Novel μ opioid antagonists derived from the μ opioid agonists endomorphin and [Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2)

Article abstract of DOI:10.1111/cbdd.13743

Hybrid analogues of the μ opioid agonists endomorphin and [Dmt¹]DALDA (H-Dmt-D-Arg-Phe-Lys-NH₂, Dmt?=?2′,6′-dimethyltyrosine) containing cis-4-amino-Pro, trans-4-amino-Pro, cis-4-aminoethyl-Pro or cis-4-guanidinylethyl-Pro in the 2 p

Full text of DOI:10.1111/cbdd.13743

PROFESSOR PETER W. SCHILLER (Orcid ID : 0000-0001-7311-869X)
Article type
: Research Article
Novel µ opioid antagonists derived from the µ opioid agonists
endomorphin and [Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2)
Saijian Shi1 | Jian Xu1 | LingLing Feng1 | Xin Fan1 | Zhen Chen1 | Yajuan Qin1 | Nga N.
Chung2 | Tingyou Li1,3* | Peter W. Schiller2,4*
1School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
2Laboratory of Chemical Biology and Peptide Research, Montreal Clinical Research Institute, 110
Pine Avenue West, Montreal, QC, Canada H2W 1R7
3Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center
for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166,
China.
4Department of Pharmacology and Physiology, Université de Montréal, Montreal, QC, Canada H3C
3J7
Correspondence
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13743
This article is protected by copyright. All rights reserved

Tingyou Li, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China;
E-mail: l_tingyou@njmu.edu.cn
Peter W. Schiller, Montreal Clinical Research Institute, 110 Pine Avenue West, Montreal, QC, Canada
H2W 1R7; E-mail: schillp@ircm.qc.ca
Abstract
Hybrid analogues of the µ opioid agonists endomorphin and [Dmt1]DALDA
(H-Dmt-D-Arg-Phe-Lys-NH2, Dmt = 2’,6’-dimethyltyrosine) containing cis-4-amino-Pro,
trans-4-amino-Pro, cis-4-aminoethyl-Pro or cis-4-guanidinylethyl-Pro in the 2-position of the peptide
sequence were synthesized. None of the compounds retained high µ opioid agonist activity and,
unexpectedly, substitution of cis-4-amino-Pro resulted in a novel class of potent µ opioid antagonists.
In particular, the compound H-Dmt-cis-4-amino-Pro-Trp-Arg-NH2 (CZ-1) turned out to be a highly
selective µ opioid antagonist with ~ 1 nM µ receptor binding affinity.
Keywords:
[Dmt1]DALDA, endomorphin, mu opioid receptor antagonists, opioid activity profiles, proline
analogues
This article is protected by copyright. All rights reserved

INTRODUCTION
Selective µ opioid receptor antagonists are important not only as pharmacological tools but also for
various clinical applications. They are commonly used as rescue agents to reverse the serious side
effects, particularly respiratory depression, induced by µ opioid agonists. Other potential therapeutic
indications of µ opioid antagonists include psychosis, Parkinson’s disease and obesity (Goodman et
al., 2007). Furthermore, peripherally restricted µ opioid antagonists may have therapeutic potential for
the treatment of opioid-induced gastrointestinal disorders.
Non-peptide µ opioid antagonists include the irreversibly binding ligands, β-FNA (Takemori et al.,
1981) and clocinnamox (Lewis et al., 1988), cyprodime (Schmidhammer et al.,1990), and NAN
(Obeng et al., 2018). CTOP And CTAP are peptidic µ opioid antagonists (Hawkins et al., 1989;
Abbruscato et al., 1997). The cyclic tetrapeptide c[-D-Arg-Phe-Lys-Dmt-] (“cyclodal”) is a selective μ
opioid antagonist with subnanomolar μ receptor binding affinity (Weltrowska et al., 2016).
Here we describe the design of hybrid analogues of the endogenous μ opioid agonists
endomorphin-1 or -2 (H-Tyr-Pro-Trp(or Phe)-Phe-NH2) (Zadina et al., 1997) and of the
dermorphin-derived μ opioid agonist [Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2; Dmt =
2’,6’-dimethyltyrosine) (Schiller et al., 2000), which led to the discovery of a novel class of potent μ
opioid antagonists. [Dmt1]DALDA is a potent µ opioid agonist with high µ opioid receptor binding
affinity (Kiµ = 0.143 nM) and high µ receptor binding selectivity (selectivity ratio µ/δ/κ = 1:14700:156)
(Schiller et al., 2000). In rat and mouse tail-flick assays [Dmt1]DALDA was a much more potent and
longer lasting analgesic than morphine with both intrathecal and subcutaneous administration
(Shimoyama et al., 2001; Neilan et al., 2001). Furthermore, [Dmt1]DALDA turned out to be a much
more effective analgesic than morphine in two animal models of neuropathic pain (Shimoyama et al.,
2012; Schiller et al., 2015).
The structural motif of [Dmt1]DALDA are alternating aromatic and basic amino acid residues. In
an effort to further explore SAR of this peptide, we designed and synthesized analogues in which the
D-Arg residue is replaced by proline derivatives that carry a basic substituent in the 4-position of the
This article is protected by copyright. All rights reserved

