Synthesis and pharmacological evaluation of novel selective MOR agonist 6β-pyridinyl amidomorphines exhibiting long-lasting antinociception

Article abstract of DOI:10.1039/c6md00450d

It was previously reported that 6β-aminomorphinan derivatives show high affinity for opiate receptors. Novel 6β-heteroarylamidomorphinanes were designed based on the MOR selective antagonist NAP. The 6β-aminomorphinanes were prepared by stereoselective Mitsunobu reaction and subsequently acylated with nicotinic acid and isonicotinic acid chloride hydrochlorides. The receptor binding and efficacy were determined in vitro and the analgesic activity was studied in vivo. The in vitro studies revealed moderate selectivity for the MOR. At least two compounds in this series exhibited a long-lasting analgesic response when administered subcutaneously and intracerebroventricularly. When the substances were given intracerebroventricularly to mice, they showed analgesic potency comparable to morphine.

Full text of DOI:10.1039/c6md00450d

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Synthesis and pharmacological evaluation of novel selective MOR agonist 6βꢀpyridinyl
amidomorphines exhibiting longꢀlasting antinociception†
Ákos Uraia, András Váradic, Levente Szőcsa, Balázs Komjátib, Valerie Le Rouzicc, Amanda
Hunkelec, Gavril W. Pasternakc, Susruta Majumdarc, Sándor Hosztafia
a Department of Pharmaceutical Chemistry, Semmelweis University, Hőgyes E. u. 9.,
Budapest Hꢀ1092, Hungary
b Department of Organic Chemistry and Technology, Budapest University of Technology and
Economics, Szent Gellért tér 4, Budapest 1111, Hungary
c Molecular Pharmacology and Chemistry Program and Department of Neurology, Memorial
Sloan Kettering Cancer Center, 1275 York Ave., New York, NY 10065, USA
†The authors declare no competing interests.
Abstract
It was previously reported that 6βꢀaminomorphinan derivatives show high affinity for opiate
receptors. Novel 6βꢀheteroarylamidomorphinanes were designed based on the MOR selective
antagonist NAP. The 6βꢀaminomorphinanes were prepared with stereoselective Mitsunobu
reaction and subsequently acylated with nicotinic acid and isonicotinic acid chloride
hydrochlorides. The receptor binding and efficacy were determined in vitro and the analgesic
activity was studied in vivo. The in vitro studies revealed moderate selectivity for MOR. At
least two compounds in this series exhibited longꢀacting analgesic response when
administered subcutaneously and intracerebroventricularly. When the substances were given
intracerebroventricularly to mice, they showed analgesic potency comparable to morphine.
The perception of pain is a consequence of several complex neurochemical processes both in
the peripheral and central nervous system. For the treatment of pain there are several classes
of drugs available, for serious or chronic malignant pains, opioids are the first choice.1
Although opioids are very effective analgesics, their use is associated with deleterious side
effects such as dependence, constipation, respiratory depression, addiction liability and
abuse.2ꢀ4

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Opioids act on the opioid receptors µ, κ, and δ, which belong to the family of Gꢀprotein
coupled receptors (GPCRs). Activation of the µꢀopioid receptor (MOR) is primarily
responsible for the desired analgesic effect. Selective MOR agonists can be useful
medications for the treatment of pain and can also serve as probes to better understand the
underlying biochemical mechanisms of MOR activation. In the 6βꢀaminomorphinanes, the
hydroxylic group in the Cꢀ6 position is substituted by an amino group while the configuration
of stereogenic Cꢀ6 carbon is inverted. Caruso et al. synthesized the first 6ꢀaminomorphinane
derivative, chloroxymorphamine, an irreversible MOR agonist.5 It was previously reported
that derivatives of 6ꢀβꢀaminomorphinans show selectivity toward opiate receptors, for
example, a selective KOR agonist (nalfurafine)6, a selective MOR antagonist (clocinnamox)7,
and a dual KOR/DOR agonist (MP1104).8 Several studies have been reported about 6βꢀ
aminomorphine derivatives developed in order to achieve higher selectivity against MOR to
mitigate the side effect of opiate analgetics.9,10
McDougall et al prepared a series of 6βꢀmorphine arylamides by the reaction of 6βꢀ
aminomorphine, with aryl and heteroaryl acid chlorides.11 These derivatives were found to
bind to MOR with significant potency. Functional assays showed that the compounds were
full MOR agonists. Li et al carried out molecular modeling studies on the MOR and a new
lipophilic binding domain was identified.12 Based on the modeling, a series of 6βꢀheteroaryl
naltrexamine analogues were designed and synthesized with 6β side chains including
nicotinic, isonicotinic, and isoquinoline carboxylic acid amides. One of the most potent and
selective to MOR of the series was the nicotinic acid derivative, NAP (1), which was chosen
as lead molecule for further studies.12 The compound was found to be a potent peripheral
MOR selective antagonist based on the in vitro and in vivo pharmacological studies.
We have previously reported the synthesis of a series of novel 6βꢀcinnamoylꢀmorphinamines
carrying various cinnamoyl side chains.13 In vitro and in vivo characterization of the
synthesized compounds revealed high affinity for MOR. Analgesic activity of 6βꢀ
cinnamoylmorphinamine (2) was found to be comparable to morphine, but with significantly
reduced respiratory depression, a major sideꢀeffect of commonly used opioids.

