Design, Synthesis, and Biological Evaluation of the Third Generation 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4′-pyridyl)carboxamido]morphinan (NAP) Derivatives as μ/κ Opioid Receptor Dual Selective Ligands

Article abstract of DOI:10.1021/acs.jmedchem.8b01158

μ opioid receptor (MOR) agonists have been widely applied for treating moderate to severe pain. However, numerous adverse effects have been associated with their application, including opioid-induced constipation (OIC), respiratory depression, and addicti

Full text of DOI:10.1021/acs.jmedchem.8b01158

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Article
Design, Synthesis, and Biological Evaluation of the Third
Generation 17-Cyclopropylmethyl-3,14#-dihydroxy-4,5#-
epoxy-6#-[(4#- pyridyl)carboxamido]morphinan (NAP)
Derivatives as Mu/Kappa Opioid Receptor Dual Selective Ligands
Yi Zheng, Samuel Obeng, Huiqun Wang, Abdulmajeed M Jali, Bharath Peddibhotla, Dwight Williams,
Chuanchun Zou, David L Stevens, William L Dewey, Hamid I Akbarali, Dana E Selley, and Yan Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01158 • Publication Date (Web): 04 Jan 2019
Downloaded from http://pubs.acs.org on January 4, 2019
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Design, Synthesis, and Biological Evaluation of the Third Generation 17-
Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4′-
pyridyl)carboxamido]morphinan (NAP) Derivatives as Mu/Kappa Opioid
Receptor Dual Selective Ligands
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Yi Zhenga, Samuel Obenga, Huiqun Wanga, Abdulmajeed M. Jalib, Bharath Peddibhotlaa,
Dwight A. Williamsa, Chuanchun Zoua, David L. Stevensb, William L. Deweyb, Hamid I.
Akbaralib, Dana E. Selleyb, Yan Zhanga*
a Department of Medicinal Chemistry, Virginia Commonwealth University, 800 E. Leigh Street,
Richmond, Virginia 23298, United States
b
Department of Pharmacology and Toxicology, Virginia Commonwealth University, 1112
East Clay Street, Richmond, Virginia 23298, United States
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Abstract
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Mu opioid receptor (MOR) agonists have been widely applied for treating moderate to severe
pain. However, numerous adverse effects have been associated with their application, including
opioid-induced constipation (OIC), respiratory depression, and addiction. Based on previous
work in our laboratory, NAP, a 6β-N-4′-pyridyl substituted naltrexamine derivative, was
identified as a peripheral MOR antagonist that may be used to treat OIC. To further explore its
structure activity relationship, a new series of NAP derivatives were designed, synthesized,
and biologically evaluated. Among these derivatives, NFP and NYP significantly antagonized
the antinociception effect of morphine. Whereas NAP acted mainly peripherally, its derivatives
NFP and NYP actually can act centrally. Furthermore, NFP produced significantly lesser
withdrawal symptoms than naloxone at similar doses. These results suggest that NFP has the
potential to be a lead compound to treat opioid abuse and addiction.
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Keywords: opioid receptor, antagonist, opioid addiction, withdrawal symptoms, dual-
selectivity.
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Introduction
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Opioid receptors belong to the Class A rhodopsin-like G-protein coupled receptor (GPCR)
family and may be classified into three main subtypes known as the μ opioid receptor (MOR),
κ opioid receptor (KOR), δ opioid receptor (DOR).1-4 Among them, MOR is the main
pharmacological target for opioid medications, such as morphine.5 After agonist binding, the
conformation change of the MOR will lead to activation of the receptor, which may further
result in opening of inwardly rectifying K+ (GIRK) channels, inhibition of the voltage-gated
Ca2+ (VGCC) channels, and inhibition of adenyl cyclase activity.6-9 These changes may further
induce a variety of changes in membrane morphology, neurons excitation, neurotransmitters
release, and expression of genes through the second messenger systems.10 The behavioral
effects of MOR activation include antinociception and addiction or abuse.11, 12 Opioid
addiction or abuse has become a global epidemic.13, 14 Moreover, opioid abuse increases the
prevalence of diseases such as HIV, tuberculosis, and hepatitis, especially for users who inject
these drugs.15-19 It is reported that over 33,000 people died due to opioid overdose in the US in
2015, 20 which has increased over 14 times in the last 20 years.21
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Currently, there are two approaches to treat opioid addiction, which are detoxification and
maintenance therapy by using opioid receptor agonists22-24, partial agonists25-27, or
antagonists.28, 29 Among them, opioid antagonists have been used as potential opioid addiction
treatments for years.28, 29 The synthesized opioid antagonists include peptide opioids and non-
peptide opioids. CTOP (Figure 1) and CTAP (Figure 1) are cyclic peptides which are highly
effective opioid antagonists with high MOR selectivity and metabolic stability, but their poor
bioavailability limited their development as clinical therapeutics.30-32 Compared to peptide
opioids, non-peptide opioid antagonists may have advantages of high bioavailability, easily
crossing the blood-brain barrier (BBB) and good metabolic stability.32 The classical opioid
antagonists used in opioid addiction treatments are naltrexone (Figure 1) and naloxone (Figure
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1). As a result of their different half-lives, naloxone is a short-acting medication while
naltrexone is used for long term treatment.33, 34 As opioid antagonists, they don’t carry the side
effects related to opioid agonists, such as addiction and respiratory suppression.35 However,
high doses of naltrexone and naloxone may induce hepatotoxicity,36, 37 cardiovascular and
pulmonary problems.38 Besides, the interactions between these compounds and the DOR or
KOR may induce mood changes39, 40 or block transmission of neurotransmitters41-43, though
more evidences are still needed to further prove them.
