Synthesis of an MIF-1 analogue containing enantiopure (S)-α- trifluoromethyl-proline and biological evaluation on nociception

Article abstract of DOI:10.1016/j.ejmech.2012.12.041

The synthesis and the effect of a novel MIF-1 analogue on nociception during acute pain in rat model are reported. The synthesis of this enantiopure trifluoromethyl group containing tripeptide was performed through a peptide coupling reaction between the HCl. Leu-Gly-NH₂ and the (S)-α-Tfm-proline. The analgesic effect of the CF₃-(MIF-1) 2 has been evaluated in vivo on rat model by paw pressure (PP) and hot plate (HP) tests and compared to the native peptide MIF-1. Highest analgesic effect was observed with CF₃-(MIF-1) 2 only in PP test. In order to study the mechanisms of nociception induced by the studied peptides, the involvement of the opioid and the nitric oxideergic systems was investigated. The results are in favor of a participation of both system since pretreatment, 20 min before injection of the CF₃-(MIF-1) 2, with the non-competitive antagonist of opiate receptors naloxone, the nitric oxide synthase (NOS) inhibitor l-N ^G-nitroarginine ester (l-NAME) or the nitric oxide (NO) donor l-arginine (l-Arg) significantly decreased the pain perception in PP and HP tests.

Full text of DOI:10.1016/j.ejmech.2012.12.041

Accepted Manuscript
Synthesis of a MIF-1 analogue containing Enantiopure (S)-α-Trifluoromethyl Proline
and Biological Evaluation on Nociception
Ibtissem Jlalia, Nathalie Lensen, Grégory Chaume, Elena Dzhambazova, Liountmila
Astasidi, Radka Hadjiolova, Adriana Bocheva, Thierry Brigaud
PII:
S0223-5234(12)00763-5
10.1016/j.ejmech.2012.12.041
EJMECH 5912
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 October 2012
Revised Date: 19 December 2012
Accepted Date: 23 December 2012
Please cite this article as: I. Jlalia, N. Lensen, G. Chaume, E. Dzhambazova, L. Astasidi, R. Hadjiolova,
A. Bocheva, T. Brigaud, Synthesis of a MIF-1 analogue containing Enantiopure (S)-α-Trifluoromethyl
Proline and Biological Evaluation on Nociception, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2012.12.041.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of a MIF-1 analogue containing Enantiopure (S)-α-Trifluoromethyl
Proline and Biological Evaluation on Nociception
Ibtissem Jlaliaa, Nathalie Lensena, Grégory Chaumea,***, Elena Dzhambazovab, Liountmila Astasidib, Radka
Hadjiolovac, Adriana Bochevac,**, Thierry Brigauda,*
a Laboratoire SOSCO, Université de Cergy-Pontoise, EA 4505, 5 Mail Gay Lussac, Neuville sur Oise, 95000
Cergy-Pontoise Cedex, France
b Department of Physiology and Clinical Physiology, Faculty of Medicine, Sofia University St. Kl. Ohridski,
Sofia, 1407, Bulgaria
c Department of Pathophysiology, Faculty of Medicine, Medical University, Sofia, Bulgaria
* Corresponding author. Tel.: +33 1 34 25 70 66
** Corresponding author. Tel.: +35 9 29 17 25 46
*** Corresponding author. Tel.: +33 1 34 25 73 87
E-mail addresses: thierry.brigaud@u-cergy.fr (T. Brigaud), аdriana_bocheva@abv.bg (A. Bocheva),
gregory.chaume@u-cergy.fr (G. Chaume).
