Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu, delta, and kappa opioid receptors

Article abstract of DOI:10.1016/j.bmc.2018.05.045

In an effort to improve biphalin's potency and efficacy at the μ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1–5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesi

Full text of DOI:10.1016/j.bmc.2018.05.045

Accepted Manuscript
Cyclic biphalin analogues with a novel linker lead to potent agonist activities at
mu, delta, and kappa opioid receptors
Michael Remesic, Giorgia Macedonio, Adriano Mollica, Frank Porreca, Victor
Hruby, Yeon Sun Lee
PII:
S0968-0896(18)30791-0
https://doi.org/10.1016/j.bmc.2018.05.045
BMC 14387
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Revised Date:
Accepted Date:
23 April 2018
17 May 2018
26 May 2018
Please cite this article as: Remesic, M., Macedonio, G., Mollica, A., Porreca, F., Hruby, V., Lee, Y.S., Cyclic biphalin
analogues with a novel linker lead to potent agonist activities at mu, delta, and kappa opioid receptors, Bioorganic
& Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.05.045
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Cyclic biphalin analogues with a novel linker lead to potent agonist activities
at mu, delta, and kappa opioid receptors
Michael Remesica, Giorgia Macedoniob, Adriano Mollicab, Frank Porrecac, Victor Hrubya and
Yeon Sun Leec*
aDepartment of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721,
USA
bDepartment of Pharmacy, University of Chieti-Pescara “G. d’Annunzio”, Chieti, Italy 66100
cDepartment of Pharmacology, University of Arizona, Tucson, Arizona 85724, USA
*Correspondence: yeon@email.arizona.edu; Tel.: 1-520-626-2820

Abstract: In an effort to improve biphalin’s potency and efficacy at the µ-(MOR) and δ-opioid
receptors (DOR), a series of cyclic biphalin analogues 1 – 5 with a cystamine or piperazine
linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-
chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all
opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate
that C-terminal flexible linkers play an important role in KOR activity compared to that of the
other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki
= 0.27, 0.46, and 0.87 nM; EC50 = 3.47, 1.45, and 13.5 nM at MOR, DOR, and KOR,
respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the
three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on
relieving pain and further studies including in vivo tests are worthwhile.

Keywords:
Biphalin
Opioid receptors
MOR/DOR/KOR agonist
Synergistic analgesic effect
Cyclic peptides

Abbreviations:
Acm, acetamidomethyl; ACN, acetonitrile; BBB, blood brain barrier; Boc, t-butyloxycarbonyl;
BOP, (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate); DAMGO,
[DAla2, NMePhe4, Gly-ol5]-enkephalin; DADLE, [DAla2, DLeu5]-enkephalin; DMF, N,N-
dimethylformamide; Dmt, 2,6-dimethyl tyrosine; DOR, δ-opioid receptor; EtOAc, ethyl acetate;
FAB-MS, fast-atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl;
HBTU, hexafluorophosphate benzotriazole tetramethyl uronium; HEK, human embryonic
kidney; HOBt, N-hydroxybenzotriazole; KOR, κ-opioid receptor; MALDI-TOF, matrix assisted
laser desorption/ionization-time of flight; MeOH, methanol; MOR, µ-opioid receptor; NMM, N-
methyl morpholine; Pen, penicillamine; RP-HPLC, reversed-phase high performance liquid
chromatography; SAR, structure-activity relationship; TFA, trifluoroacetic acid; TIS,
triisopropylsilane; TLC, thin-layer chromatography