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HO
HO
O
O
N
N
O
O
OH
N
N
H
H
N
1
2
Figure 1. Structure of 6βꢀamidomorphinanes
Here we report the design, synthesis, in vitro and in vivo evaluation of a series selective MOR
agonist, 6βꢀheteroarylamidomorphinanes, containing nicotinamide or isonicotinamide
moieties at 6β position.
The 6βꢀamino morphinans (6a,6c,12) were synthesized using the Mitsunobu reaction as
described previously.13,14 Morphine (3c) and 14ꢀhydroxyꢀdihydromorphine (3a) were
selectively protected in the Cꢀ3 position with acetic anhydride.15 The two acetyl protected
derivatives (4a,4c) and codeine (10) were reacted in the Mitsunobu reaction with phthalimide
and diisopropyl azodicarboxylate (DIAD) in the presence of triphenylphosphine to yield 6βꢀ
phthalimido morphinans (5a,5c,11). The desired 6βꢀamino morphinan derivatives (6a,6c,12)
were obtained upon treatment with hydrazine hydrate. The synthesis of 6βꢀ
aminodihydromorphine (6e) was accomplished from 6βꢀazidodihydromorphine (7). Bognar
and Makleit reported the reduction of 6βꢀazidodihydromorphine (7) by lithium aluminum
hydride in diethyl ether.16 We elaborated an efficient new method for this reduction, utilizing
hydrazine and Raney nickel in ethanol.17 With the new method, higher yields were achieved,
workꢀup was easier and the final product did not require further purification.

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AcO
HO
HO
AcO
O
a
b
c
O
O
O
N
O
R1
Me
N
N
N
R1
R1
R1
Me
Me
Me
N
H2N
HO
HO
O
3a,3c
4a,4c
6a,6c
5a,5c
R2
O
HO
HO
HO
O
O
d
e
f
O
O
O
O
6a-e
N
N
N
N
O
Me
Me
R1
R1
Me
Me
R2
N
H
R2
N
N3
H2N
H
6e
MeO
7
9a-f
8a-f
MeO
MeO
MeO
O
e
b
c
O
O
O
N
O
N
Me
O
N
N
Me
Me
Me
N
R2
N
H2N
HO
H
O
12
9g
11
10
Figure 2: Synthesis of 6βꢀamido morphinans 9aꢀg. a) acetic anhydride, NaHCO3 water r.t. 2 h;
b) benzene/ Ph3P, phthalimide, DIAD, r.t. 1 h; c) ethanol/ hydrazine monohydrate heating 3 h;
d) hydrazine monohydrate/Raney Nickel, ethanol, r.t. 2 h; e) dichloromethane / acyl chloride,
triethylamine; f) K2CO3/methanol, heating 4 h
The 6βꢀaminomorphinans (6aꢀe) were reacted with nicotinic acid chloride and isonicotinic
acid chloride in dry dichloromethane in the presence of triethylamine to yield the appropriate
6βꢀamidomorphinans. The acyl chlorides were synthesized as described in the literature.18 In
case of molecules containing a free phenolic group (5aꢀe), the ester byꢀproducts were
hydrolyzed with potassium carbonate in methanol.
R3O
O
N
O
R1
Me
8
R2
N
7
H
9a-g
Figure 4: General structure of the studied compounds.
Table 1. Structure of the studied compounds

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C7ꢀC8
bond
Compound
R1
R2
R3
single
single
double
double
single
single
double
OH
OH
H
isonicotinic
nicotinic
H
H
9a
9b
9c
9d
9e
9f
isonicotinic
nicotinic
H
H
H
H
isonicotinic
nicotinic
H
H
H
H
isonicotinic
Me
9g
The seven compounds used in in vitro and in vivo studies are summarized in Table 1. The
codeine derivative 9g showed significantly lower binding affinity compared with the
morphine derivatives, suggesting that the free phenolic hydroxyl group is necessary for
affinity at the receptor.
Compounds 9aꢀf show similar nanomolar affinity on MOR and much lower affinity on DOR
and KOR. In all cases, the difference in MOR affinities between the compounds possessing
nicotinic acid and isonicotinic acid moieties was minimal, however, there are greater
differences in affinities on DOR and KOR receptors. The two compounds 9a and 9b having
the 14ꢀhydroxy dihydromorphine skeleton showed the lowest affinity to MOR. The
dihydromorphine derivatives 9e and 9f had the highest MOR/DOR selectivity among the
studied molecules. The high MOR selectivity reported for NAP, however, was not seen in this
group. 9c and 9d had the highest affinity to MOR. 9a only differs from NAP (1) in the
substituent of the tertiary amine. The cyclopropylmethyl substituent of NAP (1) is responsible
for the antagonistic effect, on the other hand, the methyl substituent in our compounds results
in agonistic effect. These results revealed that the saturation of the double bond and the
presence of the OH group at 14 position are not beneficial in terms of binding.
Table 2. Binding affinities of compounds (9aꢀf).a
MOR
KOR
DOR
M/K
M/D
Compound
Ki (nM)
9a
7.02 ± 0.77
6.91 ± 0.88
2.00 ± 0.17
>100
>100
173.4 ± 43.6
103.7 ± 17.0
65 ± 19.0
>14.2
>14.5
39.0
24.7
15.0
32.5
9b
9c
77.9 ± 31

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9d
9e
9f
1.25 ± 0.23
3.73 ± 0.14
3.29 ± 0.36
40.59 ± 3.65
18.97 ± 5.3
>100
34.4 ± 5.00
15.2
>26.8
36.2
27.5
53.2
39.6
10.4
198.4 ± 69
130.3 ± 37
423.9 ± 88
119 ± 50.4
>100
9g
>2.5
1b
277.51 ±
7.97
0.37 ± 0.07
60.72 ± 5.58
747
163
2c
0.10 ± 0.02
4.60 ± 1.81
2.90 ± 0.66 10.26 ± 6.76
29.0
102.6
Morphine
[
a 125I]BNtxA (0.1 nM) competition binding assays were performed in membranes prepared from CHO
cells expressing mouse MOR, DOR, or KOR as previously described. Protein concentrations were
between 10ꢀ20 ꢁg/mL and incubation times were 90 minutes.19 Ki was determined by nonlinear
regression analysis (GraphPad Prism). Assays were performed at least 3 times and means ± SEM are
reported. b Values are taken from ref 12. c Values are taken from ref 13.
The [35S]GTPγSꢀbinding assay was used to characterize the functional activity of
compound 9aꢀf in vitro on opioid transfected cell lines. In the [35S]GTPγS assay, all
compounds were full agonists. 9c and 9d sharing the same morphine skeleton had the highest
potencies. The EC50 value on KOR was only determined for 9d, since no other analogs had
considerable affinity at this receptor. It is well known in the literature that the saturation of the
double bond and the presence of the 14ꢀOH group increase the affinity of morphine
derivatives. In our case, saturation of the double bond and the presence of 14ꢀOH group were
not advantageous as these modifications decreased both the affinity and activity.20,21
Table 3. In vitro efficacy of compounds in the [35S]GTPγS assay.
MOR
KOR
EC50 (nM)
Compound
EC50 (nM)
%Emax
% Emax
ndb
26.42 ± 3.17
25.7 ± 1.68
4.62 ± 0.28
2.83 ± 0.41
8.16 ± 2.64
7.98 ± 2.35
1.38 ± 0.8
19±7.0
115.07 ± 7.3
ndb
ndb
9a
9b
120.89 ± 2.55
114.36 ± 7.06
113.63 ± 4.54
114.85 ± 1.26
117.16 ± 1.98
95.3 ± 0.8
ndb
ndb
ndb
ndb
9c
9d
58.83 ± 7.49
ndb
87.88 ± 4.9
ndb
ndb
9e
9f
ndb
2
36.5 ± 19.2
ndb
106.3 ± 8.5
ndb
DAMGO
U50,488H
ndb
ndb
17±6.1
ndb