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The side effects associated with low selectivity MOR antagonists prompt researchers to
develop new opioid antagonists. β-funaltrexamine (β-FNA, Figure 1), a MOR antagonist, has
similar potency to the MOR and KOR.44b Cyprodime (Figure 1), another MOR antagonist, has
moderate selectivity to the MOR (Ki, δ/μ = 39 and Ki, κ/μ =10).45, 46 Clocinnamox (Figure 1) and
methocinamox (Figure 1) are potent MOR antagonists but with similar binding affinity to all
three opioid receptors.47 Meanwhile, it has been reported that activation of the KOR and further
dynorphin release has a relationship with mood behaviors, such as drug-related withdrawal,
aversion and relapse induced by stress.48 Studies have shown that norBNI (Ki, = 1.8 nM)
κ
increased the symptoms of opioid withdrawal in rats , while in another study with KOR knock-
out mice precipitation of opioid withdrawal was not observed.49, 50a De Marco et al studied a
new highly selective KOR partial agonist as anodyne with less harmful side effects.50b These
studies suggest that KOR probably is a potential target for drug addiction and side effects
related to mood behaviors. Collectively, highly selective and potent opioid antagonists are still
extremely desirable.
Keeping this in mind, a number of new chemical entities have been designed and synthesized
by modifying the structure of naltrexone to achieve highly selective opioid ligands.51-53 One
such compound from our lab, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(4′-
pyridylcarboxamido)morphinan (NAP) (Figure 1), a 6β-N-4′-pyridyl substituted naltrexamine
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derivative was identified as a peripherally selective MOR antagonist (Ki, δ/μ ≈ 747, Ki, δ/κ ≈ 163;
ED50 = 0.0088 mg/kg).54-56 To avoid diarrhea induced at high doses of NAP, the second
generation NAP derivatives were designed and synthesized by modifying the C(6)-pyridyl ring
system of NAP which was shown to interact with the MOR ‘address’ region. NMP, (17-
Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-{[4′-(3′-
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methylpyridyl)]carboxamido}morphinan), a second generation NAP derivative had improved
pharmacological properties compared to NAP since it not only acted as a peripherally selective
MOR antagonist but also improved motility of the gastrointestinal tract in vivo.57a, 58 Further
molecular modeling studies showed that the methyl group at 3’ position on the pyridyl group
could reinforce the interactions between NMP and the ‘address’ region of the MOR.58 Based
on these previous study results, we sought to further explore the influence on binding affinity
of 3’ position on the pyridyl ring of NAP to opioid receptors based on the “message-address”
concept.57a, 58 Thus, we designed, synthesized, and biologically evaluated a series of new
compounds as the third generation NAP derivatives. Two new compounds, NFP and NYP,
were characterized in vitro and in vivo as opioid receptor antagonists. Interestingly, these two
compounds were found to be centrally acting with less withdrawal effects compared to
naloxone, which was contrary to the peripherally acting NAP and NMP.
Results and Discussion
Molecular Design
Our molecular modeling study on NAP in the three opioid receptors demonstrated that the
C(6)-pyridyl ring of NAP may strengthen the selectivity of NAP for the MOR over the DOR
and KOR through aromatic stacking and hydrogen-bonding interactions.44 Meanwhile, a
molecular dynamics simulation study of NMP in the MOR, DOR and KOR showed that the 3’-
methyl group on the pyridyl ring may reinforce the π-π stacking interaction between the
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aromatic pyridyl ring of NMP and W3187.35 of the MOR, which resulted in the high selectivity
of NMP to the MOR over the DOR and KOR.58 Further modeling study results indicated that
the characteristics of the substituents on the 3’ position of pyridyl ring of NAP derivatives,
such as steric hindrance, electrostatic, and hydrophobic effects, may have significant effect on
the binding affinity and selectivity of NAP derivatives to the three opioid receptors.58 Therefore,
in designing the third generation NAP derivatives, substitutions with different electronic, bulky,
steric, and hydrophobic properties were introduced at the 3’ position of pyridyl ring (Figure
2).
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N
S
S
OH
N
OH
D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2
CTOP
O
O
O
S
S
O
D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2
OH
Naltrexone
OH
Naloxone
CTAP
R
N
N
N
OCH3
OCH3
H
OH
O
N
O
O
O
O
N
H
O
O
HO
O
OH
Cyprodime
OH
R = Cl, Clocinnamox
R = Me, Methocinamox
¦Â-FNA
N
N
OH
O
OH
O
O
O
N
N
H
H
N
N
OH
OH
NMP
NAP
Figure 1. MOR selective antagonists.
Compounds
R
Compounds
R
1
2
3
4
5
F
7
C2H5
CN
NO2
CF3
COOH
8
Isobutyl
Cyclopentyl
Cyclohexyl
Cycloheptyl
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COOCH3
12
Phenyl
N
OH
O
O
R
N
H
N
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OH
Compounds 1-12
Figure 2. Structures of newly designed the third generation NAP derivatives.
Chemistry
Twelve new NAP derivatives (Table 1) were synthesized. Compounds 1-6 were derivatives
with electron-withdrawing groups at the 3’ position on the pyridyl ring with a corresponding
increment in size of the substituents. Compounds 7-12 were derivatives with electron-donating
groups at the 3’ position on the pyridyl ring with different bulkiness. For compounds 1-3, 9
and 12, the 3’-substituted pyridyl carboxylic acids were from commercial resources. For
compounds 4-8, 10 and 11, the 3’-substituted pyridyl carboxylic acids had to be prepared by
ourselves as shown in Scheme 1 (Supporting information).57b,c,d In addition, 6β-naltrexamine
was synthesized as reported previously with a yield of 58% (Scheme 2).44a Then, the 4’-pyridyl
carboxylic acids were coupled with 6β-naltrexamine by EDCI/HOBt method.44, 57a After that,
the reaction crudes were treated with K2CO3 to get compounds 1-12 with reasonable yields
(Scheme 2). All compounds were then transferred to hydrochloride salt and used for in vitro
and in vivo studies.
O
SeO2, 155-185 o
C
HO
Diphenyl ether
52%
N
N
13
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HO
O
HO
O
O
O
O
O
O
MeOH
NaOH
30.3%
OH
O
O
H2SO4
80.3%
N
N
N
14
7
15
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Br
n
n
OH
n
n
n
O
SeO2, 155-185 o
Diphenyl ether
30-51%
C
O
SOCl2
Pd/C, H2
70-92%
nBuLi, -78 o
30-50%
C
Pyridine
57-70%
HO
N
N
N
N
N
16 (n = 1)
17 (n = 2)
18 (n = 3)
19 (n = 1)
20 (n = 2)
21 (n = 3)
25 (n = 1)
26 (n = 2)
27 (n = 3)
22 (n = 1)
23 (n = 2)
24 (n = 3)
Br
Benzeneboronic acid
Dioxan/H2O
Pd, K2CO3, 70 o
O
SeO2, 155-185 o
Diphenyl ether
43.5%
C
C
HO
72%
N
N
N
28
29
Scheme 1. The synthesis of 3’-substituted pyridyl carboxylic acids for the newly designed NAP
derivatives.
