Hydrazone linker as a useful tool for preparing chimeric peptide/ Nonpeptide bifunctional compounds

Article abstract of DOI:10.1021/acsmedchemlett.6b00381

The area of multitarget compounds, joining two pharmacophores within one molecule, is a vivid field of research in medicinal chemistry. Not only pharmacophoric elements are essential for the design and activity of such compounds, but the type and length o

Full text of DOI:10.1021/acsmedchemlett.6b00381

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Letter
The hydrazone linker as a useful tool for preparing
chimeric peptide/non-peptide bifunctional compounds
Jolanta Dyniewicz, Piotr FJ Lipi#ski, Piotr Kosson, Anna Le#niak, Marta Bochy#ska-Czy#,
Adriana Muchowska, Dirk Tourwé, Steven Ballet, Aleksandra Misicka, and Andrzej W. Lipkowski
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00381 • Publication Date (Web): 01 Nov 2016
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The hydrazone linker as a useful tool for preparing chimeric
peptide/non-peptide bifunctional compounds
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Jolanta Dyniewicz†*, Piotr F. J. Lipiński†, Piotr Kosson †, Anna Leśniak†, Marta BochyńskaꢀCzyż†,
Adriana Muchowska †, Dirk Tourwé§, Steven Ballet§, Aleksandra Misicka†*, Andrzej W.Lipkowski†
† Department of Neuropeptides, Mossakowski Medical Research Centre Polish Academy of Sciences, 5 Pawińskiego Str.,
02ꢀ106 Warsaw, Poland
§ Research Group of Organic Chemistry, Departments of Chemistry and Bioꢀengineering Sciences, Vrije Universiteit Brussel,
Pleinlaan 2, Bꢀ1050 Brussels, Belgium
KEYWORDS Bifunctional ligands, hydrazone linker, opioid agonists, NK1 antagonists.
ABSTRACT: The area of multitarget compounds, joining two pharmacophores within one molecule, is a vivid field of research in
medicinal chemistry. Not only pharmacophoric elements are essential for the design and activity of such compounds, but the type
and length of linkers used to connect them are also crucial. In the present contribution, we describe compound 1 in which a typical
opioid peptide sequence is combined with a fragment characteristic for neurokininꢀ1 receptor (NK1R) antagonists through a
hydrazone bridge. The compound has a high affinity for µꢀ and δꢀopioid receptors (IC50= 12.7 nM and 74.0 nM, respectively) and a
weak affinity for the NK1R. Molecular modelling and structural considerations explain the observed activities. In in vivo test,
intrathecal and intravenous administrations of 1 exhibited a strong analgesic effect, which indicates potential BBB penetration. This
paper brings an exemplary application of the hydrazone linker for fast, facile and successful preparation of chimeric compounds.
The idea of making bivalent active compounds started to
emerge as an interesting and fruitful approach in medicinal
chemistry about 40 years ago, much earlier than the existence
of membraneꢀbound receptors dimerization was discovered.1–3
Bifunctional ligands are compounds constructed by joining
two pharmacophores with a linker.4 Essential features for their
design and biological activity are not only the pharmacophoric
elements, but also the type and length of linkers used.5–7 In
case of peptides or chimeric compounds mixing peptide and
nonꢀpeptide elements, the hydrazide bridge (as in biphalin: Hꢀ
TyrꢀDꢀAlaꢀGlyꢀPheꢀNHꢀNH←Phe←Gly←DꢀAla←TyrꢀH),
piperazine, polyꢀamide or ethylene glycol residues have found
most widespread application. The hydrazone moiety had been
used in the past by Lipkowski et al. to join oxymorphone and
naltrexone fragments to peptides bearing the address portion
of enkephalin and dynorphin (1ꢀ8).8 However this strategy has