Abstract1
The synthesis and the effect of a novel MIF-1 analogue on nociception during acute pain in rat model are
reported. The synthesis of this enantiopure trifluoromethyl group containing tripeptide was performed
through a peptide coupling reaction beetween the HCl. Leu-Gly-NH2 and the (S)-α-Tfm-proline. The
analgesic effect of the CF3-MIF-1 2 has been evaluated in vivo on rat model by paw pressure (PP) and hot
plate (HP) tests and compared to the native peptide MIF-1. Highest analgesic effect was observed with CF3-
(MIF-1) 2 only in PP test. In order to study the mechanisms of nociception induced by the studied peptides,
the involvement of the opioid and the nitric oxideergic systems was investigated. The results are in favor of
a participation of both system since pretreatment, 20 min before injection of the CF3-(MIF-1) 2, with the
1
Abbreviations: S.E.M., Standard error of the mean; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride; HOBt, Hydroxybenzotriazole; BOP-Cl, Bis(2-oxo-3-oxazolidinyl)phosphonic chloride; TEA,
Triethylamine; Boc, t-Butoxycarbonyle; DCM, Dichloromethane.
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non-competitive antagonist of opiate receptors naloxone, the nitric oxide synthase (NOS) inhibitor L-NG-
nitroarginine ester (L-NAME) or the nitric oxide (NO) donor L-arginine (L-Arg) significantly decreased the
pain perception in PP and HP tests.
Keywords
Fluorinated amino acids
Fluorinated peptides
MIF-1
Nociception
Acute pain
Analgesia
Highlights
- A MIF-1 analogue containing enantiopure (S)-α-trifluoromethyl proline was synthesized
- The nociception during acute pain of the CF3-MIF-1 was evaluated in rat model
- Significant analgesic effect in paw pressure test compared to the native peptide
1. Introduction
The tripeptide PLG 1 (Pro-Leu-Gly-NH2), known as melanocyte–stimulating hormone release inhibition
factor (MIF-1), is an endogenous brain peptide that belongs to the Tyr-MIF-1 family of peptides isolated
from bovine hypothalamus and human parietal cortex. It has been shown to be involved in a wide spectrum
of physiological processes, including the development of stress [1-10]. MIF-1 demonstrates its ability to
modulate dopaminergic neurotransmission in vitro and in vivo, by probably an allosteric effect increasing
the binding affinity of agonists of dopamine receptor [10-13]. Therefore, it is considered as a promising
starting point for the development of new pharmaceutical agents for various brain disorders including
Parkinson’s disease [11,14-18], tardive dyskinesia [19,20] and depression [21,22]. MIF-1 represents also a
class of naturally occurring opiate antagonists. It does not bind to opiate receptors and it is the first peptide
known to exert anti-opioid effects [3]. In particular, MIF-1 binds to its own non-opiate sites and is able to
block respectively the analgesic effect of morphine in paw-pressure and tail-flick tests [23] and of
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enkephalin in a radiant heat tail-flick assay [24]. This peptide antagonizes also the effects of morphine in a
double-blind study in humans [25]. Because of its structural simplicity, and its relevance to activate CNS
pathways related to dopamine-, serotonin-, opioid- and noradrenergic systems, several conformationally
constrained analogues of MIF-1 have been reported in literature [26-32].
Our research group is interested in the development of convenient and scalable experimental procedures
for the stereoselective synthesis of various trifluoromethyl group containing α-amino acids (α-Tfm-AAs)
with linear [33,34], cyclic [35-37] or functionalized side chain [38]. It is well documented that proline [39-
43] and its derivatives such as pyroglutamic acid [44,45] or pseudoprolines [46,47] play a unique and
important role in the conformation of peptides and proteins resulting in the modulation of their biological
activities. Therefore, enantiopure (S)- and (R)-α-Tfm-prolines ((S)-3 and (R)-3) are very attractive target
molecules for the design of biologically active compounds. However, the use of α-Tfm-AAs remains very
limited since their incorporation into a peptide chain is still a challenge due to the low nucleophilicity of the
α-Tfm-AAs amino group. We previously reported an efficient protocol allowing the coupling at the C-
terminal position of the non-protected α-Tfm-proline or α-Tfm-alanine leading to original α-Tfm-AAs
containing dipeptides [48]. The challenging N-terminal coupling of an α-Tfm-alanine dipeptide using a
Fmoc-amino acyl chloride was also described.
