Discovery of δOpioid Receptor Full Inverse Agonists and Their Effects on Restraint Stress-Induced Cognitive Impairment in Mice

Article abstract of DOI:10.1021/acschemneuro.9b00067

The cyclopropylmethyl group in classical δopioid receptor (DOR) antagonist NTI, BNTX, and NTB was replaced with various electron-withdrawing groups to develop DOR inverse agonists. N-Benzyl NTB derivative SYK-657 was a potent DOR full inverse agonist and its potency was over 10-fold potent than that of a reference compound ICI-174,864. Intraperitoneal administration of SYK-657 induced the short-term memory improving effect in mice without abnormal behaviors.

Full text of DOI:10.1021/acschemneuro.9b00067

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Letter
Discovery of # Opioid Receptor Full Inverse Agonists and Their
Effects on Restraint Stress-induced Cognitive Impairment in Mice
Shigeto Hirayama, Takashi Iwai, Eika Higashi, Minami Nakamura, Chiharu
Iwamatsu, Kennosuke Itoh, Toru Nemoto, Mitsuo Tanabe, and Hideaki Fujii
ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019
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Discovery of  Opioid Receptor Full Inverse Agonists and
Their Effects on Restraint Stress-induced Cognitive
Impairment in Mice
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Shigeto Hirayama,†,‡ Takashi Iwai,‡,§ Eika Higashi,† Minami Nakamura,§ Chiharu Iwamatsu,†
Kennosuke Itoh,†,‡ Toru Nemoto,† Mitsuo Tanabe,‡,§ and Hideaki Fujii†,‡,*
†
Laboratory of Medicinal Chemistry, School of Pharmacy, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo,
108-8641, Japan
‡
Medicinal Research Laboratories, School of Pharmacy, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo,
108-8641, Japan
§ Laboratory of Pharmacology, School of Pharmacy, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo, 108-
8641, Japan
KEYWORDS:  Opioid receptor, DOR, inverse agonist, constitutive activity, cognitive impairment
ABSTRACT: The cyclopropylmethyl group in classical  opioid receptor (DOR) antagonist NTI, BNTX, and NTB was
replaced with various electron-withdrawing groups to develop DOR inverse agonists. N-Benzyl NTB derivative SYK-657 was
a potent DOR full inverse agonist and its potency was over 10-fold potent than that of a reference compound ICI-174,864.
Intraperitoneal administration of SYK-657 induced the short-term memory improving effect in mice without abnormal
behaviors.
G-Protein coupled receptors (GPCRs) are attractive and
important drug targets, and over 25% of all approved drugs
are known to provide their pharmaceutical effects through
GPCRs.1 The compounds interacting with GPCRs were
classically divided into two classes: agonists with positive
intrinsic activity and antagonists with no intrinsic activity.
However, after discovering the constitutive activity of
GPCRs, inverse agonists emerged and are characterized by
having negative intrinsic activity. Many investigations on
the constitutive activity of GPCRs have been undertaken
and various inverse agonists have been reported.2 The
clinical relevance of inverse agonists is also being
discussed.2 Since the peptidic  opioid receptor (DOR)
inverse agonist ICI-174,864 was discovered by Costa and
Herz,3 which was a pioneering work in the inverse agonism
field, several peptidic4,5a and non-peptidic5 DOR inverse
agonists have been reported. We also reported some 4,5-
epoxymorphinan derivatives with DOR inverse agonistic
properties (Figure 1).6 The N-substituent in the selective
DOR antagonist naltrindole (NTI)7 was converted from the
cyclopropylmethyl (CPM) group into the fluorinated ethyl
group to afford an interesting change of the functional
activity: conversion from an antagonist into an inverse
agonist (Figure 1). It is well-known that the N-substituent
in the morphinan derivatives dramatically affects their
agonist/antagonist properties, but this is the first example
that the transformation of the N-substituent in morphinan
derivatives affords the inverse agonist. Although various
DOR inverse
R
N
OH
O
N
HO
H
R = CPM (NTI): DOR antagonist
R = CH2CF3 (1a): DOR partial inverse agonist
R = CH2CHF2 (1b): DOR partial inverse agonist
R = CH2CH2F (1c): DOR partial inverse agonist
Figure 1. Structures of NTI and its derivatives with N-
fluorinated ethyl group.
