Antinociceptive and cytotoxic activity of opioid peptides with hydrazone and hydrazide moieties at the C-terminus

Article abstract of DOI:10.3390/molecules25153429

In the present contribution, we analyze the influence that C-terminal extension of short opioid peptide sequences by organic fragments has on receptor affinity, in vivo analgesic activity, and antimelanoma properties. The considered fragments were based on either N-acylhydrazone (NAH) or N⁰-acylhydrazide motifs combined with the 3,5-bis(trifluoromethyl)phenyl moiety. Eleven novel compounds were synthesized and subject to biological evaluation. The analyzed compounds exhibit a diversified range of affinities for the μ opioid receptor (MOR), rather low δ opioid receptor (DOR) affinities, and no appreciable neurokinin-1 receptor binding. In three out of four pairs, N-acylhydrazone-based derivatives bind MOR better than their N’-acylhydrazide counterparts. The best of the novel derivatives have similar low nanomolar MOR binding affinity as the reference opioids, such as morphine and biphalin. The obtained order of MOR affinities was compared to the results of molecular docking. In vivo, four tested compounds turned out to be relatively strong analgesics. Finally, the NAH-based analogues reduce the number of melanoma cells in cell culture, while their N⁰-acylhydrazide counterparts do not. The antimelanoma properties are roughly correlated to the lipophilicity of the compounds.

Full text of DOI:10.3390/molecules25153429

molecules
Article
Antinociceptive and Cytotoxic Activity of Opioid
Peptides with Hydrazone and Hydrazide Moieties at
the C-Terminus
1
Jolanta Dyniewicz 1,*, Piotr F. J. Lipin´ski 1,
*
, Piotr Kosson 2, Marta Bochyn´ska-Czyz˙ ,
Joanna Matalin´ska 1 and Aleksandra Misicka 1,3,
*
1
Department of Neuropeptides, Mossakowski Medical Research Centre Polish Academy of Sciences,
Pawin´skiego 5, 02-106 Warsaw, Poland; marta.bochynska@interia.eu (M.B.-C.);
jmatalinska@imdik.pan.pl (J.M.)
Toxicology Research Laboratory, Mossakowski Medical Research Centre Polish Academy of Sciences,
Pawin´skiego 5, 02-106 Warsaw, Poland; pkosson@imdik.pan.pl
Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
Correspondence: jdyniewicz@imdik.pan.pl (J.D.); plipinski@imdik.pan.pl (P.F.J.L.);
misicka@chem.uw.edu.pl (A.M.)
2
3
*
Academic Editors: Joanna Bojarska, Wojciech M. Wolf, Milan Remko, Piotr Zielenkiewicz,
Michele Saviano, Janusz Zabrocki and Krzysztof Kaczmarek
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 July 2020; Accepted: 25 July 2020; Published: 28 July 2020
Abstract: In the present contribution, we analyze the influence that C-terminal extension of short
opioid peptide sequences by organic fragments has on receptor affinity, in vivo analgesic activity,
and antimelanoma properties. The considered fragments were based on either N-acylhydrazone (NAH)
or N0-acylhydrazide motifs combined with the 3,5-bis(trifluoromethyl)phenyl moiety. Eleven novel
compounds were synthesized and subject to biological evaluation. The analyzed compounds exhibit
a diversified range of affinities for the
µ opioid receptor (MOR), rather low δ opioid receptor
(DOR) affinities, and no appreciable neurokinin-1 receptor binding. In three out of four pairs,
N-acylhydrazone-based derivatives bind MOR better than their N’-acylhydrazide counterparts.
The best of the novel derivatives have similar low nanomolar MOR binding affinity as the reference
opioids, such as morphine and biphalin. The obtained order of MOR affinities was compared
to the results of molecular docking. In vivo, four tested compounds turned out to be relatively
strong analgesics. Finally, the NAH-based analogues reduce the number of melanoma cells in cell
culture, while their N0-acylhydrazide counterparts do not. The antimelanoma properties are roughly
correlated to the lipophilicity of the compounds.
Keywords: analgesics; cytotoxic activity; diacylhydrazine; µ-opioid receptor; multitarget compounds;
N0-acylhydrazide; N-acylhydrazone; opioid peptides; peptidomimetics; privileged structures;
structure activity relationships
1. Introduction
N-Acylhydrazone (NAH; otherwise termed hydrazide/hydrazone; Figure 1) has been identified as
a “privileged structure” [
substituents is able to provide potent ligands for a variety of medicinally relevant molecular targets [
1
] that is a structural framework, which by being decorated with appropriate
].
2
A few marketed drugs contain the N-acylhydrazone motif in their structures, e.g., azumolene,
carbazochrome, dantrolene, nitrofurantoin, nitrofurazone, nifuroxazide, testosterone 17-enanthate
3-benzilic acid hydrazone [3]. Further compounds of this type, for example PAC-1 (a procaspase
activator with antitumour action), are being currently considered in clinical trials (NCT02355535).
Numerous other NAHs have been described and tested at the preclinical level, with some of them
Molecules 2020, 25, 3429; doi:10.3390/molecules25153429
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3429
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having potential to reach the clinic. The reported activities of N-acylhydrazone-bearing compounds
include anti-inflammatory, analgesic, antithrombotic [ ], antimicrobial [ ], and antiproliferative
(apoptosis-inducing) [ ] properties. Other authors disclosed NAHs that were -glucosidase
inhibitors [ ], non-ligand binding pocket androgen receptor antagonists [ ], or histone deacetylases
4
5,6
7
α
8
9
and phosphatidylinositol 3-kinases inhibitors [10]. A recent review on the roles of NAHs in modern
medicinal chemistry was provided by Thota et al. [3].
Figure 1. Comparison of N-acylhydrazone and N0-acylhydrazide structural motifs.
A scaffold that is closely related to N-acylhydrazone is N0-acylhydrazide (N,N0-diacylhydrazine).
Structurally, it differs from the former in having a second amide subunit instead of azomethine
(Figure 1). R1 and R2 substituents attached at the ends of both these scaffolds are separated by
an equal number of atoms; their positions may, however, be different for conformational reasons.
While N-acylhydrazones are planar [9
], the N0-acylhydrazides are usually twisted around the N-N
bond [11], but planar structures have been also observed. Both scaffolds have distinct H-bonding
opportunities. In N-acylhydrazones, there are two H-bond acceptor points (carbonyl oxygen and imine
nitrogen) spaced by an H-bond donor (amide hydrogen). In N0-acylhydrazides, there are two acceptors
(at the scaffold edges) and two donors (“inside” the motif). Due to the presence of an additional
polar carbonyl group, N0-acylhydrazides are significantly less lipophilic than the corresponding
N-acylhydrazones (by about 1.0 LogP unit). For the reasons mentioned above, consideration of the
N-acylhydrazone/N0-acylhydrazide pairs of active compounds’ analogues is an interesting and rational
approach for exploring structure-activity relationships (SAR).
Furthermore, both discussed fragments seem particularly well-suited for creating multitarget
(multivalent), modular compounds of peptide or mixed peptide–organic structures. This is foremost
due to facile synthesis, because hydrazones are readily obtained by “clicking” aldehydes with
hydrazides (e.g., peptide hydrazides). N0-acylhydrazides could be produced by an equally simple
reaction of a carboxylic acid (e.g., peptide) with a hydrazide (generated from another carboxylic
acid). Examples of using hydrazone linkage to create modular conjugates include the work by
Lipkowski et al., who joined oxymorphone and naltrexone with a portion of enkephalin or dynorphin
A [12]. Ganguly et al. demonstrated the utility of the N-acylhydrazones for attaching the chelating
moieties in radiopharmaceuticals [13]. The N0-acylhydrazide motif (usually talked about in terms of
being a “hydrazide linker”) is present in a potent opioid analgesic with a dimeric structure, biphalin
(H2N-Tyr-D-Ala-Gly-Phe-NH-NH
in numerous derivatives of biphalin [16
course of structure–activity investigations. Other dimeric opioids containing the N0-acylhydrazide
substructure include dermorphin-based analogues [23].
←
Phe
←
Gly
←
D-Ala
←
Tyr-NH2) [14
,
15]. This fragment is also found
–
20], including the cyclic ones [21
,
22] that were obtained in the
Moreover, the hydrazide linker was applied to create multifunctional compounds based on the
biphalin structure. For example, it served to append the opioid sequence with fluorescent elements,
such as dansyl [24] or 7-succinylamido-4-methyl-coumarin [25] moieties. The hydrazide linker was
also used in multitarget compounds having opioid receptor agonist and cholecystokinin receptor

