Antitrichomonal activity of δ opioid receptor antagonists, 7-benzylidenenaltrexone derivatives

Article abstract of DOI:10.1016/j.bmc.2017.06.026

The 7-benzylidenenaltrexone (BNTX) derivatives 2a–v, 3a–c, 13a–c, and 14a were synthesized from naltrexone (1) and evaluated for their antitrichomonal activity. The structure-activity-relationship studies found that 4-iodo-BNTX (2g) showed the highest act

Full text of DOI:10.1016/j.bmc.2017.06.026

Accepted Manuscript
Antitrichomonal activity of δ opioid receptor antagonists, 7-benzylidenenal-
trexone derivatives
Noriki Kutsumura, Yasuaki Koyama, Yasuyuki Nagumo, Ryo Nakajima,
Yoshiyuki Miyata, Naoshi Yamamoto, Tsuyoshi Saitoh, Naoko Yoshida,
Satoshi Iwata, Hiroshi Nagase
PII:
S0968-0896(17)30986-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.026
BMC 13809
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
10 May 2017
10 June 2017
12 June 2017
Please cite this article as: Kutsumura, N., Koyama, Y., Nagumo, Y., Nakajima, R., Miyata, Y., Yamamoto, N.,
Saitoh, T., Yoshida, N., Iwata, S., Nagase, H., Antitrichomonal activity of δ opioid receptor antagonists, 7-
benzylidenenaltrexone derivatives, Bioorganic & Medicinal Chemistry(2017), doi: http://dx.doi.org/10.1016/j.bmc.
2
017.06.026
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Antitrichomonal activity of δ opioid receptor
antagonists, 7-benzylidenenaltrexone
derivatives
Leave this area blank for abstract info.
Noriki Kutsumura, Yasuaki Koyama, Yasuyuki Nagumo, Ryo Nakajima, Yoshiyuki Miyata, Naoshi Yamamoto,
Tsuyoshi Saitoh, Naoko Yoshida, Satoshi Iwata, and Hiroshi Nagase
Ar
OH
Ar = 4-I-C6H4 (2i)
N
Antitrichomonalactivity
against T. vaginalis
MIC = 20 µM, IC50 = 10.5 µM
O
O
Morphinan skeleton is a good template
for antitrichomonal agents!
OH

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Antitrichomonal activity of δ opioid receptor antagonists, 7-benzylidenenaltrexone
derivatives
a
b
a
a
c
Noriki Kutsumura , Yasuaki Koyama , Yasuyuki Nagumo , Ryo Nakajima , Yoshiyuki Miyata , Naoshi
a
a
c
c
a,b,*
Yamamoto, Tsuyoshi Saitoh, Naoko Yoshida, Satoshi Iwata, and Hiroshi Nagase
a
International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan
School of Medicine, Keio University, 35, Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
b
c
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The 7-benzylidenenaltrexone (BNTX) derivatives 2a–v, 3a–c, 13a–c, and 14a were synthesized
from naltrexone (1) and evaluated for their antitrichomonal activity. The structure-activity-
relationship studies found that 4-iodo-BNTX (2g) showed the highest activity (IC50 = 10.5 µM)
and the affinity for the opioid receptor was less important for antitrichomonal activity against
Trichomonas vaginalis. The morphinan skeleton bearing both the double bond for a Michael
acceptor and the phenolic hydroxy group would be a specific template for development of
antitrichomonal agents. In addition, the mechanism of the antitrichomonal activity of the BNTX
derivatives may differ from that of the standard drug, metronidazole.
Available online
Keywords:
Antitrichomonal activity
Opioid receptor
BNTX
2
009 Elsevier Ltd. All rights reserved.
Morphinan
Trichomonas
4a,6
1
. Introduction
react with the thiol group of the glutathione-system,
we
considered that the antitrichomonal activity of the BNTX
derivatives could also derive from similar factors. In this report,
we describe the results of the correlations between the structures
of BNTX derivatives and their antitrichomonal activities or
between the DOR binding affinities and their antitrichomonal
activities.
Major trichomoniasis is one of the most common sexually
transmitted infections (STIs), caused by the protozoan parasite
Trichomonas vaginalis. After infection with T. vaginalis, the
main symptoms (vaginitis, cervicitis, genital pain and itching, or
1
urethral inflammation) are generally observed within a month. In
addition, serious complications such as transmission of HIV,
premature birth, cervical cancer, or prostate cancer are attributed
2. Results and Discussion
2
to this protozoiasis or co-infection with other microbes. 1-(2-
2
.1 Synthesis of BNTX derivatives and their antitrichomonal
Hydroxyethyl)-2-methyl-5-nitroimidazole (metoronidazole) is
frequently used as the standard drug therapy for trichomoniasis in
most areas of the world. Metronidazole is activated through a
single electron transfer from ferredoxin to the nitro group, acting
as a cytotoxic anion radical within the hydrogenosomes in
trichomoniasis. These ferredoxin-mediated free radicals trigger
activities
3
serious cellular damage and cell death. Since the metronidazole-
resistant Trichomonas caused by recruitment of other redox
3h
systems was first reported, the development of more effective
antitrichomonal agents with action mechanisms that differ from
metoronidazole remains a sought after objective.
Scheme 1. Synthesis of 2 and 3. Method A: Ar-CHO (6.0 equiv), piperidine
(
2.0 equiv), MeOH, 120 °C, in a sealed tube. Method B: Ar-CHO (12.0
equiv), PhCO H (2.5 equiv), iPr NEt (5.5 equiv), toluene-DMF (5/2), 120 °C.
2
2
Method C: Ar-CHO (4.0 equiv), 1 M NaOH aq. (3.8 equiv), MeOH, rt.
Recently, we reported that the δ opioid receptor (DOR)
antagonist, 7-benzylidenenaltrexone (BNTX) and its derivatives,
which are effective chloroquine-resistance (CQ) reversers for
At first, we prepared BNTX (2a) and the BNTX derivatives
2b–v from commercially available naltrexone (1) according to
4
5
5
4a
Plasmodium chabaudi, also showed antitrichomonal activity.
the previously reported synthetic methods, Methods A, B, and
7
As their CQ-resistance reversing effects were deeply related to
both the DOR antagonist activity and the existence of a Michel
acceptor structure (α,β-unsaturated ketone moiety), which can
C (Scheme 1). Regarding the structurally-converted moiety, we
mainly converted the styrene part of 2a into the substituted
arylidene part because of the ease of the assay sample supply. In
∗
Corresponding author. Tel./fax: +81-29-853-6437; e-mail: nagase.hiroshi.gt@u.tsukuba.ac.jp

addition, we also synthesized the reductive BNTX derivatives
a–c from 2a–c in order to confirm the effect by the presence or
absence of the Michel acceptor structure. All synthetic samples
were used for antitrichomonal assay after being converted into
salts of tartaric acid and those results are shown in Table 1.
has a non-substituted phenyl group, showed an MIC value of 40
µM (Entry 2), and the BNTX derivatives 2b–l bearing substituted
phenyl groups showed equal or greater activity than 2a (Entries
3–13). On the other hand, the antitrichomonal activities seemed
to be influenced partly by the position of the substituent on the
phenyl group. For example, the MIC value of 4-methoxy-BNTX
3
Table 1
Antitrichomonal activities of 2a–v and 3a–c against T. vaginalis
(
2b) was 20 µM (Entry 3), while those of 3-methoxy-BNTX (2o)
and 2-methoxy-BNTX (2v) were 80 µM and more than 320 µM,
respectively (Entries 16 and 23). Furthermore, the activities of 4-
cyano-BNTX (2j, 20 µM) and 4-trifluoromethyl-BNTX (2k, 20
µM) were higher than those of 3-cyano-BNTX (2l, 40 µM) and
the 3-trifluoromethyl- and 2-trifluoromethyl-BNTXs (2p and 2q,
8
0 µM), respectively (Entries 11 and 12 vs 13, 17 and 18). The
activities of the halogen-substituted BNTX were also quite
remarkable; all of the 4-halogenated BNTXs 2c, 2e, 2f, and 2g
showed almost the same MIC values (20 µM), whereas the
activities of 2-halogenated BNTXs 2r, 2t, and 2u, except for
sterically unhindered 2-fluorined derivative 2i, were greatly
decreased (Entries 4, 6–8, 10, 19, 21, and 22). As for other
structures, both 2m bearing a thiophene and 2n bearing a
quinoline showed 40 µM, whereas 2s bearing a furan showed 160
µM (Entries 14, 15, and 20). On the whole, the loss of activity of
the Michael acceptor by steric hindrance seemed to decrease the
antitrichomonal activity. The reduction of the double bond (the
reductive BNTX derivatives 3a–c lacking the Michael acceptor)
led to reduced activity, which also supported the importance of
the Michael acceptor (Entries 24–26). In comparison with IC50
Antitrichomonal activity
a
Entry Compounds
Method Yield (%)
b
MIC (µM)
IC50 (µM)
1
2
3
4
5
6
7
8
9
Metronidazole
―
―
99
97
94
97
75
97
14
99
78
24
25
13
83
89
99
49
26
81
86
84
54
99
73
84
89
10
40
5.2
21.7
14.1
12.8
13.2
12.7
11.2
10.5
14.2
18.6
13.2
13.7
18.0
21.6
17.2
―
2a (Ph)
A
2b (4-MeOC
2c (4-FC
2d (4-NMe
2e (4-ClC
2f (4-BrC
2g (4-IC
2h (3-FC
2i (2-FC
2j (4-CNC
2k (4-CF
2l (3-CNC
6
H
4
)
A
20
(
50% inhibitory concentration) values of relatively highly active
6
H
4
)
A
20
compounds (more than 40 µM), the 4-halogenated BNTX
derivatives showed the most potent antitrichomonal activities
2
C
6
H
4
)
A
20
(
Entries 4, 6–8).
6
H
4
)
A
20
2.2 Evaluations of antitrichomonal activities and opioid receptor
binding assays
6
H
4
)
A
20
6
H
4
)
B
20
4
6
H
4
)
A
20
In our recent malarial work, DOR antagonist 2a exhibited
potent CQ-resistance reversing effects in P. chabaudi, and the
results indicated that their interaction with DOR would be an
essential factor. Therefore, we next performed opioid receptor
binding assays for metronidazole, BNTX (2a), and the BNTX
derivatives 2b–k which showed MIC values of 20 µM. The
results are shown in Table 2.