1. Introduction
Multifunctional ligands for the µ-opioid receptor (MOR) and δ-opioid receptor (DOR) have
gained notoriety due to their promise in the realm of pain therapeutics [1]. It has been previously
demonstrated that co-administration of MOR and DOR agonists lead to an attenuation of serious
side effects caused by MOR agonists, such as tolerance, while augmenting potency and efficacy
for the receptors [2-4]. Synergistic effects from MOR and DOR agonists have also been
observed which increase analgesic efficacy and thereby allow the usage of lesser doses of MOR
agonists and decrease unwanted MOR-related side effects [5].
Figure 1. Design of new cyclic biphalin analogues
Biphalin (Fig. 1) is a homodimer of an enkephalin-based tetrapeptide linked by hydrazine that
shows high binding affinities in the nanomolar range at the MOR and DOR, and potent
antinociceptive effects with promising bioavailability including potential blood brain barrier
(BBB) penetration [6-10]. For these reasons, numerous structure-activity relationship (SAR)
studies have been performed on biphalin to develop efficacious therapeutics for the treatment of
pain, but most studies have been focused on modifications of the C-terminal linker and 2,2′-
and/or 4,4′-amino acid residues [9,11-13]. Peptide cyclization is a very efficient tool to improve
receptor specificity (lower off-target toxicity), potency, and efficacy of peptides along with
proteolytic stability and bioavailability by limiting the peptide’s dynamic nature. Limiting
conformational variants by constraints induced by formation of covalent bonds such as lactones,
lactams, and disulfides, enhances their ability to interact with target receptors. Despite the
benefits of incorporating cycles into peptides, few attempts have been made to cyclize biphalin’s
structure [9,14-17]. In those cyclic biphalin studies, L-chirality of Cys and Pen residues at

positions 2 and 2′ resulted in a significant loss in affinity and efficacy at the opioid receptors
(OR), whereas D-chirality enhanced affinity and efficacy, similar to previous SAR studies on
linear analogues [15,16].
Scheme 1. Synthesis of cyclic biphalin analogues 1-3.
Aaa: Cys for 1 & 2, Pen for 3; Bbb: Tyr for 1 & 3; Dmt for 2. i) 2.2 equiv Bop/2.2 equiv
HOBt/4.4 equiv NMM, 0 oC for 30 min & rt for 2-4 h ii) 95% TFA/5% TIS, 0 oC for 20-30 min
On the basis of these data, we sought to design and investigate a series of cyclic biphalin
derivatives of which the homodimeric opioid pharmacophores are linked through a flexible
cystamine or piperazine and 2,2′ positions are utilized for cyclization via a disulfide bond
between DPen or DCys residues. Compared to constrained aromatic linkers, the flexible
piperazine linker was shown to be more potent at the MOR and DOR, and therefore, another
flexible linker, cystamine, was also chosen and utilized in this study. Additionally, Tyr residues
at positions 1,1′ were substituted with 2,6-dimethyl-tyrosine (Dmt) residues in an effort to