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aFunctional properties were studied using agonistꢀinduced stimulation of [35S]GTPγS binding assay.
Potency is represented as EC50 (nM) and efficacy as percent maximal stimulation (%Emax) relative to
standard agonist DAMGO (MOR), or U50,488H (KOR) at 100 nM.. Protein concentration was 50
ꢁg/mL and incubation times were 90 minutes. All values are expressed as the mean ± SEM of three
separate assays performed in triplicate. bNot determined.
The antinociceptive activity of the four compounds (9cꢀf) with the highest affinity and
agonism at MOR was determined supraspinally in mice (Figure 5) in a tail flick
antinociception assay. While 9d was as potent as morphine (ED50 = 0.53 µg (0.27, 1.0)) given
icv, the other three compounds were 3ꢀ4x less potent antinociceptives. Upon subcutaneous
administration, the compounds did exhibit an antinociceptive response at 30 mg/kg (solubility
limitations didn’t permit testing at higher doses). At this high dose, 9c and 9d exhibited about
30% MPE, while 9e and 9f exhibited ~80%MPE (Figure 6A). The antinociceptive ED50 of
morphine was 2.5 mg/kg (1.8, 3.4) in the same assay. We evaluated 9e and 9f further. Both
compounds showed exceptionally long lasting antinociception. In contrast to morphine’s 3h
long antinociceptive response, the effect of 9e and 9f lasted more than 24 h subcutaneously
(Figure 6B). Similarly, upon icv dosing, unusually long antinociception was observed lasting
more than 24 h. This shows good correlation with the sc time course. Antinociception of both
9e and 9f was antagonized by βꢀFNA (Figure 6D), the mu selective antagonist, confirming
that the antinociception is mediated by mu opioid receptors.

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Figure 5. Two independent determinations of the cumulative dose–response curves were performed
on groups of mice (n = 5) for antinociception in the tail flick assay with the compounds given
intracerebroventricularly. Animals were tested 15 min later at peak effect to generate the
antinociceptive doseꢀresponse curve. Each point represents mean ± SEM for 10 mice. ED50 values (and
95% CI) were: 9c: 2.0 ꢁg (1.1, 3.6); 9d: 0.51 ꢁg (0.3, 0.9); 9e: 0.7 ꢁg (0.4, 1.2); 9f: 1.8 ꢁg (1.0, 3.2);
morphine: 0.53 ꢁg (0.27, 1.0).

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D) Reversal of Antinociception
*
*
100
80
60
40
20
0
Figure 6. A) Antinociceptive effect of compounds 9cꢀf. CD1 mice (n = 10) were given the compounds
sc at 30 mg/kg and the antinociceptive effects were assessed 30 min postꢀinjection using the radiant
heat tailꢀflick method. Results (Mean ± SEM): 9c: 38% ± 13%; 9d: 35% ± 10%; 9e: 78% ± 11%; 9f:
83% ± 11%. B) Time course of the antinociceptive effect of morphine (4.5 mg/kg), compound 9e (30
mg/kg), and 9f (30 mg/kg) following sc administration to CD1 mice. Two independent determinations
were performed on groups of mice (n = 10) for antinociception in the tail flick assay. C) Time course
of the antinociceptive effect of morphine (1 ug), compound 9e (7.5 ug), and 9f (7.5 ug) following icv
administration to CD1 mice. Two independent determinations were performed on groups of mice (n =
10) for antinociception in the tail flick assay. D) Reversal of antinociception by selective antagonists.
Groups of CD1 mice (n = 10) received 9e (30 mg/kg, sc), or 9f (30 mg/kg) with or without βꢀ
funaltrexamine (βꢀFNA; 40 mg/kg sc). βꢀFNA was administered 24 hours before agonist testing. All
antinociception testing was performed 30 min after the administration of 9e and 9f. Similar results
were observed in two independent replications. Antinociception was antagonized by βꢀFNA (twoꢀway
ANOVA followed by Bonferroni post hoc comparisons test, p<0.05). The means in each
determination were determined as percentage maximal possible effect (%MPE) [(observed latency −
baseline latency)/(maximal latency − baseline latency)] x 100. Baseline latencies were between 2ꢀ3 s
and maximal latency was 10 s to avoid tissue damage.