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
6
H
4
)
A
20
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
6
H
4
)
B
20
3
C
6
H
4
)
B
20
6
H
4
)
B
40
2m (2-thiophene)
2n (4-quinoline)
A
40
A
40
Table 2
Opioid receptor binding assay results
2o (3-MeOC
6
H
4
)
A
80
a
Compounds
IC50 (µM))
Ki (nM)
3
Entry
1
2p (3-CF
3
3
C
6
H
4
)
C
80
―
3
3
(
µ ([ H]DAMGO) δ ([ H]DPDPE) κ ([ H]U-69,593)
2q (2-CF
C
6
H
4
)
C
80
―
Metronidazole
(5.2)
Not Active
Not Active
Not Active
2r (2-BrC
2s (2-furan)
2t (2-ClC
2u (2,6-diClC
6
H
4
)
A
A
80
―
160
>320
>320
>320
80
―
2
3
4
5
6
7
8
9
2a (21.7)
2b (14.1)
2c (12.8)
2d (13.2)
2e (12.7)
2f (11.2)
2g (10.5)
2h (14.2)
11.0
5.16
20.9
17.2
6.13
5.04
19.9
10.7
12.8
32.7
192
2.81
1.80
34.5
3.72
8.22
4.19
2.15
8.37
2.94
18.1
83.9
32.8
27.8
108
6
H
4
)
A
―
6
H
3
)
A
―
28.6
78.6
70.4
126
2v (2-MeOC
3a (Ph)
6
H
4
)
A
―
2a→3a
2b→3b
2c→3c
―
3b (4-MeOC
6
H
4
)
>320
>320
―
3c (4-FC
6
H
4
)
―
a
49.0
35.1
209
Method A: Ar-CHO (6.0 equiv), piperidine (2.0 equiv), MeOH, 120 °C, In
sealed tube; Method B: Ar-CHO (12.0 equiv), PhCO H (2.5 equiv), iPr NEt
5.5 equiv), toluene-DMF (5/2), 120 °C; Method C: Ar-CHO (4.0 equiv), 1 M
NaOH aq. (3.8 equiv), MeOH, rt.
10 2i (18.6)
2j (13.2)
12 2k (13.7)
2
2
(
1
1
b
176
Minimum inhibitory concentration after incubation of each compound with
a
T. vaginalis at 37 °C for 48 h.
Ki values were obtained from radioligand-based competitive receptor
binding assays, as described in the Experimental section.
The standard drug, metronidazole showed inhibitory effects
toward the growth of T. vaginalis with a minimum inhibitory
concentration (MIC) of 10.0 µM (Entry 1). BNTX (2a), which
The results of the binding assay showed that the compounds 2
with high active antitrichomonal activities possessed relatively
higher affinity for the DOR (1.80 nM ~ 83.9 nM). However, they

also showed high affinities for both µ opioid receptor (MOR) and
κ opioid receptor (KOR). Therefore, the various standard opioid
ligands were evaluated for antitrichomonal effects (MIC values)
to examine the correlation between antitrichomonal activity and
affinity for opioid receptors (Figure 1, Table 3). The MOR
8
9
10 (DOR antagonist)
11 (KOR agonist)
80
80
1
0
12 (KOR antagonist)
>320
80
b
11
13a
8
antagonists, 1 and β-funaltrexamine (β-FNA, 4) showed MIC
1
1
2
3
13b
13c
80
values of 80 µM (Entries 1 and 2). The DOR agonist, KNT-127
80
9
10
(
(
8) and the DOR antagonists, naltrindole (NTI, 9) and naltriben
a
11
Minimum inhibitory concentration after incubation of each compound with
T. vaginalis at 37 °C for 48 h.
Opioid receptor binding assay results of 13a: MOR, Ki = 499 nM; DOR, Ki
= 100 nM; KOR, Ki = > 1000 nM
NTB, 10) showed 80 µM, but interestingly, the DOR agonists
without a morphinan skeleton, SNC-80 (5), DPDPE (6), and
deltorphin II (7) did not destroy the trichomonad perfectly at the
1
2
13
b
14
concentration of 320 µM (Entries 3–8). The KOR agonist,
nalfurafine (11) showed 80 µM MIC, but the KOR antagonist
These results indicated that (i) the affinity for opioid receptors
including for the DOR) was of limited significance for
antitrichomonal activity, contrary to the CQ-resistance reversing
activity of plasmodium falciparum, (ii) the morphinan skeleton is
a good template for development of antitrichomonal agents, and
15
(
(
1
with two morphinan skeleton), nor-binaltorphimine (nor-BNI,
2) showed more than 320 µM MIC (Entries 9 and 10). Taken
1
6
together, the data suggest that one morphinan skeleton would
play an important role to afford antitrichomonal activity, without
any relation to the DOR antagonist activity. The deficiency of
specificity in the BNTX derivatives 2 for antitrichomonal activity,
unlike the antimalarial activity of 2, might be derived from the
difference of the evolutionary stage of species between malarial
(
iii) the phenolic hydroxy group of morphinan compounds would
be necessary for antitrichomonal activity. In addition to the above
results, the reduced BNTX methyl ether 14a was also evaluated
in a similar way (Figure 2). Interestingly, the activity of 14a was
dramatically decreased, compared with 3a and 13a, even though
1
7
plasmodium and the trichomonad.
1
4a has a typical morphinan skeleton. This result supports the
In addition, the BNTX methyl ether derivatives 13a–c were
also evaluated, based on the knowledge that the block of the
phenolic hydroxy group of the morphinan derivatives decreased
idea that the phenolic hydroxy group and the double bond of
morphinan skeleton are the essential functional groups for
antitrichomonal activity.
18
the affinity for opioid receptors. The BNTX methyl ether
derivatives 13a–c partially retained the antitrichomonal activities
(
1
Entries 11–13, MIC: 80 µM), compared to those of 2a–c (Table
, Entries 2–4).
Figure 2. Structure activity relationship of 2a.
2.3 Evaluations of antitrichomonal activities against T.
mobilensis
We next examined the antitrichomonal effects of
metronidazole, BNTX (2a), and the most active BNTX derivative
g against Tritrichomonas mobilensis, which is a common
variety of the intestinal trichomonads in a colony of squirrel
monkeys. The comparison of these results with respect to those
of T. vaginalis is shown in Table 4. Unexpectedly, 2a and 2g
showed almost no activity against T. mobilensis, although
metronidazole showed an MIC value of 2.5 µM. Considering that
all compounds were effective against T. vaginalis, the
mechanism for antitrichomonal activity of 2a and 2g is possible
to differ from that of the standard drug, metronidazole.
2
1
9
Figure 1. Typical opioid ligands 4–12 and the BNTX methyl ether
derivatives 13a–c.
Table 3
Antitrichomonal activities of typical opioid ligands 4–12 and the BNTX
methyl ether derivatives 13a–c against T. vaginalis
a
Entry
Compounds
Antitrichomonal activity (MIC in µM)
1
2
3
4
5
6
7
1 (MOR antagonist)
4 (MOR antagonist)
5 (DOR agonist)
6 (DOR agonist)
7 (DOR agonist)
8 (DOR agonist)
9 (DOR antagonist)
80
80
Table 4
Antitrichomonal activities of metronidazole, 2a, and 2g against T. vaginalis
and T. mobilensis
>320
>320
>320
80
a
Antitrichomonal activity (MIC in µM)
Compounds
Against T. mobilensis
Against T. vaginalis
10 (Table 1, Entry 1)
40 (Table 1, Entry 2)
Metronidazole
2.5
2
a
> 320
80

(
chloroform/methanol or hexane/ethyl acetate) to afford the
2
g
> 320
Minimum inhibitory concentration after incubation of the indicated
compound with preparations of the trichomonads at 37 °C for 48 h.
20 (Table 1, Entry 8)
a
corresponding BNTX derivatives 2.
7
4
.1.2.3. General procedure for synthesis of 2 using Method C
3
. Conclusion
A mixture of naltrexone hydrochloride (1•HCl, 1.0 equiv),
aryl aldehyde (4.0 equiv), and 1 M aq NaOH (3.8 equiv) in
methanol (0.1 M) was stirred at room temperature. After
confirming the consumption of 1 by TLC, the reaction was
quenched with 2 M aq HCl, and then the reaction mixture was
extracted with chloroform, washed with brine, dried over MgSO ,
4
and concentrated under reduced pressure. The crude mixture was
purified by silica gel column chromatography or preparative TLC
The BNTX derivatives 2a–v, 3a–c without double bonds, the
methyl ethers 13a–c, and the BNTX methyl ether lacking a
double bond 14a were synthesized and evaluated for their
antitrichomonal activity. Among of them, 4-iodo-BNTX (2g)
showed the highest activity (IC50 = 10.5 µM). In contrast to CQ-
resistance reversing activity against P. chabaudi, the affinity for
the opioid receptor would be a minor factor for antitrichomonal
activity against T. vaginalis. However, the morphinan skeleton
(
chloroform/methanol or hexane/ethyl acetate) to afford the
corresponding BNTX derivatives 2.
itself could be
a specific structure for development of
antitrichomonal agents, and the existence of both the double bond
of a Michael acceptor moiety and the phenolic hydroxy group
could be essential for antitrichomonal activity. In addition, the
mechanism of the antitrichomonal activity of the BNTX
derivative 2 might differ from that of the standard drug,
metronidazole, resulting from the difference of the effect on T.
mobilensis. These results are expected to be useful for medicinal
chemists in the study of antitrichomonal agents.
4.1.2.4. General procedure for synthesis of 3
A mixture of 2 (1.0 equiv) and Pd/C (Degussa type, 5%, 5
mol%) in methanol (0.1 M) was stirred at room temperature
under a hydrogen atmosphere. After confirming the consumption
of 2 by TLC, the reaction mixture was filtered through a pad of
Celite, and the filtrate was concentrated under reduced pressure.
The crude residue was extracted with chloroform, washed with
4
brine, dried over MgSO , and concentrated under reduced
pressure. The crude mixture was purified by silica gel column
chromatography or preparative TLC (chloroform/methanol or
hexane/ethyl acetate) to afford the corresponding reductive
BNTX derivatives 3.
4
. Experimental section
4
4
.1. Chemistry
.1.1. General
4
.1.2.5. General procedure for synthesis of 13
All melting points were determined on a Yanaco MP melting
point (mp) apparatus and are uncorrected. Infrared spectra were
A mixture of naltrexone methyl ether (3-methoxynaltrexone,
1
recorded with a JASCO FT/IR 4100 spectrophotometer. H and
1.0 equiv), aryl aldehyde (6.0 equiv), and piperidine (2.0 equiv)
in methanol (0.1 M) was stirred at 120 °C in a sealed tube. After
confirming the consumption of the starting material by TLC, the
1
3
C NMR spectral data were obtained with JEOL JNM-ECS 400
instruments. Chemical shifts are quoted in ppm using
tetramethylsilane (δ = 0 ppm) and DMSO-d (δ = 2.49 ppm) as
the reference for H NMR spectroscopy, CDCl (δ = 77.0 ppm)
(δ = 39.5 ppm) for C NMR spectroscopy. Mass
6
reaction was quenched with saturated aq NaHCO , and then the
3
1
3
reaction mixture was extracted with chloroform, washed with
brine, dried over MgSO , and concentrated under reduced
4
1
3
and DMSO-d
6
spectra were measured with a JEOL JMS-T100LP spectrometer.