As the chemical barriers for the coupling reaction were unlocked, we decided to prepare
trifluoromethylated analogues of native peptides in order to investigate the effect of the α-Tfm-AAs on the
biological activity. Indeed, the incorporation of α-Tfm-AAs into peptides increases their chemical and
thermal stability and their resistance to degradation by proteases and enhances hydrophobicity resulting in
better affinities for lipid membranes [49-54]. Moreover, their incorporation into peptides may induce
stabilization of particular conformations and better auto-assembly [55-60]. Additionally, the trifluoromethyl
group is a label for 19F NMR studies allowing an access to pharmacokinetic and dynamic data [61-63].
The objectives of the present study were the synthesis of the enantiopure (S)-α-Tfm-proline containing
MIF-1 analogue 2 (Figure 1) and the investigation of its effect on nociception during acute pain in rat model.
An enhancement of the lipophilicity is expected since the amino group of the Tfm-proline at the N-terminal
position should not be protonated at physiological pH.
[Fig 1]
2. Results and discussion
2.1. Chemistry
Due to the stereoelectronic effects impart by the CF3 group, chemical and physical properties of the
neighboring functions are modified. The nucleophilicity and the basicity of the vicinal amino group is
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decreased while the reactivity of the acid function is increased. These properties allow efficient coupling
reactions at the C-terminal position under classical protocols without any protection of the amino group of
the α-Tfm-proline residue [48]. It should be reminded that such protection is required for native amino
acids.
The retrosynthetic pathway to CF3-(MIF-1) 2 involved a coupling reaction, at the final stage, between the
enantiopure α-Tfm-proline residue (S)-3 and the HCl.Leu-Gly-OEt 4a or the preformed HCl.Leu-Gly-NH2
dipeptide 4b (Figure 2).
[Fig 2]
Both enantiomers of the α-Tfm-proline 3 ((S)-3 and (R)-3) were prepared according to our convenient
gram-scale procedure [37,48]. The CF3-oxazolidine 5, prepared by condensation of (R)-N-Boc-
phenylglycinol with the commercially available ethyl trifluoropyruvate [36], was treated with
allyltrimethylsilane under Lewis acid activation to give, after acidic treatment, a 75:25 diastereomeric
mixture of allylmorpholinones (R,S)-6 and (R,R)-6 (Scheme 1) [64]. In the presence of iodine, 6 afforded the
pyrrolidinic bicyclic compound 7. The iodocompound 7 was hydrogenolyzed to give the corresponding
bicyclic morpholinone 8. After separation of the two diastereomers 8 by column chromatography and
deprotection with Pearlman’s catalyst, the (S)- and (R)-α-Tfm-proline [(S)-3 and (R)-3] were obtained in
enantiopure form.
[Scheme 1]
The initial strategy adopted for the synthesis of the expected CF3-(MIF-1) 2 tripeptide involved an
amidification step of the tripeptide 12 bearing an ester function at the C-terminal position.
The coupling reaction between Boc-Leu-OH 9 and HCl.Gly-OEt 10a using standard coupling reagents
such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole
(HOBt) in DMF gave the corresponding dipeptide 11a (Scheme 2). The deprotection of the N-terminal
amino group under acidic conditions led to the corresponding hydrochloride salt of the Leu-Gly-OEt
dipeptide 4a in good yield (84%). We previously reported that the coupling reaction at the C-terminal
position of a non-protected α-Tfm-proline 3 with an amino ester or a peptide chain needed a reverse addition
protocol to avoid the formation of the diketopiperazine byproduct [48]. Accordingly, by addition of one
equivalent of α-Tfm-proline (S)-3 to two equivalents of the non-fluorinated dipeptide 4a in the presence of
HOBt and EDCI, the tripeptide 12 was obtained in a reasonable good yield (62%). Surprisingly, an
amidification step using dissolved NH3 in EtOH already reported for the non-fluorinated Pro-Leu-Gly-OEt
tripeptide gave a messy mixture and an incomplete conversion when applied to the fluorinated tripeptide
analogue 12.