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Thus, these drugs do exhibit systemic antinociception, albeit at a higher dose. Their longꢀ
lasting antinociception makes these compounds exciting for further evaluation beyond
thermal pain assays such as inflammatory and neuropathic pain models. The next set of
analogs in this series will aim to optimize the potency of the compounds while maintaining
longꢀlasting antinociception.
Several studies have been reported about NAP (1) as a selective peripheral MOR antagonist
based on its in vitro/in vivo pharmacological assays and pharmacokinetic studies.12,22,23. To
our knowledge, antinociceptive Cꢀ6 arylamido morphine with a 3’ꢀpyridyl or 4’ꢀpyridyl as the
aryl moiety have not been reported before. The pharmacology on Cꢀ6 amido morphinans has
indeed been extensively studied but the SAR of novel compounds cannot be predicted based
on that of known compounds. Subtle changes in the structure can have huge impact on the in
vivo pharmacology. It is well established that compounds with a Nꢀmethyl substituent such as
morphine and oxymorphone are agonists/antinociceptives. Nꢀallyl and Nꢀcyclopropylmethyl
(NꢀCPM) leads to the antagonists naloxone and naltrexone, respectively. Some Cꢀ6 arylamido
morphinans do follow this trend where the Nꢀmethyl leads to agonists/antinociceptives.11,13
Compounds with an NꢀCPM like NAP12 from the Zhang group are antagonists and NꢀCPM
analogs24 were partial agonists. IBNtxA25 and MP 11048 are agonists/antinociceptives even
with an NꢀCPM moiety. The exceptionally long lasting antinociception seen with 9e and 9f
described in this paper has never reported and is unexpected based on the published work on
Cꢀ6 amido morphinans, suggesting the possibility of novel therapeutics with this template
after further optimization of potency.
In summary, a series of 6ꢀdesoxymorphineꢀ6βꢀacylamides containing nicotinic or isonicotinic
acid moieties were synthesized. The acyl side chains were chosen based on selective MOR
antagonists reported in the literature. We found that the synthesized compounds are MOR full
agonists with low affinity for DOR and KOR. The compounds exhibited potent
antinociception supraspinally and showed exceptionally long lasting mu opioid receptorꢀ
mediated antinociception when given subcutaneously.
Mice. Male CD1 mice (20ꢀ32 g) were obtained from Charles River Laboratories. All mice
were maintained on a 12 hour light/dark cycle with Purina rodent chow and water available ad
libitum, and housed in groups of five until testing. All animal studies were preapproved by the
Institutional Animal Care and Use Committees of the Memorial Sloan Kettering Cancer
Center in accordance with the 2002 National Institutes of Health Guide for the Care and Use
of Laboratory Animals.

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Acknowledgments
This work was supported by a research grant from the National Institute on Drug
Abuse (DA034106) to SM. This research was funded, in part, through the NIH/NCI Cancer
Center Support Grant P30 CA008748.
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112x44mm (300 x 300 DPI)

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DOI: 10.1039/C6MD00450D
Materials and methods
Drugs and Chemicals. Opiates were a gift from the Research Technology Branch of the National
Institute on Drug Abuse (Rockville, MD). IBNtxA and [125I]BNtxA were synthesized in our laboratory
as previously described.1,2 Na125I and [35S]GTPγS were purchased from PerkinꢀElmer.
Mice. Male CD1 mice (20ꢀ32 g) were obtained from Charles River Laboratories. All mice were
maintained on a 12 hour light/dark cycle with Purina rodent chow and water available ad libitum, and
housed in groups of five until testing. All animal studies were preapproved by the Institutional Animal
Care and Use Committees of the Memorial Sloan Kettering Cancer Center in accordance with the
2002 National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Radioligand Competition Binding Assays. [125I]BNtxA binding was carried out in the absence of
blockers in membranes prepared from Chinese Hamster Ovary (CHO) cells stably expressing murine
clones of MORꢀ1, DORꢀ1, and KORꢀ1, as previously described.3,4 Assays were performed at 25 °C for
90 min in 50 mM potassium phosphate buffer, pH 7.4, containing 5 mM magnesium sulfate. After the
incubation, the reaction was filtered through glassꢀfiber filters (Whatman Schleicher & Schuell,
Keene, NH) and washed three times with 3 mL of iceꢀcold 50 mM TrisꢀHCl, pH 7.4, on a
semiautomatic cell harvester. Nonspecific binding was defined by addition of levallorphan (8 ꢁM) to
matching samples and was subtracted from total binding to yield specific binding. Ki values were
calculated by nonlinear regression analysis (GraphPad Prism, San Diego, CA). Protein concentrations
were determined using the Lowry method with BSA as the standard.5
Functional assays. [35S]GTPγS binding was performed on membranes prepared from stably opioid
receptor transfected cells in the presence and absence of the indicated compound for 60 min at 30 °C
in the assay buffer (50 mM TrisꢀHCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, and 10 mM NaCl)
containing 0.05nM [35S]GTPγS; 2 ꢁg/ml each leupeptin, pepstatin, aprotinin, and bestatin; and 30µM
GDP, as previously described.6,4 After the incubation, the reaction was filtered through glassꢀfiber
filters (Whatman Schleicher & Schuell, Keene, NH) and washed three times with 3 mL of iceꢀcold
buffer (50 mM TrisꢀHCl, pH 7.4) on a semiautomatic cell harvester. Filters were transferred into vials
with 3 mL of Liquiscent (National Diagnostics, Atlanta, GA), and the radioactivity in vials was
determined by scintillation spectroscopy in a TriꢀCarb 2900TR counter (PerkinElmer Life and
Analytical Sciences). Basal binding was determined in the presence of GDP and the absence of drug.
Data was normalized to 100 nM DAMGO, DPDPE, and U50,488 for MORꢀ1, DORꢀ1, and KORꢀ1
binding, respectively. EC50 and %Emax values were calculated by nonlinear regression analysis
(GraphPad Prism, San Diego, CA).
Tail Flick Analgesia. Antinociception was determined using the radiant heat tail flick technique using
an Ugo Basile model 37360 instrument. The intensity was set to achieve a baseline between 2 and 3
seconds. Baseline latencies were determined before experimental treatments for all mice. Tail flick
analgesia was assessed quantally as a doubling or greater of the baseline latency, with a maximal 10
second latency to minimize damage to the tail. Data were also analyzed as percent maximal effect, and
similar results were observed. At each time point, MPE was calculated according to the formula: %
MPE = 100 × (test latency – control latency)/(maximal latency – control latency). Compounds were
injected subcutaneously (s.c.), and analgesia was assessed 30 min later. Intracerebroventricular dosing
(i.c.v.) was carried out as previously described.7 Briefly, the mice were anesthetized by isoflurane. A
small incision was made, and synthetic opiate analog (2 ul/mouse) was injected using a 10 uL
Hamilton syringe fitted to a 27ꢀgauge needle. Injections were made into the right lateral ventricle at
the following coordinates: 2 mm caudal to bregma, 2 mm lateral to sagittal suture, and 2 mm in
depth. Mice were tested for antinociception 15 minutes post injection.

