Synthesis and biological evaluation of compact, conformationally constrained bifunctional opioid agonist - Neurokinin-1 antagonist peptidomimetics

Article abstract of DOI:10.1016/j.ejmech.2014.12.033

A reported mixed opioid agonist - neurokinin 1 receptor (NK1R) antagonist 4 (Dmt-D-Arg-Aba-Gly-(3′,5′-(CF₃)₂)NMe-benzyl) was modified to identify important features in both pharmacophores. The new dual ligands were tested in vitro and subsequently two compounds (lead structure 4 and one of the new analogues 22, Dmt-D-Arg-Aba-β-Ala-NMe-Bn) were selected for in vivo behavioural assays, which were conducted in acute (tail-flick) and neuropathic pain models (cold plate and von Frey) in rats. Compared to the parent opioid compound 33 (without NK1R pharmacophore), hybrid 22 was more active in the neuropathic pain models. Attenuation of neuropathic pain emerged from NK1R antagonism as demonstrated by the pure NK1R antagonist 6. Surprisingly, despite a lower in vitro activity at NK1R in comparison with 4, compound 22 was more active in the neuropathic pain models. Although potent analgesic effects were observed for 4 and 22, upon chronic administration, both manifested a tolerance profile similar to that of morphine and cross tolerance with morphine in a neuropathic pain model in rat.

Full text of DOI:10.1016/j.ejmech.2014.12.033

European Journal of Medicinal Chemistry 92 (2015) 64e77
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of compact, conformationally
constrained bifunctional opioid agonist e Neurokinin-1 antagonist
peptidomimetics
,
Karel Guillemyn a, Patrycia Kleczkowska d g, Anna Lesniak d, Jolanta Dyniewicz d,
Olivier Van der Poorten a, Isabelle Van den Eynde a, Attila Keresztes b, Eva Varga b,
Josephine Lai b, Frank Porreca b, Nga N. Chung c, Carole Lemieux c, Joanna Mika e,
Ewelina Rojewska e, Wioletta Makuch e, Joost Van Duppen f, Barbara Przewlocka e,
f
d
c
ꢀ a
Jozef Vanden Broeck , Andrzej W. Lipkowski , Peter W. Schiller , Dirk Tourwe ,
,
*
Steven Ballet a
a Laboratory of Organic Chemistry, Departments of Chemistry and Bio-engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
b Department of Pharmacology, University of Arizona, 1501 N. Campbell Ave, Tucson AZ, 85724-5050, USA
c Department of Chemical Biology and Peptide Research, Clinical Research Institute, 110 Avenue Des Pins Ouest, Montreal, QC, H2W1R7, Canada
d Neuropeptide Laboratory, Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Street, PL 02-106, Warsaw, Poland
e
ꢀ
Department of Pain Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, PL 31-343, Krakow, Poland
f Animal Physiology and Neurobiology Department, University of Leuven (KU Leuven), Naamsestraat 59, 3000 Leuven, Belgium
g Department of Pharmacodynamics, Centre for Preclinical Research and Technology (CePT), Medical University of Warsaw, Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A reported mixed opioid agonist e neurokinin 1 receptor (NK1R) antagonist 4 (Dmt-D-Arg-Aba-Gly-
(30,50-(CF3)2)NMe-benzyl) was modified to identify important features in both pharmacophores. The new
dual ligands were tested in vitro and subsequently two compounds (lead structure 4 and one of the new
Received 6 October 2014
Received in revised form
26 November 2014
Accepted 19 December 2014
Available online 19 December 2014
analogues 22, Dmt-D-Arg-Aba-b-Ala-NMe-Bn) were selected for in vivo behavioural assays, which were
conducted in acute (tail-flick) and neuropathic pain models (cold plate and von Frey) in rats. Compared
to the parent opioid compound 33 (without NK1R pharmacophore), hybrid 22 was more active in the
neuropathic pain models. Attenuation of neuropathic pain emerged from NK1R antagonism as demon-
strated by the pure NK1R antagonist 6. Surprisingly, despite a lower in vitro activity at NK1R in com-
parison with 4, compound 22 was more active in the neuropathic pain models. Although potent analgesic
effects were observed for 4 and 22, upon chronic administration, both manifested a tolerance profile
similar to that of morphine and cross tolerance with morphine in a neuropathic pain model in rat.
© 2014 Elsevier Masson SAS. All rights reserved.
This paper is dedicated to the career of
Professor Andrzej W. Lipkowski.
Keywords:
Hybrid peptides
Opioid agonism
NK1R antagonism
Acute pain
Neuropathic pain
Tolerance
Abbreviations: %MPE, percent of maximal possible effect; Aba, 4-amino-2-benzazepinone scaffold; CCI, chronic constriction injury; CNS, central nervous system; D-Cit,
citrulline; DIC, N,N0-dicyclohexylcarbodiimide; DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; DML, designed multiple ligand; Dmt, 20,60-dimethyl-
-tyrosine;
DOR, opioid receptor; GPI, guinea pig ileum; HOBt, 1-hydroxybenzotriazole; i-Bu, isobutyl; i.p., intraperitoneal; i.t., intrathecal; i.v., intravenous; MOR, opioid receptor;
D-
L
d
m
MPE, maximal possible effect; MVD, mouse vas deferens; NK1, neurokinin-1; NMM, N-methylmorpholine; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; RP-
HPLC, reverse phase high pressure liquid chromatography; SP, substance P; SPPS, solid phase peptide synthesis; TBTU, O-(Benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
tetrafluoroborate; TES, triethylsilane; TMOF, trimethyl orthoformate.
* Corresponding author. Laboratory of Organic Chemistry, Departments of Chemistry and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels,
Belgium.
E-mail addresses: kguillem@vub.ac.be (K. Guillemyn), hazufiel@wp.pl (P. Kleczkowska), alesniak@imdik.pan.pl (A. Lesniak), jolantadyniewicz@gmail.com (J. Dyniewicz),
Olivier.Van.Der.Poorten@vub.ac.be (O. Van der Poorten), vandeneynde.isabelle@gmail.com (I. Van den Eynde), keratti@freemail.hu (A. Keresztes), evarga@email.arizona.edu
(E. Varga), lai@email.arizona.edu (J. Lai), frankp@email.arizona.edu (F. Porreca), NgocNga.chung@ircm.qc.ca (N.N. Chung), carole-lemieux@hotmail.com (C. Lemieux),
joamika@if-pan.krakow.pl (J. Mika), rojewska@if-pan.krakow.pl (E. Rojewska), makuch@if-pan.krakow.pl (W. Makuch), Joost.VanDuppen@bio.kuleuven.be (J. Van Duppen),
barbara.przewlocka@gmail.com (B. Przewlocka), Jozef.VandenBroeck@bio.kuleuven.be (J. Vanden Broeck), andrzej@lipkowski.org (A.W. Lipkowski), Peter.Schiller@ircm.qc.
ꢀ
ca (P.W. Schiller), datourwe@vub.ac.be (D. Tourwe), sballet@vub.ac.be (S. Ballet).
http://dx.doi.org/10.1016/j.ejmech.2014.12.033
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
65
1. Introduction
Taking earlier work on mixed opioid-NK1R peptide DMLs into
consideration [17e20], a chimeric opioid-NK1 peptidomimetic 4
(Fig. 1) was previously reported by our group [24]. The opioid
Morphine has been the prime analgesic for the treatment of
severe to moderate pain for centuries. Next to morphine, opioid
ligands such as the fentanyl family of painkillers are commonly
used in a clinical context. Despite the severe drawbacks that are
associated with these analgesics, they remain in use to date. When
opioid ligands are administered for a longer period of time, side
effects such as, for example, constipation, physical dependence and
analgesic tolerance emerge [1]. Moreover, opioids have less anal-
gesic efficacy in neuropathic pain, a pathology which is considered
to be a major healthcare burden [2e6]. Neuropathic pain can be
caused by a damaged nerve in the nervous system, be the result of
trauma, an infection, or diabetes [7], and it is characterized by an
increased sensitivity to non-noxious and noxious stimuli [8,9].
Since neuropathic pain and opioid tolerance both share the feature
of diminished morphine analgesia, a common underlying mecha-
nism could be suggested.
One explanation for the lack of long-duration opioid efficiency is
increased release and expression of certain endogenous ligands and
receptors [10,11]. Being part of a larger biological network, activa-
tion of the opioid system is compensated for by the production of
“anti-opioids” or pain-enhancing ligands, such as cholecystokinin
and substance P. These neuroplastic changes in the CNS cannot be
counteracted by the currently used treatments.
subunit in 4 is derived from the
m opioid receptor lead peptide
Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]DALDA) 5 [25], and contains a
constrained Aba (4-amino-2-benzazepinone) [26e28] moiety in
position 3. As this conformationally constrained amino acid was
also the core structure of a newly developed NK1 antagonist [24],
Ac-Aba-Gly-NMe-30,50e(CF3)2eBn (6) (corresponding to the blue
part in structure 4, Fig. 1), the combination of both ligands led to
the hybrid structure Dmt-D-Arg-Aba-Gly-NMe-30,50e(CF3)2eBn
4, a DML with overlapping pharmacophores. The in vitro bio-
logical evaluation of 4 showed that combination of the two
components gratifyingly resulted in good binding affinity for
both opioid and NK1R receptors [24]. Furthermore, the com-
pound showed antinociceptive activity after intravenous
administration in vivo, and hence the hybrid structure proved,
similarly to the parent opioid sequence Dmt-D-Arg-Aba-Gly-NH2
7 [29], capable of crossing the bloodebrain barrier (BBB). Un-
fortunately, hybrid 4 still produced analgesic tolerance in naive
animals [29]. In contrast, Vanderah and coworkers reported that
chronic administration of a peptidic DML with a similar dual
activity profile, H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-30,50-
Bn(CF3)2 8, was able to suppress antihyperalgesic tolerance [30].
We suggested that this discrepancy in results could be due to the
different pain models used in both studies. Whereas Vanderah
et al. [30] utilized a hyperalgesia animal model, our published
findings resulted from an acute pain model (tail-flick) [29]. Given
the promising results of Vanderah et al., we pursued our efforts
to develop a CNS active peptidomimetic able to attenuate opioid-
related tolerance.
For this reason, and as an alternative to the co-administration of
separate drugs, designed multiple ligands (DMLs, single chemical
entities that modulate multiple targets) with both opioid and non-
opioid activity were developed [12e14]. Among other dual activity
ligands [12], hybrid peptides bearing both an opioid and NK1R
pharmacophore (e.g. compounds 1e3 in Fig. 1) were designed by
the teams of Lipkowski [15e18] and Hruby [19,20].
In this work we report structural modifications of hybrid lead
structure 4. Various modifications (Fig. 2) were performed to: i)
increase opioid activity, ii) determine the key features for NK1R
binding and antagonism, and iii) attempt to reduce the molecular
weight of the lead structure.
In these DMLs the opioid part induces the well-established
analgesic effect that is characteristic of opioid ligands, whereas
antagonism at the NK1 receptors blocks the signals induced by an
endogenous neurotransmitter, substance P (SP), one of the prono-
ciceptive peptides involved in pain signalling [21,22]. Since pro-
longed pain states and sustained opioid administration lead to
increased secretion of SP and enhanced expression of NK1 re-
ceptors, blockage of NK1R could potentially counteract the pain-
enhancing effect induced by SP and its receptor, and eventually
lead to prolonged antinociceptive efficacy. In addition, Gonzalez
and coworkers showed that the selective neurokinin-1 receptor
antagonist CI-1021 possessed a superior side effect profile, as
compared to morphine, and may have a therapeutic use for the
treatment of neuropathic pain [23]. All of the above findings sup-
port the design and preparation of DMLs with a dual opioid ago-
nists and neurokinin-1 antagonist profile for the development of
more efficacious drugs for the treatment of acute and neuropathic
pain.
2. Results and discussion
2.1. Synthesis
Lead structure 4 was previously prepared in two main steps: a
standard solid phase synthesis using the preassembled Fmoc-Aba-
Gly-OH dipeptide [31], followed by coupling of a protected tetra-
peptide precursor to NMe-30,50-trifluoromethyl benzylamine in
solution [24].
In this work, a direct solid phase assembly of the Aba building
block was developed. This strategy avoids a separate solution phase
synthesis of the Aba-Gly and Aba-b-Ala constrained dipeptides 13
(n ¼ 1 or 2, Scheme 1).
Fig. 1. Selected examples of opioid agonist e NK1 antagonist DMLs.