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[Scheme 2]
In order to achieve the synthesis of the CF3-(MIF-1) 2, we attempted the direct condensation of Boc-Leu-
OH 9 with HCl.Gly-NH2 10b using classical (HOBt/EDCI) coupling reagents (Scheme 2). The
corresponding Boc-Leu-Gly-NH2 dipeptide 11b was obtained in moderate yield (62%) due to a tedious
chromatographic purification to remove the HOBt byproduct and DMF traces. Another protocol using
BOPCl activation in DCM was found to be more efficient giving 11b in higher yields (81%). The Boc
amino group deprotection by acidic treatment (HClg in 1,4-dioxane) followed by a coupling reaction of the
α-Tfm-proline (S)-3 under reverse addition conditions gave the tripeptide CF3-(MIF-1) 2 in a good yield
(89%).
2.2. Biology
As pain is a frequently observed symptom of various diseases and as the development of analgesic drugs
is one of the greatest achievements in medicine, we focused our investigation on the CF3-(MIF-1) 2 effects
on acute pain.
Involvement of opioidergic system in analgesic effects of CF3-MIF-1
In order to evaluate the nociceptive activity of the CF3-(MIF-1) 2 and to compare it with the native (MIF-
1) 1 peptide, we first administrated the peptide alone by intraperitoneal (i.p.) injection on male Wistar rats
and measured the analgesic effect in PP and HP tests. The first measurements were recorded 15 min after the
injection of the peptides. Alone, MIF-1 (1, 1 mg/kg, i.p.) had no analgesic effect in PP test and significantly
increased HP latency (P < 0.05) only after 15 min from the beginning of the investigated period. (Fig. 3 and
4). The results are in accordance with literature data reporting that MIF-1 had a similar action to naloxone
(Nal), a known opiate antagonist. (S)-α-Tfm-proline containing MIF-1 analogue (2, 1 mg/kg, i.p.),
developed a significant analgesic effect after 15 and 30 min in PP test (P < 0.01), and had no effect on HP
latency (Fig. 3 and 4). These results suggest that the incorporation of the CF3 group is responsible for the
analgesic effect of MIF-1 in PP test only.
We may speculate that the opposite effects are related to different receptors (mechano- and
thermoreceptors) and different pain pathways.
[Fig.3].
[Fig.4]
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In order to investigate the involvement of the opioid system in the CF3-MIF-1 peptide induced analgesia,
the non-competitive antagonist of opiate receptors naloxone (Nal, 1 mg/kg, i.p.) was tested. Pretreatment
with Nal 20 min before injection of the CF3-(MIF-1) 2 peptide significantly decreased the pain threshold (P
< 0.01) of about 50% and 40% after 15 min and 30 min respectively (Fig. 3 and 4). These results suggest the
involvement of opioid receptors in the observed CF3-MIF-1 effects.
Involvement of nitric oxideergic system in analgesic effects of CF3-MIF-1
Nitric oxide (NO) is a unique neurotransmitter, which participates in many physiological and
pathological processes including pain transmission in the organism. It is biosynthesized from L-arginine, by
the nitric oxide synthase enzyme (NOS) [65,66]. The morphological studies present evidence for the
existence of a signaling pathway between the opioidergic and the nitric oxideergic systems in the
periaqueductal gray (PAG) of the rat brain, the critical site involved for coping with different types of stress
and pain [67]. It is also known that NO is an important and many-sided regulator of a number of
physiological functions in animals. It also acts as a neurotransmitter itself and/or as a neuromodulator and
influences plastic properties of the neurons such as the phenomenon of long-lasting potentiation [68].