ring structure (Figure 1). Peptide analogues containing cis-4-amino-Pro, trans-4-amino-Pro,
trans-4-guanidino-Pro (CZ-series) and analogues containing cis-4-aminoethyl-Pro and
cis-4-guanidinylethyl-Pro (SJ-series) were synthesized. The incorporation of these Pro analogues
introduces conformational constraints in the peptide backbone and affects the positioning of their
basic substituents in the interaction with the receptor which may have interesting effects on the opioid
activity profile. Furthermore, the peptide analogues contained various natural or artificial amino acid
residues in the 3-position and a basic residue (Lys or Arg) in the 4-position as present in
[Dmt1]DALDA (Figure 2):
H-Dmt-cis-4-amino-Pro-Trp-Lys-NH2
(CZ-1, 1)
(CZ-2, 2)
(CZ-3, 3)
(CZ-4, 4)
(CZ-5, 5)
(CZ-6, 6)
(SJ-1, 7)
(SJ-2, 8)
(SJ-3, 9)
(SJ-4, 10)
(SJ-5, 11)
(SJ-6, 12)
H-Dmt-cis-4-amino-Pro-Tmp-Lys-NH2
H-Dmt-cis-4-amino-Pro-Phe-Arg-NH2
H-Dmt-cis-4-amino-Pro-Tmp-Arg-NH2
H-Dmt-trans-4-amino-Pro-Phe-Arg-NH2
H-Dmt-trans-4-guanidino-Pro-Phe-Lys-NH2
H-Dmt-cis-4-aminoethyl-Pro-Trp-Arg-NH2
H-Dmt-cis-4-aminoethyl-Pro-Tmp-Arg-NH2
H-Dmt-cis-4-aminoethyl-Pro-1-Nal-Arg-NH2
H-Dmt-cis-4-guanidinoethyl-Pro-Trp-Lys-NH2
H-Dmt-cis-4-guanidinoethyl-Pro-Tmp-Lys-NH2
H-Dmt-cis-4-guanidinoethyl-Pro-1-Nal-Lys-NH2
Because the peptides contain a modified Pro residue in the 2-positon, they can also be considered as
endomorphin-[Dmt1]DALDA hybrid peptides. .
The in vitro opioid activity profiles of the compounds were determined in opioid receptor binding
assays and the functional guinea pig ileum (GPI) and mouse vas deferens (MVD) assays.
Surprisingly, this analogue design led to the discovery of a new class of µ opioid antagonists.
2 | METHODS AND MATERIALS
2.1 | Chemistry
2.1.1 | Synthesis of proline analogues
This article is protected by copyright. All rights reserved

The five orthogonally protected Pro analogues were synthesized from commercially available
trans-4-hydroxy-L-proline. They are cis-4-BocNH-Nα-Fmoc-Pro-OH (13), trans-4-BocNH-Nα-
Fmoc-Pro-OH (14), trans-4-(diBoc-guanidino)-Nα-Fmoc-Pro-OH (15), cis-4-(2-BocNH-ethyl)-Nα-
Fmoc-Pro-OH (16) and cis-4-(2-diBoc-guanidlinoethyl)-Nα-Fmoc-Pro-OH (17).
The synthesis of cis-4-BocNH-Nα-Fmoc-Pro-OH (13) was performed following a published method
(Tamaki et al., 2001) with some modifications (Scheme 1). Briefly, the 4-hydroxyl group of
Boc-trans-Hyp-OBzl (18) was activated with mesyl chloride to give 19. Nucleophilic substitution of
the sulfonate 19 with NaN3 yielded in the cis-4-azide 20 with the chiral center at the 4 position
inverted. Boc deprotection with TFA, and Fmoc re-protection gave 22. The azide group was then
reduced by Pd-catalyzed hydrogenation to yield 23 and Boc protection gave the orthogonally
protected compound 13.
N3
HO
N3
MsO
a
c
b
COOBn
COOBn
COOBn
COOBn
N
N
N
N
H
Boc
Boc
Boc
21
18
19
20
N3
H2N
BocHN
d
e
f
COOBn
COOH
COOH
Fmoc
N
N
N
Fmoc
Fmoc
13
23
22
Scheme 1. Synthesis of cis-4-BocNH-Na-Fmoc Pro-OH (13).
Reagents and conditions (a) MsCl, pyridine, 0 °C to rt, overnight, 69%; (b) NaN3, DMF, 65 °C,
overnight, 57%; (c) TFA, DCM, 2 h, 60%; (d) Fmoc-Osu, 6% NaHCO3/1,4-dioxane, rt, overnight, 78%;
(e) Pd/C, H2 (8 atm), rt, 3 d, 30%; (f) (Boc)2O, TEA, H2O/1,4-dioxane, 0 °C to rt, overnight, 50%.
This article is protected by copyright. All rights reserved

