Novel 6β-acylaminomorphinans with analgesic activity

Article abstract of DOI:10.1016/j.ejmech.2013.09.031

Aminomorphinans are a relatively young class of opioid drugs among which substances of high in vitro efficacy and favorable in vivo action are found. We report the synthesis and pharmacological evaluation of novel 6β-acylaminomorphinans. 6β-Morphinamine a

Full text of DOI:10.1016/j.ejmech.2013.09.031

Accepted Manuscript
Novel 6β-acylaminomorphinans with analgesic activity
András Váradi, Sándor Hosztafi, Valerie Le Rouzic, Gergő Tóth, Ákos Urai, Béla
Noszál, Gavril W. Pasternak, Steven G. Grinnell, Susruta Majumdar
PII:
S0223-5234(13)00604-1
10.1016/j.ejmech.2013.09.031
EJMECH 6434
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 June 2013
Revised Date: 3 September 2013
Accepted Date: 15 September 2013
Please cite this article as: A. Váradi, S. Hosztafi, V. Le Rouzic, G. Tóth, Á. Urai, B. Noszál, G.W.
Pasternak, S.G. Grinnell, S. Majumdar, Novel 6β-acylaminomorphinans with analgesic activity,
European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.09.031.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel 6β-acylaminomorphinans with analgesic activity
András Váradia,b, , Sándor Hosztafia, Valerie Le Rouzicb, Gergı Tótha, Ákos Uraia, Béla Noszála,
Gavril W. Pasternakb, Steven G. Grinnellb, Susruta Majumdarb
a Department of Pharmaceutical Chemistry, Semmelweis University, Hıgyes E. u. 9., Budapest
H-1092, Hungary
b Molecular Pharmacology and Chemistry Program and Department of Neurology, Memorial
Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10065, USA
Corresponding author. Tel.: +36-1- 217-0891; fax: +36-1- 217-0891; e-mail:
varadi.andras@pharma.semmelweis-univ.hu

ACCEPTED MANUSCRIPT
Abstract:
Aminomorphinans are a relatively young class of opioid drugs among which substances of high
in vitro efficacy and favorable in vivo action are found. We report the synthesis and
pharmacological evaluation of novel 6β-acylaminomorphinans. 6β-Morphinamine and 6β-
codeinamine were stereoselectively synthesized by Mitsunobu reaction. The aminomorphinans
were subsequently acylated with diversely substituted cinnamic acids. In vitro binding studies on
cinnamoyl morphinamines showed moderate affinity for all opiate receptors with some selectivity
for mu opioid receptors, while cinnamoyl codeinamines only showed affinity for mu opioid
receptors. In vivo analgesia studies showed significant analgesic activity of 6β-
cinnamoylmorphinamine mediated by mu and delta receptors. The lead compound was found to
be roughly equipotent to morphine (ED50 3.13 ± 1.09 mg/kg) but devoid of the dangerous side-
effect respiratory depression, a major issue associated with traditional opioid therapy.

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Keywords:
aminomorphinan
MOR/DOR agonist
opioid
analgesia
respiratory depression
cinnamoyl morphinamine
Morphine, the principal drug of the opioid family is among the most important agents used for
the treatment of severe pain. [1] Clinically relevant effects, including various dangerous adverse
effects are predominantly mediated by mu-opioid receptors (MOR-1). The structural features of
the morphine skeleton have long been the basis of successful drug development and the
development of novel morphine-like drugs with improved efficacy, receptor selectivity and side-
effects. Aminomorphinans and their derivatives containing a nitrogen atom instead of oxygen in
position C-6 of the morphinan ring system have been used in the development of a number of
new opioids. Naloxamine and naltrexamine were the first two representatives of this family
followed by an array of compounds generated by acylation and alkylation of the C-6 amino
moiety, especially that of β-naltrexamine (β-NTA). [2] This group of compounds includes the
novel kappa-opioid receptor (KOR-1) agonist nalfurafine (TRK-820) (Remitch® in Japan) used
as a treatment for hemodialysis-related uremic pruritus [3,4] and β-funaltrexamine (β-FNA), a
covalent MOR-1 antagonist affinity label with partial KOR-1 agonist activity has been widely
used to investigate opioid receptor mechanisms. [5,6]
More recently iodobenzoylnaltrexamide (IBNtxA), a very potent analgesic with a novel
pharmacological profile devoid of most side-effects characteristic of traditional opioid agonists
due to its activity at a recently identified target (6TM/E11 sites) has been reported. [7,8] The
naloxone analog, IBNalA, is more selective for these sites and has a similar side-effect profile.
[9] _ENREF_7The equatorial β-acylamino moiety is a common structural feature of these
molecules. The C-6 amino group has been acylated by a diverse range of substituted aliphatic and

