢀhydroxy
group 65 showed very low affinity for the opioid receptor (MOR: K = 971 nM, DOR: K =
i
i
>
1,000 nM, KOR: K = 200 nM). The only 17ꢀBoc derivative 26 had a rather higher affinity for
i
the KOR (K = 184 nM) despite containing the 3ꢀmethoxy group. These data from the binding
i
assays suggest that the above obtained 3ꢀmethoxyꢀsulfonamide derivatives could remove the
serious side effects derived from the opioid receptors (addiction, constipation, respiratory
depression from MOR, and catalepsy from DOR and especially, sedation and aversion from
KOR) although the structures of these OX R antagonists were derived from the potent KOR
1
26
agonist, nalfurafine (12).
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The opioid ligands bind with opioid receptors with three main types of pharmacophore bonds:
2
6
ionic attraction, π–π interaction, and hydrogen bonding, termed the message site. The above
obtained potent OX R antagonists were hardly bound with the opioid receptors because of the
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
26a,26b,43
absence of the basic nitrogen and the phenolic hydroxy group.
The ion formed by the
protonation on the basic 17ꢀnitrogen and the acidic hydrogen derived from the 3ꢀphenolic
hydroxyl group seemed to disrupt the fitting to the orexin receptor. This information could be an
important clue for designing OX R ligands with a morphinan skeleton without affinity for opioid
1
receptors. Quite recently, Perrey et al. reported improved antagonists for OX R and the most
1
potent and selective compound in their paper showed promising K values for OX R (K ＝ 8.50
e
1
e
4
4
± 1.0 nM) and for OX R (K = >10,000 nM). On the other hand, one of our antagonists,
2
e
compound 50 showed almost equivalent activity and selectivity for OX R (OX R: K = 3.69 ±
1
1
e
4
5
0
.0376 nM, OX R: Not active).
2
Although the above antagonists were sufficiently potent and selective for OX R, even the salts
1
were not soluble in water or saline. Therefore, we tried to introduce an additional basic moiety to
the antagonists to obtain diꢀprotonated salts. The obtained diꢀhydrosulfate of 17ꢀoꢀ
dimethylaminosulfonamideꢀ6ꢀ(2ꢀpyridyl)ꢀacrylamide derivative 71 could be easily dissolved in
water (solubility: 10 mg in 50 µL saline).
Table 6. Effect of compound 71 on naloxoneꢀprecipitated withdrawal signs in morphineꢀ
dependent mice
Positive animals / Total animals
Withdrawal signals
Jumping
Saline
9 / 13
Compound 71
**
2 / 13
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Body shakes
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Rearing
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The morphine dose was increased progressively from 8 to 45 mg/kg, subcutaneously (s.c.) over a
period of 5 days. Saline or compound 71 was intraperitoneally injected 30 min before naloxone
treatment. Withdrawal signs were induced by injecting naloxone (3 mg/kg, s.c.) 2 hr after the
final morphine treatment and then were observed for 60 min. ** p<0.01 vs. Saline.
Figure 4. Morphine withdrawal is suppressed by compound 71. Naloxoneꢀprecipitated diarrhea
(a) and body weight loss (b) were suppressed by i.p. pretreatment with compound 71 (10 mg/kg)
at 30 min before naloxone challenge injection. Compound 71 was dissolved in saline. Each point
represents the mean body weight loss of 13 mice with SEM. *p<0.05: saline vs compound 71.
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We evaluated the effect of compound 71 on morphine withdrawal. As shown Table 6 and Figure
4, naloxoneꢀprecipitated withdrawal in mice with chronic morphine injection induced several
classic behavioral signs of morphine withdrawal. Of these several signs, naloxoneꢀprecipitated
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body weight loss, diarrhea, and jumping behavior were significantly suppressed by i.p.
pretreatment with compound 71 before naloxone challenge injection, indicating that the OX R
1
antagonist attenuated the expression of naloxoneꢀprecipitated morphine withdrawal. Therefore,
the newly synthesized selective OX R antagonist would be a useful tool for understanding the
1
physiological significance of the orexinergic system, and may be useful as a medicine to treat
drug dependence.
