Synthesis and Analgesic Activity of New Analogues of Tyr-MIF Including Pyrrole Moiety

Article abstract of DOI:10.1007/s10989-015-9502-8

Pain is one of many medical problems of modern society. Together with a number of other diseases such as heart attacks, strokes, tumors, etc. it ranks among the first in manifestation. There are a huge number of medical drugs more or less effective agains

Full text of DOI:10.1007/s10989-015-9502-8

Int J Pept Res Ther (2016) 22:243–248
DOI 10.1007/s10989-015-9502-8
Synthesis and Analgesic Activity of New Analogues of Tyr-MIF
Including Pyrrole Moiety
Dancho L. Danalev1 Stanislava P. Vladimirova2 Borislav P. Borisov1
•
•
•
Hristina H. Nocheva4 Adriana I. Bocheva4 Dessislava A. Marinkova1
Emilia D. Naydenova3 Valentin S. Lozanov5
•
•
•
•
Accepted: 11 December 2015 / Published online: 18 December 2015
Ó Springer Science+Business Media New York 2015
Abstract Pain is one of many medical problems of
modern society. Together with a number of other diseases
such as heart attacks, strokes, tumors, etc. it ranks among
the first in manifestation. There are a huge number of
medical drugs more or less effective against pain in a
practice. Globally, the searching of new molecules with
analgesic activity and better selectivity or greater effect at
lower doses continues. In addition, some groups trying to
improve the properties of known molecules in medical
practice as various heterocyclic compounds by modifying
one or another of their part. Other groups work on the
creation of new mimetics of natural molecules with well
established physiological activity. In this global context,
here we report the synthesis of two new compounds which
are hybrid molecules between the specifically substituted
pyrrole (Pyr) and analogues of Tyr-MIF-1 peptide. All
investigations on the analgesic activity show better activity
at the same dose than natural Tyr-MIF-1 peptide for the
analogue Pyr-Tyr-Phe-Leu-Ala-OH. Compound Pyr-Ala-
Leu-Phe-Tyr-OH has no better effect comparable to that of
the parent peptide. The obtained results clearly show that it
is essential that Tyr residue occupies N-terminal position of
MIF-1 analogue. The lack of better activity of the analogue
Pyr-Ala-Leu-Phe-Tyr-OH reveals that Pyr residue does not
influence on the analgesic activity. In addition we found
that C-terminal amide function generally presented in
natural MIF-1 is not absolutely necessary for activity.
Keywords Opioide peptides Á Pyrrole Á Pain Á Tyr-MIF-1
analogues Á Hybride structures Á Paal–Knorr reaction
Introduction
Everyday life does not pass without experiencing some kind
of pain. The pain can be caused by various factors. However,
in all cases, it represents an unpleasant experience, and in
many cases is associated with discomfort and disability.
Daily, doctors prescribe large number of painkiller drugs
which can belong to different groups—corticosteroids,
nonsteroidal anti-inflammatory drugs (NSAIDs), etc.
(Dworkin et al. 2003). Since last century studying the
properties of various heterocyclic compounds including
pyrrole (Pyr), as anti-inflammatory and anti-pain agents is
explored strongly (Lainton et al. 1995). It is interesting to
note that a number of molecules containing in its structure
Pyr heterocycle are approved as drugs with diverse activities
in medical practice (Malinka et al. 2000; Amishiro et al.
1999; Bovy et al. 1999; Fumoto et al. 1999; Carson et al.
1997; Artico et al. 2000). On the other hand, some natural
molecules including peptides can also perform a variety of
functions in the human body. They can be neurotransmitters,
neuromodulators, hormones, etc. Tyr-MIF is a tetrapeptide
well know from the literature with its opioide activity and
& Dancho L. Danalev
dancho.danalev@gmail.com
1
Biotechnology Department, University of Chemical
Technology and Metallurgy, 8 blvd. Kliment Ohridski,
1756 Sofia, Bulgaria
2
Department of Organic Synthesis and Fuels, University of
Chemical Technology and Metallurgy, 8 blvd. Kliment
Ohridski, 1756 Sofia, Bulgaria
3
Department of Organic Chemistry, University of Chemical
Technology and Metallurgy, 8 blvd. Kliment Ohridski,
1756 Sofia, Bulgaria
4
Department of Pathophysiology, Medical University of Sofia,
2 Zdrave Str., 1431 Sofia, Bulgaria
5
Department of Chemistry and Biochemistry, Medical
University of Sofia, 2 Zdrave Str., 1431 Sofia, Bulgaria
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Int J Pept Res Ther (2016) 22:243–248
good selectivity according to the l-receptors (Pan and Kastin
2007; Bocheva et al. 2000; Bocheva and Lazarova 2003).