N
N
N
OH
O
OH
O
OH
O
i. RCOOH, EDCI,
HOBt, TEA, DMF
ii. K2CO3, MeOH
i. (PhCH2)2NH, Toluene
ii. NaCNBH3, EtOH
iii. Pd/C, MeOH
O
R
O
NH2
23-65%
N
54%
H
N
OH
OH
OH
Compounds 1-12
Scheme 2. The synthesis route of the third generation NAP derivatives (compounds 1-12).
In vitro binding and functional studies were first conducted to evaluate the binding affinity,
selectivity and efficacy of the compounds to the opioid receptors. In vivo studies were further
conducted to characterize the behavioral profiles of these compounds.
In Vitro Radioligand Binding and MOR [35S]GTPγS Functional Studies
The competition radioligand binding assay was used to determine the binding affinity and
selectivity of the new NAP derivatives for the MOR, KOR, and DOR. Chinese hamster ovary
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(CHO) cell lines expressing mouse monocloned opioid receptors were used for this assay. The
MOR was labeled with [3H]naloxone, while the KOR and DOR were both labeled with
[3H]diprenorphine.
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As shown in Table 1, all the compounds maintained subnanomolar binding affinity to the
MOR except compounds 6 and 8 which showed nanomolar binding affinity. Compared to the
first and second generation lead compounds, NAP (Ki, MOR = 0.37 ± 0.07 nM) and NMP (Ki, MOR
= 0.58 ± 0.25 nM), most of the third generation derivatives still maintained high binding affinity
to the MOR. This indicated that the substituent groups with different electronic properties and
sizes at 3’ position on the pyridyl ring did not significantly affect the binding affinity of these
derivatives to the MOR. However, it is worth noting that esterification of the carboxylic acid
at the 3’ position (compound 6) and introduction of an isobutyl group at the 3’ position
(compound 8) resulted in a significant reduction in their MOR binding affinity. As mentioned
above, the structural features of substituents on the 3’ position of pyridyl ring, such as the steric
hindrance, electrostatic, and hydrophobic effects, would significantly affect the binding affinity
and selectivity of NAP derivatives to the three opioid receptors.58 Compound 6 was an
esterification product of compound 5. After esterification, the original ionic interactions
between carboxylic moiety in compound 5 and basic residues in the binding pocket of the MOR
would be weakened in compound 6 significantly so that the binding affinity of compound 6 to
the MOR was reduced. Furthermore, the isobutyl group on the pyridyl ring of compound 8 may
result in steric clashes or unfavorable interactions with hydrophilic residues in the binding
pocket that would decrease the binding affinity of compound 8 to the MOR. Interestingly,
compared to compound 8, compounds 9-12 carried even bulkier substituents on the pyridyl
ring while still maintaining high binding affinities to the MOR at the subnanomolar level. One
possible explanation of such an observation could be that the electrostatic and hydrophobic
interactions induced by the much larger substituents on the pyridyl ring (compounds 9-11) may
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facilitate the compounds to interact with the binding pocket favorably in the MOR in a different
binding mode than that of compound 8.
7
8
9
The selectivity of these compounds for the MOR over the DOR varied with different
substituent groups at 3’ position on the pyridyl ring. In general, derivatives with electron-
withdrawing groups showed higher selectivity for the MOR over the DOR than derivatives
with electron-donating groups. For example, compounds 1-6 showed more than 30-fold
selectivity for the MOR over the DOR, especially for compounds 1 (NFP, δ/μ = 435.8) and 3
(δ/μ = 562.4). Both compounds had similar selectivity for the MOR over the DOR as NMP
(δ/μ = 470). For the derivatives with electron-donating groups, compounds 7, 9, and 12 showed
50-134 fold selectivity for the MOR over the DOR while compounds 8, 10, and 11 showed 14-
20 fold selectivity for the MOR over the DOR. Thus, it seemed that introduction of bulky and
alkyl groups at position 3’ on the pyridyl ring may reduce their selectivity for the MOR over
the DOR.
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On the KOR, there was a significant improvement in the binding affinity of all the compounds
compared to NAP and NMP except for compound 8 which showed similar binding affinity to
the KOR as of NAP and NMP. Compounds 1-7 and 9-12 maintained one-digit nanomolar or
two-digit nanomolar binding affinity to the KOR. Thus, collectively, with substitution at 3’
position on the pyridyl ring, the third generation NAP derivatives seemed to act as MOR/KOR
dual-selective ligands.
Table 1. Radioligand binding results at the MOR, KOR, and DOR and [35S]GTPγS functional
assay at the MOR for the third generation NAP derivatives.