HO
HO
TsO
HO
a
b
d
c
COOBn
COOH
COOBn
COOH
N
N
H
N
N
Cbz
Cbz
Cbz
24
25
26
27
N3
H2N
BocHN
BocHN
e
f
g
COOBn
COOBn
COOBn
COOH
N
N
N
N
H
Cbz
Cbz
Cbz
28
29
30
31
BocHN
h
COOH
N
Fmoc
14
BocHN
BocHN
NHBoc
NHBoc
H2N
N
N
COOBn
j
i
k
COOH
COOBn
N
N
N
Cbz
H
Cbz
29
32
33
BocHN
NHBoc
N
COOH
Fmoc
N
15
Scheme 2. Synthesis of trans-4-BocNH-Na-Fmoc-Pro-OH (14) and trans-4-(diBoc-gunidino)-Na-
Fmoc-Pro-OH (15).
Reagents and conditions: (a) Cbz-Cl, THF/NaHCO3, rt, overnight, 84%; (b) BnBr, TEA, THF, 0 °C to
rt, overnight, 82%; (c) TsCl, TEA, DMAP, DCM, 0 °C to rt, overnight, 58%; (d) NaN3, DMF, 65 °C,
overnight, 91%; (e) PPh3, H2O, THF, reflux, overnight; (f) (Boc)2O, TEA, H2O/1,4-dioxane, 0 °C to rt,
overnight, 78%(e and f 2 steps); (g) Pd/C, H2, rt, overnight; (h) Fmoc-Osu, 6% NaHCO3/1,4-dioxane,
rt, overnight, 24%(g and h 2 steps); (i) N,N'-Di-Boc-N"-trifluoromethane sulfonyl-guanidine, DIPEA,
This article is protected by copyright. All rights reserved

dry DCM, 0 °C to rt, overnight, 55%; (j) Pd/C, H2, rt, overnight; (k) Fmoc-Osu, 6%
NaHCO3/1,4-dioxane, rt, overnight, 51% (j and k 2 steps).
For the synthesis of trans-4-BocNH-Nα-Fmoc-Pro-OH (14) (Scheme 2), cis-4-hydroxyproline (24)
was reacted with Cbz-Cl to give Nα-Cbz-Hyp-OH (25), its carboxyl group was then protected as the
benzyl ester (26) and the 4-position hydroxyl group was activated by tosylation (27). SN2
displacement in 27 with NaN3 gave 28. The azide group of 28 was then reduced by PPh3 in water and
THF. The amino group of the resulting 29 was protected with Boc-group to give 30. Simultaneous
removal of the Cbz- and Bn- groups of 30 by hydrogenation gave 31, the α-NH group of which was
protected with Fmoc to give compound 14. Compound 29 was reacted with the guanidinilating
reagent N,N'-di-Boc-N"-trifluoromethanesulfonyl-guanidine (Tamaki et al., 2001) to give 32. After
removal of the Cbz-group and re-protection with Fmoc, trans-4-(diBoc-guanidino-Nα-Fmoc-Pro -OH
(15) was obtained.
The synthesis of cis-4-(2-BocNH-ethyl)-Nα-Fmoc-Pro-OH (16) is depicted in Scheme 3.
Commercially available trans-Nα-Cbz-hydroxyproline (34) was oxidized to 35 by using a combination
of TCCA and TEMPO. Key intermediate 36 was obtained via Horner-Wadsworth- Emmons reaction
that is capable of carbon chain elongation (McCarthy, & Naylor, 2013) The reaction resulted in a pair
of isomers 36a and 36b which could be separated by silica gel column chromatography.
Hydrogenation of 36b proceeded in a diastereoselective manner to afford 37. As determined by NMR,
an 80% diastereomeric excess was observed. The cis configuration of 37 was confirmed by NOESY
measurements, where the correlation between 2-CH and 4-CH of the pyrrolidine moiety was observed
(Grygorenko et al., 2009). The α-amino group of 37 was then re-protected with Cbz (38). The carboxyl
group of 38 was selectively reduced to alcohol by IBCF/NaBH4 to give 39, the hydroxyl group of which
was then activated by reaction with TsCl to give 40. Substitution with NaN3 yielded azide 41 which
was reduced by PPh3 to give amine 42. Protecting the amino group with Boc and converting the Cbz-
to the Fmoc-group by the usual methods yielded the targeted Pro analogue 16.
This article is protected by copyright. All rights reserved