66
K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
The Aba-containing peptides were prepared using Fmoc-Gly-OH
or Fmoc- -Ala-OH linked to 2-chlorotrityl resin as the solid sup-
and MVD IC50 (4) 43.3 nM) at both MOR and DOR. Because of the
decreased binding affinity and reduced agonist potency, we
decided to replace Gly4 by a
-alanine residue [34]. The additional
methylene, present in -Ala, would shift the bulky C-terminal
benzyl group away from the opioid pharmacophore, and poten-
tially lower the steric hindrance upon receptor binding. Gratify-
ingly, the substitution improved both MOR and DOR binding 5-fold
(21, Table 1). Retained DOR binding affinity in the low nanomolar
range would be in favour of the proposed synergy of mixed MOR e
DOR activity for enhanced in vivo antinociception [35e37].
b
port, via the pathway depicted in Scheme 1. After removal of the
Fmoc protective group, a reductive amination with phthaloyl-
protected ortho-formyl phenylalanine 11 in the presence of
NaBH3CN was performed. Aldehyde 11 was obtained by reduction
of the corresponding ortho-cyano analogue following a literature
procedure [32]. The desired secondary amine was formed to a high
extent after 30 min, according to the Kaiser test. Longer reaction
times led to overalkylation. Cyclizations with TBTU as the activating
agent yielded the solid-supported aminobenzazepinones 13. To
allow further elongation of the peptide, the resin-bound 13 was
treated with hydrazine monohydrate for phthaloyl deprotection.
The remaining amino acids were coupled by standard SPPS
methods. More specifically, the coupling of Boc-Dmt-OH was per-
formed using DIC/HOBt as the coupling mixture to avoid side re-
actions, caused by the unprotected phenolic group, which occur
upon use of TBTU/DIPEA mixtures. The protected peptide acids 16
were obtained after mild acidic cleavage (1% TFA in CH2Cl2) from
the solid support. They were of sufficient purity to allow a direct
coupling with the different benzylamines in solution and thus did
not require intermediate purification. The synthesis of N-methyl
and N-isobutyl-30,50-bis(trifluoromethyl)benzylamines (18) could
be achieved by reacting methyl- or isobutylamine with commer-
cially available benzylchloride or 30,50-bis(trifluoromethyl)benzyl
chloride following a literature procedure [33]. To reduce formation
of the double alkylated product, an excess of the amine was used.
The crude amines were purified by flash chromatography to afford
the desired benzylamines in moderate yield (43e48%). Coupling of
the protected tetrapeptide analogues to the set of amines was
performed using a mixture of DIC/HOBt/DIPEA. Final deprotection
of the N-terminal Boc group and Pbf side chain protective group
was achieved with the cleavage cocktail TFA/TES/H2O (95/2.5/2.5).
Precipitation in cold ether, followed by preparative RP-HPLC puri-
fication and lyophilisation yielded pure (>95%) peptide analogues
20.
b
b
Whereas the improvement in DOR binding affinity was
translated into improved functional activity as determined in the
MVD tissue bioassay (MVD IC50 (21) 6.20 nM), in the GPI assay,
representative of MOR activation, and in comparison with 4, a
slightly lower IC50 value (11.3 nM) was determined for 21. How-
ever, the drop in NK1R binding and antagonism potency was
more dramatic (Table 1). Insertion of the additional methylene
unit proved to be detrimental for recognition at the NK1R re-
ceptor. In small peptidomimetic NK1R antagonists the two crucial
aromatic groups of the key bis-aryl motif are in close proximity of
each other [38]. The introduction of a methylene unit in 21 makes
the linker between these groups more flexible, and hence they
can move away from each other, potentially lowering binding
affinity at NK1R. Here, this modification induced a 120-fold loss in
NK1R binding and an almost two log unit drop in antagonism
(pA2 (4) 7.80 / pA2 (21) 6.04). When the trifluoromethyl groups
in 21 were removed in an attempt to lower the molecular weight
of the mixed ligands resulting in compound 22, picomolar MOR
binding was preserved (Kmi 0.08 nM), DOR binding increased by a
factor of 10 and in vitro agonism was improved 6- and 3-fold at
MOR and DOR, respectively. Removal of the CF3 groups also
improved NK1R binding affinity approximately 4 times. The in-
crease was also apparent in the pA2 values (pA2 (21) 6.04 / pA2
(22) 6.44). In a previous study [39], we demonstrated that D-Arg2
could be replaced by D-Cit, a modification that removes one
positive charge in the peptide, without loss of BBB permeability.
Hence, this modification was also carried out in the present study,
to give 23. Comparison with 22 showed that this change was well
tolerated both at the opioid receptors and at the NK1 receptor.
However, when carrying out the two modifications simulta-
neously (CF3 group removal and D-Arg/D-Cit), a dramatic loss in
NK1R binding was observed (Ki 3000 nM for 24). Again, the
opioid system was more tolerant towards this double modifica-
tion. The promising results obtained with compound 22 led to the
reinvestigation of the bis-trifluoromethyl removal in the Gly4-
bearing tetrapeptide. In compounds 25 and 26 this removal was
again well-tolerated at both MOR and DOR, but dramatically
decreased NK1R binding affinity. The N-methyl amide function at
the C-terminus was also confirmed to be of importance. Whereas
secondary amides exist almost exclusively in the trans confor-
mation, tertiary amides contain an appreciable amount of the cis
conformer, which may be the conformer that is binding to the
receptor. The cis/trans ratio of the amide bond between residue 4
and the C-terminal benzylamine moiety in lead compound 4 was
investigated by NMR analysis and was estimated to be 35:65.
Results obtained by molecular modelling also confirmed one of
the three lowest energy conformations in the NK1R pharmaco-
phore to adopt the cis amide geometry [24], and hence this
orientation could be important for NK1R activity. In an attempt to
increase the cis/trans ratio, a N-iBu group was used instead of the
N-Me motif. Although the bulkiness of the iBu group was ex-
pected to promote the cis amide bond conformation, the biolog-
ical data of compounds 27 to 30 clearly show that the presence of
the iBu group did not enhance NK1R affinity. Only one of these
ligands, compound 27, showed moderate nanomolar range NK1R
2.2. In vitro biological evaluation
In designed bifunctional ligands with high framework overlap,
the risk of encountering undesired steric encumbrance increases.
While highly merged structures take advantage of structural
commonalities in the starting compounds e they present multiple
ligands with a lower molecular weight e the close proximity of the
two pharmacophores can more readily lead to steric clashes during
the drug-target recognition event. This phenomenon was also
apparent for hybrid 4 (Table 1). Compared to the opioid parent
compound 7, creation of the opioid-NK1R hybrid ligand 4 resulted
in a significantly reduced binding affinity (Kim (7) 0.15 nM and Kid
(7) 0.60 nM / Kim (4) 0.42 nM and Kid (4) 10.4 nM) and activity (GPI
IC50 (7) 0.32 nM and MVD IC50 (7) 0.42 nM / GPI IC50 (4) 8.51 nM
Fig. 2. Overview of the investigated structural modifications.