not been applied in the following years, although hydrazone
and Nꢀacyl hydrazone (NAH) derivatives are extensively
studied in small molecules medicinal chemistry, and are often
considered to be privileged structures.9–11 From the standpoint
of designing new multivalent ligands (be it of peptidic or
chimeric character), the hydrazone function has clear benefits:
the ease of synthesis, a variety of commercially available
condensation reagents and their significant contributions in
conformationꢀactivity relationship. On the basis of the
advantages, we chose the linker for the synthesis of
bifunctional compounds. Here we report compound JZ031: Hꢀ
TyrꢀDꢀAlaꢀGlyꢀPheꢀNHꢀN=CHꢀ[3',5'ꢀ(CF3)2ꢀPh] (1) in which
the hydrazone serves to join a typical opioid peptide sequence
with a small moiety common to a neurokininꢀ1 receptor
(NK1R) antagonist pharmacophore. The opioid and NK1
receptors play a key role in transmission and modulation of
pain signal.12 It was suggested that simultaneous activation of
opioid receptors and blockade of NK1R may prevent side
effects induced by opioids administered alone such as: nausea,
analgesic tolerance, euphoria or addiction.13–15 Therefore, a lot
of attention was devoted to preparing bivalent ligands
containing opioid agonist and NK1R antagonist
pharmacophores.16–20
Compound 1 was synthesized by the method published
previously.21 Briefly, Bocꢀprotected tetrapeptide BocꢀTyrꢀDꢀ
AlaꢀGlyꢀPheꢀOH (3) was made in solution by use of DCC and
HOSu as a coupling mixture in DMF. Hydrazide BocꢀTyrꢀDꢀ
AlaꢀGlyꢀPheꢀNHꢀNH2 (2) was also obtained by the same
coupling method using an excess of hydrazine. Next,
intermediate 2 was isolated by the preparative RPꢀHPLC and
mixed with 3,5ꢀbis(trifluoromethyl)benzaldehyde in ethanol.
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Scheme 1. The synthesis of compound 1 (JZ031: H-Tyr-D-Ala-Gly-Phe-NH-N=CH-[3',5'-(CF3)2-Ph]).
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Table 1. Affinity of bifunctional ligands at MOR, DOR, and NK1R.
IC50a (nM)
Compound
Sequence
MOR
12.7 ± 0.7
4.7
DOR
74.2 ± 0.4
230
NK1
4.24 ꢁM± 0.8
n/db
1 (JZ031)
522
TyrꢀDꢀAlaꢀGlyꢀPheꢀNHꢀN=CHꢀ[3',5'ꢀ(CF3)2ꢀPh]
TyrꢀDꢀAlaꢀGlyꢀPheꢀNHꢀNH2
622
TyrꢀDꢀAlaꢀGlyꢀPheꢀNHꢀNH←Phe
0.74
15
n/db
n/db
Biphalin22
(TyrꢀDꢀAlaꢀGlyꢀPheꢀNHꢀ)2
1.4 ± 0.4
n/db
2.6 ± 0.3
n/db
Substance P
HꢀArgꢀProꢀLysꢀProꢀGlnꢀGlnꢀPheꢀPheꢀGlyꢀLeuꢀMetꢀNH2
1.09 ± 0.42
a Mean IC50 value ± SEM (n=3), b n/d (not determined)
After a few minutes, a protected hydrazone BocꢀTyrꢀDꢀAlaꢀ
GlyꢀPheꢀNHꢀN=CHꢀ[3',5'ꢀ(CF3)2ꢀPh] (4) was obtained in a
good yield. Deprotection of 4 was carried out using
trifluoroacetic acid and the final product was purified by RPꢀ
HPLC. The purity and structure was confirmed by highꢀ
performance liquid chromatography (HPLC), electrospray
ionization mass spectrometry (ESIꢀMS) and one and two
dimensional nuclear magnetic resonance methods (1D (1H,
13C, DEPT) and 2D (COSY, HSQC, HMBC) NMR) (details of
the experimental and analytical parts are described in the
Supporting Information).
compound 6, has a second protonated free amine that is able to
interact with Ile215 and Asn127. Moreover, this compound
assumes a twisted conformation of the diacylhydrazine
subunit,23 which allows the aromatic ring of the terminal Phe
to accommodate an additional interaction with Trp133. In
contrast, JZ031 1 is able to interact with neither Asp216, nor
to place the aromatic ring in the vicinity of Trp133 (the
acylhydrazone bridge is preferentially planar, or close to
planarity, and extended as shown by quantum mechanical
calculations24 and a query in PDB database (see Supporting
Information). On the other hand, the ꢀCF3 groups can
potentially contact Asn127, Tyr128, Gly131 or Ser214.