Ki (nM)
Selectivity
δ/μ κ/μ
MOR [35S]GTPγS Binding
EC50 (nM) % Emax of
Compounds
R
ΜOR
DOR
KOR
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DAMGO
NTXa
NAPa
N/A
0.26 ± 0.02
0.37 ± 0.07
117.1 ± 8.9
277.5 ± 8.0
5.15 ± 0.26
60.7 ± 5.6
450 20
ND
7.75 ± 0.20
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747
163
1.14 ± 0.38
22.72 ± 0.84
N
F
1(NFP)
2(NYP)
0.36 ± 0.02
0.87 ± 0.09
156.93 ± 7.81
46.29 ± 7.86
4.80 ± 0.26
435.8 13.3
1.20 ± 0.19
1.28 ± 0.20
34.97 ± 3.07
26.05 ± 0.74
N
N
C
2.74 ± 0.47
4.46 ± 0.11
53.2
3.1
N
NO2
N
3
4
0.17 ± 0.02
0.43 ± 0.01
95.61 ± 5.90
61.79 ± 5.0
562.4 26.5
0.53 ± 0.01
1.38 ± 0.20
39.10 ± 2.57
31.88 ± 3.39
CF3
12.53 ± 0.89 143.7 29.1
N
O
OH
N
5
6
0.76 ± 0.08
4.31 ± 0.29
55.58 ± 6.02
2.43 ± 0.19
73.2
3.2
1.83 ± 0.18
29.27 ± 1.42
O
O
N
137.31 ± 17.55 11.74 ± 1.15 31.9
2.7
13.40 ± 1.36 29.19 ± 0.84
NMPb
0.58 ± 0.25
0.32 ± 0.05
273.6 ± 1.8
96.7 ± 12.2
9.18 ± 1.36
470
166
1.52 ± 0.26
2.29 ± 0.28
30.63 ± 0.55
41.65 ± 3.14
N
7
42.86 ± 8.27
134.1 28.8
N
8
7.20 ± 0.81
148.75 ± 15.96 75.63 ± 3.87 20.7
10.5
19.70 ± 2.37 27.30 ± 0.25
N
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0.60 ± 0.06
0.78 ± 0.09
33.99 ± 3.99
11.24 ± 1.91
8.16 ± 1.17
3.32 ± 0.54
56.7
14.4
13.7
4.2
1.85 ± 0.16
2.46 ± 0.30
56.67 ± 3.14
N
10
51.59 ± 3.73
48.32 ± 3.34
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0.93 ± 0.13
0.63 ± 0.05
14.97 ± 2.44
44.83 ± 3.70
5.16 ± 0.54
3.42 ± 0.38
16.1
71.1
5.6
5.4
5.11 ± 0.16
1.46 ± 0.18
N
44.42 ± 3.12
N
a reference 44a; b reference 57a
The [35S]GTPγS functional assay was then conducted to determine the potency and relative
efficacy of the synthesized compounds at the MOR. The potency was determined as the EC50
while the efficacy was determined as the Emax relative to DAMGO, a MOR full agonist. From
these results, it seemed that all the compounds acted as partial agonists with low to moderate
efficacy. Derivatives with electron-withdrawing groups (compounds 1-6) showed 20-40%
MOR stimulation with one-digit nanomolar potency except compound 6. Meanwhile,
derivatives with electron-donating groups (compounds 7-12) showed more than 40% efficacy
with one or two-digit nanomolar potencies except compound 8 which had the lowest potency.
In Vivo Tail Immersion assay
All the newly synthesized NAP derivatives were further assessed for their acute agonistic
and/or antagonistic effects using the tail immersion assay in mice as previously reported.44, 57
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The baseline latency (control) was determined before the tested compound was injected into
the mice. For the study of agonism, the time of tail immersion was 20 min (time that morphine’s
antinociceptive effect starts to peak) after the test compound was injected (s.c.). For the study
of antagonism, the test compound was given 5 min before morphine. The tail immersion test
was then conducted 20 min after morphine was given. The percent maximum possible effects
(%MPE) was calculated for each mouse using at least five mice per drug. Briefly, the
derivatives were studied for their ability to produce antinociception or block the
antinociception produced by morphine (10 mg/kg). As shown in Figure 3A, none of these new
derivatives showed significant antinociception compared to morphine at the same dose (10
mg/kg). However, when they were tested as antagonists at the same dose (Figure 3B, 10
mg/kg), compounds 1 (NFP) and 2 (NYP) antagonized morphine’s antinociception effect
significantly. The %MPEs of morphine (10 mg/kg) in the presence of NFP and NYP (10 mg/kg)
were only 6.2 ± 2.4% and 7.8 ± 5.4%, respectively. The antagonism of NFP and NYP were
shown to be dose-dependent (Figure 4A and 4B, AD50 values of NFP and NYP were 2.82
(1.34-5.94) mg/kg with 95% confidence level (CL) and 1.75 (0.88-3.47) mg/kg with 95% CL,
respectively). In contrast to NFP and NYP, other NAP derivatives did not significantly block
morphine’s antinociception at the tested dose of 10 mg/kg. These results suggested that NFP
and NYP had the ability to cross the blood brain barrier (BBB) and block the analgesic effect
of morphine in CNS.
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It was intriguing to us that two of the third generation NAP derivatives, NFP and NYP,
crossed the BBB and antagonized morphine’s antinociception, while NAP was identified as a
P-glycoprotein substrate and failed to cross the BBB effectively. Apparently introduction of
fluoro and nitrile groups on the pyridyl ring may play an important role in improving their CNS
penetration. To be noted, about 5-15 % of drugs on the market are fluorinated compounds59, 60
and it has been shown that these compounds have improved metabolic stability and
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physicochemical properties. Meanwhile fluorination seems to enhance the CNS penetration
ability of non-CNS drugs and the efflux function of P-glycoprotein.61, 55 This may help explain
why NFP crossed the BBB while NAP did not.61 In addition, it has been reported that
introducing nitrile groups may also enhance metabolic stability and improve hydrogen bonding
interactions with residues in the orthosteric binding site of protein targets.62 Currently, there
are at least 30 drugs containing nitrile groups on the market and more than 20 drug candidates
containing nitrile groups under clinical trials.62 An example of such a drug is piritramide, an
opioid analgesic which crosses the BBB and is used for post-operation pain.63
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Figure 3. Tail immersion assay results for the third generation NAP derivatives as agonist (A)
and antagonist (B) in the presence of morphine in mice at a single dose of 10 mg/kg. Morphine
(10 mg/kg) and saline were used as positive and negative controls (n = 5, *** indicates P <
0.005).