Finally, we found that all antagonists derived from nalfurafine showed high selectivity for OX R
1
(as OX R/OX R selectivity from IC value; at least 88.4ꢀfold) as shown in Tables 1–3, although
2
1
50
many extremely low selective derivatives for OX R (some derivatives showed rather selective
1
for OX R) were observed in the course of structureꢀactivity relationship study of the other known
2
antagonists shown in Scheme 1. This result indicates that nalfurafine derivatives with the
morphinan skeleton would be specific lead compounds for developing selective OX R
1
antagonists, which would provide important information for many researchers in the field of
orexin research.
Recently, two Xꢀray crystal structures of the human OX R bound to suvorexant (1) (PDB: 4ZJ8)
1
4
6
and 6 (PDB: 4ZJC) were reported. Using these Xꢀray structures, the binding mode of 71 with
OX R was investigated by molecularꢀdocking calculations. The resulting binding mode of 71 is
1
shown in Figure 5. The morphinan skeleton of 71 was suggested to be located in the middle of
the ligandꢀbinding site of OX R (Figure 5A). The 17ꢀoꢀdimethylaminobenzene group of 71 was
1
oriented toward transmembrane helixes 2 and 3 (TM2 and TM3) to form hydrophobic
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interactions with A102 (TM2), V106 (TM2), W112 (loop between TM2 and TM3), I122 (TM3),
and P123 (TM3) of OX R (Figure 5B). On the other hand, the 2ꢀpyridyl group of 71 was
1
oriented in the opposite direction, and made hydrophobic interactions with F219 (TM5), F220
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(
TM5), I314 (TM6), and I319 (TM6). Compound 71 also used an ether oxygen of the morphinan
skeleton and a nitrogen atom of the 2ꢀpyridyl group to form two hydrogen bonds with N318
TM6) of OX R (Figure 5B). This configuration might indicate the importance of the nitrogen
(
1
atom on the pyridyl group for interactions with OX R.
1
Figure 6 compares the binding modes of 71, suvorexant (1) (DORA), and 6 (1ꢀSORA
7
representing a 117ꢀfold selectivity ). The position of the morphinan skeleton of 71 corresponds
to the 7ꢀmembered ring of 1 and the 2ꢀpyrrolidyl methylene part of 6, which were proposed to
inhibit inward movements of TM5 and TM6 relative to the rest of the TM bundle (Figures 6B
and C). The 17ꢀoꢀdimethylaminobenzene group of 71 corresponds to the 5ꢀchloroꢀ1,3ꢀ
benzoxazolꢀ2ꢀyl group of 1 and the 5ꢀphenylꢀ1,3,4ꢀoxadiazolꢀ2ꢀyl group of 6. Recently, the
46
selective OX R antagonist activity of 6 was examined from the structural point of view.
1
Comparing the binding sites of OX R and OX R, there are only two substitutions. S103 (TM2)
1
2
and A127 (TM3) of OX R were mutated to T111 (TM2) and T135 (TM3) of OX R. As both
1
2
residues of OX R are larger than those of OX R, the volume of the pocket of OX R is somewhat
2
1
2
smaller than OX R. In the literature, when the experimentallyꢀobserved pose of 6 in complex
1
with OX R was placed into the OX R structure by superimposition of the pockets, some clashes
1
2
46
with T111 (TM2) and T135 (TM3) of OX R were observed, suggesting that the volume of the
2
pocket of OX R was a much better fit to 6 than that of OX R. When we also placed 71 bound to
1
2
OX R into the pocket of OX R, a clash between the 17ꢀoꢀdimethylaminobenzene group of 71
1
2
and T111 (TM2) of OX R was observed. This observation might be a source of the selective
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OX R antagonist activity of 71 and may well explain our experimental results that the 17ꢀ
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sulfonamide motif is a key pharmacophore to afford selective OX R antagonist activity.
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Figure 5. The binding mode of 71 with the OX R determined by our docking procedure.
1
Hydrogenꢀbonding interactions are indicated by dashed lines.