Molecular docking to DOR (PDB structure: 4RWA, Figure 3
in Supporting Information) presents similar binding poses. For
all compounds, the protonated amine of Tyr1 forms a salt
bridge with Asp128 and the side chain is bent so that the
hydroxy group of Tyr1 is able to contact with His278. The rest
of the peptide chain is extended and the aromatic ring of Phe4
interacts again with Trp284. In case of DOR, however, the
hydrazide bridge protons do not seem to have an interaction
partner (DOR has Val197 in place of MOR Asp216), and this
may explain relatively low affinity of 5 for DOR. The terminal
Phe of 6 could pack in a position only slightly different than
that of Phe3 in DmtꢀTicꢀPheꢀPheꢀNH2 (a strong DOR binder
coꢀcrystallized in 4RWA structure).25 In the case of JZ031,
CF3 groups can reach carbonyl oxygens of Lys108 or Glu112
for interactions.
The compound JZ031 (1) was checked for activity both in
vitro and in vivo. First, binding affinity to MOR, DOR and
NK1 receptors was determined (Table 1). JZ031 binds with
IC50 (half maximal inhibitory concentration) of 12.7 nM and
74.0 nM to MOR and DOR in whole rat brain preparation,
respectively. JZ031’s MOR binding is lower than the one of
biphalin (TyrꢀDꢀAlaꢀGlyꢀPheꢀNHꢀ)2 or its truncated variants 5
(TyrꢀDꢀAlaꢀGlyꢀPheꢀNHꢀNH2) and 6 (TyrꢀDꢀAlaꢀGlyꢀPheꢀ
NHꢀNH←Phe). Regarding DOR binding, compound
1
exhibits significantly higher affinity than hydrazide 5, but it is
far less effective than biphalin and 6. On the other hand,
compound 1 possesses only a moderate affinity for NK1R
with IC50 = 4.24 ± 0.80 ꢁM.
The gradation of affinities for opioid receptors in the
considered molecules may be partially rationalized by
molecular modelling. When docked in MOR (PDB structure:
4DKL, Figure 1), JZ031 (1), 5 and 6 all have a very similar
overall binding mode. The protonated amine of Tyr1 forms a
salt bridge with Asp147 and the side chain is bent so that the
hydroxy group is able to interact with His297. The rest of the
peptide chain is extended and the aromatic ring of Phe4 points
towards Trp318 and His319. The hydrazide moiety of 5 and 6
contacts Asp216 by hydrogen bonding. The most potent
Regarding the weak binding affinity of JZ031 to NK1R, three
remarks can be made. First, based on the above mentioned
preferential planarity of the acylhydrazone linker, it may be
speculated that JZ031 1 is unable to acquire the appropriate
pharmacophoric arrangement of two aromatic rings without
significant distortion from the energetic minimum.26–28
Additionally, further deterioration of the binding could arise
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Figure 1. Contacts of compounds 1, 5 and 6 with the MOR binding site. On the left given are interactions of the TyrꢀDꢀAlaꢀGlyꢀPheꢀ part
common to 1, 5 and 6; on the right interactions of the Cꢀterminal parts of these molecules. The black dot marks the joint between the
common part and the rest of the molecule in each case.
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from a suboptimal distance, as one additional atom was
inserted. Finally, in the so far reported potent, bifunctional
peptides containing NK1R and opioid pharmacophores, a Trp
residue is always present.29,30 Designing JZ031, we tried to
mimic the two phenyl rings as found in numerous small
molecule NK1R antagonists.
minutes after administration (69.84% ± 19.5 MPE) and
thereafter a significant loss was observed at 120 min. (16.84%
± 9.7 MPE). Intrathecal administration of JZ031 produced
strong analgesic response already after 5 minutes (Figure 1c).