agonists have been reported,4,5 there have been almost no
investigation on in vivo pharmacological effects induced by a
DOR inverse agonist. To the best of our knowledge, only Lilly’s
research group reported that the non-peptidic DOR inverse
agonist LY255582 induced anorectic activity.5d,8 However, the
same research group later concluded that the anorexic effects
produced by LY255582 resulted from a combination of 
(MOR), DOR, and  opioid receptor (KOR) activities based on
the examination of the binding of [3H]LY255582 in mouse
brain.9 Therefore, the in vivo pharmacological effects by a
DOR inverse agonist, and perhaps the physiological role in the
constitutive activity of the DOR, remain unclear. To
investigate the influence of the N-substituent on the DOR
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inverse agonistic activity, we introduced various electron-
withdrawing groups as the N-substituents in DOR antagonists
with the morphinan skeleton like NTI because the fluorinated
ethyl
1
2
3
Scheme 1. Synthesis of NTI derivativesa
4
5
6
7
8
9
H
H
H
N
N
N
OH
OH
OH
a), b)
c), d)
O
O
O
O
N
N
MeO
MeO
e), c)
TBSO
O
H
H
4
2
f), g), c)
or
h), c)
3
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f), i)
R
N
N
OH
OH
O
1j
O
N
N
HO
HO
H
H
1d-i, k, l
F
1d: R = CH2CCl3
1e: R = CH2CH2CF3
1f: R = CH2CF2CF3
1i: R = CH2CH2OH
1k: R = CH2Ph
1l: R = CH2CH2Ph
1g: R =
1h: R =
CF3
N
N
OH
OH
b)
O
N
H
HO
O
O
HO
Naloxone
1m
aReagents and conditions: (a) 2 M HCl, MeOH, reflux; (b) PhNHNH2·HCl, AcOH, reflux; (c) 1 M BBr3, CH2Cl2, -10 ˚C; (d) TBSCl,
imidazole, DMF, rt; (e) 2-(trimethylsilyl)phenyl trifluoromethanesulfonate, TBAF, THF, -40 ˚C; (f) trichloroacetic anhydride,
CH2Cl2, rt, or R'CO2H, EDCI·HCl, DMAP, CH2Cl2, rt; (g) BH3·THF, THF, reflux; (h) RBr, K2CO3, DMF, rt; (i) BH3·THF, THF, reflux,
then 4 M HCl, rt. R = CH2R'.
routes. In a route, the N-substituent was introduced by N-
alkylation with the corresponding alkyl bromides or
amidation with the corresponding carboxylic acid or acid
anhydride
group is one of the representative electron-withdrawing
groups. Previous reports suggested that the  opioidergic
neurons may be relevant to learning memory.10,11 Therefore,
using a DOR full inverse agonist obtained in this earlier
research, we investigated in vivo pharmacological effects
induced by a DOR inverse agonist to determine if such an
agent could induce a short-term memory improving effect in
mice. Herein, we report the synthesis of DOR antagonist
derivatives bearing an electron-withdrawing group as the N-
substituent to obtain some DOR full inverse agonists. The
short-term memory improvement effect by a newly developed
DOR full inverse agonist is also described.
Scheme 2. Synthesis of BNTX and NTB derivativesa
R
N
CF3
N
OH
OH
a) - c)
f)
2
O
O
HO
O
HO
O
d),
e)
h)
7a, k
5
R
g)
RESULTS AND DISCUSSION
N
N
f),
OH
OH
Chemical Synthesis NTI Derivatives with various N-
substituents have been synthesized from noroxymorphone.12
However, we chose the other synthetic routes delineated in
scheme 1 because noroxymorphone is hardly available in our
country. The synthesis of NTI derivatives 1d–l with electron-
withdrawing groups as the N-substituent commenced with
compound 213 (Scheme 1). The treatment of compound 2 with
hydrochloric acid and subsequent Fischer indolization gave
compound 3. The objective compounds were prepared by two
h), g)
O
O
O
MeO
O
HO
8a, k
a: R = CH2CF3, k: R = Bn
6
aReagents and conditions: (a) FTAA, CH2Cl2, rt; (b) BH3·THF,
THF, reflux; (c) 48% HBr, reflux; (d) BnBr, K2CO3, DMF, rt; (e)
2 M HCl, MeOH, reflux; (f) PhCO2H, PhCHO, i-Pr2EtN,
Tol/DMF = 5/1, 140 ˚C; (g) 1 M BBr3, CH2Cl2, -10 ˚C; (h)
PhONH2·HCl, MeSO3H, EtOH, reflux.