K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
67
Scheme 1. Solid phase assembly of the constrained opioid tetrapeptide and solution phase coupling of NK1R pharmacophoric benzyl group. Reaction conditions: a) 20% 4-
methylpiperidine in DMF, 5 þ 15 min: b) 2 eq. of 11 and 4 eq. NaCNBH3 in CH2Cl2/TMOF/0.5% AcOH, 30 min; c) 3 eq. TBTU in 0.4 M NMM in DMF, 3 h; d) 6 eq. NH2NH2.H2O in
DMF, 18 h; e) 3 eq. Fmoc-D-Arg(Pbf)-OH or Fmoc-D-Cit-OH, 3 eq. TBTU in 0.4 M NMM in DMF, 1.5 h; f) 20% 4-methylpiperidine in DMF, 5 þ 15 min; g) 2 eq. Boc-Dmt-OH, 2 eq. DIC/
HOBt in DMF, 2 h; h) 1% TFA in CH2Cl2, rt, 30 min; i) 5 eq. MeNH2, 1 eq. of 17, CH2Cl2, 50 ꢀC, 16 h; j) 2 eq. DIPEA, 1.5 eq. DIC/HOBt, 1.5 eq. of 18, 15 h; k) TFA/TES/H2O 95/2.5/2.5, 3 h; l)
preparative RP-HPLC purification.
Table 1
Opioid and NK1 receptor affinity and activity of all parent and hybrid compounds.
hNK1Rb
(Ki nM)
GPIc
MVDc
MORc
DORc
Selectivity
a
Compound number
Compound
pA2
(NK1R)
(IC50 nM)
(IC50 nM)
(Ki nM)
(Ki nM)
Ki DOR/Ki MOR
4
6
7
H-Dmt-D-Arg-Aba-Gly-NMe-30,50-(CF3)2-Bn
Ac-Aba-Gly-NMe-30,50-(CF3)2-Bn
H-Dmt-D-Arg-Aba-Gly-NH2
7.80
8.40
nd
6.04
6.44
6.27
nd
nd
nd
nd
nd
0.50
27.0
nd
59.7
13.0
34.7
3000
1530
6190
35
1020
743
308
734
16050
nd
8.51
nd
43.3
nd
0.42
nd
10.4
nd
25.0
nd
0.32
11.3
1.86
2.03
0.96
5.97
4.44
19.7d
P.Ae
nd
0.42
6.20
2.16
1.06
0.33
8.64
3.50
14.5
28.7
nd
0.15
0.08
0.08
0.37
0.28
0.09
0.14
nd
nd
nd
nd
0.62
0.10
1.34
0.60
2.14
0.28
0.55
0.48
2.35
0.62
nd
nd
nd
nd
1.7
4.0
25.3
3.5
1.5
1.7
24.8
4.4
nd
nd
nd
nd
2.7
43.4
12.7
21
22
23
24
25
26
27
28
29
30
31
32
33
H-Dmt-D-Arg-Aba-
H-Dmt-D-Arg-Aba-
b
b
-Ala-NMe-30,50-(CF3)2-Bn
-Ala-NMe-Bn
H-Dmt-D-Cit-Aba-
H-Dmt-D-Cit-Aba-
b
b
-Ala-NMe-30,50-(CF3)2-Bn
-Ala-NMe-Bn
H-Dmt-D-Arg-Aba-Gly-NMe-Bn
H-Dmt-D-Arg-Aba-Gly-NH-Bn
H-Dmt-D-Arg-Aba-Gly-NiBu-30,50-(CF3)2-Bn
H-Dmt-D-Arg-Aba-Gly-NiBu-Bn
H-Dmt-D-Arg-Aba-
H-Dmt-D-Arg-Aba-
b
b
-Ala-NiBu-30,50-(CF3)2-Bn
-Ala-NiBu-Bn
nd
nd
nd
nd
nd
nd
H-Dmt-D-Arg-Phe-Sar-NMe-30,50-(CF3)2-Bn
H-Dmt-D-Arg-Phe-Sar-NMe-Bn
2.95
3.10
0.80
2.31
7.76
0.24
4.43
17.0
H-Dmt-D-Arg-Aba-
b-Ala-NH2
nd
All data of the two hybrids which were tested in vivo are presented in bold.
a
The pA2 values were calculated using Schild's equation [40].
b
Inhibitory constants (Ki) of NK1 receptor ligands, measured for the receptor prototype [3H]-SP in the presence of hNK1-CHO membranes. Results are means of three
independent experiments. Binding data were calculated using the nonlinear regression/one site competition fitting options of the GraphPad Prism Software.
c
Values represent means of 3e4 experiments. The GPI functional assay is representative of MOR activation, whereas the MVD is a DOR receptor-representative assay.
Binding affinities of compounds for MOR and DOR opioid receptors were determined by displacement of [3H]DAMGO ([D-Ala2, NMePhe4, Gly-ol5]enkephalin) and [3H]DSLET
([D-Ser2, Leu5]enkephalin-Thr6) from rat brain membrane binding sites, respectively.
d
Partial agonist, Ke ¼ 12,7 nM (
m
antagonist).
e
Ke ¼ 5.91 nM (
m antagonist); nd: not determined.
binding affinity. Due to its rather limited opioid activity, this
compound was not investigated any further and the cis/trans ratio
was not determined experimentally.
Up to this point, the requirement of the conformational
constraint, imposed by the Aba moiety, was not verified. In the
‘open ring’ compounds 31 and 32, bearing a sarcosine in position
4, the ring opening was not crucial for opioid affinity and activity,
but led to a dramatic decrease in NK1R binding affinity. This could
be explained by the flexibility of the Phe3 side chain in the ring
opened compounds, allowing it to move away from the C-ter-
minal benzyl amide. In contrast, in the Aba containing peptides,
the methylene anchorage of the aromatic side chain to the
backbone orients this side chain to the C-terminus possibly
keeping the two aromatics in the bis-aryl motif in close proximity
of each other.