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Figure 4. Dose-response studies of NFP (A) and NYP (B) as antagonists in mice. Morphine
(10 mg/kg) and saline were used as positive and negative controls (n = 5, *** indicates P <
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In Vivo Withdrawal Study
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Opioid antagonists such as naloxone and naltrexone have been applied to reverse the effects of
opioid agonists in cases of opioid overdose and in opioid addiction treatments. However, it has
been shown that naloxone and naltrexone produce significant withdrawal symptoms when
administered, which limits their use in opioid addiction treatments.64 Since NFP and NYP were
identified as opioid antagonists in vivo, a withdrawal study was conducted using morphine-
pelleted mice to determine whether these two compounds would produce withdrawal effects
similar to naloxone. In this study, somatic symptoms of opioid withdrawal (shakes, jumps, and
paw tremors) were quantified over a period of 20 minutes.55a As shown in Figure 5, NFP
produced significantly fewer wet dog shakes, jumps, and paw tremors at 10 mg/kg than
naloxone at 1 mg/kg. Meanwhile, at much higher doses (50 and 100 mg/kg) NFP produced wet
dog shakes (Figure 5A) and paw tremors (Figure 5C), but not significantly different from
those of naloxone at 1 mg/kg. More interestingly, at a high dose of 50 mg/kg NFP still produced
significantly fewer jumps (Figure 5B) than naloxone at 1 mg/kg. NYP, on the other hand,
produced significantly fewer wet dog shakes and jumps at 1 mg/kg than naloxone at 1 mg/kg,
but at doses 5 mg/kg and higher it produced wet dog shakes (Figure 6A) and jumps (Figure
6B) similar to naloxone at 1 mg/kg. Also, NYP produced similar paw tremors (Figure 6C) at
5 mg/kg similar to naloxone at 1 mg/kg.
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It has been reported that naloxone can antagonize morphine’s antinociception effect potently
(AD50 at 3.1 mg/kg) in a tail flick assay55b while it may induce significant withdraw symptom
(e.g. Figures 5 and 6, at 1 mg/kg). Compared to naloxone, NFP can effectively across BBB
entering into brain and reverse morphine’s antinociception effect at a similar potency level but
showed much lesser withdrawal symptoms. These results are very encouraging since they
suggest that NFP may has potential application in opioid addiction treatments with more
compatible patient compliance profiles compared to naloxone.
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A
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Jumps
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Paw tremors
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*
*
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Figure 5. In vivo withdrawal study of NFP in morphine-pelleted mice (n=5). The first column
in each figure represents the withdrawal effects of NFP in placebo-pelleted mice while the
second to the seventh show the withdrawal effects of naloxone (NLX, 1mg/kg) and NYP in
morphine-pelleted mice. (n = 5, * means P < 0.05, ** means P < 0.005, *** indicates P <
0.0005)
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*
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Paw tremors
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Figure 6. In vivo withdrawal study of NYP in morphine-pelleted mice (n=5). The first column
in each figure represents the withdrawal effects of NYP in placebo-pelleted mice while the
second to the seventh show the withdrawal effects of naloxone (NLX, 1mg/kg) and NYP in
morphine-pelleted mice. (n = 5, * indicates P < 0.05, ** indicates P < 0.005, *** indicates P <
0.0005).
We then further characterized NFP’s partial agonism and antagonism via concentration-
effect curves for stimulation of [35S]GTPγS binding in mMOR-CHO cells by NFP and
DAMGO alone (Figure 7A) in comparison to NFP curves in the presence in an EC90 DAMGO
concentration (Figure 7B). Naltrexone was examined as a positive control for MOR
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antagonism (Figure 7B). Relative to DAMGO (normalized Emax = 100 ± 2.6% and EC50 = 32.5
± 1.1 nM), NFP was a partial agonist with an Emax value of 34.9 ± 1.5% and EC50 value of 1.21
± 0.19 nM, in agreement with our initial screening results (Table 1). In the presence of 0.3 µM
DAMGO, NFP produced a concentration-dependent inhibition of DAMGO-stimulated
[35S]GTPγS binding, achieving maximal inhibition (minimum) at 37.7 ± 1.5% of the maximal
stimulation produced by DAMGO. Similarly, naltrexone produced concentration-dependent
inhibition, with maximal inhibition at 12.8 ± 0.4% of maxima DMAGO stimulation. Functional
Ki values were then calculated from these data. NFP and naltrexone inhibited DAMGO-
stimulated [35S]GTPγS binding with Ki values of 0.86 ± 0.09 and 0.67 ± 0.05 nM, similar to
their Ki values for MOR binding (Table 1). NFP had a relative efficacy that was approximately
35% of DAMGO and 3-fold that of naltrexone under these experimental conditions.
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Figure 7. MOR partial agonism and antagonism by NFP in mMOR-CHO cells. Ligand
concentration-effect for stimulation of [35S]GTPγS binding in membranes from mMOR-CHO
cells were determined alone (A) or in the presence of 0.3 µM DAMGO (B). Data are mean ±
SEM (n = 4-5) of the percentage of maximal stimulation (produced by a maximally-effective
DAMGO concentration of 0.3 µM).
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Modeling study
As described above, NFP (Figure 8) showed dual-selective binding affinity to the MOR and
KOR, and displayed significant selectivity over the DOR (Ki, δ/μ = 435.8). Meanwhile, NFP
acted as an MOR partial agonist with moderate efficacy (Table 1) and a KOR partial agonist
with low efficacy (EC50=13.62 ± 1.57 nM, 26.71 ± 1.94 % max effect of U50,488H), and a
DOR antagonist (EC50 not determined, 7.22 ± 1.17 % max effect of SNC80). However, the
results of its in vivo studies displayed that NFP acted as an effective opioid antagonist in the
CNS. To understand its selectivity profile to the opioid receptors, the antagonist-bound crystal
structures of MOR (PDB ID: 4DKL)64, KOR (PDB ID: 4DJH)65 and DOR (PDB ID: 4EJ4)66
were adopted from Protein Data Bank http://www.rcsb.org and NFP was docked into the three
receptors by GOLD 5.4.67, 68 The binding poses of NFP with the highest CHEM-PLP scores
from the docking studies were chosen as the optimal binding poses of the ligand in the MOR,
KOR, and DOR.
Figure 8. The chemical structure of NFP with atom notations from the docking studies.
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Similar to the lead compound NAP, NFP was designed and synthesized according to the
‘message-address’ concept where the ‘message’ moiety (epoxymorphinan moiety) was
assumed to determine its efficacy and the ‘address’ moiety (pyridyl ring) to contribute to its
selectivity (Figure 8).58 Comparing the binding poses of NFP in the MOR, KOR, and DOR
with those of NAP, we found that the identical ‘message’ moiety of NFP and NAP bound with
the same domain of the MOR, KOR, and DOR through similar interactions with the conserved
residues in the three receptors.58 Basically, the epoxymorphinan moiety formed hydrophobic
interactions with the conserved residues M3.36, W6.48 and H6.52 (superscript numbers follow the
Ballesteros-Weinstein numbering method for GPCRs69) and hydrogen bonding interactions
with Y3.33. Meanwhile, an ionic interaction was formed between the conserved D3.32 and the
nitrogen atom at 17-position of the epoxymorphinan moiety of NFP (Figure 9). Collectively,
the interactions between the ‘message’ moiety (epoxymorphinan moiety) of NFP and the
conserved residues seemed to have no significant influence on the selectivity of NFP to the
MOR, KOR, and DOR.