COOBn
N
O
HO
a
b
BnOOC
COOMe
COOMe
COOMe
+
COOMe
N
N
N
Cbz
Cbz
Cbz
Cbz
34
35
36a
36b
HO
HOOC
HOOC
d
e
c
COOMe
COOMe
COOMe
N
N
N
H
Cbz
Cbz
37
38
39
N3
H2N
TsO
g
h
f
COOMe
COOMe
COOMe
N
N
N
Cbz
Cbz
Cbz
40
41
42
BocHN
BocHN
BocHN
j
i
c
COOH
COOMe
COOH
N
N
N
H
Cbz
Cbz
43
44
45
BocHN
COOH
k
N
Fmoc
16
Scheme 3. Synthesis of cis-4-(2-BocNH-Et)-Na-Fmoc-Pro-OH (16).
Reagents and conditions: (a) TCCA, DCM, 0 °C,15 min, then TEMPO 1 h, 75%.; (b) benzyl
2-(diethoxyphosphoryl)acetate, 60% NaH, dry THF, -20 °C, 30 min, then 35, 30 min, 84%.; (c) Pd/C,
H2 (4 atm), MeOH, overnight; (d) Cbz-Cl, THF/NaHCO3, rt, overnight, 86% (c and d 2 steps); (e) NMM,
IBCF, THF, 0 °C,10 min, then NaBH4, 0 °C, 20 min, 90%.; (f) TsCl, TEA, DMAP, DCM, 0 °C to rt,
overnight, 80%; (g) NaN3, DMF, 65 °C, overnight; (h) PPh3, H2O, THF, reflux, overnight, 86% (g and h
This article is protected by copyright. All rights reserved

2 steps); (i) (Boc)2O, TEA, H2O/1,4-dioxane, 0 °C to rt, overnight, 95%; (j) LiOH, THF/H2O, rt,
overnight, 80%; (k) Fmoc-OSu, NaHCO3, acetone/water, rt, overnight, 45% (c and k 2 steps).
Starting from intermediate 42, cis-4-(2-diBoc-guanidinoethyl)-Nα-Fmoc-Pro-OH (17) was
synthesized by guanidination, followed by deprotection and reprotection strategy (Scheme 4).
NHBoc
NHBoc
BocHN
BocHN
H2N
N
N
a
b
COOMe
NHBoc
COOH
COOMe
NHBoc
N
N
N
Cbz
Cbz
Cbz
47
42
46
BocHN
BocHN
d
c
N
N
COOH
Fmoc
COOH
N
N
H
48
17
Scheme 4. Synthesis of cis-4-(2-diBoc-guanidinoethyl)-Na-Fmoc-Pro-OH (17)
Reagents and conditions: (a) N,N'-Di-Boc-N"-trifluoromethanesulfonyl-guanidine, DIPEA, dry DCM, 0
°C to rt, overnight, 63%; (b) LiOH, THF/H2O, rt, overnight, 85%; (c) Pd/C, H2 (4 atm), MeOH,
overnight; (d) Fmoc-OSu, NaHCO3, acetone/water, rt, overnight, 64%(c and d 2 steps).
2.1.2 | Peptide synthesis
Peptide synthesis was performed by the manual solid phase technique on a Rink Amide resin.
Crude peptides were purified by semi-preparative RP-HPLC. Each peptide was at least 98% pure, as
assessed by analytical RP-HPLC. Molecular weights were confirmed by MS (Table 1).
2.2 | In vitro pharmacological characterization
This article is protected by copyright. All rights reserved

2.2.1 | Opioid receptor binding assays
Opioid receptor binding studies were performed as described in detail elsewhere (Schiller et al.,
2000). Binding affinities for μ and δ receptors were determined by displacing, respectively,
[3H]DAMGO (Multiple Peptide Systems, San Diego, CA) and [3H]DSLET (Multiple Peptide Systems)
from rat brain membrane binding sites, and κ opioid receptor binding affinities were measured by
displacement of tritiated
(5α,7α,8β-(‒)-N-methyl-N-[7-(1-pyrrolidinyl-1-oxaspiro[4.5]-dec-8-yl]benzeneacetamide ([3H]U69,593)
(Lahti et al., 1985) (Amersham) from guinea pig brain membrane binding sites. Incubations were
performed for 2 h at 0° C with [3H]DAMGO, [3H]DSLET and [3H]U69,593 at respective concentrations
of 0.72, 0.78 and 0.80 nM. IC50 values were determined from log dose-displacement curves and Ki
values were calculated from the obtained IC50 values by means of the equation of Cheng and Prusoff
(1973) using values of 1.3, 2.6 and 2.9 nM for the dissociation constants of [3H]DAMGO, [3H]DSLET
and [3H]U69,593, respectively.
2.2.2 | Functional guinea pig (GPI) and mouse vas deferens (MVD) assays
The GPI (Paton, 1957) and MVD (Henderson et al., 1972) functional assays were carried out as
reported in detail elsewhere (Schiller et al., 1978; DiMaio et al., 1982). A dose-response curve was
determined with [Leu5]enkephalin as standard for each ileum and vas preparation, and IC50 values of
the compounds being tested were normalized according to a published procedure (Waterfield et al.,
1979). Ke values for antagonists were determined from the ratios of IC50 values obtained with an
agonist in the presence and absence of a fixed antagonist concentration (Kosterlitz et al., 1968). μ
antagonist Ke values were determined in the GPI assay against the μ agonist TAPP
(H-Tyr-D-Ala-Phe-Phe-NH2) (Schiller et al.,1989). κ antagonist Ke values of compounds were also
determined in the GPI assay against the κ agonist U50,488. δ antagonist Ke values were determined
in the MVD assay against the δ agonist DPDPE.
3
| Results and Discussion
This article is protected by copyright. All rights reserved