Elemental analysis was performed with a YANACO CHN-
CODER JM-10 model analyzer. Column chromatography was
carried out on silica gel (spherical, neutral, 40–50 µm, Kanto
Chemical Co., Japan). Thin layer chromatography (TLC) and
preparative TLC were performed on Merck Kieselgel 60 F254
pressure. The crude mixture was purified by silica gel column
chromatography or preparative TLC (chloroform/methanol or
hexane/ethyl acetate) to afford the corresponding BNTX
derivatives 13.
4
.1.2.6. Synthesis of 14a
(
0.25 mm) plates.
.1.2.1. General procedure for synthesis of 2 using Method A
A mixture of naltrexone hydrochloride (1•HCl, 1.0 equiv),
A mixture of 13a (145.6 mg, 0.3283 mmol) and Pd/C
5
(Degussa type, 5%, 32.7 mg, 5.8 mol%) in methanol (3.0 mL)
was stirred at room temperature for 2 days under a hydrogen
atmosphere. After confirming the consumption of 13a by TLC,
the reaction mixture was filtered through a pad of Celite
(methanol), and the filtrate was concentrated under reduced
pressure. The crude residue was extracted with chloroform (20,
4
aryl aldehyde (6.0 equiv), and piperidine (2.0 equiv) in methanol
0.1 M) was stirred at 120 °C in a sealed tube. After confirming
the consumption of 1 by TLC, the reaction was quenched with
saturated aq NaHCO , and then the reaction mixture was
extracted with chloroform, washed with brine, dried over MgSO
(
3
15, 10 mL), washed with brine, dried over MgSO , and
4
4
,
concentrated under reduced pressure. The crude mixture was
and concentrated under reduced pressure. The crude mixture was
purified by silica gel column chromatography or preparative TLC
purified
by
silica
gel
column
chromatography
(chloroform/methanol = 100/1 to 25/1) to afford 14a (108.6 mg,
(
chloroform/methanol or hexane/ethyl acetate) to afford the
74%) as a colorless amorphous solid.
corresponding BNTX derivatives 2.
20
4
.1.3.1. 7(E)-Benzylidenenaltrexone (2a)
4
a
4
.1.2.2. General procedure for synthesis of 2 using Method B
Yellow amorphous solid; IR (neat): 3271, 2921, 2817, 1683,
1379 cm ; H NMR (400 MHz, CDCl , δ) 0.11−0.15 (m, 2H),
3
−
1 1
A mixture of naltrexone benzoic acid (1•PhCO H, 1.0 equiv),
2
aryl aldehyde (12.0 equiv), benzoic acid (2.5 equiv), and
diisopropylethylamine (5.5 equiv) in toluene/DMF (5/2, 1.6 M)
was stirred at 120 °C. After confirming the consumption of 1 by
0.52−0.56 (m, 2H), 0.79−0.90 (m, 1H), 1.65 (d, J = 12.8 Hz, 1H),
2.28−2.50 (m, 5H), 2.64−2.74 (m, 2H), 3.01 (d, J = 15.6 Hz, 1H),
3.14 (d, J = 18.8 Hz, 1H), 3.22 (d, J = 6.4 Hz, 1H), 4.71 (s, 1H),
5.20 (brs, 2H), 6.64 (d, J = 8.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H),
7.26−7.38 (m, 5H), 7.67 (d, J = 2.8 Hz, 1H); HR-MS (ESI): m/z
TLC, the reaction was quenched with sat. aq NaHCO
the mixture was extracted with chloroform or diethyl ether,
washed with brine, dried over MgSO , and concentrated under
reduced pressure. The crude mixture was purified by silica gel
column chromatography or preparative TLC
3
, and then
+
4
[M + H] calcd for C H NO : 430.2018. Found: 430.2038.
2
7
28
4

2
a•tartrate: MP (dec) 167.6−169.4 °C; Anal. Calcd for
· C ·H O: C, 62.30; H, 5.90; N, 2.34. Found: C,
1H), 2.29−2.50 (m, 5H), 2.65−2.76 (m, 2H), 2.94 (d, J = 15.1 Hz,
1H), 3.15 (d, J = 18.8 Hz, 1H), 3.24 (d, J = 6.4, 1H), 4.70 (s, 1H),
4.99 (brs, 2H), 6.64 (d, J = 8.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H),
C
6
27
H
27NO
4
4
H
6
O
6
2
2.16; H, 6.04; N, 2.34.
2
1
7.20 (d, J = 8.7 Hz, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 2.8
4
.1.3.2. 7(E)-(4-Methoxybenzylidene)naltrexone (2b)
13
Hz, 1H); C NMR (100 MHz, CDCl
3
, δ) 3.7, 4.1, 9.2, 22.8, 31.7
Pale yellow amorphous solid; IR (neat): 3397, 2930, 2836,
33.6, 43.3, 47.8, 59.3, 61.5, 70.2, 89.9, 117.6, 120.1, 123.2,
−
1 1
1
2
1
1
3
(
2
676, 1378 cm ; H NMR (400 MHz, CDCl
3
, δ) 0.13−0.16 (m,
124.5, 129.7, 131.6 (× 4), 133.0, 134.0, 138.3, 139.0 143.7,
+
H), 0.53−0.55 (m, 2H), 0.83−0.86 (m, 1H), 1.66 (d, J = 11.5 Hz,
H), 2.26−2.47 (m, 5H), 2.66−2.72 (m, 2H), 3.00 (d, J = 15.6 Hz,
H), 3.15 (d, J = 18.8 Hz, 1H), 3.23 (d, J = 6.4 Hz, 1H), 3.82 (s,
H), 4.69 (s, 1H), 5.09 (brs, 2H), 6.63 (d, J = 8.2 Hz, 1H), 6.74
198.3; HR-MS (ESI): m/z [M + H] calcd for C27
H
27BrNO
4
:
508.1124. Found: 508.1115.
2
f•tartrate: MP (dec) 190.2−192.0 °C; Anal. Calcd for
·C ·0.7H O: C, 55.48; H, 5.02; N, 2.09. Found:
C, 55.54; H, 5.32; N, 2.07.
C
27
H
26BrNO
4
4
H
6
O
6
2
d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.8 Hz,
+
H), 7.66 (d, J = 1.9 Hz, 1H); HR-MS (ESI): m/z [M + H] calcd
: 460.2124. Found: 460.2142.
for C28
H30NO
5
4.1.3.7. 7(E)-(4-Iodobenzylidene)naltrexone (2g)
2
b•tartrate: MP (dec) 175.0−177.0 °C; Anal. Calcd for
·C ·1.5H O: C, 62.68; H, 5.82; N, 2.28. Found:
C, 62.45; H, 5.82; N, 2.33.
Yellow amorphous solid; IR (neat): 3358, 2924, 2830, 1685,
1312, 1062 cm ; H NMR (400 MHz, CDCl , δ) 0.12−0.16 (m,
3
2H), 0.51−0.59 (m, 2H), 0.81−0.90 (m, 1H), 1.65 (d, J = 12.8 Hz,
1H), 2.50−2.48 (m, 5H), 2.63−2.73 (m, 2H), 2.91 (d, J = 15.1 Hz,
H), 3.14 (d, J = 18.8 Hz, 1H), 3.21 (d, J = 6.0 Hz, 1H), 4.69 (s,
−
1 1
C
28
H
29NO
5
4
H
6
O
6
2
2
0
4
.1.3.3. 7(E)-(4-Fluorobenzylidene)naltrexone (2c)
1
Yellow amorphous solid; IR (neat): 3350, 2925, 2831, 1685,
316, 1158 cm ; H NMR (400 MHz, CDCl , δ) 0.11−0.60 (m,
3
1H), 5.07 (brs, 2H), 6.64 (d, J = 8.2 Hz, 1H), 6.74 (d, J = 8.2 Hz,
1H), 7.06 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 2.3 Hz, 1H), 7.70 (d, J
= 8.2 Hz, 2H); C NMR (100 MHz, CDCl , δ) 3.8, 4.3, 9.4, 22.9,
3
−
1 1
1
2
1
1
1
1
2
13
H), 0.51−0.58 (m, 2H), 0.81−0.87 (m, 1H), 1.66 (d, J = 13.3 Hz,
H), 2.25−2.48 (m, 5H), 2.65−2.74 (m, 2H), 2.95 (d, J = 15.6 Hz,
H), 3.15 (d, J = 18.8 Hz, 1H), 3.23 (d, J = 6.4 Hz, 1H), 4.71 (s,
H), 5.23 (brs, 2H), 6.64 (d, J = 8.2 Hz, 1H), 6.74 (d, J = 8.2 Hz,
H), 7.05 (dd, J = 8.7, 8.7 Hz, 2H), 7.32 (dd, J = 8.7, 5.5 Hz,
31.9, 33.8, 43.4, 48.0, 59.5, 61.7, 70.3, 90.1, 95.3, 117.5, 120.2,
124.8, 129.9, 131.8 (× 2), 133.1, 134.6, 137.7 (× 2), 138.2, 139.3,
+
143.8, 198.2; HR-MS (ESI): m/z [M + H] calcd for C27
H
27INO
4
:
556.0985. Found: 556.1007.
+
H), 7.62 (d, J = 2.8 Hz, 1H); HR-MS (ESI): m/z [M + H] calcd
2
g•tartrate: MP (dec) 190.7−192.2 °C; Anal. Calcd for
for C27
H27FNO
4
: 448.1924. Found: 448.1940.
C
27
H26INO
4
·C
H
4 6
O
6
·1.3H
2
O: C, 51.08; H, 4.78; N, 1.92. Found:
2
c•tartrate: MP (dec) 186.0−188.0 °C; Anal. Calcd for
C, 50.91; H, 5.01; N, 1.77.
C
27
H
26FNO
4
·C
4
H
6
O
6
·0.4H
2
O: C, 61.56; H, 5.47; N, 2.32. Found:
21
4
.1.3.8. 7(E)-(3-Fluorobenzylidene)naltrexone (2h)
C, 61.35; H, 5.66; N, 2.23.
5
Pale yellow amorphous solid; IR (neat): 3357, 2925, 2830,
−1 1
4
.1.3.4. 7(E)-(4-Dimethylaminobenzylidene)naltrexone (2d)
1
3
687, 1378, 1263 cm ; H NMR (400 MHz, CDCl , δ) 0.12−0.16
Pale yellow amorphous solid; IR (KBr): 3385, 2922, 2825,
670, 1639, 1260 cm ; H NMR (400 MHz, CDCl , δ) 0.13–0.15
3
(m, 2H), 0.53−0.57 (m, 2H), 0.83−0.86 (m, 1H), 1.66 (d, J = 13.3
Hz, 1H), 2.25−2.48 (m, 5H), 2.65−2.75 (m, 2H), 2.97 (d, J = 15.1
Hz, 1H), 3.15 (d, J = 18.8 Hz, 1H), 3.23 (d, J = 6.4 Hz, 1H), 4.71
(s, 1H), 5.22 (brs, 2H), 6.65 (d, J = 8.2 Hz, 1H), 6.75 (d, J = 8.2
Hz, 1H), 7.00−7.05 (m, 2H), 7.11 (d, J = 7.8 Hz, 1H), 7.33 (dd, J
-
1 1
1
(
m, 2H), 0.49–0.54 (m, 2H), 0.82–0.85 (m, 1H), 1.63 (d, J = 10.0
Hz, 1H), 2.26–2.49 (m, 5H), 2.69–2.76 (m, 2H), 2.98 (s, 6H),
.02–3.17 (m, 2H), 3.28 (d, J = 5.0 Hz, 1H), 4.68 (s, 1H), 5.27
brs, 2H), 6.60–6.64 (m, 3H), 6.73 (d, J = 8.3 Hz, 1H), 7.28 (d, J
9.2 Hz, 2H), 7.68 (d, J = 1.2 Hz, 1H); HR-MS (ESI): m/z [M +
3
(
=
= 13.8, 7.8 Hz, 1H), 7.58 (d, J = 2.3 Hz, 1H); HR-MS (ESI): m/z
+
[M + H] calcd for C27
H
27FNO
4
: 448.1924. Found: 448.1944.