Molecules 2020, 25, 3429
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antagonist [26] pharmacophores. Yet another bivalent combination in which the hydrazide bridge found
application was in hybrids of opioid agonist and neurokinin-1 receptor (NK1R) antagonists [27 28].
,
The rationale for preparing such multitarget compounds consisting of opioid sequences along with
the pharmacophoric elements required for targeting some other receptors stems from the fact that
simultaneous modulation of a few receptors involved in pain perception can provide analgesic
compounds with enhanced efficacy and fewer side effects [29–32].
In the course of our works aimed at obtaining opioid receptor agonist/neurokinin-1 receptor
antagonist chimeras, we designed a series of compounds (Figure 2, Table 1) in which opioid sequences
(
1–6) were appended (at the C-terminus) with the 3,5-bis(trifluoromethyl)phenyl moiety characteristic
for neurokinin-1 antagonists. The structural elements that enabled this fusion were the discussed
fragments: N-acylhydrazone (series ). Moreover, in the case of
) and N0-acylhydrazide (series
H-Tyr-D-Ala-Gly-Phe- sequence ( ), two additional linkers were considered ( , -NH-NH-C(=O)-CH2;
and , -NH-NH-C(=O)-NH-N=CH-). One of the compounds resulting from these efforts, JZ031 (1a),
a
b
1
c
d
has been already described [33]. In the present contribution, we extend that report by discussing the
synthesis, receptor binding, and molecular modelling of further 11 compounds. Moreover, the in vivo
antinociceptive activity of compounds 1a
–1d was tested. Finally, the cytotoxic activity of the compounds
under study was examined in a melanoma cell line, MeW155.
Figure 2. Structural variation of the reported compounds.
Table 1. Analytical data for the synthesized compounds.
ESI-MS [M + H] + m/z
LogP
Compound
tR [min]
No
Formula
Calcd
695.24
711.24
725.25
753.26
734.25
750.25
785.29
801.28
677.23
693.23
811.30
664.24
Obsvd
694.95
710.85
724.90
752.85
733.90
749.95
784.85
800.85
677.15
692.85
810.80
664.16
1a [33]
1b
1c
H-Tyr-D-Ala-Gly-Phe-NH-N=CH-3,5-(CF3)2Ph
H-Tyr-D-Ala-Gly-Phe-NH-NH-C(=O)-3,5-(CF3)2Ph
H-Tyr-D-Ala-Gly-Phe-NH-NH-C(=O)-CH2-3,5-(CF3)2Ph
H-Tyr-D-Ala-Gly-Phe-NH-NH-C(=O)-NH-N=CH-3,5-(CF3)2Ph
H-Tyr-D-Ala-Gly-Trp-NH-N=CH-3,5-(CF3)2Ph
H-Tyr-D-Ala-Gly-Trp-NH-NH-C(=O)-3,5-(CF3)2Ph
H-Tyr-D-Ala-Phe-Phe-NH-N=CH-3,5-(CF3)2Ph
H-Tyr-D-Ala-Phe-Phe-NH-NH-C(=O)-3,5-(CF3)2Ph
H-Tyr-D-Ala-Trp-NH-N=CH-3,5-(CF3)2Ph
C32H32F6N6O5
C32H32F6N6O6
C33H34F6N6O6
C33H34F6N8O6
C34H33F6N7O5
C34H33F6N7O6
C39H38F6N6O5
C39H38F6N6O6
C32H30F6N6O4
C32H30F6N6O5
C41H40F6N6O5
C32H31F6N5O4
12.91
11.82
12.10
12.53
12.88
12.03
14.20
13.40
12.94
12.25
14.09
13.25
5.55
4.5
3.54
4.19
5.48
4.43
7.74
6.69
6.09
5.04
8.03
6.03
1d
2a
2b
3a
3b
4a
4b
5a
H-Tyr-D-Ala-Trp-NH-NH-C(=O)-3,5-(CF3)2Ph
H-Tyr-Pro-Phe-Phe-NH-N=CH-3,5-(CF3)2Ph
6a
H-Tyr-Pro-Phe-NH-N=CH-3,5-(CF3)2Ph
2. Results and Discussion
2.1. Chemistry
The synthesis of the herein reported compounds (1b and 2a–6a) was carried out by (1) obtaining
the N-tert-butoxycarbonyl (Boc)-protected linear peptides in solution, (2) coupling them with the
appropriate organic fragments (Scheme 1), and (3) removing the N-protection.

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Scheme 1. Synthesis of the reported compounds. Reagents and conditions: (i) N-hydroxysuccinimide,
(HOSu, 1.1 equiv), N,N0-dicyclohexylocarbodiimide (DCC, 1 equiv), 1,1,3,3-tetramethylguanidine
(TMG, 1.1 equiv), 24 h in dimethylformamide (DMF); (ii) 3,5-bis(trifluoromethyl)benzaldehyde, 20 h in
isopropanol (i-PrOH); (iii) trifluoroacetic acid/ dichloromethane (TFA/DCM), 2 h.
In order to execute the second stage, for the analogues with the N-acylhydrazone motif in
their structures (2a, 3a, 4a, 5a, 6a), the protected peptides were converted into hydrazides by
reaction with hydrazine, and later the N-Boc-protected peptide hydrazides were condensed with
3,5-bis(trifluoromethyl)benzaldehyde.
Alternatively, hydrazides of 3,5-bis-(trifluoromethyl) benzoic acid (for the synthesis of 1b, 2b, 3b,
4b) or 3,5-bis(trifluoromethyl)phenylacetic acid (for the synthesis of 1c) were obtained and then reacted
with N-Boc-protected peptides. Finally, in the case of 1d analogue, the second stage was performed
by the reaction of the N-Boc-protected peptide with the fragment obtained from condensation of
3,5-bis(trifluoromethyl)benzaldehyde with carbohydrazide.
The purity and identity of the structures were confirmed by HPLC and ESI-MS, as well as by
NMR. The analytical data for the synthesized analogues are summarized in Table 1.
RP-HPLC retention times of compounds 1a–6a were plotted (Figure 3) against the theoretical
lipophilicity descriptor, LogP, calculated in the ACD/ChemSketch program [34]. Upon excluding
compounds 1c and 1d, a very good correlation appears, with a coefficient of determination of r2 = 0.95.
This agreement of theoretical and experimental results confirms the additive nature of lipophilicity
variation with exchanging N-acylhydrazone/N0-acylhydrazide motifs in our structures. Overall, the
hydrazones are more lipophilic, with LogP values being greater by cca 1.0 unit and elution times
greater by 0.7–1.1 min in the RP-HPLC system applied here. The remaining two linkers (present in 1c
and 1d) represent intermediate levels of lipophilicity (in terms of the retention time; note that the LogP
values do not fit this conclusion and counterintuitively suggest that 1c and 1d are even less lipophilic
than 1b.
2.2. Binding Affinity
The synthesized compounds were assayed for binding affinity to the µ- and δ-opioid (MOR
and DOR, respectively), as well as to the neurokinin-1 receptors (NK1R). The determinations were
performed in rat brain homogenates by competitive displacement of selective radioligands. The results
are shown in Table 2 as the half-maximal inhibitory concentration (IC50) with the standard error of the
mean (SEM).