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following reduction of the intermediate amides with borane.
Introduction of a phenyl group (compound 1j) was achieved
by using benzyne chemistry. Although Larock’s reaction
conditions14 furnished 87% yield, our modified reaction
conditions improved the yield (98%). The O-methyl group was
deprotected with boron tribromide. In the alternative route,
1
2
3
4
5
Table 1. Binding Affinities and Selectivities of Synthesized Compounds 1a–m, 7a, 7k, 8a, 8k, and Parent DOR Antagonists NTI,
BNTX, and BNT for the Opioid Receptorsa and Their Functional Activities for the DORb
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7
8
9
Opioid Receptor Binding
Functional Activity for
DOR
Affinity Ki, nM (95% CI)
Selectivity
Compd
R
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EC50
(95% CI)
nM
DOR
MOR
N.T.e
KOR
N.T.e
MOR/DOR KOR/DOR
Emax (%)
100c
4.66
DPDPEc
–
–
N.T.e
422
N.T.e
N.T.e
N.T.e
(2.08-10.4)
114
ICI-174,864d
-100d
N.T.e
30.7
N.T.e
14.7
N.T.e.
(67.9-192)
(215-
829)
N.D.f
0.46
NTI
CPM
66.7
32.0
pA2 = 10.1
7.5g
(0.192-
1.09)
(12.5-
75.4)
(3.16-
68.5)
(9.39-10.8)
134
45.5
1ah
1bh
1ch
1d
CH2CF3
CH2CHF2
CH2CH2F
CH2CH2CF3
>1,000
>1,000
N.C.i
N.C.i
348
N.C.i
N.C.i
204
-44.8
-48.6
-38.1
19.3
(75.4-
239)
(17.5-118)
15.5
17.2
>1,000
675
>1,000
397
(9.14-
26.4)
(7.49-39.7)
1.94
220
(1.29-
2.93)
(373-
1221)
(172-
913)
(54.6- 885)
40.2
1,050
1,490
40.3
26.1
37.1
(18.2-
88.6)
(456-
2,426)
(938-
2,353)
(6.75- 241)
31,400
484
2,090
86.0
(4,380-
224,905
)
1e
CH2CF2CF3
64.9
4.32
21.1
(154-
1,517)
(1,137-
3,845)
(3.33-2,222)
N.D.f
pA2 = 7.84
(7.14-8.53)
8.67
40.4
1,850
1,340
1f
CH2CCl3
45.8
414
33.2
31.4
12.2g
22.9
(19.0-
85.5)
(513-
6,677)
(671-
2,655)
1g
CH2(1-F-c-C3H4)
1.11
460
34.8
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(0.697-
1.76)
(233-
910)
(23.5-
51.4)
(0.295-255)
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7
8
7.47
3,440
1,090
67.6
1h
1i
CH2(1-(CF3)-c-C3H4)
461
146
26.8
21.8
(4.69-
11.9)
(1,356-
8,724)
(744-
1,594)
(9.43-484)
10.2
998
714
7.37
CH2CH2OH
97.8
70.0
9
(6.08-
17.2)
(606-
1,644)
(464-
1,098)
(0.424-128)
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N.D.f
25.5
1,260
825
1j
Ph
49.4
32.4
pA2 = 7.72
(7.07-8.38)
4.11g
(17.7-
36.6)
(609-
2,596)
(593-
1,148)
15.8
1,160
615
16.1
1k
1l
Bn
CH2CH2Ph
allyl
73.4
90.2
299
38.9
399
297
-56.5
40.3
16.2
(7.23-
34.7)
(605-
2,213)
(443-
853)
(7.31-35.4)
1.94
175
775
15.1
(1.50-
2.50)
(114-
267)
(637-
943)
(2.67-85.0)
0.61
181
101
182
1m
(0.407-
0.913)
(134-
246)
(4.46-
2,278)
(121-275)
N.D.f
3.53
10.5
28.3
BNTX
CPM
2.99
8.03
pA2 = 9.47
-10.2g
(2.38-
5.23)
(6.61-16.