In a second experimental series, NOS inhibitor L-NG-nitroarginine ester (L-NAME, 10 mg/kg, i.p.) or NO
donor L-arginine (L-Arg, 1 mg/kg, i.p.) were injected 20 min before the studied analogues. Each compound
used alone showed analgesic effects in both tests. The effect of L-NAME was more pronounced and long
lasting compared to that of L-Arg (Fig. 5-8).
The results of our investigations showed that both non fluorinated and fluorinated peptides significantly
decreased the analgesic effects of L-NAME and L-Arg in PP and HP tests during the whole investigated
period (Fig.5-8) and demonstrate that the nitric oxideergic system is also involved in the mechanisms of
nociception of the investigated peptides.
[Fig.5]
[Fig.6]
[Fig.7]
[Fig.8]
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3. Conclusions
In summary, we reported a convenient synthesis of a novel trifluoromethylated analogue of MIF-1 from
the commercially available ethyl trifluoropyruvate. Our choice was to incorporate a 2-trifluoromethylproline
in place of the native proline in order to investigate the biological activity improvement imparted by the
presence of the CF3 group. The PLG analogue (CF3-MIF-1) was synthesized in a good yield. Its involvement
on opiate and in nitric oxideergic systems was shown by in vivo acute pain experiments on rat model. The
results from this study demonstrated that the CF3-MIF-1 has significant analgesic effect in PP test (P < 0.01)
compared to the native MIF-1, but has no effect on HP latency. This study illustrates that the introduction of
an -Tfm-amino acid into a peptide can modulate its biological profile compared to the native peptide.
α
Moreover it could be anticipated that the incorporation of a trifluoromethylated amino acid at the N-terminal
position of a peptide will increase its lipophilicity.
4. Experimental Section
4.1. Chemistry
Unless otherwise mentioned, all the reagents were purchased from commercial source. All glassware was
dried in an oven at 150 °C prior to use. Ether and THF were distilled under nitrogen from
sodium/benzophenone prior to use. CH2Cl2 was distilled under nitrogen from CaH2 prior to use. 1H NMR
(400.00 MHz), 13C NMR (100.50 MHz) and 19F NMR (376.20 MHz) were measured on a JEOL ECX400
spectrometer. Chemical shifts of 1H NMR are expressed in parts per million downfield from
tetramethylsilane (δ = 0) in CDCl3. Chemical shifts of 13C NMR are expressed in parts per million downfield
from CDCl3 as internal standard (δ = 77.0). Chemical shifts of 19F NMR are expressed in parts per million
downfield from C6F6 as an internal standard (δ = –164.9). Coupling constants are reported in Hertz. Column
chromatography was performed on SDS 60Å, (40–63 µm) silica gel, employing a mixture of the specified
solvent as eluent. Thin-layer chromatography (TLC) was performed on Merck silica gel (Merck 60 PF254)
plates. Silica TLC plates were visualized under UV light, by a 10% solution of phosphomolybdic acid in
ethanol followed by heating. Mass spectra (MS) were obtained on a GC/MS apparatus HP 5973 MSD with
an HP 6890 Series GC. Ionization was obtained by electronic impact (EI 70 eV). Infrared spectra (IR) were
obtained by Fourier-transformation on BRÜCKER TENSOR 27, wavenumbers are given in cm–1. Liquid
Chromatography-Mass Spectrometry (LC-MS) analyses were done on a Shimadzu LCSM-2010 A on a
HPLC, column Alltima HP C8 3µ (Alltech), reversed phase (L= 53 mm; ID = 7 mm), PDA diodes detector
SPD-M10 A (D2, lamp from 190 to 400 nm) and light scattering detector ELSD-LT. The LC were run using
a 1 ml/min flow using a gradient between acetonitrile and water containing formic acid (0,1%): 0 to 1 min :
30% CH3CN, 1 to 5 min : from 30% to 100% CH3CN, 5 to 12 min : 100% CH3CN, 12 to 14,99 min : from
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100% to 30% CH3CN, 14,99 to 20 min : 30% CH3CN. MS spectrum was recorded between m/z = 100 to 500
at the exit of the column using an ESI ionization and positive ion mode (detector = 1.5 kV, quadripole = 5
V). Elemental analyses were performed on Perkin–Elmer CHN 2400. Optical rotations were determined
using a JASCO P1010 polarimeter. HRMS analyses were performed on a Jeol JMS-GC Mate II. Melting
points were obtained on a Büchi apparatus and are uncorrected.