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aromatic carboxylic acids, including furanylacrylic acid (nalfurafine), 3-iodobenzoic acid
(IBNtxA and IBNalA) and methylfumaric acid (β-FNA).
The cinnamoyl amides of β-NTA possess KOR-1 agonist and MOR-1 antagonist activity,
similar to β-FNA. [10-12] C-14 cinnamoyl amides (e.g. clocinnamox) and esters of various
morphinans have received significant attention for their varying MOR-1 activity (Figure 1). [13-
15] 6-arylamido morphinans have been proposed as analogues of morphine-6-O-glucuronide.
[16] Similarly, aryl naloxamides have been considered as alcohol cessation agents [17,18] and as
potential MOR-1 antagonists. [19-22] N-Naphthoyl-naltrexamide (NNTA) reportedly targets
MOR-KOR opioid dimers. [23]
Figure 1
To the best of our knowledge, C-6 acylamines of morphine and its congeners carrying ꢀ7-8
double bond and a methyl group on N-17 have not been reported. Herein, we present the
synthesis of novel C-6 acylamines of morphine and codeine analogues acylated by a series of
cinnamic acids. The synthesized compounds were evaluated for their binding affinities to mu
(MOR-1), kappa1 (KOR-1) and delta (DOR-1) opioid receptors and analgesia. Based on in vivo
results, 6β-cinnamoylmorphinamine was subjected to more detailed studies.
The 6β-amino derivatives of morphine and codeine were synthesized using the Mitsunobu-
reaction. [24] Morphine (1) was selectively acetylated in the C-3 position and subsequently
treated with phthalimide and diisopropyl azodicarboxylate (DIAD) in the presence of Ph3P.
Codeine (5) was reacted directly under the same conditions. The 6β-phthalimidoyl intermediates

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(3,6) were isolated as HCl salts and then treated with hydrazine hydrate in ethanol to yield the
desired 6β-amino derivatives (4, 7). For amidation reactions, the appropriate carboxylic acids
were treated with thionyl chloride and reacted with the 6β-amino compounds in dichloromethane
in the presence of Et3N. In case of 6β-morphinamine (4) formation of 3-O-esters could also be
detected; the amidation reaction mixture was evaporated to dryness and dissolved in methanolic
aqueous sodium carbonate solution and left to stand overnight to ensure cleavage of C-3 phenolic
esters. The crude products were purified by column chromatography using chloroform-methanol
9:1 isocratic eluent and crystallized from hexane (Scheme 1, Figure 2).
Scheme 1
HO
O
O
HO
O
O
(i)
(ii)
(iii)
O
O
O
O
N
O
N
N
O
N
O
HO
HO
N
H2N
4
2
3
1
O
O
(ii)
(iii)
O
O
O
N
N
N
O
N
H2N
HO
5
6
7
O
O
O
HO
HO
O
(iv)
(v)
O
O
O
O
N
O
N
N
N
R
N
R
N
H2N
H2N
H
H
4
8aꢀe
9aꢀe
7
Figure 2

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The compounds showed moderate to high affinity for MOR-1 with lower affinities for KOR-1
and DOR-1 (Table 1). Not surprisingly, affinities of codeinamine-derived amides (9a-e) were
significantly lower than those of the amides of morphinamine (8a-e). Derivatives 8a-e had very
similar subnanomolar affinitites for MOR-1 irrespective of the aryl ring substituent of the
acylamino moiety, while KOR-1 binding was approximately 10-fold lower, with highest KOR-
1/MOR-1 Ki ratio for 8c (55.4) and the lowest for 8e (6.8). DOR-1 receptor affinity was roughly
two magnitudes lower than MOR-1 affinity. DOR-1/MOR-1 Ki ratio for 8a was 102.6.
Cinnamoyl morphinamines in our series lacked the marked KOR-1 affinity seen with the
previously reported acylated aminomorphinans. [10]
Next, the substances were administered to mice subcutaneously to determine their in vivo
analgesic activity (Table 1, Figure 3 A). Somewhat surprisingly only the unsubstituted cinnamoyl
morphamine analog 8a (ED50 3.13 ± 1.09 mg/kg) was analgesic with potency similar to that of
morphine (4.96 ± 0.97 mg/kg s.c.). [25] Despite similar affinities against MOR-1, KOR-1 and
DOR-1 binding, substitution on the aromatic ring of the cinnamoyl acid eliminated the analgesic
activity of analogs (8b-e) at the highest dose tested, i.e. 10 mg/kg. Thus, the in vivo active
compound 8a was selected as lead compound and further characterized. The analgesic activity of
8a was partially reversed by both the MOR-1 selective antagonist β-FNA and the DOR-1
selective antagonist NTI but, interestingly, was insensitive to the KOR-1 selective antagonist
norBNI (Table 1, Figure 3 B).
Table 1