8)
(22.5-
35.7)
(6.87-12.1)
1,680
1,750
7,450
94.7
7a
CH2CF3
1.04
1.45
4.43
2.07
-48.5
-103
(578-
4,885)
(914-
3,353)
(4,666-
11,880)
(29.7-302)
444
643
919
225
7k (SYK-656)
Bn
(263-
751)
(459-
901)
(582-
1,452)
(132-383)
N.D.f
0.603
31.0
30.4
NTB
CPM
51.5
50.4
pA2 = 9.54
-1.6g
(0.384-
0.946)
(19.0-
50.5)
(23.3-
39.7)
(8.68-10.4)
128
5,820
5,630
37.3
8a
CH2CF3
Bn
45.5
22.2
44.0
9.82
-59.7
-99.0
(82.5-
199)
(3,449-
9,825)
(3,664-
8,638)
(9.38-148)
8k (SYK-657)
177
3,930
1,740
10.6
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(93.8-
334)
(2,122-
7,291)
(1,175-
2,574)
(7.16-15.6)
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aEvaluated by ability of each compound to displace [3H]DAMGO (MOR), [3H]DPDPE (DOR), or [3H]U-69,593 (KOR) binding to
the CHO cells expressing human MOR, DOR, or KOR. The data represent means of three samples. bMembranes were incubated
with [35S]GTPS and GDP with the compound. The  human recombinant cell membrane (CHO) was used in this assay. The data
represent means of four samples. DPDPE (1 M) was used as the standard DOR agonist. ICI-174,864 (10 M) was used as the
standard DOR inverse agonist. eNot tested. fNot determined. g% Stimulation at 10 M of the tested compound. hReference 6. iNot
calculated because Ki values for MOR or KOR were over 1,000 nM. 95% CI: 95% confidence interval.
c
d
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the protecting group in the phenolic hydroxy group was
changed from methyl into t-butyldimethylsilyl group. After
agonistic activity with a moderate efficacy (Emax = 40.3%),
while 1l was reported to be a full agonist in the electrically
stimulated mouse vas deferens (MVD) assays.20 Whereas N-
allyl derivative 1m was a partial agonist with a low efficacy
(Emax = 16.2%), 1m showed antagonist property in the MVD
assays.7b These differences of the observed efficacies would
stem from the distinct assay system. These results suggested
that the replacement of CPM with some electron-withdrawing
groups would convert the compound’s property from an
antagonist into an inverse agonist. The length between the
nitrogen atom and fluorine atom or phenyl group may be an
important structural determinant to exert inverse agonistic
activities (1a-c vs. 1d, 1k vs. 1j and 1l). However, N-
trifluoroethyl derivative 1a was an inverse agonist, while N-
trichloroethyl derivative 1f was an antagonist. Although N-
pentafluoropropyl derivative 1e shared the same partial
structure as N-trifluoroethyl derivative 1a, 1e was not an
inverse agonist. Moreover, although the length between the
fluorine and nitrogen atom in 1g was the same as those in 1a-
c, which were inverse agonists, N-(1-fluorocyclopropyl)methyl
derivative 1g did not exert any inverse agonistic activity. These
observations were interesting, but it is now difficult to
rationally explain this phenomenon. The conformational
distinction among the N-substituents might be also an
important determinant. Among the tested substituents, only
the fluorinated ethyl and benzyl groups afforded inverse
agonist properties. Therefore, we applied these replacement of
substituents to other DOR antagonists BNTX and NTB with
chemotypes different from that of NTI. Fascinatingly, all the
resulting compounds 7a, 7k, 8a, and 8k showed inverse
agonism. N-Trifluoroethyl derivatives 7a and 8a were partial
inverse agonists, while N-benzyl derivatives 7k (SYK-656) and
8k (SYK-657) were full inverse agonists. Particularly, SYK-657
(8k) was very potent (EC50 = 10.6 nM) and exhibited a greater
than 10-fold more potent inverse agonistic activity than did the
reference compound ICI-174,864. Unfortunately, a clear
relationship between their inverse agonistic activities and
binding affinities for the DOR was not observed.21
introducing the N-substituent by
a two step method
(amidation and reduction), the silyl group was removed by