4.1.1. General procedure for the synthesis of dipeptides (11a-11b).
Boc-Leu-OH 9 and HOBt were dissolved in DMF. The reaction mixture was cooled to 0 °C, EDCI was
added and the solution was stirred for 1 h. HCl.Gly-OEt 10a or HCl.Gly-NH2 10b and TEA were added. The
reaction mixture was stirred at r.t. for 16 h, then cooled to 0°C and the urea precipitate was removed by
filtration. The solvent was evaporated under vacuum and the crude residue was dissolved in ethyl acetate.
The organic phase was extracted with 5% NaHCO3 solution, brine, dried over MgSO4 and concentrated
under reduced pressure to afford the dipeptides 11a and 11b in 65-84%.
4.1.1.1. Boc-Leu-Gly-OEt (11a)
The dipeptide 11a was prepared following the general procedure starting from Boc-Leu-OH 9 (1.0 g, 4.3
mmol, 1.0 equiv), HOBt (0.6 g, 4.7 mmol, 1.1 equiv) in DMF (4 mL), then EDCI (0.8 g, 4.3 mmol, 1.0
equiv), HCl.Gly-OEt 10a (0.6 g, 4.3 mmol, 1.0 equiv) and TEA (1.2 mL, 8.6 mmol, 2.0 equiv). Purification
by recrystallization in ethyl acetate/petroleum ether (40:60) gave 1.14 g (84%) of pure dipeptide 11a as a
white solid. Spectroscopic data of 11a are in accordance with the literature reported data [69]. White solid;
mp 72-74 °C; IR (neat) 3290, 2954, 2868, 1754, 1661, 1246, 1166 cm−1; [α]26D −16 (c 0.5, CHCl3); 1H
RMN (400 MHz, CDCl3) δ 0.94 (d, J = 6.3 Hz, 3 H), 0.99 (d, J = 6.3 Hz, 3 H), 1.29 (t, J = 7.1 Hz, 3 H),
1.45 (s, 9 H), 1.68-1.71 (m, 3 H), 4.04 (d, J =5.0 Hz, 2 H), 4.16 (bs, 1 H), 4.18 (q, J = 7.1 Hz, 2 H), 4.85 (m,
1 H), 6.64 (s, 1 H); 13C RMN (100 MHz, CDCl3) δ 14.0, 21.8, 22.8, 24.5, 28.2, 41.1, 41.3, 52.8, 61.2, 79.8,
155.7, 169.6, 173.1; LCMS (ESI+) rt = 4.98 min, m/z = 339 [M + Na]+ (100), 261; ELSD pur. 99%, UV pur.
100%.
4.1.1.2. Boc-Leu-Gly-NH2 (11b)
HOBt activation protocol: The product was prepared following the corresponding general procedure
starting from Boc-Leu-OH 9 (2.0 g, 8.6 mmol, 1.0 equiv), HOBt (1.7 g, 12.0 mmol, 1.5 equiv) in DMF (8
mL), then EDCI (2.5 g, 13.0 mmol, 1.5 equiv), HCl.Gly-NH2 10b (2.0 g, 18.1 mmol, 2.1 equiv) and TEA
(4.2 mL, 30 mmol, 3.5 equiv) to give 1.46 g (62%) of dipeptide 11b as a white solid. Dipeptide 11b was
engaged in the next step without further purification. Spectroscopic data of 11b are in accordance with the
literature reported data [69].