68
K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
2.3. Behavioural study in rats and mice
Among the new analogues the one with a most suited profile for
in vivo evaluation was judged to be hybrid 22. This DML has a
potent and quite balanced MOR and DOR binding affinity and
agonist potency profile (sub- and low nanomolar range, resp.), a
feature believed to be beneficial to opioid antinociception
[35e37,41,42], while still showing low nanomolar NK1R binding
affinity, and a pA2 value of 6.44 (i.e. 1.5 log unit loss in antagonism
potency, compared to lead compound 4). The profile of compound
22 differs from the lead structure in terms of opioid receptor
selectivity (Kdi /Kim (4) 25 vs. Kid/Kim (22) 3.5), opioid potency (IC50 GPI
(4) 8.51 nM and IC50 MVD (4) 43.3 nM vs. IC50 GPI (22) 1.86 nM and
IC50 MVD (22) 2.16 nM), NK1R binding (Ki (4) 0.5 nM vs. Ki (22)
13 nM), and NK1R antagonist potency (pA2 (4) 7.8 vs. pA2 (22) 6.4).
The in vivo evaluation of 4 in an acute pain model by the tail-flick
assay has been published previously [29]. From these results, it
could be concluded that lead peptide 4 was able to cross the BBB, as
Fig. 4. The time dependent effect of intravenous (i.v.) administration in mice of hybrid
22 (1.08; 10.8 mol/kg), its parent compound 33 (1.21 mol/kg), and morphine (M,
12.5 mol/kg) in the tail-flick assay (6e8 animals per group). Statistical analysis has
shown significant differences between 22 (10.8 mol/kg) and morphine (***p < 0.001);
between 22 (1.08 mol/kg) and morphine (##p < 0.01) as well as between 33 and
it showed
a potent antinociceptive effect after intravenous
m
m
m
administration.
m
m
morphine ($$ p < 0.01 and $$$ p < 0.001).
2.3.1. Acute pain evaluation in rats
The potencies of two new compounds (22 and 33) were
compared to the effect of the initial lead peptide 4 and its parent
opioid structure 7, NK-1 antagonist 6 and morphine in naïve rats
(Fig. 3). All tested compounds were administered i.t. at different
doses in naïve rats and the effect was measured by the tail-flick test
30 min after administration. Both hybrids (4 and 22) and their
parent compounds (7 and 33, resp.) increase the nociceptive
threshold. The antinociceptive potencies of the parent compounds
(7 and 33) are clearly much higher in comparison with those of the
two hybrids and morphine. In contrast, the effect of the parent NK-
1 antagonist 6 was very weak as compared to the hybrids and
opioid parent compounds in naïve animals. Moreover, in the same
test and after intravenous administration, structure 6 did not show
2.3.2. Acute pain in mice
When hybrid 22 and its parent opioid compound 33 were
evaluated after i.v. administration in mice, we observed that a
10.8
nociceptive response that is approximately three times higher than
the one of morphine at a slightly lower dose of 12.5 mol/kg (Fig. 4).
For comparison, when compound 4 was administered i.v. at a dose
of 3.8 mol/kg in a previous study, it showed an antinociceptive
mmol/kg dose of compound 22 gave way to a potent anti-
m
m
potency which was 3.7 times higher than i.p. administered
morphine (not shown) [29]. From these results it appears that both
compounds, hybrids 4 and 22, have a similar potency that is su-
perior to morphine. In contrast, parent peptide 33 still showed a
any activity at all at doses up to 1.21 mmol/kg (not shown), which
potent response at a dose of 1.21 mmol/kg, which largely exceeded
can be indicative of a low BBB permeation. Interestingly, lead
structure 4 generates a toxic effect (observed as seizures and death
of animals) at the dose of 9.5 nmol (not shown in Fig. 3). Unlike
hybrid 4, the modified DML hybrid 22 did not induce a toxic effect
at this higher dosage. Our results also indicate that both 22 and 33
produced a long-lasting antinociceptive effect (2 h) after intra-
thecal administration (data not shown).
that of morphine (Fig. 4). The beneficial potency shift from 22 to 33
might be attributed to a 10-fold shift in DOR potency (see MVD
assay in Table 1). Several studies have provided evidence that
compounds with a dual MOR/DOR activity present beneficial
pharmacological effects in comparison to highly selective MOR
agonists [35].
Fig. 3. The effect of intrathecal (i.t.) administration of morphine (M, 0.35e35 nmol), hybrid 22 (0.1e11 nmol) and its parent compound 33 (opioid agonist, 0.001e0.1 nmol), hybrid 4
(0.1e1 nmol) and its parent compound 7 (opioid agonist, 0.001e0.1 nmol) and compound 6 (NK-1 antagonist 1e50 nmol) measured in tail-flick test 30 min after administration in
naïve rats (7e12 animals per group). Inter-group differences were analysed by ANOVA Bonferroni's Multiple Comparison Test; *p < 0.05; ***p < 0.001 vs. V e vehicle treated naïve
rats. %MPE ¼ percentage of maximal possible effect.

K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
69
2.3.3. Neuropathic pain in rats
was difficult to observe. At this stage the NK-1 pharmacophore
appeared to be important for activity in the von Frey assay, a sup-
position which was supported by the antiallodynic activity of
compound 6. Compound 6 was significantly more potent than the
opioid parent compounds.
In order to verify whether compounds 4 and 22 would be
capable of suppressing allodynia and hyperalgesia, induced by
chronic constriction injury (CCI) to the sciatic nerve in rat, these
hybrids were examined in this neuropathic pain model with i.t.
administration.
Seven days after CCI the two hybrids, their parent compounds,
the NK-1 antagonist and morphine were administered i.t. in rats. In
the model of neuropathic pain, the antiallodynic effect of morphine
and both parent compounds (33 and 7) is weaker as compared to
the observed effect in the acute pain assay. This is in agreement
The ability of both parent opioid peptides, the two investigated
hybrids and the NK-1R antagonist parent unit of hybrid 4 (i.e.
compound 6) to lower hyperalgesia was measured by the cold plate
test. As shown in Fig. 5B, the hybrids, but not their parent opioid
compounds, decrease hyperalgesia. In comparison to morphine and
at the equal dose of 1 nmol, the effect of the hybrid compounds is
significantly stronger. In contrast to parent compounds 33 and 7,
compound 6 is more potent in antagonizing hyperalgesia than
hybrid 4 and approximately similar to 22. Similar to the observa-
tions made in the acute pain (tail-flick) test and the allodynia test
(von Frey), the only hybrid lacking toxicity at the dose of 10 nmol i.t.
is the hybrid structure with reduced NK1R binding and potency,
compound 22 (not shown).
The ED50 values of compounds 4, 22 and morphine are pre-
sented in Table 2. In contrast to the acute pain data, a reduction of
the antinociceptive potency of morphine in the neuropathic pain
model (von Frey and cold pate) is observed. In contrast, in both tests
the hybrid compounds perform well in neuropathic and in acute
pain attenuation. The ED50 values clearly indicate that a lower dose
of 22 is needed for potent effects, as compared to 4 and morphine.
Interestingly, and contrary to the classical opioids, the hybrids
perform better in neuropathic pain attenuation. This observation
with literature in which a lower potency of
m opioids for neuro-
pathic pain treatment is described (Fig. 5A). In contrast to the pure
opioids, the two hybrids (22 and 4) were significantly more active
in this test. The strongest attenuation of allodynia was observed
after administration of hybrid 22, reaching 80% MPE at a dose of
1 nmol (30% MPE in acute pain), and hybrid 4, reaching 60% MPE
after dose of 1 nmol (30% MPE in acute pain). At a higher dose of
1 nmol, the two parent compounds 33 and 7 showed a toxic effect
(marked on Fig. 5A and B as black bar), which was not observed
after administration of hybrid compounds 22 and 4 administered at
the same dose. Hybrid 4 also showed a toxic effect at the highest
investigated dose (9.5 nmol, not shown). The highest decrease of
allodynia and lack of toxic signs were noticed for the hybrid com-
pound 22, although the highest investigated dose of 11 nmol was
weaker in antagonizing allodynia and hyperalgesia than lower
doses (not shown). This may be indicative of a weak toxic effect that
Fig. 5. The effect of intrathecal (i.t.) administration of following compounds in CCI neuropathic pain model in rats: morphine (M, 0.35e35 nmol), hybrid 22 (0.1e1 nmol), its parent
compound 33 (0.001e1 nmol), hybrid 4 (0.1e1 nmol), its parent compound 7 (0.001e1 nmol) and NK-1 antagonist 6 (0.1e1 nmol) on (A) allodynia as measured by von Frey test and
(B) on hyperalgesia as measured by cold plate test (6e12 animals per group). Both tests were made on day 7 after injury in CCI-subjected rats. V ¼ vehicle-treated CCI-exposed rats,
black bars ¼ toxic effect. Inter-group differences were analysed by ANOVA Bonferroni's Multiple Comparison Test; *p < 0.05, **p < 0.01, ***p < 0.001 vs. V-treated CCI-exposed rats.