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Figure 3. Correlation of RP-HPLC retention times of the studied compounds with theoretical LogP
values. Compounds 1c and 1d excluded from the correlation. Points are colored so that analogues with
the same peptide sequence have the same color.
Table 2. Affinity of the synthesized compounds for opioid and neurokinin receptors.
IC50 ± SEM 1 (nM)
Compound
MOR
DOR
NK1R
YaGF-
(enkephalin-derivative)
1a [33]
1b
12.73 ± 0.75
74.20 ± 0.40
413.45 ± 105.15
161.65 ± 8.75
44.04 ± 2.75
4.24 µM ± 0.80
>1000
110.64 ± 12.66
5.25 ± 1.14
1c
>1000
1d
4.73 ± 0.02
>1000
YaGW-
(enkephalin-derivative)
2a
112.01 ± 16.89
93.465 ± 32.84
914.4 ± 43.50
467.05 ± 36.95
>1000
>1000
2b
YaFF-
(TAPP)
3a
90.4 ± 9.91
391.05 ± 57.85
>1000
>1000
722.8 ± 58.2
>1000
3b
YaW-
4a
118.11 ± 40.19
>1000
621.0 ± 65.20
>1000
>1000
>1000
4b
YPFF-
(endomorphin-2)
5a
28.5 ± 9.41
>1000
>1000
YPF-
(truncated endomorphin-2)
6a
113.1 ± 7.00 >1000
>1000
1
IC50 values represent means ± SEM of at least two experiments performed in duplicates.
Contrary to the design assumptions, the majority of the compounds exhibit only very low NK1R
binding (IC50 > 1000 nM). The sole exception is compound 3a, which has a low NK1R affinity, with an
IC50 value of 722.8 nM.
Regarding the opioid receptors, the presented compounds show a diversified spectrum of affinities.
For MOR, the IC50 values are as different as a few nanomoles (1c, 1d) and more than 1000 nM (4b).

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In three of the four N-acylhydrazone–N0-acylhydrazide analogue pairs (1a vs. 1b
,
3a vs. 3b and
4a vs. 4b), the hydrazones exhibit better MOR binding (4 to 10 times). In the fourth pair (2a vs. 2b),
the MOR affinities are almost equal for both analogues.
If we compare the reported hydrazone compounds with the parent opioid sequences, for the
enkephalin derivative (1a) expanding the C-terminus with 3,5-bis(trifluoromethyl)phenylhydrazone
is rather neutral (or slightly negative) for affinity. This compound exhibits similar MOR affinity
levels as its tetrapeptide amide counterpart (H-Tyr-D-Ala-Gly-Phe-NH2, Ki = 2.8 nM [35] or
H-Tyr-D-Ala-Gly-Phe-NH-NH2, IC50 = 4.7 nM [36]). For the enkephalin-based derivative with
Trp in the 4th position (2a), the IC50 reading 112.01 nM can be considered a slight decrease in affinity
compared to the parent tetrapeptide, as H-Tyr-D-Ala-Gly-Trp-NH2 was reported to be an about 2 times
weaker as a MOR binder than the [Phe4]-variant [37], while for the hydrazones 2a and 1a the IC50 ratio
is about 10.
TAPP-based hydrazone (3a; TAPP = H-Tyr-D-Ala-Phe-Phe-NH2) binds with IC50 = 90.04 nM,
which is clearly a higher value than that recently determined for the tetrapeptide in our laboratory
(IC50 = 5.1 nM [38]). Regarding compound 4a, a similar comparison is not straightforward, as we
are not aware of any MOR binding data for amide H-Tyr-D-Ala-Trp-NH2. On the other hand,
Laskowska et al. [39] reported that tripeptide H-Tyr-D-Ala-Trp-OH has low MOR affinity with
IC50 = 977 nM, while expanding this structure by attaching trans-1-cinnamylpiperazine (Cyn)
improves the binding, so that H-Tyr-D-Ala-Trp-Cyn has an IC50 value of 77.6 nM. In this case
4a, whose IC50 value is 118.11 nM, could be considered to enjoy the very same improvement of affinity
upon attaching the 3,5-bis(trifluoromethyl)phenylhydrazone element to H-Tyr-D-Ala-Trp- sequence.
The endomorphin-2-based analogue (5a) has a slightly worse MOR affinity (IC50 = 28.5 nM) than
that of the parent tetrapeptide (IC50 = 3.9 nM [40]). The C-truncated EM-2 (H-Tyr-Pro-Phe-NH2) was
previously found to have a MOR binding affinity with an inhibition constant Ki of 46.3 [41]. Thus, in the
case of this sequence, attachment of 3,5-bis(trifluoromethyl)phenylhydrazone (6a, IC50 = 113.1 nM)
seems to be slightly adverse to MOR affinity.
The highest MOR affinities in our set were found for compounds 1c and 1d, in which
the C-terminal part is expanded by organic fragments containing N0-acetylhydrazide (1c) or
-C(=O)-NH-NH-C(=O)-NH-N=CH- substructures (1d). Particularly interesting is that 1c, being only
one methylene unit longer than 1b, exhibits significantly better MOR binding (approximately 21 times
lower IC50 value).
For DOR, half of the compounds exhibit low affinity, with IC50 > 1000 nM. As could be expected
of endomorphin-2- and TAPP-based derivatives, compounds 3a, 3b, 5a, and 6a do not exhibit any
appreciable DOR binding. In the enkephalin-based analogues, the type of C-terminal expansion
modifies the DOR affinity, which for the H-Tyr-D-Ala-Gly-Phe-NH2 was reported to be rather moderate
(Ki = 300 nM [35]). The hydrazone 1a shows IC50 = 74.2 nM (previously reported [33]). The analogue
with N0-acylhydrazide (1b) has a significantly lower DOR binding value (IC50 = 413.45 nM). Interestingly,
compounds 1c and 1d, which exhibit similarly high MOR affinity, differ with respect to DOR binding.
For the former, the determined DOR IC50 value is 161.65 nM, while for the latter the value is 44.04 nM.
Enkephalin-related compounds with Trp in the 3rd position exhibit low or at best moderate DOR affinity
(DOR IC50 values of 914.4 nM and 467.05 nM for hydrazone 2a and N0-acylhydrazide 2b, respectively).
In general, the most potent MOR ligands reported here (1c and 1d) have similarly high affinity as
typical reference opioids, be they peptides (like biphalin, MOR IC50 = 1.4 nM [33]) or non-peptides
(like morphine, IC50 = 4.02 nM [42]).
2.3. Molecular Docking
In order to rationalize the observed binding data, the reported compounds were modelled in
the µ-opioid receptor binding site. The modelling was done by building the N-acylhydrazone-,
N0-acylhydrazide-, or N0-acetylhydrazide-based fragments into the peptide structures in the MOR
binding site (PDB accession code: 6DDF [43]), followed by local search docking using AutoDock