+
H] calcd for C29
33 2 4
H N O : 473.2440, found: 473.2418.
2
h•tartrate: MP (dec) 167.3−169.0 °C; Anal. Calcd for
2
d•tartrate: MP (dec) 154.4−156.4 °C; Anal. Calcd for
C
27
H
26FNO
4
·C
4
H
6
O
6
·0.4H
2
O: C, 61.56; H, 5.47; N, 2.32. Found:
C
29
H
32
N
2
O
4
·C ·2H O: C, 60.17; H, 6.43; N, 4.25. Found: C,
4
H
6
O
6
2
C, 61.54; H, 5.75; N, 2.16.
6
0.34; H, 6.36; N, 4.35.
21
4
.1.3.9. 7(E)-(2-Fluorobenzylidene)naltrexone (2i)
2
0
4
.1.3.5. 7(E)-(4-Chlorobenzylidene)naltrexone (2e)
Pale yellow amorphous solid; IR (neat): 3357, 2925, 2831,
1687, 1388, 1152 cm ; H NMR (400 MHz, CD OD): δ (ppm)
3
−
1 1
Yellow amorphous solid; IR (KBr): 3422, 2929, 2838, 1693,
−
1 1
1
2
1
1
1
1
406, 1062 cm ; H NMR (400 MHz, CDCl
3
, δ) 0.10−0.14 (m,
0.13−0.15 (m, 2H), 0.19−0.52 (m, 2H), 0.84−0.88 (m, 1H), 1.54
(d, J = 12.8 Hz, 1H), 2.27−2.44 (m, 5H), 2.65−2.72 (m, 2H), 2.79
(d, J = 15.6 Hz, 1H), 3.15 (d, J = 18.8 Hz, 1H), 3.25 (d, J = 6.4
Hz, 1H), 4.62 (s, 1H), 6.60 (d, J = 7.8 Hz, 1H), 6.65 (d, J = 7.8
Hz, 1H), 7.08−7.16 (m, 2H), 7.30−7.36 (m, 2H), 7.58 (s, 1H).
H), 0.51−0.55 (m, 2H), 0.80−0.85 (m, 1H), 1.64 (d, J = 12.8 Hz,
H), 2.24−2.46 (m, 5H), 2.62−2.72 (m, 2H), 2.91 (d, J = 15.1 Hz,
H), 3.15 (d, J = 18.8 Hz, 1H), 3.20 (d, J = 6.4 Hz, 1H), 4.69 (s,
H), 5.14 (brs, 2H), 6.63 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz,
H), 7.25 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 7.57 (d, J
2.3 Hz, 1H); HR-MS (ESI): m/z [M + H] calcd for
The OH peak/signal was not observed; HR-MS (ESI): m/z [M +
+
+
=
H] calcd for C27
H
27FNO
4
: 448.1924. Found: 448.1943.
C
27
H
27ClNO
4
: 464.1629. Found: 464.1626.
2
i•tartrate: MP (dec) 162.9−164.7 °C; Anal. Calcd for
2
e•tartrate: MP (dec) 144.1−145.7 °C; Anal. Calcd for
C
27
H
26FNO
4
·C ·1.5H O: C, 59.61; H, 5.65; N, 2.24. Found:
4
H
6
O
6
2
C
27
H
26ClNO
4
·C ·1.2H O: C, 58.57; H, 5.45; N, 2.20. Found:
4
H
6
O
6
2
C, 39.37; H, 5.74; N, 2.20.
C, 58.49; H, 5.66; N, 2.25.
4
.1.3.10. 7(E)-(4-Cyanobenzylidene)naltrexone (2j)
Pale yellow amorphous solid; IR (KBr) 3423, 2931, 2837,
4
.1.3.6. 7(E)-(4-Bromobenzylidene)naltrexone (2f)
−
1 1
Yellow amorphous solid; IR (neat): 3357, 2925, 2830, 1685,
3
1698, 1321 cm ; H NMR (400 MHz, CDCl , δ) 0.11−0.15 (m,
2H), 0.52−0.56 (m, 2H), 0.83−0.87 (m, 1H), 1.65 (d, J = 12.8 Hz,
1H), 2.27−2.48 (m, 5H), 2.62−2.75 (m, 2H), 2.88 (d, J = 15.1 Hz,
−
1 1
1
2
402, 1071 cm ; H NMR (400 MHz, CDCl
3
, δ) 0.14−0.17 (m,
H), 0.54−0.57 (m, 2H), 0.82−0.87 (m, 1H), 1.65 (d, J = 12.8 Hz,

1
1
=
H), 3.14 (d, J = 18.8 Hz, 1H), 3.21 (d, J = 5.0 Hz, 1H), 4.69 (s,
H), 6.63 (d, J = 8.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 7.43 (d, J
4.1.3.14. 7(E)-[(4-Quinoline)methylene]naltrexone (2n)
Yellow amorphous solid; IR (KBr): 3386, 2925, 1699, 1389
8.1 Hz, 2H), 7.54 (d, J = 2.1 Hz, 1H), 7.64 (d, J = 8.1 Hz, 2H).
−1
1
13
cm ; H NMR (400 MHz, CDCl
0
2
3
, δ) 0.08−0.12 (m, 2H),
.49−0.53 (m, 2H), 0.76−0.86 (m, 1H), 1.67 (d, J = 12.8 Hz, 1H),
.23−2.44 (m, 5H), 2.57 (dd, J = 18.8, 6.4 Hz, 1H), 2.71−2.77
The OH peak/signal was not observed; C NMR (100 MHz,
CDCl , δ) 3.7, 4.1, 9.3, 22.8, 31.6, 33.6, 43.2, 48.0, 59.4, 61.7,
0.3, 90.0, 112.1, 117.7, 118.5, 120.2, 124.5, 129.6, 130.3 (× 2),
32.1 (× 2), 135.2, 137.4, 138.3, 139.7, 143.6, 198.7; HRMS-
3
7
1
(
m, 2H), 3.07−3.13 (m, 2H), 4.77 (s, 1H), 5.80 (brs, 2H), 6.62 (d,
+
J = 8.0 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 4.6 Hz,
ESI: m/z [M + H] calcd for C28
4
H
27
N
2
O
4
: 455.1971. found:
1
1
H), 7.54 (dd, J = 8.2, 7.0 Hz, 1H), 7.73 (dd, J = 8.2, 7.0 Hz,
H), 7.90 (d, J = 8.2 Hz, 1H), 7.96 (d, J = 2.3 Hz, 1H), 8.18 (d, J
55.1953.
13
2
j•tartrate: MP (dec) 186.6−188.6 °C; Anal. Calcd for
= 8.2 Hz, 1H), 8.98 (d, J = 4.6 Hz, 1H); C NMR (100 MHz,
CDCl , δ) 3.7, 4.0, 9.2, 22.7, 31.6, 33.8, 43.3, 48.3, 59.3, 61.6,
70.4, 90.0, 117.9, 120.2, 120.8, 124.1, 124.7, 126.7, 127.1,
C
28
H
26
N
2
O
4
·C ·0.5H O: C, 62.64; H, 5.42; N, 4.57. found:
4
H
6
O
6
2
3
C, 62.82; H, 5.62; N, 4.65.
1
1
29.59, 129.64, 129.9, 134.8, 137.3, 138.8, 141.5, 143.9, 147.9,
49.7, 199.0; HR-MS (ESI): m/z [M + H] calcd for C30H N O :
29 2 4
4
.1.3.11. 7(E)-(4-Trifluoromethylbenzylidene)naltrexone (2k)
+
Yellow amorphous solid; IR (KBr): 3403, 2929, 2842, 1698,
327, 1119 cm ; H NMR (400 MHz, DMSO-d , δ) 0.07−0.12
6
m, 2H), 0.41−0.49 (m, 2H), 0.79−0.83 (m, 1H), 1.42 (d, J = 11.5
Hz, 1H), 2.13 (ddd, J = 12.4, 11.5, 3.2 Hz, 1H), 2.26−2.41 (m,
H), 2.61−2.67 (m, 2H), 2.75 (d, J = 16.0 Hz, 1H), 3.05 (d, J =
8.3 Hz, 1H), 3.16 (d, 5.5 Hz, 1H), 4.64 (s, 1H), 4.89 (brs, 1H),
.58 (d, J = 8.0 Hz, 1H), 6.61 (d, J = 8.0 Hz, 1H), 7.50 (d, J = 1.8
481.2127. Found: 481.2117.
−1
1
1
(
2
n•tartrate: MP (dec) 184.8−186.6 °C; Anal. Calcd for
C
30
H
28
N
2
O
4
·C ·2.4H O: C, 60.60; H, 5.80; N, 4.16. Found:
4
H
6
O
6
2
C, 60.67; H, 5.84; N, 3.89.
4
1
6
21
4.1.3.15. 7(E)-(3-Methoxybenzylidene)naltrexone (2o)
Pale yellow amorphous solid; IR (KBr) 3379, 2927, 2833,
Hz, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.74 (d, J = 8.2 Hz, 2H), 9.26
−1 1
1
3
1685, 1377 cm ; H NMR (400 MHz, CDCl
2
1
3
, δ) 0.10−0.14 (m,
H), 0.50−0.56 (m, 2H), 0.81−0.85 (m, 1H), 1.62 (d, J = 11.0 Hz,
H), 2.26−2.46 (m, 5 H), 2.63−2.73 (m, 2 H), 3.04 (d, J = 15.6
(
3
(
3
1
4
brs, 1H); C NMR (100 MHz, DMSO-d
1.4, 34.0, 43.0, 47.1, 58.6, 60.7, 69.7, 88.6, 117.4, 119.7, 124.07
q, J = 272.2 Hz), 124.14, 125.3 (q, J = 3.8 Hz, × 2), 128.8 (q, J =
6
, δ) 3.5, 3.9, 9.1, 22.4,
Hz, 1H), 3.12 (d, J = 18.8 Hz, 1H), 3.22 (d, J = 6.0 Hz, 1H), 3.77
(s, 3H), 4.70 (s, 1H), 5.52 (brs, 2H), 6.61 (d, J = 8.2 Hz, 1H),
1.6 Hz), 130.2, 130.9 (× 2), 134.9, 136.8, 138.9 (× 2), 143.7,
+
27 3 4
97.6; HR-MS (ESI): m/z [M + H] calcd for C28H F NO :
6
1
.74 (d, J = 8.2 Hz, 1H), 6.84−6.91 (m, 3H), 7.24 (d, J = 7.8 Hz,
H), 7.59 (d, J = 2.3 Hz, 1H); HR-MS (ESI): m/z [M + H] calcd
98.1892. Found: 498.1886.
+
2
k•tartrate: MP (dec) 216.5−218.4 °C; Anal. Calcd for
NO ·C ·0.7H O: C, 58.22; H, 5.10; N, 2.12. Found:
for C28
H
30NO
5
: 460.2124. Found: 460.2123.
o•tartrate: MP (dec) 148.0−149.7 °C; Anal. Calcd for
·C ·H O: C, 61.24; H, 5.94; N, 2.23. Found: C,
C
28
H
26
F
3
4
H
4 6
O
6
2
2
C, 58.27; H, 5.39; N, 2.16.