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DOI: 10.1039/C6MD00450D
1.
2.
3.
4.
Majumdar, S.; Burgman, M.; Haselton, N.; Grinnell, S.; Ocampo, J.; Pasternak, A. R.;
Pasternak, G. W. Bioorganic & Medicinal Chemistry Letters 2011, 21, 4001.
Pickett, J. E.; Váradi, A.; Palmer, T. C.; Grinnell, S. G.; Schrock, J. M.; Pasternak, G. W.;
Karimov, R. R.; Majumdar, S. Bioorganic & Medicinal Chemistry Letters 2015, 25, 1761.
Pan, Y.ꢀX.; Xu, J.; Bolan, E.; Abbadie, C.; Chang, A.; Zuckerman, A.; Rossi, G.; Pasternak,
G. W. Molecular Pharmacology 1999, 56, 396.
Váradi, A.; Palmer, T. C.; Haselton, N.; Afonin, D.; Subrath, J. J.; Le Rouzic, V.; Hunkele, A.;
Pasternak, G. W.; Marrone, G. F.; Borics, A.; Majumdar, S. ACS Chemical Neuroscience
2015, 6, 1570.
5.
Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Journal of Biological Chemistry
1951, 193, 265.
6.
7.
Bolan, E. A.; Pan, Y.ꢀX.; Pasternak, G. W. Synapse 2004, 51, 11.
Haley, T. J.; McCormick, W. G. British Journal of Pharmacology and Chemotherapy 1957,
12, 12.
Analytical methods
All chemicals were purchased from SigmaꢀAldrich Chemicals (Darmstadt, Germany) and
1
Alfa Aesar (Ward Hill, MA) and were used without further purification. H and 13C NMR
1
spectra were recorded on a Varian VNMRS spectrometer (600 MHz for H, 150.9 MHz for
13C) equipped with a dual 5ꢀmm inverseꢀdetection gradient (IDPFG) probehead. Spectra were
recorded in CD3OD or CDCl3 at 25.0±0.1 °C and referenced to internal standard Me4Si. NMR
spectra were processed with VNMRJ 2.2C and MestReNova software (ver. 6.1.1.). The high
resolution accurate masses (HRMS) were determined with an Agilent 6230 timeꢀofꢀflight
mass spectrometer. Samples were introduced by Agilent 1260 Infinity LC system, the mass
spectrometer was operated in conjunction with a Jet Stream electrospray ion source in positive
ion mode. Reference masses of m/z 121.050873 and 922.009798 were used to calibrate the
mass axis during analysis. Mass spectra were processed using Agilent MassHunter B.02.00
software.
General synthesis of 6ꢀdezoxymorphineꢀ6βꢀamines (6aꢀb,12)
3ꢀOꢀacetyl morphine(4c) (or 3ꢀOꢀacetyl dihydromorphine(4a)) (1.15 g, 3.51 mmol) and
triphenylphosphine (1.84 g, 7.03 mmol, 2.0 eq.), phthalimide (1.3 g, 7.03 mmol, 2.0 equiv)
were dissolved in 25 mL benzene. Afterwards DIAD (1.2 ml, 6.09 mmol, 1.74 equiv) was
added dropwise. The yellowish solution was stirred for 1 hour at room temperature, then
concentrated in vacuo. To the residue a solution of 7 g tartaric acid in 35 ml water was added.
The solution washed with 4x20 ml diethyl ether, and the pH of aqueous phase was adjusted to
9 with concentrated NH3 and then extracted with 4 X 25 mL chloroform. The product was
dissolved in ethanol and was isolated as HCl salt.
Yield: 80ꢀ 87%
The HCl salt of 6βꢀphthalimido derivatives (5a,5c,11) (1.39g, 3.05 mmol) was dissolved in
ethanol 25 mL, and was added hydrazine monohydrate 0.6 mL, and was refluxed for 4 hours.
After full completion monitored by TLC 20% acetic acid (35 mL) was added and cooled on
ice bath. The white precipitate was filtered, and the solvent was evaporated under reduced
pressure. The residue was basified with concentrated NH3, and the product was extracted with