Molecules 2020, 25, 3429
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4.2.6 [44]. In the body of the paper we describe a few representative compounds (1a, 1b, 1c, 3a, 3b,
4a
, 4b), while the results for the remaining analogues are given in the Supplementary Materials. The
Supplementary Materials also contain the description of the redock validation exercise with AutoDock
4.2.6 and AutoDock Vina [45], as well as the comparison of modelling compounds 1a–6a executed with
AutoDock Vina.
In the case of compounds 1a–1c, the starting position of the peptide sequence (H-Tyr-D-Ala-
Gly-Phe-) was based on the position of H-Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) in the experimental
structure (PDB accession code: 6DDF [43]). As a result of the local docking search for our derivatives,
only minor displacements occurred in the peptide part (Figure 4A), and overall the positioning of this
part was close to that of the corresponding fragments in DAMGO (6DDF [43]). The main features of
this binding mode are (1) the canonical ionic interaction of the protonated Tyr1 amine with D147, (2) the
location of the Tyr1 phenol group close to H297, and (3) the position of the Phe4 in a hydrophobic subsite
made of side chains of several residues belonging to transmembrane helix 3 (TM3) and extracellular
loops 1 and 2 (ECL1 and ECL2). The first and the third features correspond to analogous elements of
the binding modes of small molecular MOR agonist BU72 (as found in crystal structure 5C1M [46]) or
fentanyl (as found by modelling [42,47]).
Figure 4. Binding modes of compounds 1a (green), 1b (aquamarine blue), and 1c (orange) in the MOR
binding site (yellow). The ligands are shown as colored sticks. Several residues and transmembrane
helices (relevant for each picture) are shown and labelled. Yellow dotted lines represent interactions.
(
A
) All of the compounds superimposed on DAMGO (pink, from 6DDF [43]). (
part and compounds 1a 1c together. ( ) Focus on the interactions with Y148 and compounds 1a
1b (D), and 1c (E). (F–H) Focus on the C-terminal part and compounds 1a (F), 1b (G), and 1c (H).
B
) Focus on the C-terminal
–
C
–E
(C),
Regarding the C-terminal part (Figure 4B), all three compounds (1a–1c) assume the cis-geometry
of the amide joining the peptide and the organic fragment (Figure 4C–E). This allows for the creation of

Molecules 2020, 25, 3429
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two hydrogen bonds with the Y148 phenol group. In the case of 1a, the flat hydrazone fragment (with
a co-planar ring and double bond) reaches towards the residues of ECL2 (Figure 4F). The aromatic ring
is placed close to T218, while the fluorines of the CF3 groups interact with D216 carboxylate and C217
carbonyl groups or with T218 hydroxyl and R211 guanidine moieties. On the other hand, in 1b the
twisted N(H)-NH bond causes the 3,5-bis-CF3-Ph to locate closer to TM6 (Figure 4G). One of the CF3
groups interacts with K233, K303, and V300, while the other one is exposed to the solvent. The ring
does not seem to be involved in the interactions with the binding site residues either. In the case of 1c
elongation of the fragment by one methylene (with free rotation) again allows for contact with the
ECL2 (Figure 4H). The hydrogen in the hydrazides’ N0-nitrogen participates in hydrogen bonding to
T218. Both CF3 groups form interactions with R211 and D216.
,
Overall, these differences in the binding modes of the C-terminal parts of 1a–1c form a
reasonable basis for explaining (qualitatively) the experimental differences in affinity. Let us note
that the binding model of 1a considered here is different than that we reported previously [33].
The differences are with respect to both the position of Phe4, as well as the position of the
3,5-bis(trifluoromethyl)phenylhydrazone element. We decided to embrace this novel model due
to its consistency with the experimental position of DAMGO.
Regarding the TAPP-based analogues (3a and 3b), the starting position of the peptide sequence
(H-Tyr-D-Ala-Phe-Phe-) was based on the position of TAPP reported in our recent paper, which focused
on TAPP derivatives [38]. Again, as a result of the local docking search performed for the novel
analogues, only minor displacements occurred in the peptide part (Figure 5A). The main features of
the binding mode of TAPP (3a and 3b) for the peptide fragment are: (1) the canonical ionic interaction
of protonated Tyr1 amine with D147; (2) the location of the Tyr1 phenol group close to H297; (3) the
position of the Phe4 in a hydrophobic subsite made of side chains of several residues belonging to
TM3, ECL1, and ECL2; and (4) the position of Phe3 close to N127 and H319. The first three features
are identical to those found in the enkephalin-based series described above. Moreover, TAPP has
been predicted to form a hydrogen bond to T218 via the C-terminal amide (Figure 5B). For 3a and 3b
,
in which this amide joins the peptide and the organic fragment, the need to accommodate the organic
fragment seems to impede formation of this hydrogen bond (or at least to create a certain distortion in
the interaction geometry). This corresponds well to the experimentally determined decrease in affinity
for 3a and 3b in comparison to the parent TAPP. The aromatic rings in both analogues are located close
to TM5. The CF3 groups form interactions with E229 and K233 (Figure 5C,D).
For modelling of compounds 4a and 4b, the tripeptide amide (H-Tyr-D-Ala-Trp-NH2) was first
docked and then the organic fragments were appended to the structure from docking, as with the former
compounds. After the local optimization, the obtained binding mode was common to 4a and 4b in the
peptide part, as expected. Again, the canonical ionic interaction of protonated Tyr1 amine with D147 was
formed and the Tyr1 phenol group was positioned close to H297, just as in the case of enkephalin-based
and TAPP-based derivatives (Figure 6A). The indole ring of Trp3 assumed a position close to TM3
residues I144, V143, and Y148. The phenol group of the latter is predicted to form a hydrogen bond to
the indole’s nitrogen. According to modelling, the 3,5-bis(trifluoromethyl)phenyl fragments of both
compounds are located in different subareas of the binding site. In the hydrazone-based derivative
(4a), the moiety approaches TM2 and TM7 (Figure 6B). This allows the CF3 groups to contact Y128
and N127. Moreover, the aromatic ring is involved in hydrophobic interactions with W318. In the
hydrazide-based analogue (4b), the organic part is directed towards ECL2, TM5, and TM6 (Figure 6C).
The CF3 groups interact with the sidechains of T218, K233, E229, and V300. Finally, considering all
the modelled compounds (1a, 1b, 1c, 3a, 3b, 4a, 4b, DAMGO, and TAPP), it should be noted that an
attempt to quantitatively correlate the affinities with the predicted energies in the whole set gives a
very poor correlation (Figure SM-1).

Molecules 2020, 25, 3429
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Figure 5. Binding modes of compounds 3a (palecyan) and 3b (light orange) in the MOR binding
site (yellow). The ligands are shown as colored sticks. Several residues and transmembrane helices
(relevant for each picture) are shown and labelled. Yellow dotted lines represent interactions. ( ) Both
compounds superimposed on TAPP (pink, from 6DDF [43]). ( ) Focus on the interactions with T218
and compounds 3a 3b, and TAPP together. ( ) Focus on the interactions with E229 and K233, as well
as compounds 3a (C), 3b (D), and 1c (E).
A
B
,
C,D
Figure 6. Binding modes of compounds 4a (lime) and 4b (light teal) in the MOR binding site (yellow).
The ligands are shown as colored sticks. Several residues and transmembrane helices (relevant for
each picture) are shown and labelled. Yellow dotted lines represent interactions. (
A) Both compounds
superimposed on DAMGO (pink, from 6DDF [43]). ( ) Focus on the on the interactions of the
B,C
C-terminal part, showing compounds 4a (B) and 4b (C).
2.4. In Vivo Analgesic Activity
Four of the reported compounds (1a–1d) based on the H-Tyr-D-Ala-Gly-Phe- sequence were
tested in vivo for antinociceptive activity in male Wistar rats in a tail flick test after the intrathecal (i.t.)
administration. The data for compound 1a have already been published [33]. The results are presented
in Figures 7 and 8 and Table 3.
All studied compounds exhibited time-dependent analgesic effects. The effects were also dose-dependent,
except for compound 1b, for which doses of 0.1 nmol/rat, 0.5 nmol/rat, and 1.0 nmol/rat produced
similar results. Comparing the analgesic action of the compounds administered in 0.5 nmol/rat dose,
all four analogues reach their peak activity 15 min after the injection. At this time point and with this
dose, the highest effect was observed for 1a and 1d (~ 80% of maximal possible effect, MPE). The lowest