The modification in the structure of some natural opioid
peptides, and combining them into hybrid structures with
various heterocyclic compounds is investigated as a
promising alternative to existing commercial and non-ster-
oidal antiinflammatory and opioid agents.
deprotection of small aliquot of the resin by treatment with
20 % piperidine/DMF.
General Procedure for Coupling of Amino Acids
by HBTU(TBTU)/DIPEA Method
The Fmoc-amino acid (4 eq. relative to resin loading),
HBTU (4 eq. relative to resin loading) and DIPEA
(4 eq. relative to resin loading) were dissolved in DMF.
The reaction mixture was stirred for 10 min. and added to
the peptide-resin solution in DMF. The reaction mixture
was left to rest for 2 h. The Kaiser test was made at the end
of the reaction time after consecutively washing by
4 9 25 ml of DMF and 4 9 25 ml of DCM.
In this global context, we made a design and synthesized
hybrid structures between the Pyr molecule and analogues
of Tyr-MIF in order to combine the properties of both types
of molecules and to obtain compounds with analgesic
activity. Our previous studies [unpublished results] show
that the incorporation of single amino acids in a Pyr cycle
through the reaction of Paal–Knorr is possible. Our pre-
liminary biological studies indicate that the resulting
compounds exhibit a good analgesic activity. Based on
these preliminary results, we have made the design of two
tetrapeptides, which mimic the structure of the well-known
in the literature for its analgesic properties Tyr-MIF. The
peptide moiety is a combination of those amino acids
which, in our preliminary studies in combination with a Pyr
heterocycle showed the highest analgesic activity.
Preparation of the Peptide-Resin for Cleavage
The obtained peptide-resin was washed by 39 DMF; 39
DCM; 39 i-propanol and 39 Ether to shrink the resin. The
peptide-resin was then dried under high vacuum for 4 h
over NaOH.
TFA Cleavage and Deprotection of Peptide
Materials and Methods
Chemical Synthesis
The dry peptide-resin was placed in a flask and TFA/TIS/H2O
(95:2,5:2,5) were added for 5 h. At the end of the reaction
time the obtained peptide was collected by filtration and the
removed resin was washed by 39 TFA and 39 DCM. The
obtained filtrates were collected and evaporated to dryness.
Final product is recrystallized in cold diethylether.
All amino acids and condensation agents are purchased by
IRIS Biotech, Germany. All solvents are purchased by
Valerus, Bulgaria and they are used without any prelimi-
nary treatment. TLC characteristics of aim products were
measured on Silica gel 60 F254 aluminum sheets, Merck
1.05554 at ambient temperature using chloroform as a
mobile phase.
General Procedure for Paal–Knorr Reaction
Peptide (0.10 mol) was dissolved in glacial acetic acid
(50 mL) and to the resulting solution 1,4-dicarbonyl
compound (0.10 mol) were added. The obtained solution
was refluxed. The reaction development was monitored by
TLC. After disappear of starting 1,4-dicarbonyl compound
on TLC, the mixture was poured into water. The separated
precipitate was filtered off, washed with water, dried, and
recrystallized from warm ethanol and wash with 5 % citric
acid. The reaction time was varied from 3 to 5 h depending
on the starting compounds (TLC control).
General Procedure for Attachment of Amino Acid to Wang
Resin
The Wang resin was pre-swelled with DCM for 1 h and
then thoroughly washed with DMF. Fmoc-amino acid
(4 eq. to the activated resin) is dissolved in a minimal
amount of DCM and diisopropylcarbodiimide (1.65 eq. to
the Fmoc-amino acid) is added at 0 °C. The obtained
mixture is stirred for 20 min at 0 °C. Further the solvent is
removed under vacuum and obtained crude product is
dissolved in minimal amount of DMF. The obtained solu-
tion is added to the Wang resin in the reactor for SPPS and
additional amount of 25 ml of DMF and 0.1 eq. to the resin
of dimethylaminopyridin are added. The reaction mixture
was stirred for 2 h and after the end of the reaction time it
was washed consecutively by 4 9 25 ml of DMF and
4 9 25 ml of DCM. Further the Kaiser test was made after
LC/MS Analysis
The structure of both aim compounds was proven by LC/
MS.