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DOI: 10.1039/C6MD00450D
chloroform 3 X 20 mL. The combined organic layers were washed with 10 mL brine, dried
over Na2SO4, filtered and the solvent was removed in vacuo.
Yield: 74ꢀ96%
Synthesis of 6ꢀdezoxyꢀ7,8ꢀdihydromorphineꢀ6ꢀazide (7)
3ꢀOꢀacetylꢀdihydroꢀmorphine (1.0 g, 3.04 mmol) and methanesulfonyl chloride (0.5 g, 0.33
ml, 4.37 mmol) were dissolved in pyridine. After full completion monitored by TLC, the
solvent was evaporated under reduced pressure, the residue was basified with concentrated
NH3, and the product was extracted with chloroform 3x20 mL. The combined organic layers
were washed with 10 mL brine, dried over Na2SO4, filtered and the solvent was removed in
vacuo. The product was dissolved in ethanol and was isolated as HCl salt.
Yield: 0.58 g 1.44 mmol, 47.5 %
3ꢀOꢀacetlyꢀ6ꢀmesylꢀdihydromorphine (0.58 g, 1.44 mmol) was dissolved in DMF (19 mL)
and sodium azide (1.16 g, 18 mmol) dissolved in water (3 mL) was added dropwise. This
mixture was stirred for 24 hours at 100 C̊ . after completion monitored by TLC, water (70
mL) was added and was extracted with chloroform 3 X 20 mL. The combined organic layers
were washed with 10 mL brine, dried over Na2SO4, filtered and the solvent was removed in
vacuo. The product was crystallized in solution of acetone:water 3.5:2.
Yield: 255 mg, 0.72 mmol, 50.3%
General synthesis Pyridine carbonyl chloride hydrochloride
Pyridine carboxylic acid (1.00 g, 8.12 mmol 1.0 equiv) was added in thionyl chloride (4 mL)
and 1 drop of DMF. This reaction mixture was refluxed for 4 hours, and the solvent was
evaporated under reduced pressure, 10 mL benzene was added to the residue and then
removed under reduced pressure. The crude white crystals were filtered and washed with
diethyl ether (10 mL).
Yield: 85ꢀ 98%, white crystals
Synthesis of 6ꢀdezoxyꢀ7,8dihydromorphineꢀ6βꢀamine (6e)
6ꢀdezoxyꢀ7,8ꢀdihydromorphineꢀ6βꢀazide (255 mg, 0.72 mmol 1.0 equiv.) was dissolved in
ethanol (5mL), and Raney nickel (89%) (95 mg, 1.11 mmol, 1.54 equiv.) was added. The
suspension was cooled down on ice bath, then hydrazine monohydrate (98%) (0.6 mL,12.3
mmol , 17.10 equiv.) was added dropwise. This mixture was stirred at rt for 2 h then the
Raney nickel was filtered on a pad of celite and washed with ethanol (5 mL). The filtrate was
evaporated under reduced pressure, water (5 ml) was added and basified with 10% NH3.The
product was extracted with chloroform (3 X 5 mL).The combined organic layers were washed
with brine (5 mL), dried over Na2SO4, filtered and concentrated in vacuo.
Yield: 202 mg, 0.71 mmol, 98% white solid
General synthesis of 6ꢀβꢀmorphinan amides (9g)

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6ꢀdezoxymorphineꢀ6βꢀamines (0.88 mmol, 1 equiv.) were dissolved in dichloromethane (5
mL), triethylamine (2.9 mmol, 0.4mL) was added followed by the dropwise addition of
pyridinecarbonyl choride (1.06 mmol, 150 mg, 1.2 equiv.) dissolved in dichloromethane (2
mL). This yellowish solution was stirred at room temperature for 24 h. Then the solvent was
evaporated under reduced pressure, 10 mL of distilled water was added and it was basified
with 10 % NH3 to pH=9. The crude product was extracted with 3 X 10 mL chloroform. The