[Dmt1]DALDA and endomorphin, that both can be considered as parent peptides of the CZ- and SJ-
peptides, are potent and selective µ opioid agonists. Surprisingly, none of the peptides described here
showed the expected high µ agonist potency in the GPI assay (Table 3). Most of them displayed µ
antagonist activity (compounds CZ-1, CZ-2, CZ-3, CZ-4, SJ-1, SJ-2 and SJ-3) with varying µ receptor
binding affinities (Table 2) and represent a new class of selective µ opioid antagonists. Three of the
peptides (CZ-5, SJ-4 and SJ-6) turned out to be selective µ opioid partial agonists and one of them
was a moderately potent µ opioid full agonist (CZ-6).
The most potent µ antagonists contain cis-4-amino-Pro in the 2-position of the peptide sequence
(CZ-1 CZ-4), whereas a trans-4-amino-Pro2-containing analogue (CZ-5) was identified a weak µ
partial agonist. Substitution of trans-4-guanidino-Pro in the 2-position produced the only compound
with modest µ opioid agonist activity (CZ-6). Peptides containing a Pro analogue with the amino- or
guanidino group attached to the 4-position via an ethyl linker (SJ-series) in general showed weak µ
antagonist activity or modest µ partial agonist activity. All compounds displayed weak δ receptor
binding affinity with weak δ partial agonist activity or weak δ antagonist activity, as determined in the
MVD assay. All of them showed relatively weak κ receptor binding affinity and for some of them weak
κ antagonist activity could be determined in the GPI assay (this was not possible with some of the
compounds because of very weak κ antagonist activity and was indicated as N.D. in Table 3).
Most notable was compound CZ-1 which is a potent µ opioid antagonist, showing high µ opioid
receptor binding affinity (Kiµ = 1.62 nM) and high µ receptor selectivity in the opioid receptor binding
µ
assays, and high µ antagonist activity in the functional GPI assay (Ke = 22.6 nM). In comparison with
the widely used µ opioid antagonist CTOP, CZ-1 showed about equal µ receptor binding affinity and
2-fold higher µ opioid antagonist activity in the GPI assay (Table 3). In the opioid receptor binding
assays it displayed higher µ versus δ receptor selectivity than CTOP, but lower µ versus κ receptor
selectivity (Table 2). Compound CZ-5 turned out to be a µ partial agonist with high µ receptor binding
affinity and high µ receptor binding selectivity.
4 | Conclusions
Unexpectedly, substitution of cis- 4-amino-Pro in the 2-position of the peptide sequence of
peptides structurally related to [Dmt1]DALDA and endomorphin resulted in a novel class of potent µ
opioid antagonists. In particular, the compound CZ-1 is of interest as a pharmacological tool because
This article is protected by copyright. All rights reserved

of its high µ opioid antagonist activity and high µ opioid receptor binding selectivity. A limitation of
using the opioid antagonist naloxone to reverse respiratory arrest caused by opioid overdose is its
relatively short duration of action (Collins et al., 2018; Baumann et al., 2018). Depending on the
results of future in vivo studies, CZ-1 or further analogues of the new µ opioid antagonist class
described here may also turn out to be useful to counter opioid-induced respiratory depression.
ACKNOWLEDGEMENTS
This work was supported in part by grants from the National Nature Science Foundation of China
(NSFC No. 81573280) to T.L, and supported in part by grants to Y.Q. from Jiangsu Province Funds
for Young Scientists (No. BK20171048). It was also supported in part by grants to P.W.S. from the
U.S. National Institutes of Health (DA-04443) and the Canadian Institutes of Health Research
(MOP-89716).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon
reasonable request.
ORCID
Peter W. Schiller ID 0000-0001-7311-869X
Nga N. Chung ID 0000-0001-7293-4855
REFERENCES
Abbruscato, T.J., Thomas, S.A., Hruby, V.J., & Davis, T.P. (1997). Blood-brain barrier permeability
and bioavailability of a highly potent and mu-selective opioid receptor antagonist, CTAP: comparison
with morphine. The Journal of Pharmacology and Experimental Therapeutics, 280, 402-409.
This article is protected by copyright. All rights reserved