Instrumentation
Analyses were carried out on Q ExactiveÒ hybrid quad-
rupole-Orbitrap mass spectrometer equipped with
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Int J Pept Res Ther (2016) 22:243–248
245
TurboFlowÒ HPLC system, PAL autosampler and HESIÒ
electrospray ionization module (ThetmoScientific Co,
USA). Data acquisition and processing were carried out
Pressure was applied to the hind-paw and the pressure
(g) required to elicit nociceptive response (squeak and
struggle) was taken as the mechanical nociceptive thresh-
old. A cut-off value of 500 g was used to prevent damage
of the paw (Picture 1).
Ò
with XCalibur 2.4 software package.
Chromatographic Conditions
Hot-Plate (HP) Test Description
Column: synchronis C18, 1.7 lm (50 9 2.1 mm) (Thet-
moScientific Co, USA); mobile phase: A = 10 mM
ammonium hydrogencarbonate in water, B = A/acetoni-
trile (10/90, v/v); flow rate: 300 ll/min; gradient: 10 % B
for 2 min; 10–90 % B for 15 min; 90 % B for 2 min;
90–10 % B for 1 min and equilibration at 10 % for 3 min.
Injection volume: 10.0 ll.
The latency of response to pain was measured from the
moment of placing an animal on a metal plate (heated to
55 0.5 °C) to the first signs of pain (paw licking,
jumping). The cut-off time observed to prevent paw dam-
age was 30 s (Picture 2).
Statistical Analysis
Mass Spectrometry Conditions
The results for both methods were statistically assessed by
one-way analysis of variance ANOVA followed by t test
comparison. Values are mean S.E.M. Values of p B 0.05
were considered to indicate statistical significance.
Full-scan spectra over the m/z range 150–2000 were
acquired in positive ion mode at resolution settings at
70,000. All MS parameters were optimized for sensitivity
to the target analytes using the instrument control software
program. Q Exactive parameters were: spray voltage
4.0 kV, sheath gas flow rate 35, auxiliary gas flow rate 8,
spare gas flow rate 2, capillary temperature 280 °C, probe
heater temperature 300 °C and S-lens RF level 50.
Experimental Part
Synthesis of Pyr-Tyr-Phe-Leu-Ala-OH (1)
H-Tyr-Phe-Leu-Ala-OH (0.10 mol) was dissolved in glacial
acetic acid (50 mL) and to the resulting solution 2-[2-(4-
chlorophenyl)-2-oxo-ethyl]-3-oxo-butyric acid ethyl ester
(0.10 mol) was added. The obtained solution was refluxed
for 3–5 h (TLC control). After disappear of starting 2-[2-(4-
chlorophenyl)-2-oxo-ethyl]-3-oxo-butyric acid ethyl ester
on TLC, the mixture was poured into water. The separated
precipitate was filtered off, washed with water, dried,
recrystallized from warm ethanol and wash with 5 % citric
Biological Studies
Animals
All the experiments were carried out on male Wistar rats
(180–200 g). Animals were housed in groups of 8 per cage
and kept under a normal 12 h light/dark cycle and
22 2 °C temperature, with free access to food and water.
Tyr-MIF-1 (was obtained from Sigma) and two newly
synthesized compounds Pyr-Tyr-Phe-Leu-Ala-OH (1) and
Pyr-Ala-Leu-Phe-Tyr-OH (2) were dissolved in sterile
saline solution (0.9 % Na Cl) and injected intraperitoneally
(i.p.). All drugs were administrated at a dose 1 mg/kg.
All the experiments were conducted between 9.00 and
12.00 a.m., and according to the ‘‘Principles of laboratory
animal care’’ 9 (NIH Publication No. 85-23, revised 1985),
and the rules of the the Animal Care and Use Committee of
the Medical University of Sofia.