combined organic layers were washed with brine (10 mL), and dried over Na2SO4. The
solvent was removed under reduced pressure and the obtained yellowish oil was purified by
column chromatography (chloroform:methanol 9:1).
General synthesis of Nꢀ(6ꢀdeoxymorphineꢀ6βꢀyl)ꢀ acylꢀamide (9aꢀf)
6ꢀdezoxymorphineꢀ6βꢀamines (0.88 mmol, 1 equiv) were dissolved in dichloromethane (5
mL), and triethylamine (2.9 mmol, 0.4mL) was added followed by the dropwise addition of
pyridinecarbonyl choride (1.06 mmol, 150 mg, 1.2 equiv.). This yellowish solution was
stirred at room temperature for 24 h. Then the solvent was evaporated under reduced pressure,
and the residue was dissolved in methanol (5 mL). Then K2CO3 (244 mg, 1.76 mmol, 2.0
equiv.) was added to the solution then it was refluxed for 4 h, then it was stirred overnight at
room temperature. 10 ml of distilled water was added and it was basified with 10 % NH3 to
pH=9. The crude product was extracted with 3 X 10 mL chloroform. The combined organic
layers were washed with brine (10 mL), and dried over Na2SO4. The solvent was removed
under reduced pressure and the obtained yellowish oil was purified by column
chromatography (chloroform:methanol 9:1).
Nꢀ(6ꢀdezoxyꢀ14αꢀhydroxyꢀ7,8ꢀdihydromorphineꢀ6βꢀyl)isonicotinamide (9a):1HꢀNMR (600
MHz, CD3OD): δ= 8.71 (d, J = 6.1 Hz, 2H,ArꢀH), 7.82 (dd, J = 4.5, 1.6 Hz, 2H,ArꢀH), 6.67 (d, J =
8.1 Hz, 1H,Hꢀ2), 6.63 – 6.61 (m, 1H,Hꢀ1), 4.63 (d, J = 7.8 Hz, 1H,Hꢀ5), 3.92 (ddd, J = 12.7, 7.7, 4.9
Hz, 1H,Hꢀ6), 3.23 – 3.18 (m, 1H,Hꢀ9), 2.84 (d, J = 5.5 Hz, 1H,Hꢀ10), 2.63 (d, J = 5.8 Hz, 1H), 2.49
(dd, J = 11.6, 4.7 Hz, 1H), 2.41 (s, 2H,ꢀNꢀCH3), 2.30 (td, J = 12.4, 5.0 Hz, 1H), 2.24 – 2.15 (m, 1H),
2.03 – 1.93 (m, 1H), 1.73 – 1.67 (m, 1H), 1.62 (dt, J = 13.5, 3.3 Hz, 1H), 1.55 (td, J = 13.2, 3.3 Hz,
1H), 1.44 (dd, J = 12.3, 3.0 Hz, 1H), 1.30 (s, 1H).13C (150 MHz, CD3OD):δ= 166.20, 149.20, 142.79,
141.91, 140.49, 131.31, 123.80, 121.81, 119.28, 116.83, 91.43, 70.46, 64.84, 62.40, 52.34, 45.85,
41.44, 29.95, 23.86, 21.09, 9.06. . Yield:80%,mp: 233ꢀ235
̊C MS: calc: 408.1923, found: HRꢀMS
[M+ H+]408.1934, C23H23N3O4
Nꢀ(6ꢀdezoxyꢀ14αꢀhydroxyꢀ7,8ꢀdihydromorphineꢀ6βꢀyl)nicotinamide (9b): 1HꢀNMR (600 MHz,
CD3OD): δ= 9.01 (d, J = 1.7 Hz, 1H,ArꢀH), 8.70 (dd, J = 4.9, 1.5 Hz, 1H,ArꢀH), 8.30 – 8.25 (m,
1H,ArꢀH), 7.56 (ddd, J = 8.0, 4.9, 0.7 Hz, 2H,ArꢀH), 6.68 (d, J = 8.1 Hz, 1H,Hꢀ2), 6.63 (d, J = 8.1
Hz, 1H,Hꢀ1), 4.65 (d, J = 7.8 Hz, 1H,Hꢀ5), 4.59 (s, 1H,Hꢀ6), 3.92 (ddd, J = 12.7, 7.8, 4.9 Hz, 1H,Hꢀ
9), 3.22 (d, J = 18.7 Hz, 1H,Hꢀ10), 2.91 (d, J = 7.3 Hz, 2H), 2.69 (dd, J = 18.5, 5.5 Hz, 1H), 2.54 (d, J
= 7.6 Hz, 1H), 2.45 (s, 4H,ꢀNꢀCH3), 2.32 (td, J = 12.3, 4.8 Hz, 1H), 2.26 – 2.20 (m, 1H), 2.02 – 1.94
(m, 2H), 1.74 – 1.69 (m, 1H), 1.63 (dt, J = 13.2, 3.2 Hz, 1H), 1.56 (td, J = 13.3, 3.2 Hz, 2H), 1.48 –
1.43 (m, 1H), 1.30 (s, 1H), 1.20 (t, J = 7.3 Hz, 2H).13C (150 MHz, CD3OD):δ= 167.31, 151.17,
147.53, 142.47, 140.22, 135.19, 131.07, 123.79, 120.39, 118.73, 117.38, 110.34, 91.12, 71.01, 67.14,
53.45, 52.03, 47.84, 45.85, 29.71, 28.05, 23.86, 21.33. Yield:86%,mp: 199ꢀ202
̊C MS: calc:
408.1923, found: HRꢀMS [M+ H+]408.1520, C23H23N3O4
Nꢀ(6ꢀdezoxymorphineꢀ6βꢀyl)isonicotinamide (9c): 1HꢀNMR (600 MHz, CD3OD):
= 4.7 Hz, 1H,ArꢀH), 7.57 (d, J = 4.9 Hz, 1H,ArꢀH), 7.08 (d, J = 6.3 Hz, 1H,ArꢀH), 6.63 (d, J = 8.0
δ= 8.62 (d, J