Baumann, M.H., Kopajtic, T.A., & Madras, B.K. (2018). Pharmacological research as a key
component in mitigating the opioid overdose crisis. Trends in Pharmacological Sciences, 39, 995-998.
Cheng, Y.C., & Prusoff, W.H. (1973). Relationship between the inhibition constant (KI) and the
concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction.
Biochemical Pharmacology, 85, 3099-3102.
Collins, F.S., Koroshetz, W.J., & Volkow, N.D. (2018). Helping to end addiction over the long term.
The research plan for the NIH HEAL Initiative. Journal of the American Medical Association, 320,
129-130.
Di Maio, J., Nguyen, T.M.-D., Lemieux, C., Schiller, P.W. (1982). Synthesis and pharmacological
characterization in vitro of cyclic enkephalin analogues: effects of conformational constraints on
opiate receptor selectivity. Journal of Medicinal Chemistry, 25, 1432-1438.
Goodman, A.J., Le Bourdonnec, B., & Dolle, R.E. (2007) Mu opioid receptor antagonists: recent
developments. ChemMedChem, 2, 1553-1570.
Grygorenko, O.O., Komarov, I.V., Cativiela, C. (2009). A novel approach to 2,4-ethanoproline.
Tetrahedron: Asymmetry, 20, 1433-1436.
Hawkins, K.N., Knapp, R.J., Lui, G.K., Gulya, K., Kazmierski, W., Wan, Y.P., Pelton, J.T., Hruby, V.J.,
& Yamamura, H.I. (1989). [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H-CTOP, a potent
and highly selective peptide for mu opioid receptors in rat brain. The Journal of Pharmacology and
Experimental Therapeutics, 248, 73-80.
Henderson, G,, Hughes, J., & Kosterlitz, H.W. (1972). A new example of a morphine sensitive
neuroeffector junction. British Journal of Pharmacology, 46, 764-766.
Kosterlitz, H.W. &, Watt, A.Z. (1968). Kinetic parameters of narcotic agonists and antagonists with
particular reference to N-allylnoroxymorphone (naloxone). British Journal of Pharmacology and
Chemotherapy, 33, 266-276.
Lahti, R.A., Michelson ,M.M., McCall, J.M., & von Voigtlander, P.F. (1985). 3H-U-69,593 a highly
selective ligand for the κ receptor. European Journal of Pharmacology, 109, 281-284.
This article is protected by copyright. All rights reserved

Lewis, J.W., Smith, C.F.C., McCarthy, P.S., Walter, D.S., Kobylecki, R., Myers, M., Haynes, A.S.,
Lewis, C.J., & Waltham, K. (1988). New 14-aminomorphinones and codeinones. NIDA Research
Monograph, 90, 136-143.
McCarthy, T.D., & Naylor, A. (2013). Heterocyclic compounds and methods for their use. International
Patent WO2013/102242(A1).
Neilan, C.L., Nguyen, T.M-D., Schiller, P.W., & Pasternak, G.W. (2001). Pharmacological
characterization of [Dmt1]DALDA, a highly potent and selective μ-opioid peptide. European
Journal of Pharmacology, 419,15-23.
Obeng, S., Jali, A., Zheng, Y., Wang, H., Schwienteck, K.L., Chen, C., Stevens, D.L., Akborali, H,I.,
Dewey, W.L., Banks,M.L., Liu-Chen, L.-Y., Selley, D.E., & Zhang, Y. (2018). Characterization of
17-cycloproylmethyl=3.14β-dihydroxy-4,5α-epoxy-6α(indole-7-carboxamido)morphinan INAN) as a
novel opioid receptor modulator of opioid use disorder treatment. ACS Chemical Neuroscience, 10,
2518-2532.
Paton, W.D.M. (1957). The action of morphine and related substances on contraction and on
acetycholine output of coaxially stimulated guinea pig ileum. British Journal of Pharmacology, 12,
119-127.
Schiller, P.W., Lipton, A., Horrobin, D.F., & Bodanszky, M. (1978). Unsulfated C-terminal 7-peptide of
cholecystokinin: a new ligand of the opiate receptors. Biochemical and Biophysical Research
Communications, 85, 1332-1338.
Schiller, P.W., Nguyen, T.M.-D., Chung, N.N., & Lemieux, C. (1989). Dermorphin analogues carrying
an increased positive net charge in their “message” domain display extremely high μ opioid receptor
selectivity. Journal of Medicinal Chemistry, 32, 698-703.
Schiller, P.W., Nguyen, T.M-D., Berezowska, I., Dupuis, S., Weltrowska, G.,, Chung N.N., & Lemieux,
C. (2000). Synthesis and in vitro opioid activity profiles of DALDA Analogues. European Journal of
Medicinal Chemistry, 35, 895-901.
This article is protected by copyright. All rights reserved