acidic conditions. Allyl derivative 1m was prepared from
naloxone by Fisher indolization. We applied some N-
substituents, which provided DOR inverse agonist activity
mentioned below, to the other DOR antagonist 7-
benzylidenenaltrexone (BNTX)15 and naltriben (NTB)16 having
structures that differed from NTI. The synthesis of BNTX and
NTB derivatives are shown in Scheme 2. 2,2,2-Trifluoroethyl
and benzyl groups were introduced in compound 2 by
previously reported methods17 and subsequently treated with
48% hydrobromic acid and 2 M hydrochloric acid to give
compounds 5 and 6, respectively. According to the reported
methods,16,18 BNTX and NTB derivatives 7 and 8 were obtained
from respective compounds 5 and 6. Finally, all the objective
compounds were converted to the hydrochloride or
camphorsulfonate for pharmacological evaluation of the
compounds.
Binding affinity and functional activity The binding
affinities of the prepared compounds 1d−m, 7a, 7k, 8a, and 8k
were evaluated by competitive binding assays according to the
previously reported methods19 (Table 1). For the purpose of
comparison, the results of the parent DOR antagonists NTI,
BNTX, and NTB are also shown. All the NTI derivatives 1
except 1a and 1e strongly bound to the DOR with Ki values of
double digit or lower nanomolar range and most derivatives
showed selectivity for the DOR comparable to or higher than
that of the parent compound NTI. These observations agreed
with the previously reported results.6 Compared to the NTI
derivatives 1, the binding affinities and selectivities for the
DOR of BNTX derivatives 7 and NTB 8 variants were poorer.
We next assessed the functional activities of the compounds
1d−m, 7a, 7k, 8a, and 8k for the DOR by [35S]GTPS binding
assays according to the previously reported methods19 (Table
1), and we compared the results to those obtained with the
parent DOR antagonists NTI, BNTX, and NTB. The Emax values
were calculated using the maximum response of reference
compounds DPDPE and ICI-174,864 for the compounds with
the positive and negative intrinsic activities, respectively.
Concerning NTI derivatives 1, only the N-benzyl derivative 1k
showed inverse agonistic activity with the Emax value of -56.5%,
which was comparable to a little higher efficacy compared
with those of N-trifluoroethyl and N-difluoroethyl derivatives
1a and 1b. Compounds 1f and 1j were DOR antagonists. Other
derivatives, except for 1f, 1j, and 1l, were agonists with almost
no or low efficacy. N-Phenethyl derivative 1l exhibited partial
The Effects on Restraint Stress-Induced Cognitive
Impairment With a potent DOR full inverse agonist (SYK-
657) in hand, we sought to define the in vivo pharmacological
effects induced by such an inverse agonist. It was previously
reported that learning memory was impaired by DOR
agonists.10 In addition, preproenkephalin knockout mice,
which cannot produce the endogenous DOR agonists
enkephalins, were resistant to chronic stress, which induces
memory impairment.11 Thus, we evaluated the influence of
SYK-657, which had almost no activities toward the MOR and
KOR,22 on repeated restraint stress-induced memory
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impairment. Short-term memory performance was assessed
memory was improved. SYK-657 did not affect total arm
entries, which are indices of movement (Figure 2). Moreover,
no abnormal behaviors in mice injected with SYK-657 were
observed in the open-field test (Figure S1, Supporting
1
2
3
4
by recording spontaneous alternation behavior in a Y-maze.
The restraint stress caused cognitive impairment.
Intraperitoneal administration of SYK-657 significantly
increased % alteration, which meant that the short-term
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Figure 2. The effects of SYK-657 camphorsulfonate on restraint stress-induced cognitive impairment in mice.