increase potency and efficacy at the ORs [18,19]. As a result, a series of nonselective
MOR/DOR/κ-opioid receptor (KOR) agonists were designed and synthesized (Fig. 1).
2. Results
The designed cyclic biphalin analogues were prepared by stepwise liquid phase peptide synthesis
using the Nα-Boc-chemistry approach (Scheme 1, Table 1). During the stepwise chain
elongations, intermediate peptides were isolated by routine precipitation using common organic
solvents, typically diethyl ether. A work-up process using basic (5% NaHCO3) and subsequent
acidic aqueous solutions (5% citric acid) removed excess amounts of unreacted Nα-Boc-amino
acids and coupling reagents from the reaction mixture and allowed intermediate peptides to be
isolated by via precipitation. Cyclization of Acm-protected linear hexapeptides were performed
using an I2 solution prior to Nα-Boc-group deprotection due to sluggishness caused by Nα-free
amino groups. After cyclization, the cyclic Nα-Boc-hexapeptides were deprotected and coupled
with Nα-Boc-Tyr-OH or Nα-Boc-Dmt-OH. By following the synthetic scheme, we were able to
accelerate disulfide bond formation and avoid serious side reactions caused by iodination of
tyrosine or Dmt residues. In our first trial to cyclize a linear octapeptide using I2, the iodinated
cyclic octapeptide was obtained as a major product. To avoid the unwanted side reaction, an Nα-
Boc-protected linear hexapeptide was cyclized first, and then coupled with Nα-Boc-Tyr-OH or
Nα-Boc-Dmt-OH. After the final chain elongation and deprotection, crude products were isolated
by preparative RP-HPLC to afford more than 98% purity of cyclic analogues 1 - 5 in overall 20-
40% yields.
To evaluate an analogue’s biological activity, in vitro binding and functional assays were
performed at the MOR, DOR, and KOR. Ki determinations, receptor binding profiles, and
agonist functional data (EC50 and Emax) were generously provided by the National Institute of
Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2013-00017-C
(NIMH PDSP) [20-22].
Table 1. Analytical Data of Cyclic Biphalin Analogues
(M + H)+a
HPLCb
tR (min)
17.7
Structure
Molecular Formula
aLogPc
Calculated
1025.4014
1081.4640
1081.4640
1091.3612
1147.4238
Observed
c2,2′(Tyr-DCys-Gly-Phe)2-piperazine
c2,2′(Dmt-DCys-Gly-Phe)2-piperazine
c2,2′(Tyr-DPen-Gly-Phe)2-piperazine
c2,2′(Tyr-DCys-Gly-Phe)2-cystamine
c2,2′(Dmt-DCys-Gly-Phe)2-cystamine
C50H60N10O10S2
C54H68N10O10S2
C54H68N10O10S2
C50H62N10O10S4
C54H70N10O10S4
1025.40081
1081.46647
1081.46206
1091.35949
1147.42226
1.62
1.92
2.56
1.68
2.11
1
2
3
4
5
18.3
18.7
20.4
21.2
aFAB-MS (JEOL Hx110 sector instrument) or MALDI-TOF (Brucker Ultraflex III).
bPerformed on a Hewlett Packard 1100 [C-18, Vydac, 4.6 mm x 250 mm x 5 µm, 10-100% acetonitrile containing 0.1% TFA within 45 min, 1
mL/min].

chttp://www.vcclab.org/lab/alogps/ [23].

Table 2. Binding Data of Ligands 1-5a
MOR [3H]DAMGOb
DOR [3H]DADLEc
KOR [3H]U69593d
pKi
Ki (nM)
8.9
pKi
Ki (nM)
27
pKi
Ki (nM)
19
8.05 ± 0.07
8.60 ± 0.08
8.54 ± 0.07
8.63 ± 0.07
9.57 ± 0.07
7.6 ± 0.1
8.7 ± 0.1
8.7 ± 0.1
8.4 ± 0.2
9.3 ± 0.1
7.73 ± 0.08
8.31 ± 0.04
8.25 ± 0.04
7.76 ± 0.05
9.06 ± 0.05
1
2.5
2
4.9
2
2.9
1.9
5.6
3
2.4
4.1
18
4
0.27
12 + 2
0.46
4.6 + 0.2
0.87
270 + 15
5
Biphalin6
616
5.2 + 0.5
1.9 + 0.2
257 + 25
aMembrane preparations were made from transient HEK cells expressing the appropriate receptor.
bKd = 1.73 ± 0.14 nM.
cKd = 1.85 ± 0.15 nM.
dKd = 1.07 ± 0.10 nM. 6: c2,2′(Tyr-DPen-Gly-Phe-NH-)2.

Table 3. Functional Data of Ligands 1 - 5: Agonist Modea
MOR
EC50 (nM)
513
DOR
EC50 (nM)
245
KOR
EC50 (nM)
339
pEC50
Emax (%)b
54
pEC50
Emax (%)c
77
pEC50
Emax (%)d
6.29 + 0.08
6.95 + 0.05
6.72 + 0.09
7.06 + 0.09
8.46 + 0.14
6.61 + 0.86
7.93 + 0.06
6.57 + 0.04
7.57 + 0.06
8.84 + 0.15
6.47 + 0.06
7.20 + 0.05
6.57 + 0.15
6.87 + 0.08
7.87 + 0.12
92
82
62
99
98
1
2
3
4
5
112
58
117
90
63.1
191
86
269
206
91
269
87.1
90
26.9
135
3.47
119
1.45
106
13.5
aMOR, DOR, and KOR expressed from HEK cells.
bCompared to DAMGO (pEC50 = 8.94).
cCompared to DADLE (pEC50 = 7.36).
dCompared to Salvinorin A (pEC50 = 9.43).