Molecules 2020, 25, 3429
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value was found for compound 1c (31
At 120 min post-injection (0.5 nmol/rat), none of the compounds had an analgesic effect significantly
different than the control, although the plot for 1d seemed to “tail” towards the right side, suggesting
±
6% MPE), while the analogue 1b exhibited 65
±
6% MPE.
this compound may have longer duration of action. In fact, for a 2 nmol/rat dose, as much as 39
MPE can be measured for 1d, even at 120 min after administration. If the area under the time course
curve (AUC, Figure 7, the column on the right) is considered, compounds 1a 1b, and 1d had similar
AUC values (for 0.5 nmol/rat dose) of 4483 592, 3421 673, and 5732 1161 units, respectively.
The same parameter for 1c was significantly lower (1417 ± 730.1).
±
10%
,
±
±
±
Figure 7. Analgesic responses of compounds 1a (A,B), 1b (C,D), 1c (E,F), and 1d (G,H), as well as
morphine (MF). In the left column given is the time course curve of the effect. In the right column
are column plots of the area under the time course curve (AUC) for different doses. Results were
either analyzed with two-way ANOVA followed by the Bonferroni post-hoc test (
A
,
C
,E
,G) or one-way
ANOVA followed by the Dunnett post-hoc test ( ) and plotted as the mean
Asterisks (*) indicate a significant difference compared with saline-injected animals. Note: * p < 0.033,
** p < 0.002, and *** p < 0.001.
B
,
D
,
F
,
H
±
SEM (n = 4–8).

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Figure 8. Comparison of the area under the time course curve (AUC) with a 0.5 nmol/rat dose for
compounds 1a 1d. Results were analyzed with one-way ANOVA followed by the Tukey post-hoc
–
test. Asterisks (*) indicate a significant difference on comparison of particular pair of columns. Note:
* p < 0.033, and ** p < 0.002.
Table 3. The effective dose (ED50) of compounds 1a–1d.
Cmpd
1a
Compound
ED50 ± SEM (nmol/rat) 1
0.18 ± 0.05
H-Tyr-D-Ala-Gly-Phe-NH-N=CH-3,5-(CF3)2Ph
H-Tyr-D-Ala-Gly-Phe-NH-NH-C(=O)-3,5-(CF3)2Ph
H-Tyr-D-Ala-Gly-Phe-NH-NH-C(=O)-CH2-3,5-(CF3)2Ph
H-Tyr-D-Ala-Gly-Phe-NH-NH-C(=O)-NH-N=CH-3,5-(CF3)2Ph
n./a 2
1b
1c
0.56 ± 0.04
0.32 ± 0.04
1d
1
ED50 (half maximal effective dose)
±
standard error of the mean, n = 4–8; 2 n/a—not applicable, as there was no
dose dependency the value could not be established.
The highest antinociceptive effects found for each compound were 80
±
9% MPE (15 min,
7%
MPE (15 min, 4 nmol/rat, 1d). In terms of the effective dose (ED50, Table 3), the hydrazone 1a was
0.5 nmol/rat, 1a), 65
±
6% MPE (15 min, 0.5 nmol/rat, 1b), 85
±
7% MPE (15 min, 1 nmol/rat, 1c), 99
±
found to be the most active (ED50 = 0.18
0.56 ± 0.04 and 0.32 ± 0.04, respectively.
±
0.05 nmol/rat). For the analogues 1c and 1d, the values were
Overall, the antinociceptive action (in the tail flick test, after i.t. administration) of the tested
compounds was relatively high, comparable to that we have previously found for morphine (3 nmol/rat)
in the same test [33] (in terms of MPE and the peak activity time point). On the other hand, the observed
antinociception was weaker than in the case of the potent, dimeric opioid peptide biphalin [48],
for which lower doses than tested here were enough to exert similar activity. It should be noted that
while these good antinociceptive effects of 1a
opioid agonists, the analgesic efficacy of the compound 1b is somehow anomalous, given that this
analogue has the weakest MOR and DOR affinities out of the 1a 1d subseries. Perhaps an action via
, 1c, and 1d are what one would expect of high-affinity
–
some receptor other than those tested herein is responsible for this effect. This speculation requires
further investigation. Finally, let us note that the lack of the dose dependency for compound 1b could
stem from the fact that perhaps all the tested doses were at the plateau of the dose–response curve.
In order to verify this, the in vivo determination ought to be done with a wider range of concentrations.
2.5. Cytotoxicity
The neurokinin-1 receptor antagonists are known to exhibit cytotoxic effects [49–52], in particular
in cancers, as many types of cancer cells overexpress the NK-1R. These effects were found both in vitro
and in vivo.
Contrary to the design assumptions, our compounds do not have high NK1R affinity. Still, since we
have recently found [53] that a chimeric opioid-antitachykinin peptide, AWL3020, exhibits some

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significant cytotoxicity in a few cancer cell lines, despite having a very low NK1R affinity (IC50~70
we tested the influence of compounds 1a 6a on human melanoma cells. The determination was
performed by counting the cells of the MeW155 cell line after incubating them with 50 M of the tested
compounds. The results (presented as % of the control value) are shown in Figure 9A.
µM),
–
µ
Figure 9. The results of cytotoxicity evaluation. (A) Viability of MeW155 cells after incubation with
the tested compounds (as % of the control value). The results were analyzed with repeated measures
one-way ANOVA followed by the Dunnett post-hoc test. Asterisks (*) indicate a significant difference
compared to control. Note: * p < 0.033, and *** p < 0.001. (B) Correlation of melanoma cell line MeW155
viability (%) with the theoretical LogP value. Point 3b excluded from correlation.
It turned out that all N-acylhydrazone-based derivatives (1a
decrease the number of melanoma cells. On the other hand, the analogues with N0-acylhydrazide motif
1b 2b 3b 4b) display no effect. Similarly, no effect was found for the longest derivatives (1c and
, 2a, 3a, 4a, 5a, 6a) significantly
(
,
,
,
1d). The largest decrease in cell numbers was found for analogues 3a and 5a, for which only a few
intact MeW155 cells were observed under the microscope (~ 2 % of the control value). For compounds
2a
control, respectively.
Interestingly, the cytotoxic action of compounds 3a and 5a (and furthermore also of 2a
is significantly stronger than that of aprepitant (78 4%), reinforcing the notion that the observed
,
1a
,
4a and 6a, the following values were determined: 20
±
4%; 39
±
4%; 65
±
10%; 53
±
3% of the
,
1a, and 6a)
±
activity is not associated with the NK1R. Moreover, these compounds influence the melanoma cells
significantly more strongly than the mentioned AWL3020 compound, for which the very same type of
assay found 88% of the control value at 50 µM (this analogue was much more potent in other cell lines).
The ordering of the cytotoxic effects found for our analogues is not associated with either MOR
or DOR affinities. Some imperfect relationship may be found between these cytotoxic effects and
LogP (Figure 9B). For such a correlation, upon exclusion of 3b, the coefficient of determination is
equal to 0.76. Roughly, the more lipophilic the compound, the more it decreases the number of cells.
This may be indicative of some non-specific (non-receptor-mediated) cytotoxic effects being at work
here. Otherwise, it is also possible that the observed cytotoxic effects of the hydrazones could be
exerted via some intracellular (and not a membrane one) molecular target, e.g., a receptor. In that case,
a factor limiting the activity (irrespective of the affinity for this hypothetical target) could be the ability
of the compounds to cross the cellular membrane. A speculative candidate for such a target may be
the opioid growth factor receptor (OGFr), which binds [Met5]-enkephalin (also called opioid growth
factor, OGF), and upon the interaction with this opioid peptide inhibits proliferation of cells [54].
Further research shall be required to investigate this possibility. Furthermore, the cytotoxicity should
be tested on other cancer cell lines, as well as on normal cell lines, in order to determine the selectivity
of the compounds. Another important step will be assaying the effect on cells that the tetrapeptide
amides and 3,5-bis-CF3-Ph fragments have on their own.