70
K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
indicates the potential value of these hybrids as drug candidates for
neuropathic pain therapy.
potent NK1 antagonist (KiNK1R (4) 0.5 nM vs. KiNK1R (22) 13 nM
and pA2 (4) 7.8 vs. pA2 (22) 6.4), yet it is more active than 4. This
finding is however in agreement with prior work [43]. When
testing a combination of opioid peptides with a tachykinin antag-
onist we have shown that a low dose (not antinociceptive when
applied individually) of tachykinin antagonist strongly potentiated
opioid antinociception. Increasing the dose of the SP antagonist
resulted in neurotoxic side effects rather than potentiation of
antinociception.
2.3.4. Development of tolerance in neuropathic pain in rats
In this study the development of tolerance was observed after
chronic administration of morphine and compounds 4 and 22,
which were injected i.t. repeatedly for 6 days (once daily) starting
from day 7 after CCI and lasting until day 13. The effect of the
investigated compounds is similar to the long-term profile of
morphine as demonstrated by the time course curves (Fig. 6A and
C). However, in spite of the same time required for the development
of tolerance with morphine, hybrid compound 22 exhibits the most
potent antinociceptive effect, as demonstrated by the area under
curve values in both tests (Fig. 6B and D).
Also, an important result in this work is that in naïve rats the
antinociceptive potencies of the hybrid compounds are weaker
than those of the parent compounds (e.g. 0.1 nmol of 22 ¼ 8.9 %MPE
vs 33 ¼ 99.7 %MPE in i.t. dosed rats), but in the neuropathic pain
model, the potencies of the two parent compounds, lacking the NK-
1R pharmacophore, were much lower, as was also the case with
morphine. This confirms the known effect of a decrease in efficacy
of opioids in the treatment of neuropathic pain and points to the
potential value of hybrids that act more strongly in neuropathic
pain attenuation. Although the NK-1 pharmacophores in hybrid 4
and 22 could not attenuate analgesic tolerance development, the
results suggest that antagonism at pronociceptive systems is a very
important factor in improving the analgesic effects of opioids in
neuropathic pain. The data of compound 6 (i.e. parent NK-1 phar-
macophore of hybrid 4) confirmed this assumption, but also earlier
studies demonstrating that NK-1R antagonists are highly effective
in attenuating hyperalgesia in experimental rodent neuropathy
models [44,45]. We demonstrated in the present study that the
parent NK-1 antagonist pharmacophore was efficient in reversing
neuropathic pain behavioural effects in CCI rats and only showed a
minimal effect in the acute pain model (tail-flick) in naïve rats after
intrathecal administration. Additionally, the NK-1R pharmacophore
6 was not active at all in the latter assay after i.v. administration in
mice, indicating that the parent NK-1R pharmacophore alone is not
able to cross the BBB, in contrast to the hybrids in which the opioid
unit may serve as a BBB transport vector (cf. activity of 4 versus 6).
Despite promising preclinical results on rodents, NK-1 antagonists
have been shown to lack efficacy in human neuropathy [46,47].
Nonetheless, NK-1 antagonists may prove useful in combination
therapies or by use of hybrid compounds that contain both opioid
and NK-1 pharmacophores, as this may ensure simultaneous drug
action and may improve therapy as compared to the administration
of a single general analgesic.
2.3.5. Cross tolerance between morphine and hybrid compounds 22
and 4 in neuropathic pain model in rat
In order to check cross tolerance between the hybrid com-
pounds and morphine, a cross tolerance study was conducted
(Fig. 7). Starting from day 7 after CCI, animals were given daily
injections of the hybrid compounds or morphine. Tolerance de-
velops within 6 days, until day 13 after CCI (Fig. 6). The animals, that
had been rendered tolerant after subsequent administrations of 4
and 22, were administered with a single dose of morphine on the
13th day of the experiment. The lack of a ‘morphine effect’ after
chronic administration of the tested hybrids shows cross tolerance
between these compounds. Moreover, in rats which were rendered
tolerant to morphine, a single dose of the hybrid 22 was also not
effective.
3. Conclusions
Several bifunctional ligands were synthesised and tested in vitro
and in vivo in acute and neuropathic pain models. The conforma-
tionally constrained peptidomimetic ligands turned out to have
subnanomolar binding affinity and high agonist potency at the
MOR and DOR receptors, and good to moderate binding affinity and
antagonist activity at the neurokinin-1 receptor. The best in vitro
activity profiles were displayed by reference compound 4 and by
the newly synthesized hybrid 22. When compound 22 was tested
in vivo it showed an activity in the acute pain model (i.v. adminis-
tration) which appeared to be similar to the previously determined
activity of hybrid 4 [29], and approximately 3 times higher than the
one of morphine (Fig. 4). Both bifunctional compounds were also
tested in two in vivo models for neuropathic pain (tactile and
thermal stimuli). Both ligands were significantly more potent than
morphine in neuropathic rats at day 7 after chronic constriction
injury (CCI) in the von Frey test (tactile allodynia). The effect was
even more pronounced in the cold plate test (thermal hyper-
algesia), where 22 was more potent than 4, and much more potent
than morphine. Moreover, it is remarkable that the most active
compound in the neuropathic pain models (22) is not the most
4. Materials and methods
4.1. General
Thin-layer chromatography (TLC) was performed on glass
plates precoated with silica gel 60F254 (Merck, Darmstadt, Ger-
many) using the mentioned solvent systems. Mass Spectrometry
(MS) was done on a Micromass Q-Tof Micro spectrometer with
electrospray ionisation (ESI). Data collection and spectrum anal-
ysis was done with Masslynx software. Analytical RP-HPLC was
performed using a Waters 717plus Autosampler, a Waters 1525
Binary HPLC Pump and a Waters 2487 Dual Absorbance Wave-
length Detector (Milford, MA), with a Grace (Deerfield, IL) Vydac
Table 2
Calculated ED50 values for effect of hybrids 4 and 22 compared to morphine (M) in
acute pain (tail-flick test) and neuropathic pain (von Frey and cold plate tests) as
measured on naïve rats (acute pain) and neuropathic pain models at day 7 after CCI.
RP C18 column (25 cm ꢁ 4.6 mm ꢁ 5
mm) using UV detection at
215 nm. The mobile phase is a mixture of water and acetonitrile
and contains 0.1% TFA. The used gradient runs from 3 to 100%
acetonitrile in 20 min at a flow rate of 1 ml/min. Preparative RP-
HPLC purification was done on a Gilson (Middleton, WI) HPLC
system with Gilson 322 pumps, controlled by the software pack-
age Unipoint, and a reversed phase C18 column (Discovery®BIO
Response (ED50) with 95% confidence limits [nmol i.t.]
Drugs
Acute pain
Neuropathic pain
von Frey
Tail-flick
Cold plate
M
4
22
0.49 (0.27e0.89)
1.66 (1.11e2.51)
2.78 (1.80e4.30)
6.25 (2.0e19.44)
1.05 (0.65e1.69)
0.24 (0.18e0.32)
377 (130e1093)
1.14 (0.74e1.77)
0.34 (0.25e0.46)
SUPELCO Wide Pore C18 column, 25 cm ꢁ 2.21 cm, 5
mm) with the
same conditions as the analytical RP-HPLC but with a flow rate of

K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
71
Fig. 6. The development of tolerance to antiallodynic effect measured by (A) the von Frey test and (C) antihyperalgesic effect measured by the cold plate test after repeated once
daily (6 days) intrathecal (i.t.) injections of morphine (M, 3.5 nmol), hybrid compound 4 (1 nmol) and 22 (1 nmol) from 7th day until 13th day after CCI (12e16 animals per group).
The results are also presented as area under curve (AUC) in (B) von Frey test and (D) cold plate test. V ¼ vehicle-treated CCI-exposed rats. Inter-group differences were analysed by
ANOVA Bonferroni's Multiple Comparison Test; *p < 0.05 vs V-treated CCI-exposed rats.
20 ml/min. After purification, the purity of all compounds was
evaluated as being more than 95% by analytical RP-HPLC. All
fractions were lyophilised using a Flexy-Dry lyophilizer (FTS Sys-
tems, Warminster, PA). 1H and 13C NMR spectra were recorded at
250 and 63 MHz on a Bruker Avance 250 spectrometer or at 500
and 125 MHz on a Bruker Avance II 500 (Bruker Corp, Billerica,
MA). Trimethylsilane (TMS) or residual solvent signals are used as
internal standard. The solvent used is mentioned in all cases, and
the abbreviations used are as follows: s (singlet), d (doublet), dd
(double doublet), t (triplet) and m (multiplet).
4.3. Peptide synthesis including Aba structures
4.3.1. Reductive amination
The reductive amination was executed after Fmoc deprotection
of the first amino acid. The resin was first swollen in 0.5% AcOH in
TMOF/CH2Cl2 for 30 min and filtered. Two equivalents of Phth-
ortho-formyl phenylalanine [32] were dissolved in the same
mixture and 4 eq. of NaBH3CN was dissolved in a minimum volume
of DMF. Both solutions were added to the resin and the reaction
vessel was shaken for 30 min. The course of the reaction was
monitored after this time by the Kaiser test. When the test was
positive, the reaction was left for another 30 min. The monitoring is
repeated until the reaction remains complete, which is indicated by
a light red colour of the Kaiser test due to the presence of the
secondary amine.
4.2. General peptide synthesis
All peptides were synthesized manually by Fmoc-based solid
phase peptide synthesis (SPPS) on 2-chlorotritylchloride resin
(0.15 mmol scale). The first amino acid (Fmoc-Gly-OH or Fmoc-b-
Ala-OH) was loaded onto the resin by use of 2 eq. Fmoc-protected
amino acid with 4 eq. DIPEA in CH2Cl2 for 2 h. The remaining
chlorines were substituted by treatment of the resin with a mixture
of MeOH/CH2Cl2/DIPEA (2:17:1) during 4 times 5 min. For normal
couplings, a 3-fold excess of the Fmoc-protected amino acids
(Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Cit-OH) and 3-fold excess of
coupling reagent (TBTU) in 0.4 NMM in DMF was used for 1.5 h.
Fmoc deprotection was carried out by treatment of the resin with
20% 4-methylpiperidine in DMF for 5 and 15 min. After every re-
action step, the resin was washed with DMF (3ꢁ 1 min), iPrOH (3ꢁ
1 min) and CH2Cl2 (3ꢁ 1 min).
4.3.2. Cyclisation towards the Aba building block (13)
An excess of 3 eq. TBTU is added to the resin and shaken for 3 h.
4.3.3. Phthaloyl deprotection to 14
The resin was treated with 6 eq of hydrazine monohydrate in
DMF for 18 h.
4.3.4. Boc-Dmt-OH coupling
An excess of 2 eq. Boc-Dmt-OH and 2 eq DIC/HOBt in DMF was
added to the peptide-resin. The reaction vessel was shaken for 3 h.