Binding affinities at the ORs were determined for ligands 1 - 5 using membranes prepared from
transient human embryonic kidney (HEK) cells. Competition binding assays were performed
against radiolabeled ligands [3H]DAMGO, [3H]DADLE, and [3H]U69593 for MOR, DOR, and
KOR, respectively (Table 2). In these assays, interestingly, all ligands showed balanced binding
affinities (selectivity < 8-fold) at the three subtype receptors due to enhanced KOR affinity (Ki:
0.87 - 19 nM). Biphalin interacts with MOR and DOR strongly but not with KOR (Ki = 270
nM), and its cyclic analogue 6 with a disulfide bond at positions 2 and 2’ was shown to retain the
same biological profile as biphalin [16]. Based on this previous SAR, the enhanced KOR binding
affinity is considered to be attributed to linker modifications from a hydrazine to a piperazine or
a cystamine.
Overall, ligands linked via a cystamine (4 and 5), which is a more flexible and dynamic motif,
showed slight affinity improvements relative to ligands with a piperazine linker (1 and 2).
Modification of Tyr residues at positions 1 and 1′ with Dmt also improved binding affinities at
the ORs in ligands 2 and 5. Ligand 3 substituted DPen for DCys, a more constrained residue due
to the presence of geminal methyl groups on Cβ, and enhanced DOR binding affinity more so
than that of MOR and KOR (14-fold at DOR vs 3-fold for both MOR and KOR). It was shown
that a bulky disulfide bond and the geminal methyl groups of a Pen residue limit backbone
flexibility and increase DOR activity in DPDPE. Ligand 3 might resemble the DPDPE structure
and thus its increased DOR activity. It turned out that ligand 5 with Dmt at positions 1 and 1′
with a cystamine linker is the most potent lead, which displayed a subnanomolar range of
binding affinities at the three ORs.
Opioid functional activity was explored by employing a split luciferase cAMP assay using HEK
cells that stably express the respective OR (Table 3). For agonist modes, efficacies were
compared to DAMGO, DADLE, and Salvinorin A at MOR, DOR, and KOR, respectively.
Antagonist mode was run using naltrexone, naltrindole, and GNTI as reference ligands for the
MOR, DOR, and KOR, respectively. Ligands 1 – 5 did not show antagonist activities at the ORs.
Overall, ligands’ functional activities correlated with their binding affinities at the ORs, and most
ligands exhibited high efficacy at the MOR, DOR, and KOR. Ligand 5, which showed binding
affinities in the subnanomolar range at the ORs, was the most potent MOR/DOR/KOR agonist
with the same high efficacies (Emax=119%, 106%, and 98% for MOR, DOR, and KOR,
respectively) relative to the reference ligands. In these functional assays, it was again observed
that the linkers piperazine and cystamine aided ligands 1-5 to interact with the KOR, and
therefore, all ligands except for 3 achieved high efficacies (Emax: 82 - 99%) relative to Salvinorin.
The ligands containing piperazine with DCys (1 and 2) exhibited lower efficacy at the MOR
compared to the DOR and KOR (Table 3). Ligand 3, which incorporated the DPen residues as a
bridge, turned out to be an extremely efficacious agonist at the DOR relative to DADLE (Emax
=
206%). Despite encouraging Emax results, EC50 values achieved low potency (240 nM). Ligands
4 and 5 gave more promising outcomes which had cystamine and DCys. Replacement of Tyr