The analgesic curve has a characteristic bellꢀshape with the
peak at 15 minutes postꢀinjection for all examined doses.
Animals injected with 1 exhibited analgesia equipotent to that
of morphineꢀtreated animals, but at much lower dose. Here, 2
nmol/kg was enough to produce the same magnitude of peak
analgesia as morphine at a dose of 12 nmol/kg, reaching 80.31
% ± 8.59 MPE at 15 minutes postꢀinjection. A further dose
increase (4 nmol/kg) did not improve the analgesic response.
Ligand 1 retained the peak activity up to 30 minutes postꢀ
administration, whereas morphine sustained peak analgesia up
to 60 min. In the case of both compounds, a major decline in
the antinociceptive effect was observed 120 minutes postꢀ
injection.
The high affinity of JZ031 for the opioid receptors prompted
us to test it for analgesic activity in vivo. The compound exerts
strong, timeꢀ and doseꢀdependent analgesic effect following
both systemic i.v. (intravenous) and central i.th. (intrathecal)
administration. The effect was measured in plantar (i.v.) and
tailꢀflick (i.v. and i.th.) tests (see Supporting Information for
experimental details). The results are presented in Figure 2. At
the supraspinal level, JZ031 evoked the strongest analgesic
response 30 minutes after i.v. administration of 80 µmol/kg
dose, reaching 75.41% ± 8.27 MPE (maximal possible effect)
(Figure 1a). This effect diminished after 60 minutes and
dropped to baseline values after 120 minutes. Here, our
compound was less potent than morphine. However, at the
spinal level (Figure 1b), the analgesic response quickly
The analgesic effect of JZ031 (1) in dose 2 nmol/kg is weaker
than that of biphalin which between 5 and 30 min. after
injection reaches 90ꢀ100% MPE and decreases to 68% MPE at
60 min.31 This is consistent with lower affinity of compound 1
for MOR and DOR.
reached 84.81%
±
15.00 MPE at
5
minutes after
administration of JZ031, whereas in the case of morphine no
effect was observed at this particular time point. Thus, 1
produces an impressive rapid onset of analgesia but the effect
decreases faster than the response to morphine does. In the
plantar test, only the administration of the highest 80µmol/kg
dose produced most effective and longꢀlasting analgesia. The
peak analgesic response was recorded as early as 5 minutes
postꢀinjection and lasted up to 30 minutes being 94.26% ±
4.00 MPE. A decline in analgesic effect was reported 60
Taking into account the fast onset of action obtained in tailꢀ
flick test after i.v. administration, it may be speculated that
this effect is caused by fast BBB penetration of our ligand.
The similar onset of morphine and JZ031 after i.th.
administration and higher lipophilicity of our ligand
(compared to morphine) support this explanation.
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(JZ031) exhibited good analgesic effects in animal models and
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a remarkably fast onset of antinociception was noticed, even
after i.v. administration. This report is an exemplary
application of the hydrazone linker for a fast and facile
preparation of bifunctional chimeric compounds.
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ASSOCIATED CONTENT
Supporting Information
Experimental procedures for the synthesis, binding experiments,
in vivo tests and computational analysis, as well as compound
characterization data are available free of charge via the Internet
at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
* Aleksandra Misicka, eꢀmail: misicka@chem.uw.edu.pl.
* Jolanta Dyniewicz, eꢀmail: jolantadyniewicz@gmail.com
Author Contributions
DJ synthesized all compounds, DJ, LA, MA participated in
binding assays, DJ, KP, LA carried out in vivo tests, PFJL carried
out modeling study. The manuscript was written through
contributions of all authors. All authors have given approval to the
final version of the manuscript.
Funding Sources
The research was supported by: National Science Centre (NCN)
Poland, PRELUDIUM grant DECꢀ2011/03/N/ST5/04725 and a
scholarship from the European Social Fund, Human Capital
Operational Programme for the execution of the project “Support
for bio tech med scientists in technology transfer”; (UDAꢀ
POKL.08.02.01ꢀ14ꢀ041/09). The work was coꢀfinanced with the
European Union funds by the European Social Fund.