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Figure 9. The docking studies results of NFP in the MOR (a, PDB ID: 4DKL), KOR (b, PDB
ID: 4DJH), and DOR (c, PDB ID: 4EJ4), respectively, with the highest CHEM-PLP scores.
NFP atoms: carbon (magenta); the key residue atoms in MOR: carbon (green); the key residue
atoms in KOR: carbon (orange); the key residue atoms in DOR: carbon (light blue); oxygen
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(red); nitrogen (blue). The red dashed lines represent the electrostatic interactions. The yellow
dashed lines represent the hydrogen bonds.
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The fluorine atom on the pyridyl ring (‘address’ moiety) of NFP is a strong electron
withdrawing group which would weaken the capability of the nitrogen atom on the pyridyl ring
of NFP to keep a proton compared to the case of NAP and NMP.58 In consequence, the nitrogen
atom on the pyridyl ring of NFP might form electrostatic interactions with the conserved
residue E2295.35 in the MOR, D2235.35 in the KOR, and D2105.35 in the DOR. In addition, the
pyridyl ring of NFP could also form hydrophobic interactions with the conserved hydrophobic
residue LECL2 and FECL2 (Table S1). Thus, the ‘address’ moiety (pyridyl ring) of NFP seemed
to bind to the same domain (termed ‘address’ domain) in the MOR, KOR, and DOR. However,
several non-conserved residues located at this ‘address’ domain (T218ECL2, T2255.31, and
L2325.38 in the MOR, S211ECL2, Y2195.31, and M2265.38 in the KOR and M199ECL2, S2065.31
,
and T2135.38 in the DOR, Figure 9) could also form direct interactions with the ‘address’
moiety (pyridyl ring) of NFP.
As the distances shown in Figure 9, the electrostatic interaction between the nitrogen atom
on the pyridyl ring of NFP and E2295.35 in the MOR was much stronger than those in the
NFP/KOR and NFP/DOR complexes, which may help to explain the highest affinity of NFP
to the MOR. For the NFP/KOR complex, the non-conserved Y2195.31 and S211ECL2 could also
form hydrogen bond with the nitrogen atom on the pyridyl ring of NFP, which may facilitate
the binding of NFP to the KOR. However, for the NFP/DOR complex, no additional interaction
being observed between the ‘address’ moiety of NFP and the residues located in the ‘address’
domain of DOR except the possibly weakest electrostatic one from D2105.35 (Table S1).
Overall, the different interactions between the ‘address’ moiety of NFP and the ‘address’
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domain of the MOR, KOR, and DOR helped explain the binding affinity profile observed for
NFP in the three receptors.
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Conclusions
Based on previous studies from our group, the third generation 6β-N-4′-pyridyl substituted
naltrexamine derivatives were designed, synthesized and evaluated in both in vitro and in vivo
assays. The in vitro competition assays showed that the third generation compounds bearing
various substitutions at the 3’ position of pyridyl ring carried a different selectivity profile than
NAP. In that, the third generation compounds were typically MOR/KOR dual-selective while
NAP was highly MOR selective. Most of the new compounds retained their binding affinity to
the MOR with subnanomolar level and one-digit nanomolar binding affinity to the KOR.
Meanwhile, most of the compounds had low to moderate efficacies at the MOR with one-digit
nanomolar potencies. Among these derivatives, NFP and NYP significantly antagonized the
antinociception produced by morphine in a dose dependent fashion. The in vivo studies showed
that NFP produced significantly lesser withdrawal symptoms than naloxone at similar doses.
These findings suggest that NFP may serve as a lead compound to develop novel MOR/KOR
dual selective ligand for treating opioid addiction and abuse.
Experimental
Chemistry
General methods
Reagents were purchased from either Sigma-Aldrich or Alfa Aesar. TLC analyses were
carried out on the Analtech Uniplate F254 plates. Chromatographic purification was conducted
1
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on silica gel column (230-400 mesh, Merck). H (400 MHz) and C (100 MHz) nuclear
magnetic resonance (NMR) spectra were recorded at ambient temperature with
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tetramethylsilane as the internal standard on a Varian Mercury 400 MHz NMR spectrometer.
Melting points were recorded with OptiMelt Automated Melting Point System (Fisher
Scientific). IR spectra were obtained with Nicolet iS10 instrument (Thermo Scientific).
Applied Bio Systems 3200 Q trap with a turbo V source for TurbolonSpray was used for MS
analysis. HPLC analysis was performed by a Varian ProStar 210 system on Agilent Microsorb-
MV 100-5 C18 (250 mm × 4.6 mm) at 210 nm with water/acetonitrile (25/75, 30/70 or 35/65,
0.01% TFA in water) at 0.8 mL/min over 30 min. The purity of the third generation NAP
derivatives were determined by above analytical methods and their purity were identified as
≥95%.
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General procedure
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride
(2.5
eq),
hydrobenzotriazole (2.5 eq), 4 Å molecular sieves, and trimethylamine (5 eq) were added to a
solution of the carboxylic acid (2.5 eq) in DMF on an ice-water bath under N2 protection. After
30 min, a solution of 6β-naltrexamine (1 eq) in DMF was added dropwise. The mixture was
kept stirring overnight at room temperature and filtered the next day. The filtrate was then
concentrated under reduced pressure to remove the solvent. The residue was dissolved in
MeOH and potassium carbonate (2 eq) was added to the mixture. The reaction mixture was
stirred overnight at room temperature. Next day, the mixture was concentrated and the residue
was purified with silica gel column using methanol/dichloromethane (1% NH3H2O) as the
solvent system to obtain the target compounds.