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Table 1
Receptor binding and in vivo analgesia data of selected 6 -acylaminomorphinansa
β
Compound
Affinity (Ki, nM)
Analgesia (ED50, mg/kg, s.c. )
MOR-1
KOR-1
DOR-1
0.10 ± 0.02
0.15 ± 0.03
0.19 ± 0.09
0.74 ± 0.12
0.12 ± 0.006
4.73 ± 1.64
2.90 ± 0.66
1.97 ± 0.01
10.52 ± 0.90
5.43 ± 1.38
0.81 ± 0.09
>100
10.26 ± 6.76
9.38 ± 1.53
14.52 ± 6.82
15.26 ± 1.74
5.15 ± 0.75
>100
3.13 ± 1.09
>10
8a
8b
8c
8d
8e
9a
9b
>10
>10
>10
>10
3.81 ± 0.15
5.44 ± 0.93
>100
>100
>100
>100
>100
>100
>10
>10
>10
>10
9c
25.16 ± 13.02 >100
9d
3.74 ± 0.83
4.60 ± 1.81b
>100
9e
4.96 ± 0.96c
morphine
a Competition studies were performed with the indicated compounds against 125I-BNtxA (0.1 nM)
in membranes from CHO cells stably expressing the indicated cloned mouse opioid receptors. Ki
values were calculated from the IC50 values [26] and represent the means ± SEM of at least three
independent replications. 125IBNtxA KD values for MOR-1, KOR-1, DOR-1 sites were 0.11, 0.03
and 0.24, respectively. b Values from the literature. [7] c Values from the literature. [25]

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Figure 3
A
B
C
The [35S]GTPγS-binding assay was used to characterize the functional activity of 8a on in vitro
opioid transfected cell lines (Table 2). 8a produced near maximum stimulation in all cell lines
when compared with the MOR-1 agonist DAMGO, DOR-1 agonist DPDPE and KOR-1 agonist
U50,488H suggesting that it acts as a full agonist at all three opioid receptors. 8a showed a 25-
fold and 4-fold selective potency for MOR-1 and DOR-1 over the KOR-1 in this assay which
partially explains why in spite of high, nanomolar affinity for KOR-1 it does not exert KOR-
mediated agonism in vivo, as shown by the in vivo behavior assay in which 8a analgesia could be
antagonized by selective MOR-1 and DOR-1 antagonists but not by KOR-1 antagonist. Since in

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vivo behavior is a better predictor of functional activity, it is safe to say that analgesia of 8a is
mediated by agonism at both MOR-1 and DOR-1.
Perhaps the most serious, potentially life-threatening side-effect of opiate therapy is respiratory
depression. We tested the effect of 8a on the respiratory rate of CD1 mice and found that at doses
approximately four times the ED50 morphine (20 mg/kg) caused the expected marked respiratory
depression, while after treatment with even a high, 8-times the ED50 dose of 8a (24 mg/kg) no
significant difference was found in respiratory rate compared with saline (Figure 3 C).
Significantly reduced risk of respiratory depression has been reported in the literature for MOR-
1/DOR-1 mixed opioid agonists, including DPI-3290 and the peptide MMP-2200. [27, 28]
Table 2
Table 2
a
Opioid receptor efficacy of
Compound
8a
EC50 (nM)
% stimulation
KOR-1
MOR-1
KOR-1
DOR-1
MOR-1
DOR-1
1.38 ± 0.8 36.5 ± 19.2 9.1 ± 3.0
95.3 ± 0.8
106.3 ± 8.5
101.7 ± 2.8
8a
ndb
ndb
ndb
2.05 ± 0.6
DPDPE
DAMGO
U50,488H
ndb
ndb
5.6 ± 3
ndb
21.7 ± 4.6
a Efficacy data were obtained using agonist induced stimulation of [35S]GTPγS binding assay. Efficacy is
represented as EC50 (nM) and percent maximal stimulation relative to standard agonist DAMGO (MOR-1),
DPDPE (DOR-1), or U50,488H (KOR-1) at 100 nM. All values are expressed as the mean ± SEM of three
separate assays performed in triplicate. b Not determined
In conclusion we have synthesized a series of novel 6β-cinnamoyl morphinamines carrying
various cinnamoyl side chains. Characterization of compounds in vitro and in vivo revealed high
affinity for MOR-1 receptors. Analgesic activity of 6β-cinnamoylmorphinamine was found to be
comparable to morphine, but without causing respiratory depression, a major side-effect of
commonly used opioids. Functional assays and reversal of analgesia by selective antagonists
revealed an interesting receptor activity profile. The results suggest that new 6β-acylamino
derivatives of morphine and its congeners are promising new opioids worthy of further
investigation.
Acknowledgements:

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This work was supported, in part, by research grants from the National Institute on Drug Abuse
(DA034106-01) to SM, (DA06241, DA02165, and DA07242) to GWP, a Core Grant from the
National Cancer Institute to MSKCC (CA08748) as well as the Technology Development Fund
of Memorial Sloan-Kettering Cancer Center.
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Captions and titles
Figure 1. Structures of pharmacologically important aminomorphinans.
Figure 2. Acyl side chains of the synthesized 6β-cinnamoyl morphinamines and
codeinamines.
Figure 3. Pharmacology of 6β-cinnamoylmorphinamine 8a. (A) Analgesia: Cumulative dose-
response curves were carried out on groups of mice (n = 10) with 8a at the indicated doses (s.c.)
and analgesia tested 30 min later at peak effect. The ED50 value was 3.13 ± 1.09 mg/kg in CD1
mice by using the radiant heat tail-flick assay. Results were evaluated as %MPE [(observed
latency − baseline latency)/(maximal latency − baseline latency)] and shown as the average of
each group (n = 10). (B) Sensitivity of 8a to opioid antagonists: Groups of mice (n = 6) received
a fixed dose of 8a (5 mg/kg, s.c.) alone or with NorBNI (10 mg/kg, s.c.), β-FNA (40 mg/kg, s.c.),
NTI (20 mg/kg, s.c.). β-FNA and NorBNI were given 24h before 8a while NTI was given 15 min
before 8a. Tail flick analgesia was measured 30 min after 8a. Similar results were observed in
two independent replications. 8a analgesia is insensitive to NorBNI while analgesia is partially
antagonized by both β-FNA and NTI (ANOVA followed by Bonferroni multiple comparison test
(p < 0.05). (C) Respiratory rate. Animals were randomly assigned to receive saline (n = 3), 8a (24
mg/kg, n = 3), or morphine (20 mg/kg, n = 3). Each animal’s baseline average breath rate was
measured every 5 min for 25 min before drug injection, and breath rates after drug injection are
expressed as a percent of baseline. 8a did not depress respiratory rate and was not significantly
different from saline at any time point, whereas morphine decreased respiratory depression in
comparison with both saline and 8a (p < 0.05) as determined by repeated-measures ANOVA
followed by Bonferroni multiple-comparison test.
Scheme 1. Synthesis of 6β-cinnamoyl morphinamines and codeinamines. (i) acetic anhydride,
NaHCO3, H2O, r.t., 1 h. (ii) phthalimide, DIAD, Ph3P, benzene, r.t., 2h. (iii) hydrazine hydrate,
EtOH. (iv) 1.1 eq. acyl chloride, Et3N, CH2Cl2, r.t., 2 h. (v) 1.1 eq. acyl chloride, Et3N, CH2Cl2,
r.t., 2 h. then Na2CO3, H2O, MeOH, r.t., 12h.

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•
•
•
•
•
We designed and synthesized the 6β-acylamino derivatives of morphine and codeine
The synthesis of 10 novel potentially analgesic compounds is reported
The compounds were tested for opioid receptor binding and in vivo analgesia
6β-Cinnamoylmorphinamine were shown to possess MOR-1/DOR-1 analgesia
6β-Cinnamoylmorphinamine did not cause respiratory depression

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Table 1
Receptor binding and in vivo analgesia data of selected 6β-acylaminomorphinansa
Compound
Affinity (Ki,
nM)
Analgesia (ED50, mg/kg,
s.c.)
MOR-1
KOR-1
DOR-1
0.10 ± 0.02
2.90 ± 0.66
10.26 ± 6.76
3.13 ± 1.09
>10
8a
8b
8c
8d
8e
9a
0.15 ± 0.03
0.19 ± 0.09
0.74 ± 0.12
0.12 ± 0.006
4.73 ± 1.64
1.97 ± 0.01
10.52 ± 0.90
5.43 ± 1.38
0.81 ± 0.09
>100
9.38 ± 1.53
14.52 ± 6.82
15.26 ± 1.74
5.15 ± 0.75
>100
>10
>10
>10
>10
3.81 ± 0.15
5.44 ± 0.93
>100
>100
>100
>100
>100
>100
>10
>10
>10
>10
9b
9c
25.16 ± 13.02 >100
9d
3.74 ± 0.83
4.60 ± 1.81b
>100
9e
4.96 ± 0.96c
morphine
aCompetition studies were performed with the indicated compounds against 125I-BNtxA (0.1
nM) in membranes from CHO cells stably expressing the indicated cloned mouse opioid
receptors. Ki values were calculated from the IC50 values [26] and represent the means ± SEM
of at least three independent replications. 125IBNtxA KD values for MOR-1, KOR-1, DOR-1
b
c
sites were 0.11, 0.03 and 0.24, respectively. Values from the literature. [7] Values from the
literature. [25]

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Table 2
Opioid receptor efficacy of 8aa
Compound
%
EC50 (nM)
stimulation
MOR-1
KOR-1
DOR-1
MOR-1
KOR-1
DOR-1
1.38 ± 0.8 36.5 ± 19.2 9.1 ± 3.0
95.3 ± 0.8
106.3 ± 8.5
101.7 ± 2.8
8a
ndb
ndb
ndb
2.05 ± 0.6
DPDPE
DAMGO
ndb
ndb
5.6 ± 3
ndb
21.7 ± 4.6
U50,488H
aEfficacy data were obtained using agonist induced stimulation of [35S]GTPγS binding assay. Efficacy
is represented as EC50 (nM) and percent maximal stimulation relative to standard agonist DAMGO
(MOR-1), DPDPE (DOR-1), or U50,488H (KOR-1) at 100 nM. All values are expressed as the mean ±
SEM of three separate assays performed in triplicate. b Not determined