BOP-Cl activation protocol: Boc-Leu-OH 9 (9.5 g, 41.1 mmol), HCl.GlyNH2 10b (5.0 g, 45.2 mmol, 1.1
equiv) and TEA (17.6 mL, 127 mmol, 3.1 equiv) were mixed in DCM (1.7 L) and the resulting solution was
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stirred for 10 min. BOP-Cl (13 g, 51 mmol, 1.25 equiv) was then added and the reaction mixture was stirred
at r.t. overnight, quenched with HCl aqueous solution (1 M, 500 mL). The layers were separated and the
organic phase was washed with a saturated NaHCO3 aqueous solution (2 x 500 mL), brine (500 mL), dried
over MgSO4 and concentrated under reduced pressure to afford 10.1 g (81%) of the crude dipeptide 11b
which was engaged in the next step without further purification. White solid; mp 70-72 °C; IR (neat) 3282,
2956, 1657, 1246, 1162 cm−1; [α]27D −15 (c 0.5, CHCl3); 1H RMN (400 MHz, CDCl3, 323 K) δ 0.93 (d, J =
6.7 Hz, 3 H), 0.96 (d, J = 6.7 Hz, 3 H), 1.41 (s, 9 H), 1.50 (m, 1 H), 1.56-1.76 (m, 2 H), 3.80 (dd, J = 17.0,
5.0, 1 H), 4.00 (dd, J = 17.0, 5.0 Hz), 5.31 (bs, 1 H), 6.09 (bs, 1 H), 6.75 (bs, 1 H), 7.26-7.36 (m, 2 H); 13C
RMN (100 MHz, CDCl3) δ 21.8, 22.8, 24.7, 28.3, 40.8, 42.7, 53.6, 80.3, 156.3, 172.4, 173.7; LCMS (ESI+)
rt = 3.74 min, m/z = 310 [M + Na]+ (100), 232, 188; ELSD pur. 99%, UV pur. 100%.
4.1.2. General procedure for the Boc deprotection.
Boc-Leu-Gly-OEt 11a or Boc-Leu-Gly-NH2 11b was dissolved in 1,4-dioxane and was treated at r.t. for
40 min with dry HCl gas. The reaction mixture was stirred at r.t. until disappearance of the starting material
(monitoring by TLC), then the solvent was removed under vacuum to afford quantitatively the crude
dipeptides 4a and 4b which were engaged in the next step without further purification.
4.1.2.1. HCl.Leu-Gly-OEt (4a)
The product was prepared following the corresponding general procedure starting from Boc-Leu-Gly-OEt
11a (315 mg, 1 mmol) in 1,4-dioxane (5 mL) to give 260 mg (quantitative) of dipeptide 4a. Pale yellow oil;
IR (neat) 3240, 3080, 2960, 2850, 1735, 1671, 1205, 870 cm−1; [α]29D +12.7 (c 0.75, MeOH); 1H RMN (400
MHz, DMSO-d6) δ 0.89 (d, J = 7.1 Hz, 3 H), 0.91 (d, J = 7.1 Hz, 3 H), 1.19 (t, J = 7.1 Hz, 3 H), 1.46-1.64
(m, 2 H), 1.72 (m, 1 H), 3.80 (m, 1 H), 3.85 (dd, J = 17.0, 5.7 Hz, 1 H), 3.96 (dd, J = 17.0, 6.0 Hz, 1 H),
4.10 (q, J = 7.1 Hz, 2 H), 8.29 (bs, 3 H), 9.04 (t, J = 5.2 Hz, 1 H); 13C RMN (100 MHz, DMSO-d6): δ 13.9,
22.1, 22.3, 22.4, 23.2, 50.5, 60.5, 66.3, 169.0, 169.4; LCMS (ESI+) rt = 1.40 min, m/z = 217 [M + H −
HCl]+ (100); ELSD pur. 100%, UV pur. 100%.