17-Cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[(3-fluoro-4-pyridyl)
acetamido]morphinan (compound 1, NFP)
Compound 1 was synthesized as shown in the general procedure with a yield of 48%.
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1H NMR (400 MHz, CDCl3) δ 8.94 (s, 1 H), 8.74 (d, J = 8.08 Hz, 1 H), 8.58 (m, 1 H), 8.43 (m,
1 H), 7.46 (t, J = 5.6 Hz, 1H), 6.51 (d, J=8.1 Hz, 1H), 6.45 (d, J=8.1 Hz, 1H), 4.77 (s, 1 H),
4.45 (d, J=8.1 Hz, 1 H), 3.52 (m, 1H), 2.90 (m, 2 H), 2.47 (m, 2 H), 2.21 (m, 2 H), 2.02 (m, 1
H), 1.87 (m, 1 H), 1.73 (m, 1 H), 1.48 (m, 1 H), 1.37 (m, 1 H), 1.21 (m, 1 H), 1.15 (m, 1 H),
0.73 (m, 1 H), 0.35 (m, 2 H), 0.01 (m, 2 H).
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13C NMR (100 MHz, DMSO-d6) δ 161.54, 156.46, 153.90, 146.23, 146.18, 142.06, 140.46,
139.10, 138.86, 131.24, 131.05, 130.93, 123.52, 123.23, 118.47, 117.12, 90.39, 69.52, 61.75,
58.38, 51.90, 47.06, 43.66, 30.32, 29.97, 24.33, 22.19, 9.22, 3.65, 3.52.
19F NMR (75 MHz, DMSO-d6) δ -192.29.
IR (diamond, cm−1) Vmax 2942.19, 2829.33, 1655.64, 1529.02, 1487.78, 1454.25, 1416.41,
1322.93, 1238.29, 1204.29, 1186.88, 1152.58, 1131.78, 1100.14, 1034.82, 1017.27, 985.87,
942.11, 919.01, 898.28, 882.36, 799.14, 785.35, 763.19, 746.38, 667.00.
Mass Spectrum C26H28FN3O4 m/z calc. 465.2064 obs. 466.2159 [M + H]+. Mp 277.6-280.6 oC.
17-Cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[(3-cyano-4-pyridyl)
acetamido]morphinan (compound 2, NYP)
Compound 2 was synthesized as shown in the general procedure with a yield of 48%.
1H NMR (400 MHz, DMSO-d6) δ 9.15-9.13 (m, 2 H), 8.94 (s, 1 H), 7.92 (d, J = 3.6 Hz, 1 H),
7.44 (s, 1 H), 7.31 (s, 1 H), 7.18 (s, 1 H), 6.77 (d, J = 8.08 Hz, 1 H), 6.69 (d, J = 8.08 Hz, 1 H),
5.06 (d, J = 8.24 Hz, 1 H), 3.38-3.33 (m, 3 H), 3.11-3.06 (m, 3 H), 2.9 (m, 1 H), 1.93-1.90 (m,
1 H), 1.51-1.43 (m, 3 H), 1.10-1.09 (m, 1 H), 0.67 (m, 1 H), 0.60 (m, 1 H), 0.51 (m, 1 H), 0.42
(m, 1 H).
13C NMR (100 MHz, DMSO-d6) δ 167.45, 167.09, 156.63, 144.66, 142.37, 142.15, 139.45,
129.84, 125.95, 121.26, 120.30, 118.68, 117.50, 87.36, 70.14, 62.25, 57.32, 52.30, 47.03, 46.66,
30.59, 27.74, 23.65, 21.51, 6.32, 5.70, 3.24.
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IR (diamond, cm−1) Vmax 3111.05, 2087.54, 1721.23, 1626.30, 1501.34, 1467.64, 1371.61,
1322.77, 1079.34, 1031.03, 858.34, 731.40.
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Mass Spectrum C27H28N4O4 m/z calc. 472.2111 obs. 474.2049 [M + 2H]+. Mp 222.8-224.5 oC.
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17-Cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[(3-nitro-4-pyridyl)
acetamido]morphinan (compound 3)
Compound 3 was synthesized as shown in the general procedure with a yield of 52%.
1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1 H), 8.94 (m, 1 H), 8.87 (m, 1 H), 7.53 (m, 1 H),
6.50 (d, J=8.1 Hz, 1H), 6.44(d, J=8.1 Hz, 1H), 4.35(d, J=7.8 Hz, 1H), 3.42 (m, 1H), 2.90 (m, 2
H), 2.49 (m, 2 H), 2.22 (m, 2 H), 2.01 (m, 1 H), 1.88 (m, 1 H), 1.70 (m, 1 H), 1.57 (m, 1 H),
1.39 (m,1 H), 1.19 (m, 2 H), 0.73 (m, 1 H), 0.35 (m, 2 H), 0.01 (m, 2 H).
13C NMR (100 MHz, DMSO-d6) δ 163.07, 154.58, 145.17, 142.56, 142.01, 140.49, 139.18,
131.16, 123.46, 122.82, 118.53, 117.14, 90.25, 69.52, 61.69, 58.32, 54.85, 52.11, 47.04, 43.66,
30.28, 29.84, 23.57, 22.18, 9.11, 3.68, 3.48.
IR (diamond, cm−1) Vmax 3072.08, 2952.94, 2820.03, 1681.02, 1639.45, 1615.13, 1567.61,
1550.22, 1531.35, 1451.63, 1393.77, 1357.98, 1348.34, 1329.88, 1277.14, 1262.58, 1253.30,
1241.33, 1227.99, 1189.63, 1147.14, 1121.29, 1035.17, 984.65, 945.33, 931.99, 922.82,
915.19, 896.65, 882.54, 858,32, 828.95, 806.17, 763.88, 740.60, 732.83, 721.00, 672.69.
Mass Spectrum C26H28N4O4 m/z calc. 492.2009 obs. 493.2087 [M + H]+. Mp 266.46-269 oC.
17-Cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[(3-triflurometyl-4-pyridyl)
acetamido]morphinan (compound 4)
Compound 4 was synthesized as shown in the general procedure with a yield of 51%.