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Figure 6. (A) Chemical structures of 71, suvorexant (1), and 6. (B) Superimposition of 71
(purple) and 1 (green) in the ligandꢀbinding site of OX R. (C) Superimposition of 71 (purple)
1
and 6 (orange) in the ligandꢀbinding site of OX R.
1
CONCLUSIONS
The antagonistic activity of nalfurafine (12) for OX R was discovered and improved from K
1
i
value: 250 to 1.36 nM by modification of the 17ꢀnitrogen substituent and the 3ꢀhydroxy group,
and the selectivities of the obtained derivatives were not active for OX R. The oꢀsubstituted
2
benzenesulfonamide derivatives showed tendency for more potent antagonistic activities than mꢀ
and pꢀsubstituted benzenesulfonamide derivatives. The most stable conformations of 50 and 52
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and the superimpositions of the lowꢀenergy conformers of 50 and 52 were performed.
Interestingly, thus obtained 17ꢀsulfonamide derivatives shown in Table 5 showed almost no
affinity to opioid receptors which means that the OX R antagonists would be expected to have
1
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few side effects derived from opioid receptors. One of the most potent OX R antagonists
1
attenuated the physical dependence of morphine via i.p. injection and would be expected to
clarify the pharmacological activities of OX R. Furthermore, the antagonists could be applied to
1
the treatment of opioid addiction.
Finally, the nalfurafine derivatives with the morphinan skeleton could serve as specific lead
compounds to develop OX R selective antagonists, which would provide important information
1
for many researchers in the field of orexin research.
EXPERIMENTAL SECTION
Chemistry.
General. All melting points were determined on a Yanaco MP melting point apparatus and are
1
uncorrected. Infrared spectra were recorded with a JASCO FT/IR 4100 spectrophotometer. H
1
3
and C NMR spectral data were obtained with JEOL JNMꢀECS 400 instruments. Chemical
1
shifts are quoted in ppm using tetramethylsilane (δ = 0 ppm) as the reference for H NMR
13
spectroscopy, CDCl (δ = 77.0 ppm) and pyridineꢀd (δ = 135.5 ppm) for C NMR
3
5
spectroscopy. Mass spectra were measured with a JEOL JMSꢀT100LP spectrometer. The purity
≥95%) of the assayed compounds was determined by analytical HPLC or elemental analysis.
(
Analytical HPLC were performed on a Shimadzu LCꢀ2040C 3D instrument, equipped with
XbridgeꢀC18 3.5 ꢁm, 4.6 x 150 mm column, with PDA detection at 254 nm, at column
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temperature of 40 °C. Elemental analyses were performed with a JꢀSCIENCE LAB micro corder
JM10. Column chromatography was carried out on silica gel (spherical, neutral, 40–50 µm,
Kanto Chemical Co. or packed column, 40 µm, Yamazen Co.), NHꢀsilica gel (40–75 µm, Fuji
Silysia Chemical Ltd.) and DIOLꢀsilica gel (40–75 µm, Fuji Silysia Chemical Ltd.).
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2,2,2-Trichloroethyl
hexahydro-3'H,7a'H-spiro[[1,3]dioxolane-2,7'-[4,12]methanobenzofuro[3,2-e]isoquinoline]-
'-carboxylate (21)
(4'R,4a'S,7a'R,12b'S)-4a'-acetoxy-9'-methoxy-1',2',4',4a',5',6'-
3
To a suspension of naltrexone hydrochloride (20) (20 g, 52.9 mmol) in DMF (150 mL) were
added K CO (18.3 g, 132 mmol) and MeI (3.65 mL, 58.5 mmol) and stirred at room
2
3
temperature for 11 h under an argon atmosphere. The reaction was quenched with H O (200 mL)
2
and the mixture was extracted with Et O (300 mL × 2, 200 mL). The organic layer was washed
2
with H O (200 mL) and brine, dried over Na SO , and concentrated under reduced pressure to
2
2
4
afford a crude product as a colorless solid. To a solution of the crude product in toluene (150
mL) were added pꢀTsOH·H O (14.3 g, 75.2 mmol) and ethylene glycol (16.7 mL, 299 mmol),
2
and the mixture was refluxed with a DeanꢀStark apparatus for 17 h under an argon atmosphere.