C
28
H29NO
5
4
H
6
O
6
2
4
.1.3.12. 7(E)-(3-Cyanobenzylidene)naltrexone (2l)
61.16; H, 6.16; N, 2.26.
Yellow amorphous solid; IR (KBr): 3381, 2925, 2830, 1689,
321 cm ; H NMR (400 MHz, CDCl , δ) 0.13−0.18 (m, 2H),
3
4.1.3.16. 7(E)-(3-Trifluoromethylbenzylidene)naltrexone (2p)
−1
1
1
0
2
3
2
1
9
1
1
Pale yellow amorphous solid; IR (neat): 3360, 2926, 2831,
.54−0.58 (m, 2H), 0.83−0.88 (m, 1H), 1.66 (d, J = 13.3 Hz, 1H),
.26−2.51 (m, 5H), 2.68−2.77 (m, 2H), 2.88 (d, J = 15.1 Hz, 1H),
.16 (d, J = 18.8 Hz, 1H), 3.26 (d, J = 6.0 Hz, 1H), 4.60 (brs,
H), 4.71 (s, 1H), 6.67 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 8.0 Hz,
−1
1
1
0
689, 1331, 1125 cm ; H NMR (400 MHz, CDCl
3
, δ)
.12−0.16 (m, 2H), 0.53−0.56 (m, 2H), 0.81−0.89 (m, 1H), 1.65
(
d, J = 12.8 Hz, 1H), 2.25−2.46 (m, 5H), 2.63−2.75 (m, 2H), 2.92
1
3
(d, J = 15.6 Hz, 1H), 3.15 (d, J = 18.3 Hz, 1H), 3.21 (d, J = 6.4
Hz, 1H), 4.71 (s, 1H), 6.65 (d, J = 8.2 Hz, 1H), 6.75 (d, J = 8.2
3
H), 7.47−7.61 (m, 5H); C NMR (100 MHz, CDCl , δ) 3.7, 4.2,
.2, 22.8, 31.6, 33.6, 43.3, 47.9, 59.3, 61.5, 70.4, 89.8, 112.7,
17.8, 118.4, 120.3, 124.4, 129.4, 129.5, 132.0, 132.8, 134.4,
Hz, 1H), 7.46−7.66 (m, 5H). The OH peak/signal was not
13
observed; C NMR (100 MHz, CDCl
3
, δ) 3.7, 4.1, 9.3, 22.8,
34.7, 136.3, 137.1, 138.3, 143.7, 198.8; HR-MS (ESI): m/z [M +
+
31.6, 33.6, 43.3, 47.9, 59.3, 61.6, 70.3, 90.0, 117.6, 120.2, 123.8
q, J = 272.2 Hz), 124.5, 125.3 (q, J = 3.8 Hz), 126.7 (q, J = 3.8
H] calcd for C28
H
27
N
2
O
4
: 455.1971. Found: 455.1949.
(
2
l•tartrate: MP (dec) 165.8−167.3 °C; Anal. Calcd for
Hz), 128.9, 129.7, 130.9 (q, J = 32.6 Hz), 133.0, 134.2, 135.8,
138.2, 138.3, 143.7, 198.7; HR-MS (ESI): m/z [M + H] calcd for
+
C
28
H
26
N
2
O
4
·C ·0.5H O: C, 62.45; H, 5.44; N, 4.55. Found:
4
H
6
O
6
2
C, 62.21; H, 5.60; N, 4.41.
C
28
H
27
F
3
4
NO : 498.1892. Found: 498.1890.
4
.1.3.13. 7(E)-[(2-Thiophene)methylene]naltrexone (2m)
2p•tartrate: MP (dec) 151.5−153.2 °C; Anal. Calcd for
C
28
H
26
F
3
NO ·C ·0.9H O: C, 57.90; H, 5.13; N, 2.11. Found:
4
4
H
6
O
6
2
Yellow amorphous solid; IR (KBr): 3422, 2923, 1676, 1413
−
1
1
C, 57.98; H, 5.27; N, 2.06.
cm ; H NMR (400 MHz, DMSO-d
6
, δ) 0.09−0.17 (m, 2H),
0
2
3
4
.44−0.54 (m, 2H), 0.84−0.90 (m, 1H), 1.46 (d, J = 11.0 Hz, 1H),
4.1.3.17. 7(E)-(2-Trifluoromethylbenzylidene)naltrexone (2q)
.13−2.38 (m, 5H), 2.64−2.70 (m, 2H), 2.84 (d, J = 16.9 Hz, 1H),
.10 (d, J = 18.3 Hz, 1H), 3.26 (d, J = 6.4 Hz, 1H), 4.56 (s, 1H),
.95 (brs, 1H), 6.56 (s, 2H), 7.20 (dd, J = 5.0, 3.9 Hz, 1H), 7.56
Pale yellow amorphous solid; IR (neat): 3366, 2925, 2831,
−1
1
1
0
694, 1316, 1164 cm ; H NMR (400 MHz, CDCl
3
, δ)
.09−0.13 (m, 2H), 0.49−0.57 (m, 2H), 0.78−0.88 (m, 1H), 1.64
(
(
d, J = 3.9 Hz, 1H), 7.70 (s, 1H), 7.87 (d, J = 5.0 Hz, 1H), 9.22
brs, 1H); C NMR (100 MHz, DMSO-d
13
(d, J = 12.8 Hz, 1H), 2.21−2.44 (m, 5H), 2.57 (dd, J = 18.8, 6.4
Hz, 1H), 2.69−2.77 (m, 2H), 3.07−3.12 (m, 2H), 4.73 (s, 1H),
6
, δ) 3.5, 3.9, 9.2, 22.7,
1.9, 35.1, 42.9, 47.0, 58.8, 60.7, 69.1, 88.4, 117.4, 119.7, 124.2,
28.2, 128.4, 160.4, 131.8, 132.2, 134.8, 137.8, 138.9, 143.8,
3
1
1
4
5
.23 (brs, 2H), 6.62 (d, J = 8.2 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H),
+
7.36 (d, J = 7.3 Hz, 1H), 7.42 (dd, J = 7.6, 7.3 Hz, 1H), 7.53 (dd,
J = 7.8, 7.6 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.73 (s, 1H);
95.0; HR-MS (ESI): m/z [M + H] calcd for C25
H26NO
4
S:
13
C
36.1583. Found: 436.1566.
NMR (100 MHz, CDCl , δ) 3.8, 3.9, 9.3, 22.8, 31.5, 33.4, 43.2,
3
2
m•tartrate: MP (dec) 185.8−187.8 °C; Anal. Calcd for
S·C ·1.2H O: C, 57.36; H, 5.54; N, 2.31. Found:
48.2, 59.4, 62.0, 70.4, 90.2, 114.5, 120.1, 124.5, 126.1 (q, J = 4.8
Hz), 127.4 (q, J = 246.3 Hz), 128.3, 129.3 (q, J = 29.7 Hz),
129.7, 130.7, 131.4, 133.9, 135.1, 136.2, 138.3, 143.7, 199.0;
C
25
H
25NO
4
4
H
6
O
6
2
C, 57.34; H, 5.46; N, 2.28.

+
13
HR-MS (ESI): m/z [M + H] calcd for C28
H
27
F
3
NO
4
: 498.1892.
peak/signal was not observed; C NMR (100 MHz, CDCl
3
, δ)
Found: 498.1892.
3.8, 3.9, 9.2, 22.8, 31.6, 34.4, 43.2, 48.2, 59.4, 61.7, 70.1, 89.9,
1
1
17.6, 120.1, 124.49, 124.53, 127.9 (× 2), 129.6, 129.7, 133.2,
2
q•tartrate: MP (dec) 163.4−165.2 °C; Anal. Calcd for
NO ·C ·0.4H O: C, 58.70; H, 5.05; N, 2.14. Found:
C, 59.05; H, 5.43; N, 2.18.
34.3, 137.1, 138.23, 138.27, 143.6, 198.3; HR-MS (ESI): m/z
C
28
H
26
F
3
4
H
4 6
O
6
2
+
[
M+H] calcd for C27
H26Cl
2
NO
4
: 498.1239. Found: 498.1225.
2
u•tartrate: MP (dec) 168.5−170.2 °C; Anal. Calcd for
NO ·C ·1.2H O: C, 55.56; H, 5.02; N, 2.09.
4
.1.3.18. 7(E)-(2-Bromobenzylidene)naltrexone (2r)
C
27
H
25Cl
2
4
4
H
6
O
6
2
Colorless amorphous solid; IR (KBr): 3376, 2923, 1692, 1042
Found: C, 55.50; H, 5.03; N, 2.08.