1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1 H), 9.04(s, 1 H), 9.02 (m, 1 H), 8.98 (m, 1 H), 7.56
(m, 1 H), 6.75 (d, J=8.1 Hz, 1H), 6.66 (d, J=8.1 Hz, 1H), 4.62(d, J=8.1 Hz, 1H), 3.38 (m, 1H),
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3.04 (m, 2 H), 2.83 (m, 2 H), 2.43 (m, 2 H), 2.16 (m, 1 H), 1.82 (m, 2H), 1.84 (m, 1 H), 1.62
(m, 1 H), 1.41 (m, 1 H), 0.86 (m, 1 H), 0.76 (m, 2 H), 0.53 (m, 2 H).
13C NMR (100 MHz, DMSO-d6) δ 164.68, 154.10, 147.20, 143.25, 142.09, 141.39,129.55,
123.01, 122.22, 121.83, 121.17, 119.33, 117.98, 89.34, 69.64, 61.60, 60.67, 51.54, 45.67, 32.18,
29.30, 27.69, 22.97, 21.96, 13.95, 5.71, 5.05.
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19F NMR (75 MHz, DMSO-d6) δ -57.80.
IR(diamond, cm−1) Vmax: 3216.30, 2935.48, 2831.91, 1676.72, 1607.94, 1557.26, 1502.39,
1471.37, 1452.47, 1422.76, 1377.05, 1328.16, 1281.32, 1194.14, 1132.27, 1081.31, 1054.16,
1038.57, 1027.12, 999.69, 982.16, 927.80, 890.56, 858.47, 821.56, 785.26, 720.13, 691.36,
675.16.
Mass Spectrum C27H28F3N3O4 m/z calc. 515.2032 obs. 516.2089 [M + H]+. Mp 224.9-227.5
oC.
17-Cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[(3-carboxyl-4-pyridyl)
acetamido]morphinan (compound 5)
Compound 5 was synthesized as shown in the general procedure with a yield of 23%.
1H NMR (400 MHz, DMSO-d6) δ 9.14 (d, J = 0.84 Hz, 1 H), 9.12 (d, J = 4.76 Hz, 1 H), 8.99
(s, 1 H), 7.91 (d, J = 1.08 Hz, 1 H), 7.90 (d, J = 0.92 Hz, 1 H), 6.62-6.52 (m, 2 H), 4.99 (d, J =
8.08 Hz, 1 H),4.92 (s, 1 H), 3.91-3.82 (m, 1 H), 3.16 (d, J = 5.24 Hz, 1 H), 3.03 (t, 2 H), 2.97
(s, 1 H), 2.63-2.60 (m, 1 H), 2.57-2.53 (m, 1 H), 2.40-2.30 (m, 2 H), 2.24-2.18 (m, 1 H), 2.02-
1.95 (m, 1 H), 1.59-1.55 (m, 1 H), 1.18-1.44 (m, 1 H), 1.41-1.34 (m, 1 H), 1.26-1.23 (m, 1 H),
0.87-0.82 (m, 1 H), 0.48-0.46 (m, 2 H), 0.13-0.11 (m, 2 H).
13C NMR (100 MHz, DMSO-d6) δ 167.65, 167.25, 156.53, 144.44, 141.89, 141.13, 139.43,
125.90, 119.61, 117.53, 88.10, 70.04, 62.06, 58.80, 52.80, 49.03, 48.08, 44.14, 31.23, 22.66,
4.26, 4.00.
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IR (diamond, cm−1) Vmax 3345.73, 3039.98, 2478.91, 2079.72, 1782.60, 1725.96, 1651.70,
1630.27, 1614.51, 1556.21, 1505.72, 1455.55, 1398.02, 1361.26, 1263.68, 728.87, 708.62
Mass Spectrum C27H29N3O6 m/z calc. 491.2056 obs. 474.2033 [M-CH3]+. Mp 271.2-273.7 oC.
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17-Cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[(3-methyl formate-4-pyridyl)
acetamido]morphinan (compound 6)
Compound 6 was synthesized as shown in the general procedure with a yield of 32%.
1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1 H), 9.12 (s, 1 H), 9.08 (s, 1 H), 9.00 (d, J = 3.68
Hz, 1 H),7.91 (d, J = 3.68 Hz, 1 H), 7.76 (d, J = 3.52 Hz, 1 H), 7.31 (s, 1 H), 7.26 (d, d, J =
8.24 Hz, 1 H), 7.00 (d, J = 8.24 Hz, 1 H), 5.16 (7.26 (d, J = 8.24 Hz, 1 H)), 3.83 (s, 3H), 3.55
(s, 1 H), 3.50 (s, 1H), 3.36-3.35 (m, 1 H), 3.27-3.24 (s, 1 H), 3.23 (s, 2 H), 3.16-3.12 (m, 1 H),
2.95 (m, 1 H), 2.66-2.63 (m, 1 H), 1.99-1.95 (m, 1 H), 1.57-1.44 (m, 3 H), 1.23-1.19 (m, 1 H),
1.13 (m, 1 H), 0.69 (m, 1 H), 0.62 (m, 1 H), 0.54 (m, 1 H), 0.45 (m, 1 H).
13C NMR (100 MHz, DMSO-d6) δ 167.04, 165.51, 163.83, 156.57, 154.31, 150.66, 146.23,
144.61, 139.14, 134.01, 131.22, 130.14, 123.75, 122.67, 120.93, 117.47, 89.86, 70.03, 61.97,
57.38, 53.57, 52.41, 49.14, 46.62, 30.53, 27.58, 24.12, 21.11, 6.29, 5.70, 3.29.
IR (diamond, cm−1) Vmax 3554.62, 2953.39, 2088.05, 1716.47, 1633.27, 1557.57, 1489.97,
1446.28, 1367.76, 1290.11, 1263.40, 1234.61, 1193.08, 1152.84, 1128.11, 1101.15, 1076.56,
1032.71, 995.14, 960.71, 938.56, 914.77, 892.11, 855.03, 834.94, 797.66, 780.76, 732.58,
685.21, 662.07
Mass Spectrum m/z calc. C28H31N3O6 505.2213 obs. 506.2227 [M + H]+. Mp 210.5-212.3 oC.
17-Cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[(3-ethyl-4-pyridyl)
acetamido]morphinan (compound 7)
Compound 7 was synthesized as shown in the general procedure with a yield of 61%.
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