ACKNOWLEDGMENT
Thanks to Barbara Nowicka and Zdzisława Kowalska for the help
with laboratory work. Molecular modelling was supported by
Computational Grant G63ꢀ10 from the Interdisciplinary Centre for
Mathematical and Computer Modelling (ICM) at the University
of Warsaw. Świerk Computing Centre of the National Centre for
Nuclear Research in Świerk, Poland is also acknowledged for
generous allotment of computer time. Project subject carried out
with the use of CePT infrastructure financed by the European
Union – the European Regional Development Fund within the
Operational Programme “Innovative economy” for 2007ꢀ2013.
SB and DT acknowledge the Research Foundation – Flanders
(FWO Vlaanderen) for the financial support.
Figure 2. Analgesic responses of 1 (JZ031) in four different doses
compared to morphine (MF) expressed as % MPE (means ±
SEM); n=6–8. Significance: *p < 0.05; **p < 0.01 ***p < 0.001
as compared to morphine. a) i.v. administration, plantar test b) i.v.
administration, tailꢀflick test. c) i.th. administration, tailꢀflick test.
In conclusion, we presented 1 (JZ031), a chimeric compound
in which a typical opioid peptide sequence is fused with a
fragment characteristic of the neurokininꢀ1 receptor antagonist
pharmacophore by means of a hydrazone bridge. The
compound has high affinity for MOR and DOR, but it is only
a weak binder of NK1R. Molecular modelling suggests that 1
(JZ031) preserves interactions with MOR and DOR, typically
observed for opioid peptides, and the [3',5'ꢀ(CF3)2ꢀPh]
fragment is able to find additional contacts in the binding sites.
The weak NK1R binding may be explained in terms of a
larger distance between pharmacophoric aromatic rings (here
we refer to pharmacophores derived for small molecule
antagonists26–28) and a reduced flexibility of the hydrazone
bridge. Furthermore, our compound lacks a Trp residue which
is found in all soꢀfar published potent bifunctional compounds
of this kind (chimeric peptides). Gratifyingly, compound 1
ABBREVIATIONS
BBB, blood-brain barrier, Boc, tert-butyloxycarbonyl, DCC,
dicyclohexylcarbodiimide, DMF, dimethylformamide, DOR, δ-
opioid receptor, HOSu, N-hydroxysuccinimide, i.th., intrathecal,
i.v., intravenously, NK1R, neurokinin-1 receptor, MF, morphine,
MOR, µ-opioid receptor, MPE, maximal possible effect, NAH, N-
acyl hydrazone, NMR, nuclear magnetic resonance RP-HPLC,
reverse phase high pressure liquid chromatography, SP,
substance P, TFA, trifluoroacetic acid.
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The hydrazone linker as a useful tool for preparing chimeric
peptide/non-peptide bifunctional compounds
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Jolanta Dyniewicz†*, Piotr F. J. Lipiński†, Piotr Kosson †, Anna Leśniak†, Marta BochyńskaꢀCzyż†,
Adriana Muchowska †, Dirk Tourwé§, Steven Ballet§, Aleksandra Misicka†*, Andrzej W.Lipkowski†
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Scheme 1. The synthesis of compound 1 (JZ031: H-Tyr-D-Ala-Gly-Phe-NH-N=CH-[3',5'-(CF3)2-Ph]).
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Figure 1. Contacts of compounds 1, 5 and 6 with the MOR binding site. On the left given are interactions of
the Tyr-D-Ala-Gly-Phe- part common to 1, 5 and 6; on the right interactions of the C-terminal parts of these
molecules. The black dot marks the joint between the common part and the rest of the molecule in each
case.
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Figure 2. Analgesic responses of 1 (JZ031) in four different doses compared to morphine (MF) expressed as
% MPE (means ± SEM); n=6–8. Significance: *p < 0.05; **p < 0.01 ***p < 0.001 as compared to
morphine. a) i.v. administration, plantar test b) i.v. administration, tail-flick test. c) i.th. administration, tail-
flick test.
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Table of contents
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