.1.3.22. 7(E)-(2-Methoxybenzylidene)naltrexone (2v)
Pale yellow amorphous solid; IR (KBr): 3390, 2931, 1682,
−
1
1
cm ; H NMR (400 MHz, CDCl
3
, δ) 0.10−0.14 (m, 2H),
21
4
0
2
1
.51−0.56 (m, 2H), 0.80−0.88 (m, 1H), 1.65 (d, J = 12.8 Hz, 1H),
.23−2.46 (m, 5H), 2.61 (dd, J = 18.8, 6.4 Hz, 1H), 2.74 (dd, J =
1.7, 4.3 Hz, 1H), 2.79 (d, J = 15.1, 1H), 3.09−3.16 (m, 2H), 4.73
−
1 1
1376 cm ; H NMR (400 MHz, CDCl
3
, δ) 0.10−0.13 (m, 2H),
(
(
2
s, 1H), 6.62 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H),7.18
ddd, J = 6.2, 5.5, 2.5 Hz, 1H), 7.28−7.32 (m, 2H), 7.59−7.61 (m,
0.5 0−0.54 (m, 2H), 0.80−0.86 (m, 1H), 1.63 (d, J = 12.8 Hz,
1H), 2.23−2.46 (m, 5H), 2.61−2.73 (m, 2H), 2.93 (d, J = 15.1 Hz,
1H), 3.11 (d, J = 18.8 Hz, 1H), 3.19 (d, J = 6.4 Hz, 1H), 3.79 (s,
3H), 4.71 (s, 1H), 5.07 (brs, 1H), 6.61 (d, J = 8.2 Hz, 1H), 6.74
(d, J = 8.2 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.91 (dd, J = 7.3,
6.4 Hz, 1H), 7.17 (d, J = 6.4 Hz, 1H), 7.30 (dd, J = 8.3, 7.3 Hz,
13
H). The OH peak/signal was not observed; C NMR (100 MHz,
, δ) 3.8, 4.0, 9.3, 22.8, 31.5, 33.4, 43.3, 48.1, 59.4, 61.8,
0.4, 90.2, 117.5, 120.1, 124.5, 125.0, 126.9, 129.7, 130.0, 130.7,
32.9, 134.0, 135.5, 138.3, 139.1, 143.7, 199.1; HR-MS (ESI):
CDCl
7
1
3
+
m/z [M + H] calcd for C27
5
H
27BrNO
4
: 508.1124. Found:
1H), 7.82 (d, J = 2.8 Hz, 1H). The OH peak/signal was not
+
08.1099.
observed; HR-MS (ESI): m/z [M + H] calcd for C28
H
30NO
5
:
4
60.2124. Found: 460.2111.
2v•tartrate: MP (dec) 162.3−163.9 °C; Anal. Calcd for
· C ·2.3H O: C, 59.03; H, 6.13; N, 2.15. Found:
2
r•tartrate: MP (dec) 165.8−167.7 °C; Anal. Calcd for
C
27
H
26BrNO
4
·C ·0.5H O: C, 55.78; H, 4.98; N, 2.10. Found:
4
H
6
O
6
2
C, 51.63; H, 5.26; N, 2.13.
C
28
H29NO
5
4
H
6
O
6
2
C, 59.06; H, 6.13; N, 1.99.
.1.3.23. 7α-Benzylnaltrexone (3a)
Colorless amorphous solid; IR (KBr): 3423, 2925, 1719, 1459
4
.1.3.19. 7(E)-[(2-Furyl)methylene]naltrexone (2s)
4
Pale yellow amorphous solid; IR (KBr): 3420, 2929, 1679,
−1
1
1
0
2
3
5
3
3
591 cm ; H NMR (400 MHz, CDCl , δ) 0.16−0.18 (m, 2H),
−
1 1
.56−0.58 (m, 2H), 0.88−0.89 (m, 1H), 1.67 (d, J = 11.0 Hz, 1H),
.32−2.50 (m, 5H), 2.70−2.76 (m, 2H), 3.16 (d, J = 18.8 Hz, 1H),
.33 (d, J = 5.0 Hz, 1H), 3.39 (d, J = 16.5 Hz, 1H), 4.68 (s, 1H),
.31 (brs, 1H), 6.47 (s, 1H), 6.62 (d, J = 8.2 Hz, 1H), 6.67 (d, J =
.2 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.41 (s, 1H), 7.53 (s, 1H).
3
cm ; H NMR (400 MHz, CDCl ): δ (ppm) 0.09−0.12 (m, 2H),
0.49−0.54 (m, 2H), 0.79−0.82 (m, 1H), 1.39 (dd, J = 13.1, 13.1
Hz, 1H), 1.56 (dd, J = 12.8, 3.2 Hz, 1H), 1.84 (dd, J = 13.1, 4.3
Hz, 1H), 2.13 (ddd, J = 11.9, 11.9, 3.2 Hz, 1H), 2.29−2.53 (m,
5H), 2.67 (dd, J = 11.9, 4.6 Hz, 1H), 3.00 (d, J = 18.8 Hz, 1H),
3.10 (d, J = 5.5 Hz, 1H), 3.18 (dd, J = 14.7, 5.5 Hz, 1H), 3.43 (m,
1H), 4.78 (s, 1H), 5.82 (brs, 2H), 6.58 (d, J = 8.1 Hz, 1H), 6.74
13
The OH peak/signal was not observed; C NMR (100 MHz,
CDCl , δ) 3.7, 4.2, 9.2, 23.0, 31.8, 33.9, 43.4, 47.5, 59.4, 61.5,
9.6, 89.5, 112.4, 117.6, 118.6, 120.0, 124.5, 126.6, 127.5, 129.9,
3
1
3
6
1
(d, J = 8.1 Hz, 1H), 7.14−7.28 (m, 5H); C NMR (100 MHz,
CDCl ): δ (ppm) 3.7, 4.0, 9.3, 22.5, 30.4, 34.2, 37.4, 43.6, 45.7,
51.8, 59.1, 61.8, 70.1, 91.3, 118.1, 119.8, 124.2, 126.1, 128.4 (×
), 128.9, 129.0 (× 2), 138.9, 139.6, 143.5, 210.2; HR-MS (ESI):
+
38.3, 143.7, 145.3, 151.8, 196.7; HR-MS (ESI): m/z [M + H]
: 420.1811. Found: 420.1819.
3
calcd for C25H26NO
5
2
2
s•tartrate: MP (dec) 150.1−151.2 °C; Anal. Calcd for
·C ·1H O: C, 59.28; H, 5.66; N, 2.38. Found: C,
+
m/z [M+H] calcd for C27
4
H30NO : 432.2175. Found: 432.2154.
C
25
H
25NO
5
4
H
6
O
6
2
5
9.22; H, 5.72; N, 2.55.
3a•tartrate: MP (dec) 157.8−159.7 °C; Anal. Calcd for
2
1
C
27
H
29NO
4
·C ·1.1H O: C, 61.91; H, 6.23; N, 2.33. Found:
4
H
6
O
6
2
4
.1.3.20. 7(E)-(2-Chlorobenzylidene)naltrexone (2t)
C, 61.84; H, 6.40; N, 2.33.
Colorless amorphous solid; IR (KBr): 3411, 2924, 1688, 1047
−
1
1
4.1.3.24. 7α-(4-Methoxybenzyl)naltrexone (3b)
cm ; H NMR (400 MHz, CDCl
3
, δ) 0.10−0.14 (m, 2H),
0
2
1
1
1
.51−0.55 (m, 2H), 0.80−0.88 (m, 1H), 1.65 (d, J = 12.6 Hz, 1H),
Colorless amorphous solid; IR (neat): 3357, 2927, 1722, 1455
cm ; H NMR (400 MHz, CDCl
3
, δ) 0.09−0.13 (m, 2H),
−
1
1
.23−2.46 (m, 5H), 2.62 (dd, J = 18.8, 6.4 Hz, 1H), 2.73 (dd, J =
1.7, 4.3 Hz, 1H), 2.81 (d, J = 15.1 Hz, 1H), 3.12 (d, J = 18.8 Hz,
H), 3.16 (d, J = 6.0 Hz, 1H), 4.74 (s, 1H), 6.63 (d, J = 8.2 Hz,
H), 6.75 (d, J = 8.2 Hz, 1H), 7.22−7.30 (m, 3H), 7.40 (d, J = 6.9
0.50−0.54 (m, 2H), 0.80−0.83 (m, 1H), 1.36 (dd, J = 13.3, 13.3
Hz, 1H), 1.56 (d, J = 12.8 Hz, 1H), 1.84 (dd, J = 13.3, 4.1 Hz,
1H), 2.13 (ddd, J = 12.1, 12.1, 3.4 Hz, 1H), 2.23−2.54 (m, 5H),
2.68 (dd, J = 11.9, 4.1 Hz, 1H), 3.01 (d, J = 18.3 Hz, 1H),
3.08−3.13 (m, 2H), 3.38 (m, 1H), 3.77 (s, 3H), 4.76 (s, 1H), 6.58
(d, J = 8.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 8.5 Hz,
Hz, 1H), 7.68 (d, J = 2.3 Hz, 1H). The OH peak/signal was not
+
4
observed; HR-MS (ESI): m/z [M + H] calcd for C27H27ClNO :
4
64.1629. Found: 464.1638.
2
H), 7.06 (d, J = 8.5 Hz, 2H). The OH peak/signal was not
2
t•tartrate: MP (dec) 175.5−176.3 °C; Anal. Calcd for
13
observed; C NMR (100 MHz, CDCl
3
1
3
, δ) 3.8, 4.0, 9.3, 22.5,
0.4, 33.3, 37.3, 43.6, 45.9, 51.8, 55.2, 59.1, 61.8, 70.1, 91.3,
13.8 (× 2), 118.1, 119.8, 124.1, 128.9, 129.9 (× 2), 131.5, 139.0,
C
27
H
26ClNO
4
·C ·0.7H O: C, 59.42; H, 5.37; N, 2.24. Found:
4
H
6
O
6
2
C, 59.51; H, 5.61; N, 2.26.
+
4
.1.3.21. 7(E)-(2,6-Dichlorobenzylidene)naltrexone (2u)
143.5, 157.9, 210.5; HR-MS (ESI): m/z [M + H] calcd for
C
28
H
32NO
5
: 462.2281. Found: 462.2267.
Colorless amorphous solid; IR (KBr): 3422, 2925, 1698, 1047
−
1
1
cm ; H NMR (400 MHz, CDCl
3
, δ) 0.09−0.10 (m, 2H),
3b•tartrate: MP (dec) 150.1−151.9 °C; Anal. Calcd for
0
2
7
1
.49−0.51 (m, 2H), 0.78−0.81 (m, 1H), 1.64 (d, J = 10.5 Hz, 1H),
C
28
H31NO
5
·C ·1.8H O: C, 61.20; H, 6.52; N, 2.23. Found:
4
H
6
O
6
2
.19−2.43 (m, 6H), 2.61 (dd, J = 18.6, 6.2 Hz, 1H), 2.74 (d, J =
.3 Hz, 1H), 3.06−3.11 (m, 2H), 4.74 (s, 1H), 6.63 (d, J = 8.2 Hz,
H), 6.75 (d, J = 8.2 Hz, 1H), 7.17−7.36 (m, 4H). The OH
C, 61.28; H, 6.53; N, 2.18.
4
.1.3.25. 7α-(4-Fluorobenzyl)naltrexone (3c)

Colorless amorphous solid; IR (KBr): 3448, 2926, 1719, 1509
δ) 3.7, 4.1, 9.3, 22.8, 32.0, 33.7, 43.3, 47.5, 56.8, 59.4, 61.6, 70.0,
89.7, 114.8, 115.5 (d, J = 22.0 Hz, × 2), 119.5, 125.2, 130.3,
131.3 (d, J = 2.9 Hz,), 132.0, 132.1 (d, J = 8.6 Hz, × 2), 138.9,
−
1
1
3
cm ; H NMR (400 MHz, CDCl , δ) 0.10−0.13 (m, 2H),
0
.51−0.55 (m, 2H), 0.79−0.85 (m, 1H), 1.39 (dd, J = 13.3, 12.8
Hz, 1H), 1.56 (dd, J = 12.8, 2.3 Hz, 1H), 1.82 (dd, J = 13.1, 4.3
Hz, 1H), 2.13 (ddd, J = 12.1, 12.1, 3.7 Hz, 1H), 2.27−2.54 (m,
142.5, 145.4, 162.8 (d, J = 250.1 Hz), 197.2; HR-MS (ESI): m/z
+
[M + H] calcd for C28
H
29FNO
4
: 462.2081. Found: 462.2086.