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Supplementary material for
Novel 6β-acylaminomorphinans with analgesic activity
András Váradi, Sándor Hosztafi, Valerie Le Rouzic, Gergı Tóth, Ákos Urai, Béla Noszál, Gavril
W. Pasternak, Steven G. Grinnell, Susruta Majumdar
1. Experimental procedures
1.1.
Materials and methods
All chemicals were purchased from Sigma-Aldrich Chemicals (Darmstadt, Germany) and Alfa
Aesar (Ward Hill, MA) and were used without further purification. 1H and 13C NMR spectra were
recorded on a Varian VNMRS spectrometer (600 MHz for 1H, 150.9 MHz for 13C) equipped with
a dual 5-mm inverse-detection gradient (IDPFG) probehead. Spectra were recorded in DMSO-d6
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 time-of-flight 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. Biological data were
analyzed by Graphpad Prism (Graphpad Software, La Jolla, CA).
1.2.
Chemical synthesis
1.2.1. Synthesis of 3-O-acetylmorphine (2):
The compound was synthesized according to the literature method.[1] Morphine hydrochloride
(1) (0.90 g, 2.8 mmol) was dissolved in the mixture of water (100 mL) and sodium hydrogen
carbonate (15 g) and acetic anhydride (4 x 1.5 mL) was added in 10 minute intervals and stirred
for further 15 min at room temperature. The solution was then extracted with chloroform. The
organic phase was separated, dried over anhydrous Na2SO4 and evaporated to dryness to yield
0.90 g of 2 (98% yield). Pale yellow oil.
1.2.2. General procedure for the synthesis of 6β-aminomorphinans (4, 7):
The compounds were synthesized according to the literature method.[2] Morphine derivative (2
or 5) (0.90 g) was dissolved in benzene (20 mL), Ph3P (1.50 g), phthalimide (0.90 g) and
diisopropyl azodicarboxylate (DIAD) (1.05 mL) were added and stirred for 1 hour at room
temperature. The mixture was evaporated under reduced pressure and a solution of tartaric acid (5
g) and water (25 mL) was added and the resulting slurry was extracted with diethyl ether. The
aqueous phase was basified (pH = 9) with 10% aq. NH3 and extracted with chloroform. The
organic phase was dried and evaporated to dryness. The resulting pale yellow oil (crude 4, 7) was
dissolved in ethanol (30 mL) and 36% aq. HCl was added (0.1 ml). The white precipitate was
filtered and washed with ethanol to yield white crystalline HCl salts of phthalimide derivatives 3

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and 6. The HCl salt of phthalimide (3 or 6) derivative was dissolved in ethanol (15 mL) and
hydrazine hydrate was added (0.5 mL) and the solution refluxed for 2 hrs. The solution was
subsequently poured into 20% aq. acetic acid solution after cooling (20 mL) and the white
precipitate was filtered. Ethanol was evaporated from the filtrate and basified (pH = 9) with 10%
aq. NH3 and extracted with chloroform. The organic phase was dried and evaporated to dryness
to yield the crude 6β-aminomorphinans 4 and 7 (45-50 % yields).
1.2.3. General procedure for acylation of morphinamine derivatives (8a-e):
The appropriate carboxylic acid (1.2 molar equivalents) was mixed with benzene (5 mL) and
thionyl chloride (0.5 mL) and refluxed for 1 h. The solution was evaporated to dryness under
reduced pressure and then dissolved in dichloromethane (10 mL). 6β-morphinamine (4) (1.0
molar equivalent) and triethylamine (0.4 mL) were added and stirred for 2 hrs at room
temperature. The solvent was evaporated under reduced pressure, methanol (10 mL) and Na2CO3
(0.5 g) were added and the mixture stirred overnight. The resulting slurry was neutralized with 20
% aq. acetic acid and extracted with chloroform. The organic phase was dried and evaporated to
dryness. The crude products were purified by column chromatography using chloroform-
methanol 9:1 isocratic eluent and crystallized from hexane to yield crystalline pale yellow
products 8a-e (20-60% yields).
1.2.4. General procedure for acylation of codeinamine derivatives (9a-e):
The method is identical to the synthesis of 8a-e, except that the hydrolysis with methanolic
Na2CO3 is left out. Pale yellow crystals (29-50% yields).
1.3. Receptor-Binding Assays:
Competition-binding assays in CHO cells stably expressing MOR-1 (mu), DOR-1 (delta) or
KOR-1 (kappa) were performed at 25°C in potassium phosphate buffer (50 mM; pH 7.4), with
the inclusion of MgSO4 (5 mM) in the MOR-1 assays. All competition assays were carried out
using 125I-BNtxA as described.[3] Specific binding was defined as the difference between total
binding and nonspecific binding, determined in the presence of levallorphan (8 ꢀM). Protein
concentrations were between 30-40 ꢀg/mL and incubation times were 90 minutes.
Specific
binding was defined as the difference between total binding and nonspecific binding, determined
in the presence of levallorphan (8 ꢀM). Protein concentration was determined as described by
Lowry et al. [4] using bovine serum albumin as the standard.
1.4. Tail Flick Analgesia Assays:
Male CD-1 mice (25-35 g; Charles River Breeding Laboratories, Wilmington, MA) were
maintained on a 12-hr light/dark cycle with Purina rodent chow and water available ad libitum.
Mice were housed in groups of five until testing. All animal experiments were reviewed and
approved by the Institutional Animal Care and Use Committee of Memorial Sloan-Kettering
Cancer Center. Analgesia was determined using the radiant heat tail-flick technique [5] using a