Schiller, P.W., Nguyen, T.M.-D., Saray, M., Poon, A.W.H., Laferrière, A., & Coderre, T.J. (2015). The
bifunctional µ opioid agonist/antioxidant [Dmt1]DALDA is a superior analgesic in an animal model of
complex regional pain syndrome type I. ACS Chemical Neuroscience, 6, 1789-1793.
Schmidhammer, M., Smith, C.F.C., Erlach, D., Koch, M., Krassnig, R., Schwetz, W., & Wechner, C.
(1990). Synthesis and biological evaluation of 14-alkoxymorphinans. 3. Extensive studies on
cyprodime-related compounds. Journal of Medicinal Chemistry, 33, 1200-1206.
Shimoyama, M., Shimoyama, N., Zhao, G.-M., Schiller, P.W,, & Szeto, H.H. (2001). Antinociceptive
and respiratory effects of intrathecal H-Tyr-D-Arg-Phe-Lys-NH2 (DALDA) and [Dmt1]DALDA. Journal
of Pharmacology and Experimental Therapeutics, 297, 364-371.
Shimoyama, M., Schiller, P.W., Shimoyama, N., Toyama, S., & Szeto, H.H. (2012). Superior
analgesic effect of H-Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA), a multifunctional opioid peptide,
compared to morphine in a rat model of neuropathic pain. Chemical Biology and Drug Design, 80,
771-774.
Takemori, E., Larson, D.L., & Portoghese, P.S. (1981). The irreversible narcotic antagonistic and
reversible agonistic properties of the fumarate methyl ester derivative of naltrexone. European
Journal of Pharmacology, 70, 445-451.
Tamaki, M., Han, G., Hruby, V.J. (2001). Practical and efficient synthesis of orthogonally protected
constrained 4-guanidinoprolines. Journal of Organic Chemistry, 66,1038-1042.
Waterfield, A.A., Leslie, F.M., Lord, J.A.H., Ling, N., & Kosterlitz, H.W. (1979). Opioid activities of
fragments of β-endorphin and of its leucine65 analogue. Comparison of the binding properties of
methionine- and leucine-enkephalin. European Journal of Pharmacology, 58, 11-18.
Weltrowska, G., Nguyen, T.M.-D., Chung, N.N., Wood, J., Ma, X., Guo, J., Wilkes, B.C., Ge, Y.,
Laferrière, A., Coderre, T.J., & Schiller, P.W. (2016). A cyclic tetrapeptide (“cyclodal”) and its
mirror-image isomer are both high-affinity µ opioid receptor antagonists. Journal of Medicinal
Chemistry, 59, 9243-9254.
Zadina, J.E., Hackler, L., Ge, L.-J., & Kastin, A. J. (1997). A potent and selective endogenous
antagonist for the μ-opiate receptor. Nature, 386, 499-502.
This article is protected by copyright. All rights reserved

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the
end of this article.
FIGURE LEGENDS
Figure 1. Proline analogues containing a basic residue in the 4-position.
Figure 2. Structures of endomorphin-[Dmt1]DALDA hybrid peptides containing Pro analogues in the
2-position.
This article is protected by copyright. All rights reserved

Table 1. Analytical parameters [Dmt1]DALDA analogues
compd peptide
Formula
TOF-MS
(M-3TFA+H)+
Obsd.
HPLCa
(tR min)
H-Dmt-cis-4-amino-Pro-Trp-Lys-NH2
C39H49F9N8O11
C40H54F9N7O11
C37H48F9N9O11
C40H54F9N9O11
C37H48F9N9O11
C38H50F9N9O11
C41H53F9N10O11
C42H58F9N9O11
C43H54F9N9O11
C42H55F9N10O11
635.4
12.61
13.5
1
2
H-Dmt-cis-4-amino-Pro-Tmp-Lys-NH2
H-Dmt-cis-4-amino-Pro-Phe-Arg-NH2
H-Dmt-cis-4-amino-Pro-Tmp-Arg-NH2
H-Dmt-trans-4-amino-Pro-Phe-Arg-NH2
H-Dmt-trans-4-guanidino-Pro-Phe-Lys-NH2
H-Dmt-cis-4-aminoethyl-Pro-Trp-Arg-NH2
H-Dmt-cis-4-aminoethyl-Pro-Tmp-Arg-NH2
H-Dmt-cis-4-aminoethyl-Pro-1-Nal-Arg-NH2
H-Dmt-cis-4-guanidinoethyl-Pro-Trp-Lys-NH2
638.4
624.4
11.94
13.59
12.03
11.85
12.43
13.69
13.2
3
666.4
4
624.4
5
638.4
6
691.7
7
694.7
8
702.7
9
705.5
12.34
13.55
13.2
10
11
12
H-Dmt-cis-4-guanidinoethyl-Pro-Tmp-Lys-NH2 C43H60F9N9O11
H-Dmt-cis-4-guanidinoethyl-Pro-1-Nal-Lys-NH2 C44H56F9N9O11
708.5
716.5
a Performed on a Sunfire™ Prep C18 column (4.6×150 mm) with a linear gradient of 95-10% solvent A (0.05%
TFA in H2O) over 30 min at a flow rate of 1.2 ml/min.
This article is protected by copyright. All rights reserved