5
3
1
H), 2.68 (dd, J = 11.9, 4.6 Hz, 1H), 3.02 (d, J = 18.3 Hz, 1H),
.10−3.17 (m, 2H), 3.34−3.42 (m, 1H), 4.76 (s, 1H), 5.74 (brs,
H), 6.59 (d, J = 8.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 6.93 (dd,
1
3c•tartrate: MP (dec) 146.9−148.6 °C; Anal. Calcd for
·C ·1.8H O: C, 59.68; H, 5.88; N, 2.17. Found:
C, 59.66; H, 5.84; N, 2.13.
C
28
H
28FNO
4
4
H
6
O
6
2
J = 8.7, 8.7 Hz, 2H), 7.11 (dd, J = 8.7, 5.5 Hz, 2H). The OH
peak/signal was not observed; C NMR (100 MHz, CDCl
3
7
1
1
3
3
, δ)
4.1.3.29. 7α-Benzyl-3-methoxynaltrexone (14a)
.8, 4.0, 9.3, 22.5, 30.5, 33.5, 37.4, 43.6, 45.9, 51.9, 59.1, 61.8,
0.1, 91.4, 115.1 (d, J =21.1 Hz, × 2), 118.0, 119.8, 124.2, 128.7,
Colorless amorphous solid; IR (neat): 3407, 2926, 1725, 1499
−
1
1
3
cm ; H NMR (400 MHz, CDCl , δ) 0.09−0.13 (m, 2H),
30.4 (d, J =8.6 Hz, × 2), 135.2, 138.9, 143.4, 161.4 (d, J =243.5
+
0.50−0.55 (m, 2H), 0.78−0.84 (m, 1H), 1.38 (dd, J = 13.3, 12.8
Hz, 1H), 1.57 (dd, J = 12.8, 2.8 Hz, 1H), 1.83 (dd, J = 12.8, 4.1
Hz, 1H), 2.10 (ddd, J = 12.4, 12.2, 3.9 Hz, 1H), 2.28−2.55 (m,
5H), 2.67 (dd, J = 11.5, 4.6 Hz, 1H), 3.01 (d, J = 18.3 Hz, 1H),
3.10 (d, J = 6.0 Hz, 1H), 3.20 (dd, J = 14.2, 5.5 Hz, 1H),
3.36−3.44 (m, 1H), 3.93 (s, 3H), 4.76 (s, 1H), 5.15 (brs, 1H),
Hz), 209.7; HR-MS (ESI): m/z [M + H] calcd for C27
4
H
29FNO
4
:
50.2081. Found: 450.2074.
3
c•tartrate: MP (dec) 143.0−144.0 °C; Anal. Calcd for
C
27
H28FNO
4
· C ·1.2H O: C, 59.94; H, 5.91; N, 2.25. Found:
4
H
6
O
6
2
C, 60.02; H, 5.93; N, 2.27.
6
5
.61 (d, J = 8.0 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 7.15−7.26 (m,
H); C NMR (100 MHz, CDCl , δ) 3.8, 4.0, 9.4, 22.5, 30.5,
3
4
.1.3.26. 7(E)-Benzylidene-3-methoxynaltrexone (13a)
13
Colorless amorphous solid; IR (KBr): 3394, 2927, 2833, 1686
34.2, 37.6, 43.6, 45.7, 51.7, 56.9, 59.1, 61.8, 69.9, 91.2, 115.0,
−
1
1
cm ; H NMR (400 MHz, CDCl
3
, δ) 0.12−0.14 (m, 2H),
119.3, 124.9, 126.0, 128.3 (× 2), 129.0 (× 2), 129.5, 139.7, 143.0,
+
0
2
3
4
8
.53−0.56 (m, 2H), 0.82−0.85 (m, 1H), 1.67 (d, J = 12.8 Hz, 1H),
144.9, 208.7; HR-MS (ESI): m/z [M + H] calcd for C28
H
32NO
4
:
.24−2.46 (m, 5H), 2.66−2.73 (m, 2H), 2.99 (d, J = 15.6 Hz, 1H),
.13 (d, J = 18.8 Hz, 1H), 3.21 (d, J = 6.4 Hz, 1H), 3.88 (s, 3H),
.69 (s, 1H), 4.89 (brs, 1H), 6.67 (d, J = 8.2 Hz, 1H), 6.73 (d, J =
446.2331. Found: 446.2341.
1
4a•tartrate: MP (dec) 120.1−122.0 °C; Anal. Calcd for
·C ·1.8H O: C, 61.20; H, 6.52; N, 2.23. Found:
1
3
C
28
H
31NO
4
4
H
6
O
6
2
.2 Hz, 1H), 7.31−7.38 (m, 5H), 7.67 (d, J = 2.7 Hz, 1H);
, δ) 3.7, 4.1, 9.3, 22.9, 31.9, 33.8, 43.3,
7.5, 56.8, 59.4, 61.6, 70.0, 89.7, 114.8, 119.5, 125.2, 128.3 (×
), 128.8, 130.2 (× 2), 130.4, 132.1, 135.2, 140.2, 142.5, 145.4,
C
C, 61.28; H, 6.53; N, 2.18.
NMR (100 MHz, CDCl
3
4
2
1
4
4.2. Pharmacology
+
4.2.1. Antitrichomonal assay
4
97.4; HR-MS (ESI): m/z [M + H] calcd for C28H30NO :
44.2175. Found: 444.2158.
The effect of test compounds on Trichomonas vaginalis was
determined as MIC in diamond TYI-S-33 medium with added
1
3a•tartrate: MP (dec) 135.3−137.1 °C; Anal. Calcd for
·C ·1.2H O: C, 62.47; H, 6.13; N, 2.28. Found:
1
0% heat-inactivated adult bovine serum and neutral red (Sigma–
C
28
H
29NO
4
4
H
6
O
6
2
Aldrich Co.; 10 mg/L). The medium was prepared in Milli-Q
C, 62.54; H, 6.39; N, 2.19.
water and distributed into test tubes (4.0 mL each). T. vaginalis
4
4
.1.3.27. 7(E)-(4-Methoxybenzylidene)-3-methoxynaltrexone
13b)
with 100 µL of preculture medium (1 × 10 bodies/mL) was
(
inoculated into each tube. Serial dilutions in duplicate of the test
compounds were added separately to the tubes after filtration for
sterilization. Saline was added to the control tube. The test tubes
were incubated at 37 °C for 48 h. After incubation, whether the
parasites were alive (as spherical shapes) or dead (not observed)
in the tubes was determined with Neubauer’s blood cell counter
Pale yellow amorphous solid; IR (KBr): 3423, 2927, 1677,
509 cm ; H NMR (400 MHz, CDCl , δ) 0.12−0.16 (m, 2H),
3
−1
1
1
0
2
3
3
1
=
.53−0.56 (m, 2H), 0.81−0.88 (m, 1H), 1.67 (d, J = 11.5 Hz, 1H),
.26−2.47 (m, 5H), 2.68−2.74 (m, 2H), 2.99 (d, J = 15.6 Hz, 1H),
.17 (d, J = 18.3 Hz, 1H), 3.23 (d, J = 6.4 Hz, 1H), 3.81 (s, 3H),
.87 (s, 3H), 4.68 (s, 1H), 4.89 (brs, 1H), 6.67 (d, J = 8.2 Hz,
H), 6.73 (d, J = 8.2 Hz, 1H), 6.88 (d, J = 8.7 Hz, 2H), 7.31 (d, J
22
under an invert microscope (NIKON, Japan). IC50 values were
calculated from inhibition-concentration curves. The effect on
Tritrichomonas mobilensis was determined in the same way as T.
vaginalis.
13
8.7 Hz, 2H), 7.66 (d, J = 2.3 Hz, 1H); C NMR (100 MHz,
, δ) 3.6, 4.1, 9.3, 22.9, 32.0, 34.0, 43.3, 47.3, 55.3, 56.8,
9.4, 61.5, 69.9, 89.7, 113.8 (× 2), 114.8, 119.4, 125.3, 127.8,
29.9, 130.5, 132.3 (× 2), 140.3, 142.5, 145.5, 160.2, 196.7; HR-
CDCl
3
5
1
4.2.2. Opioid Receptor Binding.
+
Chinese hamster ovary (CHO) cells stably expressing human
µ, δ, or κ opioid receptors were purchased from ChanTest Co.;
these cell membranes were used for opioid receptor binding
assays. Binding affinities for µ, δ, or κ opioid receptors by the
MS (ESI): m/z [M + H] calcd for C29
74.2284.
H
32NO
5
: 474.2281. Found:
4
1
3b•tartrate: MP (dec) 137.7−138.8 °C; Anal. Calcd for
3
C
29
H
31NO
5
·C ·1.7H O: C, 60.58; H, 6.22; N, 2.14. Found:
4
H
6
O
6
2
test compounds were measured by displacement of [ H]-
3
3
C, 60.27; H, 6.20; N, 2.45.
DAMGO, [ H]-DPDPE, or [ H]-U69,593 (each 2 nM),
respectively. Nonspecific binding was measured in the presence
of 10 µM unlabeled DAMGO, DPDPE, or U-69,593.
Radioactivity in the test samples was determined by a MicroBeta
scintillation counter with 96-well microplate (PerkinElmer). The
4
.1.3.28. 7(E)-(4-Fluorobenzylidene)-3-methoxynaltrexone (13c)
Pale yellow amorphous solid; IR (neat): 3583, 2927, 1685,
−1
1
1
0
2
3
4
8
3
507 cm ; H NMR (400 MHz, CDCl , δ) 0.13−0.14 (m, 2H),
.54−0.55 (m, 2H), 0.82−0.86 (m, 1H), 1.67 (d, J = 11.9 Hz, 1H),
.25−2.47 (m, 5H), 2.66−2.72 (m, 2H), 2.93 (d, J = 15.1 Hz, 1H),
.16 (d, J = 18.8 Hz, 1H), 3.22 (d, J = 6.0 Hz, 1H), 3.87 (s, 3H),
.68 (s, 1H), 4.91 (brs, 1H), 6.67 (d, J = 8.2 Hz, 1H), 6.74 (d, J =
.2 Hz, 1H), 7.05 (dd, J = 8.7, 8.7 Hz, 2H), 7.32 (dd, J = 6.7, 5.5
value of each test sample was calculated as [(T
× 100, where T is the nonspecific binding, T is the [ H]-labeled
ligand binding in the presence of various concentrations of test
1 0 2 0
– T )/ (T – T )]
3
0
1
-
5
-11
3
compounds (10 – 10 M), and T
2
is the [ H]-labeled ligand
binding in the absence of respective test compounds. Sigmoidal
13
3
Hz, 2H), 7.61 (d, J = 1.8 Hz, 1H); C NMR (100 MHz, CDCl ,

concentration–response curves and K
Prism software (version 6.05).
i
values were calculated by
12. Calderon, S. N.; Rothman, R. B.; Porreca, F.; Flippen-Anderson, J.
L.; McNutt, R. W.; Xu, H.; Smith, L. E.; Bilsky, E. J.; Davis, P.;
Rice, K. C. J. Med. Chem. 1994, 37, 2125–2128.