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machine from (Ugo Basile; model number 37360). The intensity was set to achieve a baseline
between 2-3 sec. The latency to withdraw the tail from a focused light stimulus was measured
electronically using a photocell. Baseline latencies (2.0-3.0 sec) were determined before
experimental treatments for all animals as the mean of two trials. Post-treatment tail-flick
latencies were determined as indicated for each experiment, and a maximal latency of 10 sec for
tail-flick was used to minimize tissue damage. Results were evaluated as %MPE [(observed
latency − baseline latency)/(maximal latency − baseline latency)] and shown as the average of
each group (n = 10). Drugs were given subcutaneously and cumulative dose-response
experiments carried out with two independent assays with each group (n=10). The combined
results presented as the ED50 with 95% confidence limits (n=20) presented. Analgesia was
defined quantally as a doubling, or greater, of the baseline latency. Similar results were obtained
analyzing the data in a graded response manner. Analgesic ED50 values and confidence limits
were determined using non-linear regression analysis GraphPad Prism.
1.5. [35S]GTPγS-Binding Assay:
[35S]GTPγS binding was performed on membranes prepared from transfected cells in the
presence and absence of the indicated opioid for 60 min at 30°C in the assay buffer (50 mM Tris-
HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, and 10 mM NaCl) containing 0.05nM [35S]GTPγS
and 30MGDP, as previously reported.[6,7] After the incubation, the reaction was filtered through
glass-fiber filters (Whatman Schleicher & Schuell, Keene, NH) and washed three times with 3 ml
of ice-cold 50 mM Tris-HCl, pH 7.4, on a semiautomatic cell harvester. Filters were transferred
into vials with 5 ml of Liquiscent (National Diagnostics, Atlanta, GA), and the radioactivity in
vials was determined by scintillation spectroscopy in a Tri-Carb 2900TR counter (PerkinElmer
Life and Analytical Sciences). Basal binding was determined in the presence of GDP and the
absence of drug.
1.6. Respiratory Depression Assay
Respiratory rate was assessed in awake, freely moving, adult male CD1 mice with the MouseOx
pulse oximeter system (Starr Life Sciences), as previously reported.[8] Each animal was
habituated to the device for 30 min and then tested. A 5-s average breath rate was assessed at 5-
min intervals. A baseline for each animal was obtained over a 25-min period before drug
injection, and testing began at 15 min post-injection and continued for a period of 35 min. Groups
of mice (n = 3) were treated subcutaneously with either saline or morphine (20 mg/kg) or 8a (24
mg/kg). Morphine was given at doses approximately four times its analgesic ED50, while 8a was
administered at 8 times its ED50. Groups were compared with repeated-measures ANOVA
followed by Bonferroni multiple-comparison test.
2. Compound data
2.1.
6β-Cinnamoylmorphinamine (8a, C26H26N2O3)