residues with Dmt in ligand 5 resulted in the discovery of a potent MOR/DOR/KOR agonist with
a low nanomolar range of EC50 values (3.47 nM, 1.45 nM, and 13.5 nM at the MOR, DOR, and
KOR, respectively) and high efficacies (119%, 106%, and 98% at the MOR, DOR, and KOR,
respectively).
3. Discussion
A series of cyclic biphalin analogues 1 – 5 with a cystamine or piperazine linker at the C-
terminus was designed and synthesized by solution phase synthesis using Boc-chemistry, which
resulted in the discovery of potent MOR/DOR/KOR agonists. Both disulfide bridge and linker
variants resulted in mixed opioid agonist profiles due to additional KOR agonist activity. It
appeared that ligands which employed cystamine as a linker afforded more potent agonist
profiles, whereas those with piperazine had attenuated effects and were non-selective. The series
showed mostly nanomolar range of high affinities at KOR coupled with high agonist efficacies
and mixed potencies. Considering that biphalin is MOR/DOR selective and our ligands
demonstrated non-selective affinities and activities across all ORs, the recovery of KOR
interaction may be attributed to the termini modifications to a cyclic structure. Based on the
message-address concept of enkephalin, the N-terminal Tyr or Dmt residues are in the message
region which correlate with the ligand’s efficacy, and the C-terminal linker is a part of the
address region which modifies ligand selectivity [23,24]. It has been suggested that dynorphin,
an endogenous KOR agonist, accomplishes KOR selectivity through several basic amino acids at
the C-terminus, and enkephalin, a DOR agonist, can be modified at the C-terminus to acquire
KOR activity and selectivity. Our SAR results seemed to support the explanation. Based on the
biological profile of hydrazine linked cyclic analogue 6, improved KOR activities were
considered due to cystamine and piperazine linkers in the address region. Apparently, the more
flexible linear linker cystamine (4 and 5), was preferred to piperazine (1, 2, and 3) for all three
subtype ORs. Introduction of the constrained Tyr derivative Dmt at positions 1 and 1′ enhanced
opioid activities in ligands 2 and 5 as demonstrated in many previous studies. Ligand 5 was the
most potent, non-selective agonist with high efficacies and affinities at the MOR, DOR, and
KOR, but it is worthwhile to note that ligand 3 with bulky DPen residues at positions 2 and 2′
showed the highest agonist efficacy at the DOR. This suggests DPen’s ability to selectively
augment receptor interaction at the DOR as shown in DPDPE.
4. Material and methods
All amino acid derivatives except Boc-Dmt-OH (RSP Amino Acid Analogues, Inc,) were
purchased from NovaBiochem or AAPPTec. Coupling reactions were monitored via TLC with
the following mobile phase: CHCl3/MeOH/AcOH = 90:10:1 (vol/vol/vol) with ninhydrin spray
used for detection. Analytical RP-HPLC was performed on a Hewlett Packard 1100 [C-18,
Vydac, 4.6 mm x 250 mm, 5 m, 10-100% of acetonitrile containing 0.1% TFA within 45 min, 1