72
K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
Fig. 7. Cross tolerance of hybrid compounds 22 and 4 with morphine measured in neuropathic pain. The influence of repeated 6 days i.t. administration of morphine (M, 3.5 nmol),
hybrid 22 (1 nmol) and hybrid 4 (1 nmol) on the effect of a single dose of morphine (M, 3.5 nmol), or 22 (1 nmol) was measured on day 13 after CCI in rats (6e8 animals per group).
The von Frey (A, B, C) and cold plate (D, E, F) tests were conducted 30 min after a single dose of vehicle, morphine or 22 administration. The data are presented as %MPE S.E.M.
Inter-group differences were analysed by ANOVA Bonferroni's Multiple Comparison Test; ***P < 0.001 indicates a significant difference compared with group which received
repeated vehicle and then a single dose of vehicle (V þ V-treated CCI-exposed animals); $$$P < 0.001 indicates a significant difference compared with group which received repeated
vehicle and then single dose of morphine (V þ M; A, B, D, E) or 22 (Vþ22; C, F).
4.3.5. Cleavage from the solid support
injected on a Gilson preparative RP-HPLC. Fractions were collected
and combined and lyophilised. The peptides were obtained as
white powders with a purity of >95% as determined by analytical
HPLC. The structures were confirmed by high-resolution electro-
spray mass spectrometry.
The fully protected peptide was cleaved from the resin with 1%
TFA in DMF for 30 min. The filtrate was concentrated and added to
cold ether. The precipitated peptide was then dissolved in aceto-
nitrile/H2O and lyophilised to get the compounds as a powder.
4.4. Coupling of the benzylamines to the peptide acids and final
deprotection
4.6. Synthesis of benzylamine derivates
4.6.1. N-isobutyl benzylamine (18a)
The crude protected peptide was dissolved in a minimal amount
of CH2Cl2. The solution was cooled in an ice-bath and DIPEA (2 eq.),
DIC/HOBt (1.5 eq. each) are added. The mixture was stirred for
30 min at 0 ꢀC. Then the amine (1.5 eq.) was added to the solution
and the reaction mixture stirred again for 30 min with cooling. The
reaction was left to warm up to room temperature. When the
mixture of all the reagents does not dissolve sufficiently, some
drops of DMF are added and a clear solution is obtained. The re-
action mixture was left to react for 15 h. At completion, the solvent
was evaporated and the residue was treated with TFA/TES/H2O
(95:2.5:2.5) for 3 h, after which the solvent was removed in vacuo.
Isobutylamine (1.5 ml, 5 eq.) is dissolved in 16 ml methanol in a
two-headed round bottom flask equipped with magnetic stirrer,
reflux column and septum. The reaction mixture is heated to 50 ꢀC
in an oil bath. Next, benzyl chloride (347 ml, 1 eq.) is dissolved in
16 ml methanol and added drop wise (during 1 h). The mixture is
stirred overnight at 50 ꢀC and the solvent is evaporated. The residue
is dissolved in 28 ml CH2Cl2 and washed with a 20% NaOH-solution
(3 ꢁ 60 ml) and water (2 ꢁ 60 ml). The organic phase is dried with
MgSO4, filtered en evaporated. After evaporation, a pale yellow oil is
obtained. The purification is done with flash chromatography
(Davisil LC60A, 40e63
mm) with hexane/ethyl acetate 9:1. After
evaporation of the solvent, 1 eq. of TFA is added to make a salt and
the solution was lyophilised. A white to yellow powder is obtained
with a yield of 43%. N-isobutylbenzylamine. Yield: 48% (504 mg, TFA
salt); Formula: C11H17N; MW: 163.26 g/mol; TLC Rf ¼ 0.26 (EtOAc/
chex 1:1); HPLC: tR ¼ 9.4 min; MS (ESþ): 164 [MþH]þ; 1HNMR
4.5. Purification
The crude peptides were dissolved in H2O and acetonitrile was
added until complete dissolving was observed. The solution was

K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
73
(250 MHz, CDCl3):
CH), 2.64 (2H, m, CHeCH2), 3.90 (2H, s, NHeCH2-Bn), 7.30e7.42
(5H, m, arom. H). 13CNMR (63 MHz, CDCl3):
(ppm) 20.0 (CH3), 26.0
(CH), 51.5 (CH2-Bn), 53.9 (CHeCH2eNH), 129.0 (CH arom.), 129.4
(CH arom.), 130.2 (CH arom.), 130.4 (Cq arom.).
d
(ppm) 0.92 (6H, d, CH3, J ¼ 6.7 Hz), 1.97 (1H, m,
4.7.7. H-Dmt-D-Arg-Aba-GlyeNHeBn (26)
Preparative HPLC yielded the desired compound (white powder,
22%). HPLC: tR ¼ 11.2 min. TLC Rf 0.66 (EBAW). HRMS (ESPþ) found
m/z 671.3694 [MþH]þ, C36H46N8O5 requires 671.3664.
d
4.7.8. H-Dmt-D-Arg-Aba-Gly-N-i-Bu-30,50e(CF3)2eBn (27)
Preparative HPLC yielded the desired compound (white powder,
32%). HPLC: tR ¼ 14.3 min. TLC Rf 0.62 (EBAW). HRMS (ESPþ) found
m/z 863.4044 [MþH]þ, C42H52F6N8O5 requires 863.4037.
4.6.2. N-isobutyl-30,50-bistrifluoromethyl benzylamine (18b)
Isobutylamine (1.5 ml, 5 eq.) is dissolved in 24 ml methanol in a
two-headed round bottom flask equipped with magnetic stirrer,
reflux column and septum. The reaction mixture is heated to 50 ꢀC
in an oil bath. Next, 30,50-trifluoromethylbenzylchloride (1 eq.,
528 mg) is dissolved in 20 ml methanol and added drop wise
(during 1 h). The mixture is stirred overnight at 50 ꢀC and the
solvent is evaporated. The residue is dissolved in 150 ml dichloro-
methane and washed with a 20% NaOH-solution (3 ꢁ 90 ml) and
water (2 ꢁ 90 ml). The organic phase is dried with MgSO4, filtered
en evaporated. After evaporation, a light yellow oil is obtained. The
purification is done with flash chromatography (Davisil LC60A,
4.7.9. H-Dmt-D-Arg-Aba-Gly-N-i-Bu-Bn (28)
Preparative HPLC yielded the desired compound (white powder,
15%). HPLC: tR ¼ 12.5 min. TLC Rf 0.61 (EBAW). HRMS (ESPþ) found
m/z 727.4304 [MþH]þ, C40H54N8O5 requires 727.4290.
4.7.10. H-Dmt-D-Arg-Aba-b
-Ala-N-i-Bu-30,50e(CF3)2eBn (29)
Preparative HPLC yielded the desired compound (white powder,
12%). HPLC: tR ¼ 14.6 min. TLC Rf 0.64 (EBAW). HRMS (ESPþ) found
m/z 877.4240 [MþH]þ, C43H54F6N8O5 requires 877.4194.
40e63 mm) with a gradient going from hexane/ethyl acetate 9:1 to
7.5:2.5. After evaporation of the solvent, TFA is added to make a salt
and lyophilised. A white to yellow powder is obtained with a yield
of 48%. N-isobutyl-30,50-bistrifluoromethyl benzylamine. Yield: 43%
(354 mg, TFA salt); Formula: C13H15F6N; MW: 299.26 g/mol;
Rf: ¼ 0.65 (EtOAc/petroleum ether 1:1), TLC: Rf ¼ 0.17 (EtOAc/
hexane 1:9); HPLC: tR ¼ 13.3 min; MS (ESþ): 300 [MþH]þ; 1HNMR
4.7.11. H-Dmt-D-Arg-Aba-b-Ala-N-i-Bu-Bn (30)
Preparative HPLC yielded the desired compound (white powder,
21%). HPLC: tR ¼ 12.8 min. TLC Rf 0.57 (EBAW). HRMS (ESPþ) found
m/z 741.4465 [MþH]þ, C41H56N8O5 requires 741.4446.
(250 MHz, CD3OD):
d
(ppm) 1.06 (3H, d, CH3, J ¼ 6.7 Hz), 2.08 (1H,
4.7.12. H-Dmt-D-Arg-Phe-Sar-NMe-30,50e(CF3)2eBn (31)
Preparative HPLC yielded the desired compound (white powder,
24%). HPLC: tR ¼ 13.7 min. TLC Rf 0.70 (EBAW). HRMS (ESPþ) found
m/z 823.3728 [MþH]þ, C39H48F6N8O5 requires 823.3724.
m, CH), 2.99 (2H, m, CHeCH2), 4.43 (2H, s, NHeCH2-Bn), 8.11e8.20
(3H, m, arom. H). 13CNMR (63 MHz, CD3OD):
d
(ppm) 20.3 (CH3),
27.3 (CH), 51.3 (CH2-Bn), 56.5 (CHeCH2eNH), 124.6 (q, CF3,
1J ¼ 272.0 Hz), 132.1 (CH arom.), 133.4 (q, Cq arom., 2J ¼ 33.7 Hz),
135.7 (CH arom.).
4.7.13. H-Dmt-D-Arg-Phe-Sar-NMe-Bn (32)
Preparative HPLC yielded the desired compound (white powder,
31%). HPLC: tR ¼ 11.5 min. TLC Rf 0.66 (EBAW). HRMS (ESPþ) found
m/z 687.3949 [MþH]þ, C37H50N8O5 requires 687.3983.
4.7. Peptide characterization
4.7.1. H-Dmt-D-Arg-Aba-Gly-NMe-30,50e(CF3)2eBn (4)
Preparative HPLC yielded the desired compound (white powder,
34%). HPLC: tR ¼ 14.0 min. TLC Rf 0.72 (EBAW). HRMS (ESPþ) found
m/z 821.3536 [MþH]þ, C39H46F6N8O5 requires 821.3568.
4.7.14. H-Dmt-D-Arg-Aba-b-Ala-NH2 (33)
Preparative HPLC yielded the desired compound (white powder,
49.5%). HPLC: tR ¼ 10.1 min. TLC Rf 0.44 (EBAW). HRMS (ESPþ)
found m/z 595.3394 [MþH]þ, C30H43N8O5 requires 595.3351.
4.7.2. H-Dmt-D-Arg-Aba-b
-Ala-NMe-30,50e(CF3)2eBn (21)
Preparative HPLC yielded the desired compound (white powder,
17%). HPLC: tR ¼ 14.0 min. TLC Rf 0.67 (EBAW). HRMS (ESPþ) found
m/z 835.3708 [MþH]þ, C40H48F6N8O5 requires 835.3724.
4.8. Functional NK1R assay [48]. Cell line and cell culture conditions
The Chinese hamster ovary K1 (CHO-K1) cell line, stably
expressing human NK1 receptor (hereafter referred to as CHO-
NK1 cells), was transfected with an apoaequorin expression vec-
tor (pER2) using Fugene6 (Roche Applied Science). The cell line
and expression vector were obtained from Euroscreen (Belgium).
The CHO-NK1 cells were cultured in sterile DMEM/HAM's F12
medium (Sigma) supplemented with 10% foetal bovine serum,
4.7.3. H-Dmt-D-Arg-Aba-b-Ala-NMe-Bn (22)
Preparative HPLC yielded the desired compound (white powder,
34%). HPLC: tR ¼ 11.7 min. TLC Rf 0.64 (EBAW). HRMS (ESPþ) found
m/z 699.3955 [MþH]þ, C38H50N8O5 requires 699.3977.
4.7.4. H-Dmt-D-Cit-Aba-b
-Ala-NMe-30,50e(CF3)2eBn (23)
100 IU/mL penicillin, 100 mg/mL streptomycin, and 400 mg/mL G
Preparative HPLC yielded the desired compound (white powder,
21%). HPLC: tR ¼ 14.6 min. TLC Rf 0.72 (EBAW). HRMS (ESPþ) found
m/z 836.3515 [MþH]þ, C40H47F6N7O6 requires 836.3565.
418 (Geneticin, Gibco) at 37 ꢀC with 5% CO2 and were trypsinized
every 3 days.
4.8.1. Aequorin charging protocol
4.7.5. H-Dmt-D-Cit-Aba-b-Ala-NMe-Bn (24)
Transfected cells in the midlog phase were detached by
changing the growth medium for PBS buffer supplemented with
5 mM EDTA (pH 8). The cells were spun down and incubated for 4 h
at a concentration of 5 ꢁ 106 cells/mL in DMEM-F12 medium
without phenol red (Gibco) supplemented with 0.1% BSA (BSA
Preparative HPLC yielded the desired compound (white powder,
34%). HPLC: tR ¼ 12.1 min. TLC Rf 0.70 (EBAW). HRMS (ESPþ) found
m/z 700.3790 [MþH]þ, C38H49N7O6 requires 700.3817.
4.7.6. H-Dmt-D-Arg-Aba-Gly-NMe-Bn (25)
medium) and 5 mM coelenterazine h (Molecular Probes). After
Preparative HPLC yielded the desired compound (white powder,
63%). HPLC: tR ¼ 11.7 min. TLC Rf 0.67 (EBAW). HRMS (ESPþ) found
m/z 685.3788 [MþH]þ, C37H48N8O5 requires 685.3820.
coelenterazine loading, the cells were diluted 10-fold in the same
medium and incubated for an additional period of 30 min. The cells
were mildly shaken during the incubation periods.