Nociceptive Tests
Paw-Pressure Test (Randall–Sellito Test)
Description
The changes in the mechanical nociceptive threshold of the
rats were measured by analgesimeter (Ugo Basile).
Picture 1 Paw pressure (PP) test
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Int J Pept Res Ther (2016) 22:243–248
Synthesis of Pyr-Ala-Leu-Phe-Tyr-OH (2)
H-Ala-Leu-Phe-Tyr-OH (0.10 mol) was dissolved in glacial
acetic acid (50 mL) and to the resulting solution 2-[2-(4-
chlorophenyl)-2-oxo-ethyl]-3-oxo-butyric acid ethyl ester
(0.10 mol) was added. The obtained solution was refluxed
for 3–5 h (TLC control). After disappear of starting 2-[2-(4-
chlorophenyl)-2-oxo-ethyl]-3-oxo-butyric acid ethyl ester
on TLC, the mixture was poured into water. The separated
precipitate was filtered off, washed with water, dried,
recrystallized from warm ethanol and wash with 5 % citric
acid. The obtained product is yellow powder with Rf = 0.61
and yield of 60 %. MS [M ? H?] calculated 758.30824,
found 759.31982. tR (HPLC) = 7.09 min.
Results and Discussion
Our previous investigation on the hybrid molecules includ-
ing Pyr heterocycle and single amino acids reveals good
activity of some compounds. Based on this study here we
made design of two Tyr-MIF analogues H-Tyr-Phe-Leu-
Ala-OH and H-Ala-Leu-Phe-Tyr-OH in order to include
them in Pyr heterocycle and to study their analgesic activity
as well as to reveal some relationships related to the impor-
tance of C-terminal amide function for analgesic activity.
Picture 2 Hot plate (HP) test
acid. The obtained product is light brown powder with
Rf = 0.58 and yield of 64 %. MS [M ? H?] calculated
758.30824, found 759.31915. tR (HPLC) = 6.47 min.
CH3
NH2
O
CH3
O
H
N
H
O
O
N
N
H
OH
O
CH3COOH
3-5 h, reflux
O
O
CH3
CH3
+
CH3
Cl
O
OH
O
O
CH3
CH3
O
CH3
N
CH3
O
H
N
H
Cl
N
N
OH
H
O
O
CH3
OH
Fig. 1 Synthesis of Pyr-Tyr-Phe-Leu-Ala-OH (1)
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Int J Pept Res Ther (2016) 22:243–248
247
O
O
NH2
CH3
O
O
H
N
H
N
CH3
CH3COOH
3-5 h, reflux
+
N
H
CH3
OH
CH3
Cl
O
O
O
O
CH3
OH
O
O
CH3
CH3
N
O
O
H
N
H
N
Cl
CH3
N
H
CH3
OH
O
O
CH3
OH
Fig. 2 Synthesis of Pyr-Ala-Leu-Phe-Tyr-OH (2)
The aim peptides were synthesized by standard solid
phase peptide synthesis (SPPS) on Wang-polystyrene resin
by means of Fmoc/t-Bu strategy.
We met difficulties with the coupling of Fmoc-Leu-OH
in both aim peptides and the condensation reaction was
repeated three times changing coupling reagents from
TBTU, through diisopropylcarbodiimide to HBTU.
The obtained tetrapeptides after deblocking of Fmoc-
function of N-terminal amino acid by treatment with 10 %
pyperidine/DMF were released of the polymere by treat-
ment with cocktail TFA/TIS/H2O. During this procedure
OH group of Tyr was also deprotected of t-Bu function.
Both final tetrapeptide were crystallized in cold diethyl
ether and further reaction of Paal–Knorr was used for Pyr
heterocycle formation in solution starting from the neces-
sary 1,4-dicarbonyl compound according schemes pre-
sented on Figs. 1 and 2.
Fig. 3 Effects of Tyr-MIF-1 and the newly synthesized compounds
Pyr-Tyr-Phe-Leu-Ala-OH (1) and Pyr-Ala-Leu-Phe-Tyr-OH (2) in a
dose 1 mg/kg measured with PP test. Mean values S.E.M. are
presented. ***p \ 0.001 relative to control; ???p \ 0.001 relative
to Tyr-MIF-1;?p \ 0.05 relative to Tyr-MIF-1
Structures of two aim hybrid structures are proven by
LC/MS.