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Yield: 21%. 1H NMR (600 MHz, CDCl3) δ 7.61 (d, J = 15.7 Hz, 1H), 7.40 (m, 2H), 7.30 (m, 2H),
6.69 (d, J = 7.6 Hz, 1H), 6.50 (m, 3H), 5.78 (d, J = 9.3 Hz, 1H), 5.59 (d, J = 9.3 Hz, 1H), 4.83
13
(m, 1H), 4.57 (m, 1H), 2.51 (s, 3H). C NMR (151 MHz, CDCl3) δ 166.05, 144.56, 141.63,
138.96, 134.49, 131.89, 129.78, 129.55, 128.75, 127.90, 124.85, 120.02, 119.45, 117.36, 92.70,
59.27, 50.16, 47.17, 43.70, 42.66, 39.28, 34.92, 20.34. HRMS (M+H)+ calcd for C26H26N2O3
415.2022, found 415.2031.
2.2.
6β-(4-chlorocinnamoyl)-morphinamine (8b, C26H25ClN2O3)
1
Yield: 38%. H NMR (600 MHz, CDCl3) δ 7.56 (d, J = 16.0 Hz, 1H), 7.47 (d, J = 7.8 Hz, 2H),
7.31 (d, J = 7.8 Hz, 2H), 6.68 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 16.0 Hz, 1H), 6.52 (d, J = 8.1 Hz,
1H), 5.89 – 5.82 (m, 1H), 5.64 (d, J = 9.9 Hz, 1H), 4.71 (d, J = 9.0 Hz, 4H), 4.52 (d, J = 5.3 Hz,
13
1H), 2.71 (d, J = 9.5 Hz, 3H). C NMR (151 MHz, CDCl3) δ 166.32, 144.39, 139.62, 139.26,
135.29, 133.22, 129.75, 128.96, 128.81, 128.79, 128.68, 128.61, 122.77, 120.70, 119.40, 117.47,
92.20, 59.64, 49.51, 47.11, 42.91, 41.32, 37.63, 33.54, 22.45, 20.71. HRMS (M+H)+ calcd for
C26H25ClN2O3 449.1626, found 449.1630.
2.3.
6β-(4-(trifluoromethyl)cinnamoyl)-morphinamine (8c, C27H25F3N2O3)
1
Yield: 39%. H NMR (600 MHz, CDCl3) δ 7.59 (d, J = 15.7 Hz, 1H), 7.50 (d, J = 8.3 Hz, 2H),
7.47 (d, J = 8.2 Hz, 2H), 6.70 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 15.7 Hz, 1H), 6.51 (d, J = 8.1 Hz,
1H), 5.79 (dd, J = 6.4, 3.0 Hz, 1H), 5.60 (d, J = 9.9 Hz, 1H), 4.82 (s, 1H), 4.58 (t, J = 6.4 Hz, 1H)
2.54 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 165.70, 144.87, 139.97, 139.29, 138.24, 131.82,
131.58, 131.37, 129.66, 129.05, 128.18, 125.93, 124.81, 123.07, 119.79, 117.84, 92.86, 59.73,
50.42, 47.51, 43.90, 42.76, 39.31, 34.97, 20.73. HRMS (M+H)+ calcd for C27H25F3N2O3 483.189,
found 483.1873.
2.4.
6β-(4-(methoxy)cinnamoylamino)-morphinamine (8d, C27H28N2O4)
1
Yield: 41%. H NMR (600 MHz, CDCl3) δ 7.51 (d, J = 15.5 Hz, 1H), 7.29 (d, J = 8.3 Hz, 2H),
6.72 (d, J = 8.3 Hz, 2H), 6.66 (d, J = 8.0 Hz, 1H), 6.47 (d, J = 8.0 Hz, 1H), 6.37 (d, J = 15.5 Hz,
1H), 5.72 (s, 1H), 5.53 (d, J = 9.7 Hz, 1H), 4.81 (s, 1H), 4.55 (t, J = 6.1 Hz, 1H), 3.74 (s, 3H),
2.48 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 166.67, 161.00, 144.99, 141.20, 139.49, 131.65,
129.91, 129.67, 129.16, 127.59, 124.88, 119.58, 118.21, 117.80, 114.30, 92.89, 59.45, 55.46,
50.34, 47.40, 43.90, 42.71, 39.27, 35.01, 20.72. HRMS (M+H)+ calcd for C27H28N2O4 445.2122,
found 445.2138.
2.5.
6β-(3-nitrocinnamoyl)-morphinamine (8e, C26H25N3O5)
1
Yield: 21%. H NMR (600 MHz, CDCl3) δ 8.38 (d, J = 1.8 Hz, 1H), 8.19 (dd, J = 8.2, 1.4 Hz,
1H), 7.92 (d, J = 7.7 Hz, 1H), 7.67 (t, J = 7.9 Hz, 1H), 7.58 (d, J = 15.8 Hz, 1H), 6.91 (d, J = 15.8
Hz, 1H), 6.53 (d, J = 8.0 Hz, 1H), 6.42 (d, J = 8.1 Hz, 1H), 5.81 – 5.74 (m, 1H), 5.64 (dd, J = 9.8,
1.6 Hz, 1H), 4.66 (s, 1H), 4.40 (t, J = 6.7 Hz, 1H), 2.44 (s, 3H). 13C NMR (151 MHz, CDCl3) δ
163.90, 148.02, 144.22, 138.65, 136.65, 136.23, 133.64, 129.90, 129.70, 128.05, 124.63, 123.18,