The short-term memory performance was assessed using the Y-maze test. (A) The effects on % alteration. % Alteration was
calculated by the following equation: % alternation = {(number of alternations)/(total number of arm entries - 2)}×100 (%). (B)
The effects on the total entries. The numbers in parentheses show the sample size. *p<0.05, **p<0.01, ***p<0.001 (One-way analysis
of variance followed by Bonferroni’s multiple comparison test)
useful probe to explore pharmacology mediated by the DOR,
Information). The short-term memory improving effect by
especially constitutively active DOR.
SYK-657 was reversed by the DOR antagonist NTI in a dose-
dependent manner. NTI itself had no effects on the learning
memory (Figure S2, Supporting Information). This result
METHODS
strongly indicated that the short-term memory improving
effect by SYK-657 was mediated by the DOR. Although these
outcomes suggested that the restraint stress might shift the
receptor equilibrium between active and inactive forms
toward the active form to provide higher constitutive activity
of the DOR, further investigation would be required.
17-Benzyl-6,7-didehydro-4,5α-epoxy-
benzofurano[2',3':6,7]morphinan-3,14β-diol
camphorsulfonate (SYK-657 camphorsulfonate). Under Ar, to
solution of SYK-657-3O-Me ether (S16, Supprting
(8k)
a
Information)(107 mg, 0.23 mmol) in CH2Cl2 (4 mL) solution
was added 1.0 M solution of BBr3 in CH2Cl2 (1.4 mL, 1.4 mmol)
at -10 ºC and stirred at the same temperature for 1 hr. To the
reaction mixture was added 25% ammonia solution and stirred
for 30 min, and then extracted with CHCl3. Combined organic
layers were washed with brine and dried over anhydrous
Na2SO4. After removing the solvent in vacuo, the obtained
crude product was purified by silica gel chromatography to
give SYK-657 (8k) (53 mg, 52%) as a colorless oil;. IR (neat) cm-
1: 3303, 2924, 2832, 1615, 1453, 1218, 1117, 747. 1H NMR (400 MHz,
DMSO-d6): δ 1.57 (br d, J = 11.2 Hz, 1H), 2.21 (ddd, J = 3.1, 11.8,
11.8 Hz, 1H), 2.33 (ddd, J = 4.6, 12.4, 12.4 Hz, 1H), 2.40 (d, J = 16.3
Hz, 1H), 2.46-2.55 (m, 1H), 2.61 (d, J = 16.3 Hz, 1H), 2.72 (dd, J =
6.3, 18.7 Hz, 1H), 3.00 (d, J = 6.3 Hz, 1H), 3.24 (d, J = 18.7 Hz,
1H), 3.68 (s, 2H), 4.79 (br s, 1H), 5.53 (s, 1H), 6.53 (d, J = 8.1 Hz,
1H), 6.56 (d, J = 8.1 Hz, 1H), 7.16-7.21 (m, 1H), 7.22-7.36 (m, 4H),
7.38-7.47 (m, 3H), 7.54 (d, J = 8.3 Hz, 1H), 9.14 (br s, 1H). 13C
NMR (100 MHz, CDCl3): δ 23.1, 28.7, 31.1, 43.2, 48.9, 59.1, 62.1,
72.9, 84.4, 111.5, 116.0, 117.3, 119.2, 129.7, 122.4, 124.8, 124.9, 127.5,
127.6, 128.6, 129.0, 130.0, 137.6, 139.1, 142.9, 147.6, 155.5. HR-MS
(ESI): Calcd for C29H26NO4[M+H]+: 452.1862. Found: 452.1873.
In conclusion, based on our previous results that N-
fluorinated ethyl NTI derivatives 1a-c showed DOR inverse
agonistic activities, we designed and synthesized some NTI
derivatives 1d-m with various electron-withdrawing groups as
N-substituents to find N-benzyl derivative 1k as a DOR partial
inverse agonist. Such transformation enabling the conversion
of a compound’s functional activity from an antagonist into an
inverse agonist was applicable to other DOR antagonists
BNTX and NTB having structures different from that of NTI.