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3. Materials and Methods
3.1. Chemistry
All amino acids derivatives, reagents, and solvents were obtained from commercial suppliers
and were used without further purification. N-Boc-protected compounds were purified on Merck
230–400 mesh silica gel 60 (Merck, Poland) by column chromatography. TLC analyses were carried out
on silica gel plates (silica gel 60 F254, Merck, Poland); observed using UV light and visualized with 1%
ninhydrin solution in MeOH. The target compounds (1b
Hitachi HPLC system with a reverse-phase column KROMASIL C8 (20
was 0.1% trifluoroacetic acid (TFA) in water/acetonitrile. The obtained compounds were analyzed on a
Shimadzu LC-MS system (Shim-pol, Poland) with a Jupiter 4 m Proteo 90 Å (250 4.6 mm, 4 m)
–
6a) were purified by RP HPLC on a Merc
×
250 mm). The mobile phase
µ
×
µ
column using a solvent system containing 0.05% formic acid (FA) in water/acetonitrile. For selected
compounds, nuclear magnetic resonance (NMR) spectra (1H, 13C, DEPT, COSY, HSQC, HMBC) were
also recorded with a Varian VNMRS at 600 MHz.
3.2. Synthesis
N-Boc-protected peptide fragments (as carboxylic acids) were obtained according to solution
phase strategy described previously by Lipkowski [55] and were used in subsequent steps without
further purification.
3.2.1. General Method for Synthesis of Intermediate Hydrazides
N-Boc-protected peptide, 3,5-bis(trifluoromethyl)phenylacetic acid or 3,5-bis(trifluoromethyl)benzoic
acid (1 equiv) were dissolved in dimethylformamide (DMF), N-hydroxysuccinimide (HOSu) (1.1 equiv)
was added, then the mixture was cooled to 0–5 ◦C. Further, N,N0-dicyclohexylocarbodiimide (DCC)
(1 equiv) was added and the reaction mixture was stirred for 30 min with cooling, then for 2 h at room
temperature. Then, hydrazine hydrate (10 equiv) was added and the reaction mixture was stirred for
20 h. After filtering the N,N0-dicyclohexylourea (DCU) precipitate, the filtrate was concentrated under
reduced pressure. The resulting products were precipitated by adding 15% KHCO3, then were filtered
off, washed with 5% KHCO3 and water, dried, and used without further purification. The yields of
crude products were 46–97%.
3.2.2. General Method for Synthesis of N-acylhydrazone-Type Analogues (2a, 3a, 4a, 5a, 6a)
To the solution of 3,5-bis-(trifluoromethyl)benzaldehyde (1 equiv) in isopropanol (i-PrOH) an
appropriate N-protected peptide hydrazide (1 equiv) was added, then the reaction was left overnight.
The resulting precipitate was filtered off (or alternatively, the solvent was evaporated) under reduced
pressure. The precipitate was washed with water and dried. The product was purified on silica gel with
the appropriate solvent mixtures (ethyl acetate/hexane or ethyl acetate/methanol; exact compositions
are given in Supplementary Materials). Yields after purification were 20–60%.
The same procedure was used for the synthesis of monocarbohydrazone (yield = 54%) of
3,5-bis-(trifluoromethyl)benzaldehyde required for the synthesis of compound 1d. Monocarbohydrazone
was recrystallized from water/ethanol (1:1).
3.2.3. General Method for Synthesis of Hydrazide Type of Analogues (1b, 1c, 1d, 2b, 3b, 4b)
An appropriate N-protected peptide (1 equiv) was dissolved in DMF, HOSu (1.1 equiv) was added,
then the mixture was cooled to 0–5 ◦C. Then, DCC (1 equiv) was added and the reaction mixture was
stirred for 30 min with cooling and for 2 h at room temperature. To the reaction mixture an appropriate
hydrazide (1.1 equiv) solution in DMF with 1,1,3,3-tetramethylguanidine (TMG) (1.1 equiv) was added
and the reaction was left for 20 h. Precipitated N,N0-Dicyclohexylurea (DCU) was filtered off, the filtrate
was condensed under reduced pressure, and the residue was poured into 15% citric acid. The precipitate