1
3. Clark, J. A.; Itzhak, Y.; Hruby, V. J.; Yamamura, H. I.; Pasternak,
G. W. Eur. J. Pharmacol. 1986, 128, 303–304.
Acknowledgments
1
4. Erspamer, V.; Melchiorri, P.; Falconieri-Erspamer, G.; Negri, L.;
Corsi, R.; Severini, C.; Barra, D.; Simmaco, M.; Kreil, G. Proc.
Nat. Acad. Sci. USA 1989, 86, 5188–5192.
This work was supported by JSPS KAKENHI (Grant-in-Aid
for Young Scientists (B)) Grant Number 15K16553 (N.K.),
1
5. (a) Nagase, H.; Fujii, H. Curr. Pharm. Des. 2013, 19, 7400–7414;
1
7K13259 (N.K.), and TORAY Industries, Inc. We thank Dr. S.
(
(
b) Nagase, H.; Fujii, H. Top. Curr. Chem. 2010, 299, 187–237;
c) Nagase, H.; Fujii, H. Top. Curr. Chem. 2010, 299, 29–62; (d)
Hirayama and H. Fujii (Kitasato University) for supporting the
study on opioid receptor binding.
Kawai, K.; Hayakawa, J.; Miyamoto, T.; Imamura, Y.; Yamane,
S.; Wakita, H.; Fujii, H.; Kawamura, K.; Matsuura, H.; Izumimoto,
N.; Kobayashi, R.; Endo, T.; Nagase, H. Bioorg. Med. Chem. 2008,
References and notes
1
6, 9188–9201; (e) Nagase, H.; Hayakawa, J.; Kawamura, K.;
Kawai, K.; Takezawa, Y.; Matsuura, H.; Tajima, C.; Endo, T.
Chem. Pharm. Bull. 1998, 46, 366–369.
6. (a) Takemori, A. E.; Ho, B. Y.; Naeseth, J. S.; Portoghese, P. S. J.
Pharmacol. Exp. Ther. 1998, 246, 255–258; (b) Portoghese, P. S.;
Nagase, H.; Lipkowski, A. W.; Larson, D. L.; Takemori, A. E. J.
Med. Chem. 1988, 31, 836–841.
7. (a) Barclay, V. C.; Kennedy, D. A.; Weaver, V. C.; Sim, D.;
Lloyd-Smith, J. O.; Read, A. R. Am. Nat. 2014, 184, S47–S57; (b)
Adl, S. M.; Simpson, A. G. B.; Lane, C. E.; Lukeš, J.; Bass, D.;
Bowser, S. S.; Brown, M. W.; Burki, F.; Dunthorn, M.; Hampl,
V.; Heiss, A.; Hoppenrath, M.; Gall, L. L.; Lynn, D. H.; Mcmanus,
H.; Mitchell, E. A. D.; Mozley-stanridge, S. E.; Parfrey, L. W.;
Pawlowski, J.; Rueckert, S.; Shadwick, L.; Schoch, C. L.; Smirnov,
A.; Spiegel, F. W. J. Eukaryot. Microbiol. 2012, 59, 429–493; (c)
O’Neill, J. B.; Pert, C. B.; Ruff, M. R.; Smith, C. C.; Higgins, W.
J.; Zipser, B. Brain Res. 1988, 450, 303–315.
8. (a) Granier, S.; Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F.
S.; Weis, W. I.; Kobilka, B. K. Nature 2012, 485, 400–405; (b)
Uwai, K.; Uchiyama, H.; Sakurada, S.; Kabuto, C.; Takeshita, M.
Bioorg. Med. Chem. 2004, 12, 417–421.
9. (a) Midlej, V.; Pereira-Neves, A.; Kist, L. W.; Bogo, M. R.;
Benchimol, M. Vet. Parasitol. 2011, 182, 171–180; (b) Pindak, F.
F.; Pindak, M. M.; Gardner, W. A. Jr.; Abee, C. R. J. Clin.
Microbiol. 1988, 26, 1460–1463; (c) Pindak, F. F.; Pindak, M. M.;
Abee, C. R.; Gardner, W. A. Jr.; Am. J. Primatol. 1985, 9, 197–
1
.
(a) Harp, D. F.; Chowdhury, I. Eur. J. Obstet. Gynecol. Reprod.
Biol. 2011, 157, 3–9; (b) Nanda, N.; Michel, R. G.;
Kurdgelashvili, G.; Wendel, K. A. Expert Rev. Anti-infe. 2006, 4,
1
1
1
25–135.
2
.
(a) Ghosh, I.; Muwonge, R.; Mittal, S.; Banerjee, D.; Kundu, P.;
Mandal, R.; Biswas, J.; Basu, P. J. Clin. Virol. 2017, 89, 43–48;
(
4
b) Menezes, C. B.; Frasson, A. P.; Tasca, T. Microb. Cell 2016, 3,
04–418; (c) Zhu, Z.; Davidson, K. T.; Brittignham, A.;
Wakefield, M. R.; Bai, Q.; Xiao, H.; Fang, Y. Med. Oncol. 2016,
3, 115; (d) Secor, W. E.; Meites, E.; Starr, M. C.; Workowski, K.
3
A. Am. J. Trop. Hyg. 2014, 90, 800–804; (e) Spence, A. R.;
Rousseau, M.-C.; Karakiewicz, P. I.; Parent, M.-É. BJU Int. 2014,
1
14, E90–E98; (f) Stark, J. R.; Judson, G.; Alderete, J. F.;
Mundodi, V.; Kucknoor, A. S.; Giovannucci, E. L.; Platz, E. A.;
Sutcliffe, S.; Fall, K.; Kurth, T.; Ma, J.; Stampfer, M. J.; Mucci, L.
A. J. Natl. Cancer Inst. 2009, 101, 1406–1411; (g) Sutcliffe, S.;
Alderete, J. F.; Till, C.; Goodman, P. J.; Hsing, A. W.; Zenilman, J.
M.; De Marzo, A. M.; Platz, E. A. Int. J. Cancer 2009, 124, 2082–
1
1
2
087.
3
.
(a) Hrdý, I.; Cammack, R.; Stopka, P.; Kulda, J.; Tachezy, J.
Antimicrob. Agents Chemother. 2005, 49, 5033–5036; (b)
Cudmore, S. L.; Delgaty, K. L.; Hayward-McClelland, S. F.;
Petrin, D. P.; Garber, G. E. Clin. Microbiol. Rev. 2004, 17, 783–
7
93; (c) Vidakovic, M.; Crossnoe, C. R.; Neidre, C.; Kim, K.;
Krause, K. L.; Germanas, J. P. Antimicrob. Agents Chemother.
003, 47, 302–308; (d) Rasoloson, D.; Tomková, E.; Cammack,
2
05.
2
2
0. Nan, Y.; Upadhyaya, S. P.; Xu, W.; Hughes, K. E.; Dunn, W. J.
R.; Kulda, J.; Tachezy, J. Parasitology 2001, 123, 45–56; (e)
Upcroft, P.; Upcroft, J. A. Clin. Microbiol. Rev. 2001, 14, 150–
III; Bauer, L.; Bhargava, H. N.; Doss, G. A. J. Heterocyclic. Chem.
1
996, 33, 399–407.
1
64; (f) Johnson, P. J. Parasitol. Today 1993, 9, 183–186; (g)
2
2
1. Ohkawa, S.; Portoghese, P. S. J. Med. Chem. 1998, 41, 4177–4180.
2. Garber, G. E.; Sibau, L.; Ma, R.; Proctor, E. M.; Shaw, C. E.;
Bowie, W. R. J. Clin. Microbiol. 1987, 25, 1275–1279.
Quon, D. V. K.; d’Oliveira, C. E.; Johnson, P. J. Proc. Natl. Acad.
Sci. USA 1992, 89, 4402–4406; (h) Lossick, J. G. In
Trichomonads Parasitic in Humans; Honigberg, B. M., Ed.;
Springer-Verlag, New York, 1990, pp 324-341; (i) Yarlett, N.;
Yarlett, N. C.; Lloyd, D. Biochem. Pharmacol. 1986, 35, 1703–
1
708; (j) Lloyd, D.; Yarlett, N.; Yarlett, N. C. Biochem.
Pharmacol. 1986, 35, 61–64.
4
.
(a) Miyata, Y.; Fujii, H.; Uenohara, Y.; Kobayashi, S.; Takeuchi,
T.; Nagase, H. Bioorg. Med. Chem. Lett. 2012, 22, 5174–5176; (b)
Miyata, Y.; Fujii, H.; Osa, Y.; Kobayashi, S.; Takeuchi, T.;
Nagase, H. Bioorg. Med. Chem. Lett. 2011, 21, 4710–4712.
Kutsumura, N.; Nakajima, R.; Koyama, Y.; Miyata, Y.; Saitoh, T.;
Yamamoto, N.; Iwata, S.; Fujii, H.; Nagase, H. Bioorg. Med.
Chem. Lett. 2015, 25, 4890–4892.
5
6
.
.
(a) Famin, O.; Krugliak, M.; Ginsburg, H. Biochem. Pharmacol.
1
999, 58, 59–68; (b) Srivastava, P.; Puri, S. K.; Kamboj, K. K.;
Pandey, V. C. Trop. Med. Int. Health 1999, 4, 251–254; (c)
Ginsburg, H.; Famin, O.; Zhang, J.; Krugliak, M. Biochem.
Pharmacol. 1998, 56, 1305–1313; (d) Dubois, V. L.; Platel, D. F.
N.; Pauly, G.; Tribouley-Duret, J. Exp. Parasitol. 1995, 81, 117–
1
24.
7
8
9
.
.
.
Portoghese, P. S.; Garzon-Aburbeh, A.; Nagase, H.; Takemori, A.
E. J. Med. Chem. 1991, 34, 1292–1296.
Sayre, L. M.; Larson, D. L.; Takemori, A. E.; Portoghese, P. S. J.
Med. Chem. 1984, 27, 1325–1335.
Nagase, H.; Nemoto, T.; Matsubara, A.; Saito, M.; Yamamoto, N.;
Osa, Y.; Hirayama, S.; Nakajima, M.; Nakao, K.; Mochizuki, H.;
Fujii, H. Bioorg. Med. Chem. Lett. 2010, 20, 6302–6305.
1
1
0. Portoghese, P. S.; Sultana, M.; Nagase, H.; Takemori, A. E. J.
Med. Chem. 1988, 31, 281–282.
1. Sofuoglu, M.; Portoghese, P. S.; Takemori, A. E. J. Pharmacol.
Exp. Ther. 1991, 257, 676–680.