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Hz, 1H,Hꢀ2), 6.49 (d, J = 8.0 Hz, 1H,Hꢀ1), 5.75 (s, 1H,Hꢀ7), 5.65 (d, J = 9.8 Hz, 1H,Hꢀ8), 4.77 (s,
1H,Hꢀ5), 4.55 (d, J = 5.7 Hz, 1H,Hꢀ6), 3.37 (s, 1H,Hꢀ9), 3.09 (s, 1H, amide ꢀNH), 3.01 (d, J = 18.4
Hz, 1H,Hꢀ10), 2.60 (dd, J = 14.7, 7.8 Hz, 1H), 2.42 (s, 1H,ꢀNꢀCH3), 2.33 (d, J = 12.8 Hz, 1H), 2.10 –
1.98 (m, 1H), 1.72 (d, J = 12.1 Hz, 0H), 1.37 – 1.19 (m, 1H), 1.04 (dd, J = 18.9, 11.8 Hz, 1H).13C (150
MHz, CD3OD):δ= 165.97, 149.43, 143.90, 142.22, 140.49, 128.78, 128.54, 125.46, 121.82, 121.01,
119.67, 117.61, 91.43, 75.21, 51.22, 46.17, 42.27, 32.48, 30.26, 24.96, 17.13. Yield:87%,mp: 160ꢀ
162 ̊
C MS: calc: 390.1817, found: HRꢀMS [M+ H+]390.1800, C23H23N3O3
Nꢀ(6ꢀdezoxymorphineꢀ6βꢀyl)nicotinamide: (9d)1HꢀNMR (600 MHz, CDCl3):δ= 9.14 (s, 1H,Arꢀ
H), 8.71 (d, J = 4.5 Hz, 1H,ArꢀH), 8.14 (d, J = 7.9 Hz, 1H,ArꢀH), 7.38 (dd, J = 7.8, 4.9 Hz, 1H,Arꢀ
H), 6.85 (s, 1H,amide ꢀNH), 6.68 (d, J = 8.1 Hz, 1H,Hꢀ2), 6.52 (d, J = 8.1 Hz, 1H,Hꢀ1), 5.82 – 5.75
(m, 1H,Hꢀ7), 5.69 (d, J = 9.9 Hz, 1H,Hꢀ8), 4.87 (s, 1H,Hꢀ5), 4.53 (t, J = 6.2 Hz, 1H,Hꢀ6), 3.40 (s,
1H,Hꢀ9), 3.23 (d, J = 17.6 Hz, 1H), 3.03 (d, J = 18.5 Hz, 1H,Hꢀ10), 2.93 (dd, J = 14.2, 7.1 Hz, 1H),
2.64 (dd, J = 12.0, 4.1 Hz, 1H), 2.47 (s, 3H,ꢀNꢀCH3), 2.39 (d, J = 6.2 Hz, 1H), 2.36 (d, J = 5.6 Hz,
1H), 2.12 (td, J = 12.5, 4.8 Hz, 1H), 1.79 (d, J = 11.0 Hz, 1H).13C (150 MHz, CDCl3):δ= 165.34,
151.96, 147.77, 143.89, 138.83, 135.50, 132.70, 129.65, 129.57, 127.98, 125.45, 124.03, 119.60,
117.39, 92.78, 58.99, 51.79, 47.04, 45.85, 44.51, 43.09, 35.26, 20.45.Yield:80%,mp: 127ꢀ129
̊C MS:
calc: 390.1817, found: HRꢀMS [M+ H+]390.1825, C23H23N3O3
Nꢀ(6ꢀdezoxyꢀ7,8ꢀdihydromorphineꢀ6βꢀyl)isonicotinamide (9e): 1HꢀNMR (600 MHz,
CD3OD):δ= 8.71 (d, J = 6.1 Hz, 1H,ArꢀH), 7.81 (d, J = 6.1 Hz, 1H,ArꢀH), 6.66 (d, J = 8.1 Hz, 1H,Hꢀ
2), 6.62 (d, J = 8.1 Hz, 1H,Hꢀ1), 4.60 (d, J = 8.0 Hz, 1H,Hꢀ5), 3.79 (ddd, J = 12.6, 7.9, 4.5 Hz, 1H,Hꢀ
6), 3.17 (s, 1H, Hꢀ9), 3.07 (d, J = 18.5 Hz, 1H,Hꢀ10), 2.88 (dd, J = 14.3, 7.2 Hz, 1H), 2.57 (dd, J =
12.2, 4.1 Hz, 1H), 2.48 (dd, J = 18.5, 5.5 Hz, 1H), 2.43 (s, 2H,ꢀNꢀCH3), 2.24 (ddt, J = 11.8, 7.6, 3.9
Hz, 1H), 1.94 – 1.89 (m, 1H), 1.89 – 1.84 (m, 1H), 1.72 – 1.68 (m, 1H), 1.64 (dd, J = 13.2, 2.8 Hz,
1H), 1.49 – 1.44 (m, 1H), 1.16 – 1.12 (m, 1H).13C (150 MHz, CD3OD):δ= 165.97, 149.43, 142.79,
141.67, 140.80, 129.33, 124.59, 121.26, 119.05, 117.38, 91.98, 71.02, 59.86, 52.02, 46.17, 43.08,
41.98, 34.46, 28.29, 23.86, 19.66. Yield:88%,mp: 196ꢀ198
̊C MS: calc: 392.1973, found: HRꢀMS
[M+ H+]392.1983, C23H25N3O3
Nꢀ(6ꢀdezoxyꢀ7,8ꢀdihydromorphineꢀ6βꢀyl)nicotinamide (9f): 1HꢀNMR (600 MHz,
CD3OD):δ= 9.01 (s, 1H,ArꢀH), 8.70 (d, J = 4.8 Hz, 1H,ArꢀH), 8.27 (d, J = 7.9 Hz, 1H,ArꢀH), 7.57
(dd, J = 7.9, 5.0 Hz, 1H,ArꢀH), 6.67 (d, J = 8.0 Hz, 1H,Hꢀ2), 6.63 (d, J = 8.3 Hz, 1H,Hꢀ1), 4.61 (d, J
= 7.9 Hz, 1H,Hꢀ5), 3.83 – 3.77 (m, 1H,Hꢀ6), 3.17 (s, 1H,Hꢀ9), 3.07 (d, J = 18.4 Hz, 1H,Hꢀ10), 2.60 –
2.55 (m, 1H), 2.49 (dd, J = 18.3, 5.3 Hz, 1H), 2.44 (s, 2H,ꢀNꢀCH3), 2.25 (dd, J = 12.0, 7.8 Hz, 2H),
1.70 (d, J = 11.4 Hz, 1H), 1.65 (d, J = 10.3 Hz, 1H), 1.50 – 1.43 (m, 1H), 1.30 (d, J = 9.6 Hz, 1H),
1.13 (d, J = 7.9 Hz, 1H).13C (150 MHz, CD3OD): δ= 166.22, 151.41, 147.54, 142.79, 140.81, 135.50,
131.08, 129.09, 124.58, 123.48, 118.73, 116.83, 91.67, 67.38, 59.55, 52.34, 45.86, 42.85, 41.74,
34.70, 27.74, 24.10, 19.90.Yield:74%,mp: 181ꢀ184 C
̊ MS: calc: 392.1973, found: HRꢀMS [M+
H+]392.1977, C23H25N3O3
Nꢀ(6ꢀdezoxyꢀ3ꢀmethylꢀmorphineꢀ6βꢀyl)isonicotinamide: (9g) 1HꢀNMR (600 MHz, CDCl3):δ= 8.72
(d, J = 5.7 Hz, 1H,ArꢀH), 7.60 (dd, J = 4.5, 1.5 Hz, 1H,ArꢀH), 6.68 (d, J = 8.2 Hz, 1H,Hꢀ2), 6.57 (d, J
= 8.2 Hz, 1H,Hꢀ2), 6.27 (d, J = 6.8 Hz, 1H,amideꢀNH), 5.92 – 5.87 (m, 1H,Hꢀ7), 5.70 (dd, J = 9.7,
1.7 Hz, 1H,Hꢀ8), 4.88 (s, 1H,Hꢀ5), 4.61 (t, J = 6.4 Hz, 1H,Hꢀ6), 3.86 (s, 1H,ꢀOꢀCH3), 3.37 (dd, J =
5.6, 3.2 Hz, 1H,Hꢀ9), 3.06 (d, J = 18.2 Hz, 1H,Hꢀ10), 2.58 (dt, J = 14.8, 7.4 Hz, 1H), 2.45 (s, 2H,ꢀNꢀ
CH3), 2.39 – 2.36 (m, 1H), 2.35 – 2.32 (m, 1H), 2.05 (td, J = 12.5, 5.0 Hz, 1H), 1.83 (dt, J = 13.2, 6.6
Hz, 1H). 13C (150 MHz, CDCl3):δ= 165.11, 150.30, 146.11, 141.92, 141.16, 133.29, 130.44, 127.99,
126.56, 120.71, 118.49, 114.30, 92.23, 59.31, 56.77, 50.36, 46.73, 46.02, 43.94, 43.03, 40.55, 39.77,
35.56, 19.65. .Yield:89 %,mp: 98ꢀ101 ̊
C MS: calc: 404.1973 found: HRꢀMS [M+ H+] 404.1585,
C24H25N3O3