74
K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
4.8.2. Aequorin luminescence assay
(GraphPad, San Diego, CA). Log IC50 values for each test compound
were determined from nonlinear regression. The inhibition con-
stant (Ki) was calculated from the antilogarithmic IC50 value by the
Cheng and Prusoff equation [50,51].
A dilution series of peptide agonist (SP was purchased from
Sigma) ranging from 10ꢂ11 to 10ꢂ4 M was distributed in a white 96-
well plate. For investigating antagonism, the synthetic compounds
were added to these wells to obtain the desired concentrations
(ranging from 10ꢂ8 to 10ꢂ4 M). One negative control sample (BSA
medium only) was included in each row of the 96-well plate. The
plate was loaded in a “Multimode Reader Mithras, LB940” (Bert-
hold). The wells were screened one by one, and each measurement
4.10. Functional GPI and mouse vas deferens (MVD) assays
The GPI and MVD bioassays were carried out as described in
detail elsewhere [52,53]. A doseeresponse curve was determined
with [Leu [5]]enkephalin as standard for each ileum and vas
preparation, and IC50 values of the compounds being tested were
normalized according to a published procedure [54].
started at the moment of injection of 50 mL of the coelenterazine-
loaded cell suspension, containing 2.5 ꢁ 104 cells. Light emission
was measured every second for 30 s after which 50 mL of 10 nM ATP
solution (positive control) was injected. Each measurement was
carried out in duplicate. Light emission was recorded for an addi-
tional period of 10 s per well, and the data were presented in
relative light units (RLU).
4.11. Opioid receptor binding assays
Opioid receptor binding studies were performed as described in
detail elsewhere [52]. Binding affinities for
m and d opioid receptors
4.8.3. Data analysis
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. Incubations were
performed for 2 h at 0 ꢀC with [3H]DAMGO and [3H]DSLET at
respective concentrations of 0.72 and 0.78 nM. IC50 values were
determined form log-dose displacement curves, and Ki values were
calculated from the IC50 values by means of the equation of Cheng
and Prusoff [50], using values of 1.3 and 2.6 nM for the dissociation
constants of [3H]DAMGO and [3H]DSLET, respectively.
Luminescence data (peak integration) were calculated using
MikroWin 2000 software (Berthold), which was linked to the
Microsoft Excel program. All statistical and curve-fitting analyses
were performed using Prism 4.0 (GraphPad) software. Data are
expressed in percentage (% RLU) of the maximal luminescence that
was detected with 10ꢂ4 M SP (without antagonist). The competitive
nature of antagonism was evaluated using the Schild plot method
[40]. All antagonists analysed in this study provided linear regres-
sion plots and were considered competitive. The pA2 values were
calculated using Schild's equation [49].
4.12. In vivo analgesic test in naive rats and mice
4.9. hNK1/CHO cell membrane preparation and radioligand binding
assay
The pain threshold to a thermal stimulus was assessed using
tail-flick latency evoked by noxious thermal stimulation as deter-
mined with a tail-flick analgesic meter (Analgesia Meter; Ugo
Basile, Comerio, Italy) as described previously [55e57]. The tail-
flick was used to measure spinal nociceptive responses to
thermally-induced pain using male C57BI6 mice and Wistar rats.
Mice (weighing 25e28 g) or rats (250e300 g) were maintained on a
normal lightedark cycle and testing occurred during the light
phase. Different doses of the drug were dissolved in saline or water
for injection and injected intravenously (i.v.) in mice or intrathe-
cally (i.t.) in rats. The effect was assessed at the following time point
post-injection: 5, 15, 30, 60, 120, 180 min. The thermal stimulus
emitted by a light bulb was applied to the tail at two-thirds of its
length until the animal vigorously withdrew the tail. The baseline
response was approx. 2 s and the maximal exposure was set to 7 s
(mice) and 9 s (rats) to avoid tissue damage. All experimental
procedures used in this animal testing followed the guidelines on
ethical standards for the investigation of experimental pain in an-
imals and were approved by the Animal Research Committees of
the Medical Research Centre, Polish Academy of Sciences and were
approved by the local Bioethics Committee (Krakow, Poland). All
in vivo activity was determined as a percentage of the maximal
Recombinant hNK1/CHO cells were grown to confluency in
37 ꢀC, 95% air and 5% CO2, humidified atmosphere, in a Forma
Scientific (Thermo Forma, OH) incubator in Ham's F12 medium
supplemented with 10% foetal bovine serum, 100 IU/mL penicillin,
100 mg/mL streptomycin, and 500 mg/mL geneticin. The confluent
cell monolayers were then washed with Ca2þ, Mg2þ-deficient
phosphate-buffered saline (PD buffer) and harvested in the same
buffer containing 0.02% EDTA. After centrifugation at 2700 rpm for
12 min, the cells were homogenized in ice-cold 10 mM TriseHCl
and 1 mM EDTA, pH 7.4, buffer. A crude membrane fraction was
collected by centrifugation at 18000 rpm for 12 min at 4 ꢀC, the
pellet was suspended in 50 mM Tris-Mg buffer, and the protein
concentration of the membrane preparation was determined by
using Bradford assay. Six different concentrations of the test com-
pound were each incubated, in duplicates, with 20 mg of membrane
homogenate, and 0.4 nM [3H]SP (135 Ci/mmol, PerkineElmer,
United States) in 1 mL final volume of assay buffer (50 mM Tris, pH
7.4, containing 5mMMgCl2, 50
m
g/mL bacitracin, 30
M phenylmethylsulfonylfluoride) SP at
M was used to define the nonspecific binding. The samples
mM bestatin,
10
10
mM captopril, and 100 m
m
possible
effect
(%MPE)
and
was
calculated
as:
%
were incubated in a shaking water bath at 25 ꢀC for 20 min. The [3H]
SP concentration and the incubation time were selected based on
the studies of Yamamoto et al. [20] The reaction was terminated by
rapid filtration through Whatman grade GF/B filter paper (Gai-
thersburg, MD) presoaked in 1% polyethyleneimine, washed four
times each with 2 mL of cold saline, and the filter bound radioac-
tivity was determined by liquid scintillation counting (Beckman
LS5000 TD). The media and chemicals listed above were purchased
from Sigma (SigmaeAldrich, St. Louis, MO) unless otherwise stated.
MPE ¼ [posttreatment latency/cut-off latency] ꢁ 100.
4.13. In vivo analgesic tests in neuropathic pain model in rats
4.13.1. Animals
Male Wistar rats (300e350 g) from Charles River (Sulzfeld,
Germany) were housed in cages lined with sawdust under a stan-
dard 12/12 h light/dark cycle (lights on at 08.00 h) with food and
water available ad libitum. All experiments were performed ac-
cording to the recommendations of the International Association
for the Study of Pain (IASP) [58] and the National Institutes of
Health Guide for the Care and Use of Laboratory Animals and were
approved by the local Bioethics Committee (Krakow, Poland).
4.9.1. Data analysis
Analysis of data collected from three independent experiments
performed in duplicates is done using GraphPad Prizm 4 software