The investigation started 10 min after i.p. injection of
Tyr-MIF-1, Pyr-Tyr-Phe-Leu-Ala-OH (1) and Pyr-Ala-
Leu-Phe-Tyr-OH (2) all et a dose 1 mg/kg i.p. Tyr-MIF-1
applied alone significantly increased the pain threshold
(p \ 0.001) in PP test (Fig. 3) and the hot plate latency in
HP test (Fig. 4). The analgesic effect was well pronounced
and time dependent only in PP test.
The analogue Pyr-Ala-Leu-Phe-Tyr-OH (2) has an
analgesic effect comparable to that of the Tyr-MIF-1
peptide only on the 10th min (Fig. 3).
Our results showed that only analogue Pyr-Tyr-Phe-
Leu-Ala-OH (1) significantly increased the pain threshold
comparable to the control and Tyr-MIF-1.
The new analogue Pyr-Tyr-Phe-Leu-Ala-OH (1) on the
10th (p \ 0.05) and 20th (p \ 0.001) min of investigation
significantly increased the pain threshold compared to that
of control and Tyr-MIF-1. The analgesic effect is the most
significant of the 20th min (Fig. 3).
In HP test Tyr-MIF-1 (p \ 0.001) and analogue Pyr-Tyr-
Phe-Leu-Ala-OH (1) (p \ 0.001) significantly increased the
HP-latency during the whole investigated period, while the
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received some financial support for analgesic activity determination
by the Grant 42/2014 with Medical University of Sofia.
Compliance with Ethical Standards
Conflict of Interest Stanislava P. Vladimirova, Dessislava A.
Marinkova, Emilia D. Naydenova and Valentin S. Lozanov declare
that they have no conflict of interest.
Human and Animal Rights and Informed Consent Hristina H.
Nocheva and Adriana I. Bocheva declare that all the experiments with
animals described in this article were conducted between 9.00 and
12.00 a.m., and according to the ‘‘Principles of laboratory animal
care’’ 9 (NIH publication No. 85-23, revised 1985), and the rules of
the the Animal Care and Use Committee of the Medical University of
Sofia.
Fig. 4 Effects of Tyr-MIF-1, compounds Pyr-Tyr-Phe-Leu-Ala-OH
(1) and Pyr-Ala-Leu-Phe-Tyr-OH (2) measured by hot plate test.
Mean values S.E.M. are presented. ***p \ 0.001 relative to
control; ???p \ 0.001 relative to Tyr-MIF-1
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Conclusions
Two peptides analogues of Tyr-MIF-1 were synthesized by
SPPS by means of Fmoc/t-Bu strategy. They were further
included in their N-terminus in Pyr heterocycle. Studies on
the analgesic activity of the two newly synthesized mole-
cules show better activity at the same dose than natural
Tyr-MIF-1 peptide for Pyr-Tyr-Phe-Leu-Ala-OH. Com-
pound Pyr-Ala-Leu-Phe-Tyr-OH has no better effect
comparable to that of the parent peptide. The obtained
results clearly show that it is essential that Tyr residue
occupies N-terminal position of MIF-1 analogue. The lack
of better activity of the analogue Pyr-Ala-Leu-Phe-Tyr-OH
reveals that Pyr moiety does not influence on the analgesic
activity. In addition we found that C-terminal amide
function generally presented in natural MIF-1 is not
absolutely necessary for activity.
Fumoto Y, Eguchi T, Uno H, Ono N (1999) Synthesis of pyrrolostatin
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Acknowledgments Borislav P. Borisov has received some financial
support for its Ph.D. thesis development by the Grant No.
BG051PO001-3.3.06-0059, financed by the European Social Fund
and Operational Programme Human Resources Development
(2007–2013) and co-financed by Bulgarian Ministry of Education and
Science. Dancho L. Danalev has received some financial support for
chemical synthesis of aim compounds by the Grant 11385 with Sci-
entific Fund of University of Chemical Technology and Metallurgy of
Sofia, Bulgaria. Adriana I. Bocheva and Hristina H. Nocheva have
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123