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120.89, 118.50, 116.41, 91.86, 58.43, 48.93, 46.42, 43.32, 42.37, 35.16, 28.91, 19.76. HRMS
(M+H)+ calcd for C26H25N3O5 460.1867, found 460.1857.
2.6.
6β-Cinnamoylcodeinamine (9a, C27H28N2O3)
Yield: 32%. 1H NMR (600 MHz, CDCl3) δ 7.62 (d, J = 15.7 Hz, 1H), 7.45 (m, 2H), 7.32 – 7.27
(m, 3H), 6.67 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 6.48 (d, J = 15.7 Hz, 1H), 5.88 (ddd, J
= 9.1, 5.9, 3.0 Hz, 1H), 5.59 (dd, J = 9.8, 1.5 Hz, 1H), 4.86 (s, 1H), 4.61 (t, J = 6.7 Hz, 1H), 3.84
(s, 3H), 2.50 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 165.64, 146.06, 142.43, 141.26, 134.80,
131.62, 130.07, 129.61, 129.48, 129.25, 128.75, 128.67, 127.80, 127.72, 126.00, 120.45, 119.01,
114.04, 92.51, 77.29, 77.07, 76.86, 59.18, 58.15, 56.71, 49.54, 46.93, 43.72, 42.47, 39.20, 35.06,
20.58, 18.42. HRMS (M+H)+ calcd for C27H28N2O3 429.2173, found 429.2176.
2.7.
6β-(4-chlorocinnamoyl)-codeinamine (9b, C27H27ClN2O3)
1
Yield: 32%. H NMR (600 MHz, CDCl3) δ 7.56 (d, J = 15.6 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H),
7.29 (d, J = 8.4 Hz, 2H), 6.66 (d, J = 8.2 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 6.39 (d, J = 15.6 Hz,
1H), 5.85 (ddd, J = 9.1, 5.8, 3.0 Hz, 1H), 5.62 (dd, J = 9.7, 1.1 Hz, 1H), 4.85 (s, 1H), 4.56 (t, J =
6.5 Hz, 1H), 3.83 (s, 3H), 2.43 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 165.48, 146.14, 142.45,
140.18, 135.66, 133.42, 132.72, 130.49, 129.21, 129.14, 127.00, 121.05, 119.11, 113.94, 92.63,
59.25, 56.84, 50.07, 47.11, 44.14, 43.12, 40.27, 35.96, 20.42, 18.61. HRMS (M+H)+ calcd for
C27H27ClN2O3 463.1783, found 463.1782.
2.8.
6β-(4-(Trifluoromethyl)cinnamoyl)-codeinamine (9c, C28H27F3N2O4)
1
Yield: 50%. H NMR (600 MHz, CDCl3) δ 7.67 (d, J = 15.7 Hz, 1H), 7.61 (s, 4H), 6.74 (d, J =
8.2 Hz, 1H), 6.63 (d, J = 15.7 Hz, 1H), 6.61 (d, J = 8.2 Hz, 1H), 5.97 (m, 1H), 5.58 (d, J = 10.8
13
Hz, 1H), 4.90 (s, 1H), 4.69 (s, 1H), 3.88 (s, 3H), 2.81 (s, 3H). C NMR (151 MHz, CDCl3) δ
165.27, 146.27, 143.18, 139.80, 138.48, 130.49, 130.02, 129.36, 128.21, 125.97, 125.95, 124.25,
123.23, 119.51, 114.77, 92.38, 60.33, 56.96, 49.65, 47.74, 43.41, 42.28, 38.48, 34.20, 21.33.
HRMS (M+H)+ calcd for C28H27F3N2O4 497.2047, found 497.2058.
2.9.
6β-(4-(Methoxy)cinnamoyl)-codeinamine (9d, C28H30N2O4)
1
Yield: 50%. H NMR (600 MHz, CDCl3) δ 7.58 (d, J = 15.5 Hz, 1H), 7.41 (d, J = 8.0 Hz, 2H),
6.85 (d, J = 8.0 Hz, 2H), 6.66 (d, J = 8.0 Hz, 1H), 6.54 (d, J = 8.0 Hz, 1H), 6.27 (d, J = 15.5 Hz,
1H), 5.90 (m, 1H), 5.62 (d, J = 9.4 Hz, 1H), 4.86 (s, 1H), 4.56 (s, 1H), 3.85 (s, 3H), 3.80 (s, 3H),
13
2.44 (s, 3H). C NMR (151 MHz, CDCl3) δ 165.90, 160.88, 146.05, 142.26, 141.01, 132.37,
130.36, 129.36, 129.27, 127.44, 126.78, 118.85, 117.83, 114.22, 113.86, 92.49, 59.09, 58.23,
56.73, 55.31, 49.75, 46.94, 43.94, 42.93, 40.07, 35.77, 20.28, 18.42. HRMS (M+H)+ calcd for
C28H30N2O4 459.2278, found 459.2280.
2.10.
6β-(3-Nitrocinnamoyl)-codeinamine (9e, C27H27N3O5)

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Yield: 44%. 1H NMR (600 MHz, CDCl3) δ 8.36 (s, 1H), 8.20 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 7.8
Hz, 1H), 7.70 (d, J = 15.6 Hz, 1H), 7.56 (t, J = 7.9 Hz, 1H), 6.69 (d, J = 8.2 Hz, 1H), 6.57 (d, J =
8.2 Hz, 1H), 6.54 (d, J = 15.6 Hz, 1H), 5.90 (s, 1H), 5.67 (d, J = 9.7 Hz, 1H), 4.88 (s, 1H), 4.59
13
(t, J = 6.4 Hz, 1H), 3.87 (s, 3H), 2.50 (s, 3H). C NMR (151 MHz, CDCl3) δ 164.72, 148.87,
146.23, 142.64, 139.08, 136.76, 134.14, 132.81, 130.34, 130.15, 129.27, 126.71, 124.27, 123.48,
121.97, 119.24, 114.14, 92.45, 59.48, 56.95, 50.23, 47.25, 44.14, 43.14, 40.28, 35.92, 20.59.
HRMS (M+H)+ calcd for C27H27N3O5 474.2023, found 474.2026.
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[7]
[8]

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Figure S1. 1H NMR of 8a.

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Figure S2. 13C NMR of 8a.