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was filtered off and washed with 5% citric acid and water. If the product did not precipitate, it was
isolated by extraction with ethyl acetate. The organic phase was washed with 5% citric acid (3 ) and
water (3 ), dried with anhydrous MgSO4, then the solvent was evaporated. The product was purified
×
×
on silica gel using proper solvents (ethyl acetate/hexane or ethyl acetate/methanol; exact compositions
are given in Supplementary Materials). Yields after purification were between 16 and 42%.
The N-protected precursors obtained by the above procedures were next deprotected by TFA,
and the resulting crude compounds were purified by preparative HPLC if necessary and analyzed by
ESI-MS (see Table 1) and NMR (details in Supplementary Materials). Representative examples of NMR
assignments for compounds containing each type of C-terminal organic fragment are given below.
The remaining NMR data for five other compounds are given in the Supplementary Materials.
Compound 1b (H-Tyr-D-Ala-Gly-Phe-NH-NH-C(=O)-3,5-(CF3)2Ph)
1H NMR(600 MHz, DMSO)
NH-Ala), 8.51 (1HAr), 8.38 (s, 1HAr), 8.29 (2H, NH, Tyr), 8.21 (d, J = 8.62 Hz, 1H, NH-Phe), 8.16 (t,
J = 5.97Hz, 1H, NH-Gly), 7.24–7.29 (m, 3H -Phe), 7.28 (1HAr), 7.18 (2H -Phe), 6.99 (d, J = 8.63 Hz,
2H -Tyr), 6.67 (d, J = 8.62 Hz, 2H -Tyr), 4.66 (J = 3.32 Hz, 1H -Phe), 4.30 (m, J = 7.30 Hz, 1H -Ala),
3.96 (1H -Tyr), 3.74 (d, J = 5.97 Hz, 1H -Gly), 3.59 (d, J = 5.64 Hz, 1H -Gly), 3.09 (1H -Phe),
2.83 (1Hβ–Phe), 2.81 (2Hβ–Tyr), 1.02 (d, J = 6.8 Hz, 3Hβ-Ala).
δ: 10.48 (d, J = 2.32 Hz, 2H, NH-NH), 9.35 (br. s, 1H, OH-Tyr), 8.52 (1H,
δ,
ε
ε
δ
ε
α
α
α
α
α
β
13C NMR(150 MHz, DMSO)
: 172.2 (C=O, Ala), 171.0 (C=O, Ar), 171.0 (C=O, Phe), 168.8 (C=O, Gly),
162.9 (C=O, Tyr), 157.1 (C –Tyr), 138.0 (C –Phe), 131.1 (C –Tyr), 129.8 (C –Phe), 129.8 (2C –Phe),
128.8 (CAr), 126.9 (C –Phe), 126.2 (CAr), 125.2 (C–Tyr), 124.4 (CAr), 122.6 (CAr), 115.8 (C –Tyr),
54.2 (C –Tyr), 53.1 (C –Phe), 48.6 (C –Ala), 41.9 (C –Gly), 38.4 (C –Phe), 36.8 (C –Tyr), 19.4 (C –Ala).
Compound 1c (H-Tyr-D-Ala-Gly-Phe-NH-NH-C(=O)-CH2- 3,5-(CF3)2Ph)
1H NMR(600 MHz, DMSO)
: 10.50 (2H, NH-NH), 9.33 (br. s, 1H, OH-Tyr), 8.55 (1HAr), 8.40 (1H,
NH-Ala), 8.20 (2H, NH-Tyr), 8.10 (m, 1H, NH-Phe), 8.20 (m, 1H, NH-Gly), 8.03 (1HAr), 7.96 (1HAr),
7.20–7.27 (m, 5HAr-Phe), 7.00 (2H -Tyr), 6.66 (2H -Tyr), 4.60 (1H -Tyr), 4.30 (q, J = 6.9 Hz, 1H -Ala),
4.01 (1H -Phe), 3.89 (s, 2H, C(=O)-CH2-), 3.50–3.80 (2H -Gly), 3.02 (d, J = 3.8 Hz, 1H -Phe),
2.89 (1Hβ–Tyr), 2.86 (1Hβ–Tyr), 2.87 (d, J = 3.8 Hz, 1Hβ–Phe), 1.05 (d, J = 6.7 Hz, 3Hβ-Ala).
13C NMR (150 MHz, DMSO)
: 172.3 (C=O, Ala), 171.9 (C=O, Ar), 170.7 (C=O, Gly), 168.2 (C=O,
Phe), 167.0 (C=O, Tyr), 156.7 (C –Tyr), 137.8 (C –Phe), 130.6 (C –Tyr), 129.5 (2C –Phe), 128.0 (CAr),
127.2 (CAr), 126.8 (C –Phe), 124.9 (C –Tyr), 124.5 (CAr), 121.0 (CAr), 115.6 (C –Tyr), 53.6 (C –Tyr),
δ
ζ
γ
δ
ζ
ζ
ε
ε
α
α
α
α
β
β
β
δ
δ
ε
α
α
α
α
β
δ
ζ
γ
δ
δ
ζ
γ
ε
α
52.4 (Cα–Phe), 47.8 (Cα–Ala), 41.2 (Cα–Gly), 38.1 (Cβ–Phe), 35.7 (Cβ–Tyr), 19.1 (Cβ–Ala).
Compound 1d (H-Tyr-D-Ala-Gly-Phe-NH-NH-C(=O)-NH-N=CH- 3,5-(CF3)2Ph)
1H NMR(600 MHz, DMSO)
NH-N=), 8.52 (1H, N=CH), 8.51 (1H, NH-Ala), 8.22 (2H, NH-Tyr), 8.15 (m, 1H, NH-Phe), 8.14 (m,
1H, NH-Gly), 8.04 (1HAr), 7.27 (1HAr), 7.27 (d, J = 7.3 Hz, 2H -Phe), 7.24 (t, J = 7.3 Hz, 2H -Phe),
7.17 (t, J = 7.02, 1H -Phe), 6.99 (d, J = 8.54 Hz, 2H -Tyr), 6.67 (d, J = 8.55 Hz, 2H -Tyr), 4.62 (1H -Phe),
4.30 (q, J = 7.02 Hz, 1H -Ala), 3.96 (t, J = 7.6 Hz, 1H -Tyr), 3.56–3.72 (2H -Gly), 3.07 (1H -Phe),
2.92 (1Hβ–Tyr), 2.88 (1Hβ–Tyr), 2.83 (1Hβ–Phe), 1.03 (d, J = 7.02 Hz, 3Hβ-Ala).
δ: 9.96 (2H, NH-NH), 9.33 (br. s, 1H, OH-Tyr), 8.55 (1HAr), 8.53 (1H,
δ
ε
ζ
δ
ε
α
α
α
α
β
13C NMR (150 MHz, DMSO)
: 171.7 (C=O, Ala), 171.7 (C=O, Phe), 171.2 (C=O, Ar), 168.1 (C=O, Gly),
167.6 (C=O, Tyr), 156.5 (C –Tyr), 137.7 (C–N=C), 137.7 (C –Phe), 130.7 (C –Tyr), 129.1 (2C –Phe),
128.4 (2C –Phe), 128.0 (CAr), 127.2 (CAr), 126.3 (C –Phe), 125.1 (C –Tyr), 127.2 (CAr), 122.3 (CAr),
115.1 (C –Tyr), 53.4 (C –Tyr), 52.3 (C –Phe), 48.5 (C –Ala), 41.8 (C –Gly), 38.4 (C –Phe), 36.7 (C –Tyr),
18.3 (Cβ–Ala).
δ
ζ
γ
δ
δ
ε
ζ
γ
ε
α
α
α
α
β
β

Molecules 2020, 25, 3429
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3.3. Binding Affinity Determinations
The binding affinity of compounds 1b
–
6a for MOR, DOR, and NK1R was determined in competitive
radioligand binding assays according to the method previously described [23,53]. The specific
radioligands were [3H]DAMGO (DAMGO, [Tyr1-3,5-3H])), [3H][Ile5,6]DELT II ([Ile5,6]Deltorphin II,
[Tyrl-3,5-3H]) and [3H]Substance P (Substance P, [Leu-3,4,5-3H]), for experiments with MOR, DOR,
and NK1R, respectively. The first two were obtained from Isotope Laboratory, Biological Research
Centre, Institute of Biochemistry (Szeged, Hungary) [56], whereas the latter was purchased from
Perkin Elmer (Perkin Elmer Inc., Krakow, Poland). Membrane fractions of rat brain homogenate were
incubated at 25 ◦C for 60 min in the presence of radioligands (0.5 nM) specific for each receptor and
increasing concentrations of the tested compounds. To measure non-specific binding, 10
µM naloxone
was used as the competitor for opioid receptors and 10 M cold Substance P was used for NK1R.
µ
The reactions were carried out in assay buffer containing Tris-HCl (pH 7.4) with an addition of bovine
serum albumin (BSA) and protease inhibitors (bacitracin, bestatin, captopril). After the incubation,
the binding reactions were terminated by rapid filtration with M-24 Cell Harvester (Brandel/USA)
through GF/B Whatman glass fiber strips (presoaked with 0.5% PEI in order to minimize non-specific
binding). Radioactivity retained on the filters was measured in MicroBeta LS on a Trilux scintillation
counter (PerkinElmer, Santa Clara, CA, USA). The experiments were repeated at least two times in
duplicate. The IC50 value for each compound was determined using GraphPad Prism [57].
3.4. Molecular Docking
Prior to molecular docking, the ability of AutoDock 4.2.6 [44] and AutoDock Vina [45] to
reproduce the binding pose of DAMGO in the 6DDF structure [43] was checked (results shown in the
Supplementary Materials). Since AutoDock 4.2.6. performed better, we chose to focus on this program.
Molecular docking of the studied compounds to MOR was performed using the local search
protocol in AutoDock 4.2.6 [44]. The initial binding poses of the compounds in the MOR binding site were
prepared manually by building the N-acylhydrazone-, N0-acylhydrazide-, or N0-acetylhydrazide-based
fragments into the peptide structures. The positions of the peptide sequences were based on the
experimental positions [43] of corresponding structural elements in DAMGO (1a-d) or based on the
results of molecular modelling [38] for TAPP tetrapeptide amide (3a and 3b) and for other derivatives
(2a–b, 4a–b, 5a, 6a).
The receptor structure was the activated MOR structure (PDB accession code: 6DDF [43]).
The G-protein was removed from the receptor structure and the protonation states were set as expected
at physiological pH. The ligands and the protein structure were processed in AutoDock Tools 4 [44
]
following standard routines. The ligands were considered flexible (except for amide bonds of the
peptide backbone and the C-terminal part) and the receptor was set as rigid. For modelling of the
flexibility of the C-terminal part, several conformers of this part were manually prepared and submitted
to the modelling procedure (with the peptide part flexible and the C-terminal organic fragment rigid).
The docking box (90
×
104
×
106 points, spaced by 0.375 Å) was set around the position of DAMGO in
the 6DDF structure [43] and its size was extended so that it covered the receptor binding pocket and
the binding pocket entry. Grids were calculated with AutoGrid. The local search parameters were
set as follows: 1000 runs, population of 300 individuals, 500 iterations of Solis and Wets local search,
sw_rho search space parameter 10.0. The results were clustered and representative poses from several
top-scored clusters subject to visual inspection. Molecular graphics were prepared in PyMOL [58].
3.5. In Vivo Analgesic Activity after Intrathecal Administration
Analgesic activity of compounds 1b–1d was measured in the tail flick test after intrathecal
administration to rats, according to a procedure thoroughly described before [33]. First, adult male
Wistar rats weighing 200–250 g were prepared for intrathecal administration by the catherization
method described previously by Yaksh and Rudy [59]. Each experimental group consisted of 4–8 rats.