N-Trifluoroethyl derivatives 7a and 8a were DOR partial
inverse agonists, while N-benzyl derivatives SYK-656 (7k) and
SYK-657 (8k) showed DOR full inverse agonistic activities. The
potency of SYK-657 was over 10-fold greater than that of a
reference compound ICI-174,864. SYK-657 administered
intraperitoneally induced a short-term memory improving
effect in mice without abnormal behaviors. Its effect was
blocked by DOR antagonist NTI. SYK-675 is expected to be a
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To a solution of SYK-657 (8k) in MeOH was added (-)-10-
camphorsulfonic acid at ºC to give SYK-657
camphorsulfonate; mp (dec.): 241.8-243.2 ˚C. Anal. Calcd for
C29H25NO4·CSA·0.8H2O: C, 67.09; H, 6.15; N, 2.01. Found: C,
67.05; H, 6.24; N, 2.00.
(1) (a) Overington, J. P., Al-Lazikani, B., and Hopkins, A. L.
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(2006) How many drug targets are there? Nat. Rev. Drug
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Evaluation of effects on restraint stress-induced cognitive
impairment. All of the experimental protocols used in this
study were approved by the Institutional Animal Care and Use
Committee of Kitasato University, and were carried out in
accordance with the guidelines of the National Institutes of
Health and the Japanese Pharmacological Society. ddY (5-
week, 24-29 g) Male mice (Japan SLC Inc., Shizuoka, Japan)
were used. The detailed procedures for habituation and Y-
maze test were described in the Supporting Information. The
mice were subjected to 5 days (day-1 to -5) of repeated restraint
for 2 hr per day in 50 mL tubes with multiple air wholes. On
day-6, short-term memory performance was assessed by
recording spontaneous alternation behavior during a single
session in a Y-maze, which is based on the tendency of rodents
to enter an arm of the Y-maze that was not explored in the last
two choices.23 SYK-657 camphorsulfonate was suspended in a
0.5% Arabic gum solution, and administered 60 min before the
Y-maze test. NTI hydrochloride was dissolved in phosphate-
buffered saline, and administered 15 min before SYK-657
camphorsulfonate administration.
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on the
ACS Publications website at at http://pubs.acs.org.
Synthetic procedures and characterization of all other
compounds (PDF).
(5) (a) Labarre, M., Butterworth, J., St-Onge, S., Payza, K.,
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Potent and Selective Phenylmorphan-Based Inverse Agonists
of the Opioid  Receptor. J. Med. Chem. 49, 5597–5609. (f)
Cheng, K., Kim, I. J., Lee, M. –J., Adah, S. A., Raymond, T. J.,
Bilsky, E. J., Aceto, M. D., May, E. L., Harris, L. S., Coop, A.,
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(2007) Opioid ligands with mixed properties from substituted
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AUTHOR INFORMATION
Corresponding Author
*E-mail: fujiih@pharm.kitasato-u.ac.jp.
ORCID
Hideaki Fujii: 0000-0002-0049-0084.
Author Contributions
S.H., T.I., and E.H. contributed equally to this work. H.F. and
K.I. oversaw and designed the chemistry. M.T. and T.I.
oversaw and designed in vivo pharmacology. E.H., C.I., and
T.N. synthesized compounds. S.H. performed in vitro assays.
T.I. and M.N. executed in vivo pharmacological assays. S.H.
and T.I. analyzed the data. H.F. and T.I. wrote the manuscript.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
BNTX, 7-benzylidenenaltrexone; CPM, cyclopropylmethyl;
DOR, δ opioid receptor; GPCR, G-protein coupled receptors;
KOR,  opioid receptor; MOR,  opioid receptor; MVD, mouse
vas deferens; NTB, naltriben; NTI, naltrindole.
(6) Nemoto, T., Iihara, Y., Hirayama, S., Iwai, T., Higashi, E.,
Fujii, H., and Nagase, H. (2015) Naltrindole derivatives with
fluorinated ethyl substituents on the 17-nitrogen as  opioid
receptor inverse agonists. Bioorg. Med. Chem. Lett. 25, 2927–
2930.
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