mL/min] and preparative RP-HPLC on a Hewlett-Packard 1100 [C-18, Microsorb-MV, 10 mm x
250 mm, 10 µm]. Mass spectra were obtained using FAB-MS (JEOL HX110 sector instrument)
or MALDI-TOF. aLogP values were calculated by ALOGPS 2.1 program [25,26].
Ligands 1 - 5 were prepared via stepwise synthesis using Nα-Boc chemistry starting from either
piperazine (1 - 3) or cystamine (4 and 5). Nα-Boc-Phe-OH (2.2 equiv), BOP (2.2 equiv), and
HOBt (2.2 equiv) were dissolved in DMF and cooled in an ice bath for 10 min. NMM (4.4
equiv) was added dropwise to the reaction mixture and warmed to room temperature with
additional stirring for 2-4 hours. After confirming the disappearance of the Nα-amino group by
TLC, the mixture was diluted with 10-fold volume of EtOAc and washed with 5% NaHCO3
(weight/vol) three times, 5% citric acid (weight/vol) two times, brine, and water, consecutively.
The combined organic layers were dried under anhydrous Na2SO4 and filtered. The filtrate was
concentrated under reduced pressure and the residual oil was triturated with diethyl ether to
afford the corresponding Nα-Boc-peptide as a white powder. The Nα-Boc group was deprotected
by a TFA solution containing 5% TIS (vol/vol) at 0 °C for 20 - 30 minutes. Deprotection was
monitored by TLC. After completion of chain elongation to the bridging amino acid residue
(Boc-DPen(Acm)-OH or Boc-DCys(Acm)-OH), the Nα-Boc protected linear hexapeptides were
subjected to a disulfide bond formation using I2. A 0.06 M iodine solution was added dropwise to
a linear hexapeptide solution (0.1 g peptide in 1 L of 20% MeOH in water (vol/vol)) until a light-
yellow color persisted and left overnight. The progress of disulfide bond formation was
monitored by analytical RP-HPLC. After completion of the disulfide bond, the reaction mixture
was quenched using ascorbic acid and evaporated under reduced pressure. The concentrated
reaction mixture was triturated with diethyl ether to give a Nα-Boc-protected cyclic hexapeptide
as a powder. The formed powder was subjected to Nα-Boc-deprotection and final coupling with
Nα-Boc-Tyr-OH or Nα-Boc-Dmt-OH. Final crude cyclic peptides were isolated by preparative
RP-HPLC (20-70% ACN within 25 mins) to afford pure cyclic biphalin analogues as white
powders in overall yields of 20 - 40%. Purity was validated as >98% by analytical HPLC (10-
100% ACN in water containing 0.1 % TFA (vol/vol) in 45 min).
Radioligand binding competition assays and the split luciferase cAMP assays were performed in
the Roth lab through the NIMH Psychoactive Drug Screening Program. Experimental details can
be found at the PDSP website [20-22].
5. Conclusions
Cyclization of biphalin, a MOR/DOR agonist, along with a linker modification, resulted in the
discovery of a highly potent MOR/DOR/KOR agonist (5), which is considered to have potential
synergistic effects between the three opioid receptors. Our studies demonstrated that the
replacement of Tyr residues at positions 1 and 1′, a common message region of the ORs, with
Dmt residues, improved opioid potency at the ORs and linker modification at the C-terminus, the
address region of the ORs, resulted in an increase in KOR activity. A synergistic analgesic effect

is advantageous due to reducing necessary dosages to relieve pain and therefore attenuate opioid
related side effects. However, utilizing KOR agonists to relieve pain is not typically suggested
because of serious side effects such as dysphoria. Many studies evidenced that biased KOR
agonists without β-arrestin recruitment can avoid such unwanted side effects. Since dysphoria is
considered a side effect corresponding to the central nervous system, some studies also suggested
that KOR agonists that do not penetrate the BBB but instead act with the peripheral nervous system
could be another solution. Therefore, even with a concern regarding the exploitation of KOR
agonists, having a balanced MOR/DOR/KOR agonist, if one can localize its action to the PNS, would
benefit through efficient pain relief by dose reduction and thus avoiding serious side effects
associated with the other ORs. From this point of view, ligand 5 exhibited therapeutic potential and
further studies including in vivo tests are worthwhile.
Conflicts of Interest
The authors declare no conflict of interest.
Acknowledgments
This work has been supported by grants from the Proof of Concept program (UA15-178) of the
University of Arizona and U.S. Public Health Services, NIH, and NIDA (P01DA006248).
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22. The NIMH PDSP is directed by Bryan L. Roth at the University of North Carolina at
Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda MD, USA.
https://pdspdb.unc.edu/pdspWeb/
23. Takemori, A.E.; Portoghese, P.S. Annu. Rev. Pharmacol. Toxicol. 1992, 32, 239-269.
24. Portoghese, P.S.; Sultana, M.; Takemori, A.E. J. Med. Chem. 1990, 33, 1714-1720.
25. Tetko, I. V.; Tanchuk, V. Y. J. Chem. Inf. Comput. Sci. 2002, 42, 1136-45.
26. http://www.vcclab.org/lab/alogps/