K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
75
4.13.2. Implantation of intrathecal cannulas
4.14. Chemicals administration
The rats were chronically implanted with i.t. catheters according
to Yaksh and Rudy [59] under pentobarbital anaesthesia (60 mg/kg;
ip) as described previously [56,57]. The rats were placed on a ste-
reotaxic table (David Kopf), and a sterile catheter (PE 10, INTRA-
MEDIC, Clay Adams, Becton Dickinson and Company, Rutherford,
NJ, USA.), flushed with sterile water prior to insertion, was carefully
introduced through the atlanto-occipital membrane to the sub-
arachnoid space at the rostral level of the spinal cord lumbar
enlargement (L4-L6).
The chemicals used were obtained from the following sources:
morphine hydrochloride (Polfa Kutno, Poland). All drugs were dis-
solved in sterile water (water for injection). All drugs were injected
i.t. at a volume of 5 ml, followed by an injection of 10 ml of distilled
water to flush the catheter. For acute pain, studies were carried out
on day 7 after catheter implantation and 30 min after drug admin-
istration. In rats drugs were administered in a single i.t. injection at
the following doses: morphine (0.35, 3.5, 35 nmol), 22 (0.1, 1,
11 nmol), 33 (0.001, 0.01, 0.1 nmol), 4 (0.1, 0.5,1 nmol), 7 (0.001, 0.01,
0.1 nmol), and 6 (1,10, 50 nmol). In mice drugs were administered in
4.13.3. Chronic constriction injury
a single i.v. injection at the following doses: morphine 12.5
mmol/kg,
A chronic constriction injury (CCI) was produced according to
Bennett and Xie [60] as described previously [46,47]. The right
sciatic nerve was exposed under sodium pentobarbital anaesthesia
(60 mg/kg; i.p.). Four ligatures (4/0 silk) were made around the
nerve distal to the sciatic notch with 1 mm spacing until a brief
twitch in the respective hind limb was observed. After CCI, all an-
imals developed allodynia and hyperalgesia.
22 1.08 and 10.8 mol/kg, 33 1.21 mol/kg. In neuropathic pain
m
m
model morphine (0.35, 3.5, 35 nmol), 22 (0.1, 0.4,1 nmol), 33 (0.001,
0.01, 0.1,1 nmol), 4 (0.1, 0.5,1 nmol), 7 (0.001, 0.01, 0.1,1 nmol) and 6
(0.1, 0.5, 1 nmol) were administered in a single i.t. injection on day
7e14 after CCI and the measurements were performed 30 min after
drug administration. The control groups received vehicle (water for
injection) injections according to the same schedule. The CCI to the
sciatic nerve was performed 5e7 days after catheter implantation.
After completion of the experiment, the animals were killed with an
overdose of pentobarbital (i.p.).
4.13.4. Behavioural tests in neuropathic pain model
Two different behavioural tests were used to assess analgesic
potency in neuropathic pain. The von Frey test evaluates tactile
(mechanical) allodynia. After placing the animals in a plastic cage
with the floor made from metal mesh, a mechanical stimulus is
applied to the surface of the hind paw sole by use of the von Frey
filament. In the cold plate test which measures thermal hyper-
algesia, the rats are placed on a cold plate of 5 ꢀC and latency of
reaction is assessed. In both methods, the time of reaction was
measured automatically. The obtained signal is expressed as a
percentage of the maximal potential effect (%MPE).
4.15. Data analysis
The behavioural data (6e16 rats per group) are presented as the
percentage of the maximal possible antinociceptive effect (%
MPE
SEM), which was calculated according to the following
equation: % MPE ¼ [(TL-BL)/(CUT-OFF-BL)] ꢁ 100%, where BL was
the baseline latency and TL was the latency obtained after drug
injection. The results of the experiments were statistically evalu-
ated using one-way analysis of variance (ANOVA). The differences
between the treatment groups throughout the study were further
analysed with Bonferroni post-hoc tests.
4.13.4.1. von Frey test. Mechanical allodynia in rats with CCI was
measured using an automatic von Frey apparatus (Dynamic Plantar
Anesthesiometer Cat. No. 37400, Ugo Basile Italy). The animals were
placed in plastic cages with wire net floors. They were acclimatised
to this environment for approximately 3 min prior to testing. The
von Frey filament was applied to the midplantar surface of the hind
paw, and the measurements were taken automatically, as described
previously by Mika et al. [57] The strength of the von Frey stimuli
ranged from 0.5 to 26 g in the rats.
Author information
Author contributions
KG, OVDP and IVDE were in charge of ligand synthesis. AK, EV, JL,
FP, JVD, JV contributed by providing the NK1 binding and antago-
nism data. NNC, CL and PWS provided in vitro opioid data. PK, AL
and AWL performed the tail-flick test in mice and JM, ER, WM and
BP performed acute and neuropathic pain study in rats. The
manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
4.13.4.2. Cold plate test. To assess the threshold to a cold stimulus
in rats with CCI, the Cold/Hot Plate Analgesia Meter (No. 05044,
Columbus Instruments, USA) was used, as described previously by
Mika et al. [57] The rats were placed on the cold plate (5 ꢀC), and the
time until the shake of the hind paw was recorded. The cut-off
latency for this test was 30 s. In all cases, the injured paw was
the first to react.
Funding sources
The work of SB, DT and PWS was supported by a collaboration
4.13.4.3. Tolerance development. Tolerance development was car-
ried out in neuropathic pain model. Day seven after CCI was the first
day of tested drugs i.t. injections which were continued up to 13th
day. In this study morphine (3.5 nmol), 4 (1 nmol) and 22 (1 nmol)
were administered i.t. once a day and the behavioural tests were
carried out 30 min after drug administration.
ꢁ
ꢀ
convention between the Ministere du Developpement Econo-
ꢀ
mique, de l'Innovation et de l'Exportation du Quebec and the
Research Foundation e Flanders (FWO Vlaanderen) (PSR-SIIRI-417).
The research of PWS was also supported by grants CIHR (MOP-
89716), and the NIH (DA-004443). Supported by Institute of Phar-
macology statutory founds (WM) and grant from National Science
Center Poland NCN2012/06/A/NZ4/00028 for (BP and JM).
4.13.4.4. Cross tolerance. Cross tolerance measurement was con-
ducted one day after tolerance development completion. When the
animals developed tolerance to analgesic effect of 4 and 22 a single
dose of morphine was injected to check if there is a cross tolerance
between these compounds and morphine. We also checked
whether the hybrid 22 will be effective in rats in whom tolerance to
morphine was induced.
Acknowledgements
Dr. Cecilia Betti is acknowledged for in-house reproduction of
the total solid phase assembly methodology depicted in Scheme 2.
We thank Dr. Emeric Miclet and Dr. Isabelle Correia for the NMR
analysis of hybrid 4.

76
K. Guillemyn et al. / European Journal of Medicinal Chemistry 92 (2015) 64e77
[24] S. Ballet, D. Feytens, K. Buysse, N.N. Chung, C. Lemieux, S. Tumati, A. Keresztes,
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