Table 2. Opioid receptor binding affinities of Dmt1DALDA analoguesa
Binding affinities
Selectivity ratio
Kiμ
nM
Ki nM
Kiκ nM
μ//κ
Compound
CZ-1
CZ-2
CZ-3
CZ-4
CZ-5
1.62 ± 0.23
9.73 ± 0.57
9.72 ± 1.37
2.38 ± 0.49
1.55 ± 0.15
1520 ± 160
210 ± 4
108 ± 19
152 ± 1
1/938/67
1/22/16
1
2
3
4
5
2430 ± 80
39.0 ± 0.5
8230 ± 960
64.2 ± 4.2
85.7 ± 25.3
163 ± 6
1/250/7
1/16/36
1/5310/105
CZ-6
96.6 ± 15.4
7590 ± 570
457 ± 72
1/79/5
6
SJ-1
SJ-2
300 ± 14
1160 ± 140
389 ± 20
607 ± 132
1/4/2
7
8
84.0 ± 5.2
3820 ± 280
1/5/45
SJ-3
SJ-4
99.8 ± 15.2
14.1 ± 1.7
1.91 ± 0.16
45.3 ± 6.7
1.30 ± 0.23
5750 ± 1970
1410 ± 270
679 ± 210
727 ± 27
1670 ± 100
444 ± 129
149 ± 21
1/58/17
1/100/31
1/355/78
1/16/5
9
10
11
12
SJ-5
SJ-6
240 ± 24
CTOP
258 ± 59
14900 ± 500
1/198/11500
a Mean of 3-4 determinations ± SEM.
This article is protected by copyright. All rights reserved

Table 3. GPI and MVD assay of Dmt1DALDA analoguesa
GPI
MVD
IC50 (nM)
Keμ (nM)b Ke (nM)c
IC50 (nM)
K
(nM)d
κ

Compound
e
CZ-1
22.6 ± 4.3
33.5 ± 6.3
211 ± 19
1160 ± 160
3660 ± 180
1700 ± 90
970 ± 260
N.D.
6380 ± 160
1660 ± 250
1
2
CZ-2
CZ-3
CZ-4
CZ-5
CZ-6
SJ-1
3
3060 ± 370 (IC20)
16.8 ± 3.0
463 ± 29
4
5
1420 ± 70 (IC30)
1220 ± 210
3160 ± 840 (IC20)
4370 ± 270 (IC25)
PAe
N.D.
6
4840 ± 130
1440 ± 240
1520 ± 80
N.D.
7
SJ-2
N.D.
4410 ± 600
8
SJ-3
N.D.
PA
450 ±70 (IC20)
PA
9
SJ-4
1380 ± 170 (IC30)
N.D.
10
11
12
SJ-5
2310 ± 440
40.9 ± 2.7
1490 ± 10
N.D.
SJ-6
1820 ± 110 (IC20)
348 ± 23 (IC30)
PA
CTOP
6940 ± 580
b
a Mean of 3-4 determinations ± SEM. Determined against TAPP.
c Determined against U50,488. d Determined against DPDPE. e PA = partial agonist.
This article is protected by copyright. All rights reserved

cbdd_13743_f1.docx
HN
HN
NH2
H2N
H2N
COOH
COOH
COOH
N
N
N
H
H
H
cis-4-amino-Pro-OH
trans-4-amino-Pro-OH
trans-4-gunidino-Pro-OH
HN
HN
NH2
H2N
COOH
COOH
N
H
N
H
cis-4-(2-aminoethyl)-N-Fmoc-Pro-OH
cis-4-(2-gunidinoethyl)-N-Fmoc-Pro-OH
This article is protected by copyright. All rights reserved

cbdd_13743_f2.docx
H
H2N
NH
H2N
N
CH2
CH2
HO
:
NH2
R1
H2N
CH2
NH2
NH
NH
O
O
R2
O
CH2
CH2
H
2C
N
H
O
N
N
:
:
R2
H
N
NH2
H
R1
R3
H
N
NH
NH2
R3
H
2C
NH2
H
2C
This article is protected by copyright. All rights reserved