Molecules 2020, 25, 3429
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Next, spinally mediated analgesia was assessed in the tail flick test utilizing the Plantar Test and Tail
Flick Analgesia Meter apparatus (IITC Life Science Inc., Los Angeles, CA, USA). Withdrawal latency
was measured in triplicate. The cut-off latencies were set at 7 s to avoid burns. The measurements were
performed before the administration of the tested compound (time 0) and 5, 15, 30, 60, and 120 min
after injection. The control group received injections of 0.9% NaCl. Data from in vivo studies are
presented as means
±
SEM and were analyzed using two-way repeated measures of ANOVA followed
by Dunnett-corrected multiple comparisons. Significance was defined as * p < 0.033, ** p < 0.002,
*** p < 0.001. Data were analyzed using GraphPad Prism [57]. The responses were expressed as a
percentage of a maximum possible effect (%MPE), calculated as ((T1
−
T0)/(T2
−
T0))
×
100, where T0 and
T1 are latencies before and after drug injection, respectively, and T2 is the cut-off time. All experimental
procedures were approved by The Local Committee for Ethics in Animal Experiments, permission
no. 46/2013.
3.6. Evaluation of the Compounds’ Influence on Melanoma Cells
The effect that the compounds 1a–6a exert on the melanoma cell number (in cell culture) was tested
on a MEW155 human melanoma tumor cell line obtained from the Maria Skłodowska-Curie Memorial
Cancer Centre and Institute of Oncology in Warsaw. The cells were plated in 24-well plates (5000 cells
per well) and incubated for 24 h in Eagle’s medium containing 10% fetal bovine serum and 1% solution
of penicillin and streptomycin. After the cells had reached 80% confluence, the tested compounds
(dissolved in 1% DMSO) were added at a concentration of 50 µM. Control cells were treated with 1%
DMSO only. After 96 h of incubation, the cell suspension was first lysed in lysis buffer (ReagentA,
ChemoMetec A/S) and then stabilized in stabilization buffer (ReagentB, ChemoMetec A/S). The number
of dead cells was quantified in a propidium iodide cassette with the use of a NucleoCounter® NC-100
™
(ChemoMetec A/S) cell counter. The tests were performed in triplicate. The results were normalized so
that the values obtained for the control were 100%.
4. Conclusions
In conclusion, the presented research dealt with the question of how the C-terminal extension
of short opioid peptide sequences influences the biological properties. The extending moieties
we considered were organic fragments consisting of 3,5-bis(trifluoromethyl)phenyl moiety and
N-acylhydrazone (series
a
) or N0-acylhydrazide (series
b) motifs. Additionally, two compounds (1c and
1d) contained longer elements. Eleven novel analogues (1b–6a) based on opioid peptides sequences
such as enkephalin, TAPP, endomorphin-2, or their truncated variants were synthesized.
The compounds were tested for affinity to MOR, DOR, and NK1R. Regarding the MOR binding,
the presented analogues show a diversified range of affinities. Both low nanomolar IC50 values
(1c and 1d) and values greater than 1000 nM (4b) were observed in the set. In three of the four
N-acylhydrazone–N0-acylhydrazide analogue pairs, the hydrazones exhibited better MOR binding
(4 to 10-times). The most potent MOR ligands studied here (1c and 1d) contain the longer fragments
with N0-acetylhydrazide- or -C(=O)-NH-NH-C(=O)-NH-N=CH- motifs. With respect to DOR affinity,
most of the novel compounds do not have appreciable binding. Still, the enkephalin-based analogues
show DOR affinity that varies depending on the C-terminal fragment. None of the prepared analogues
has measurable NK1R binding, except for one compound (3a) that showed rather low affinity.
The observed experimental MOR affinities were compared to the results of molecular docking. The in
silico models provide a rational basis for the design of novel compounds.
Four derivatives based on H-Tyr-D-Ala-Gly-Phe- sequence (1a–1d) were tested in vivo for
antinociceptive activity. All four analogues turned out to be fairly strong analgesics, with the previously
published compound 1a having the lowest ED50.
Finally, we have tested the cytotoxic properties of our derivatives (1a
melanoma cell line. At a concentration of 50 M, the analogues containing the N-acylhydrazone motif
significantly affected the cell viability, decreasing the number of cells in the culture. In particular,
–6a) on the MeW155 human
µ

Molecules 2020, 25, 3429
17 of 20
compounds 3a and 5a killed almost all cells in the culture. The impact on cells was not correlated with
either opioid or neurokinin-1 receptor affinities. On the other hand, the effect seems rather to be related
to the compounds’ lipophilicity.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/15/3429/s1:
Figure SM-1. Plot of predicted free energies of binding versus the experimental affinities. Validation of the docking
procedure performed with AutoDock 4.2.6 and AutoDock Vina. Table SM-1. Quality of binding pose prediction
(Auto Dock 4.2.6). Table SM-2. Quality of binding pose prediction (AutoDock Vina). Results of molecular
modelling for selected compounds. Table SM-3. Comparison of docking poses found by AutoDock and AutoDock
Vina. NMR assignments for selected compounds.
Author Contributions: Conceptualization, J.D. and A.M.; methodology, J.D. and P.F.J.L.; investigation, J.D., P.F.J.L.,
P.K., M.B.-C., and J.M.; writing—original draft preparation, J.D. and P.F.J.L.; writing—review and editing, J.D.,
P.F.J.L., and A.M.; supervision, A.M.; project administration, J.D.; funding acquisition, J.D. All authors have read
and agreed to the published version of the manuscript.
Funding: This work was supported by National Science Centre (NCN) Poland, PRELUDIUM grant
DEC-2011/03/N/ST5/ 04725.
Acknowledgments: The paper is dedicated to the memory of the late Professor Andrzej W. Lipkowski from
Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland, who inspired us to undertake
´
this research problem. The calculations were performed at Swierk Computing Centre, National Centre for Nuclear
´
Research, Swierk, Poland. Prof. Géza Tóth (Biological Research Centre, Hungarian Academy of Sciences, Szeged,
Hungary) is gratefully acknowledged for providing tritiated radioligands.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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©
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