After cooling to room temperature, the reaction mixture was basified with K CO (12 g) and
2
3
saturated aqueous NaHCO solution (80 mL), and extracted with CHCl (300, 200, 100 mL). The
3
3
organic layer was washed with brine, dried over Na SO , and concentrated under reduced
2
4
pressure to afford a crude product as a colorless solid. The crude product was suspended in Ac O
2
(200 mL) and the mixture was refluxed for 1 h under an argon atmosphere. After cooling to room
temperature, the reaction mixture was concentrated under reduced pressure and azeotropically
dried with toluene three times, then CHCl three times to afford a crude product as a brown
3
amorphous. To a solution of the crude product in 1,1,2,2ꢀtetrachloroethane (200 mL) were added
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K CO (46 g, 333 mmol) and 2,2,2ꢀtrichloroethyl chloroformate (45.8 mL, 333 mmol), and the
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mixture was stirred at 140 °C for 14 h under an argon atmosphere. The reaction mixture was
cooled to room temperature and H O (200mL) was added. The mixture was extracted with
2
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CHCl (200 mL, 100 mL × 2). The organic layer was washed with brine, dried over Na SO , and
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concentrated under reduced pressure. The crude residue was purified by column chromatography
on silica gel (EtOAc : nꢀhexane = 3 : 1 to 1 : 3) to afford compound 21 (24.3 g, 82% in 4 steps)
as a yellow amorphous.
ꢀ1 1
IR (film) 1744, 1713 cm ; H NMR (400 MHz, CDCl ) δ = 1.46–1.59 (m, 3H), 1.76–1.87 (m,
3
1H), 2.05 (s, 1.5H), 2.07 (s, 1.5H), 2.33–2.45 (m, 1H), 2.73–3.00 (m, 3H), 3.10 (ddd, J = 18.4,
5.6, 5.6 Hz, 1H), 3.77–3.84 (m, 1H), 3.87–3.95 (m, 1H), 3.89 (s, 3H), 3.97–4.08 (m, 2H), 4.17–
4.24 (m, 1H), 4.60 (s, 1H), 4.66 (d, J = 12.0 Hz, 0.5H), 4.68 (d, J = 12.0 Hz, 0.5H), 4.87 (d, J =
2.0 Hz, 0.5H), 4.91 (d, J = 12.0 Hz, 0.5 H), 5.60–5.66 (m, 1H), 6.65 (d, J = 8.4 Hz, 0.5H), 6.67
1
1
3
(
d, J = 8.4 Hz, 0.5H), 6.80 (d, J = 8.4 Hz, 1H).; C NMR (100 MHz, CDCl ) δ = 21.95, 22.0,
3
12.5, 23.5, 28.5, 28.6, 29.0, 31.5, 31.9, 37.6, 37.9, 48.0, 18.2, 51.7, 51.8, 56.5, 74.9, 75.0, 81.0,
81.2, 93.2, 95.5, 95.8, 108.0, 114.3, 118.9, 123.5, 123.7, 128.6, 128.7, 142.77, 142.8, 146.2,
+
146.2, 153.8, 154.0, 169.3, 169.5.; HRMS–ESI (m/z): [M + Na] calcd for C H Cl NO Na,
2
4
26
3
8
5
84.0622; found, 584.0638.
4'R,4a'S,7a'R,12b'S)-9'-Methoxy-1',2',3',4',5',6'-hexahydro-4a'H,7a'H-
spiro[[1,3]dioxolane-2,7'-[4,12]methanobenzofuro[3,2-e]isoquinolin]-4a'-ol (22)
(
To a suspension of compound 21 (10 g, 17.8 mmol) in DMSO (100 mL) was added 12 M
aqueous KOH solution (50 mL) and the mixture was stirred for 6 h at 110 °C under an argon
atmosphere. After cooling to room temperature, the reaction mixture was adjusted to pH 10 with
saturated aqueous NH Cl solution (100 mL) and extracted with a mixed solution, iꢀPrOH :
4
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