4.1.2.2. HCl.Leu-Gly-NH2 (4b)
The product was prepared following the corresponding general procedure starting from Boc-Leu-Gly-
NH2 11b (5 g, 17.4 mmol) in 1,4-dioxane (95 mL) to give 3.89 g (quantitative) of dipeptide 4b. Colorless
hygroscopic oil; IR (neat) 3380, 3220, 3130, 2955, 1726, 1670, 1647, 1507, 1264 cm−1; [α]29D +29.0 (c 1.3,
MeOH); 1H RMN (400 MHz, D2O) δ 0.76 (d, J = 5.5 Hz, 3 H), 0.77 (d, J = 5.5 Hz, 3 H), 1.45-1.63 (m, 3 H),
3.72 (d, J = 16.9 Hz, 1 H), 3.83 (d, J = 16.9 Hz, 1 H), 3.88 (m, 1 H); 13C RMN (100 MHz, D2O) δ 21.5,
22.0, 24.1, 40.0, 42.3, 52.3, 171.2, 173.6; LCMS (ESI+) rt = 4.98 min, m/z = 188 [M + H − HCl]+ (100);
ELSD pur. 99%, UV pur. 100%.
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4.1.3. General procedure for the synthesis of the tripeptides (12) and (2).
HCl.Leu-Gly-OEt 4a or HCl.Leu-Gly-NH2 4b was dissolved in DMF. TEA was added and the solution
was stirred at 0 °C for 1 h, followed by the addition of HOBt and EDCI to the reaction mixture. After
stirring for 20 min at 0 °C, (S)-(αTfm)-Proline was added. The resulting mixture was warmed to r.t. for 72 h,
then cooled to 0 °C and the urea precipitate was removed by filtration. The DMF was evaporated under
reduced pressure to dryness and the corresponding crude mixture was diluted with ethyl acetate and water.
The layers were separated and the aqueous phase was extracted with ethyl acetate. The combined organic
phases were evaporated and the crude product was purified by reverse phase chromatography (gradient H2O/
CH3CN). The residue was dissolved in 5% NaHCO3 aqueous solution, extracted with ethyl acetate (3 x),
dried over MgSO4 and concentrated to give the tripeptides 12 and 2 in respectively 62% and 89% yield.
4.1.3.1. (S)-(α−Tfm)-Pro-Leu-Gly-OEt (12)
The product was prepared following the corresponding general procedure starting from HCl.Leu-Gly-OEt
4a (278 mg, 1.1 mmol, 2 equiv) in DMF (3 mL), TEA (260 µL, 1.9 mmol, 3.4 equiv), HOBt (110 mg, 0.8
mmol, 1.5 equiv), EDCI (157 mg, 0.8 mmol, 1.5 equiv), (S)-(α−Tfm)-Proline (100 mg, 1 equiv).
Purification by reverse phase chromatography (100% H2O then 5% gradient H2O/CH3CN) gave 127 mg
(62%) of tripeptide 12. Colorless hygroscopic oil; IR (neat) 3309, 2957, 2871, 1747, 1655, 1284, 1158 cm−1;
[α]25D −58.0 (c 1.0, MeOH); 1H RMN (400 MHz, CD3OD) δ 0.93 (d, J = 5.3 Hz, 3 H), 0.96 (d, J = 4.6 Hz, 3
H), 1.26 (t, J = 7.1 Hz, 3 H), 1.57-1.67 (m, 3 H), 1.73 (m, 1 H), 1.89 (m, 1 H), 2.21-2.27 (m, 2 H), 3.01-3.09
(m, 2 H), 3.85 (d, J = 17.4 Hz, 1 H), 4.00 (d, J = 17.4 Hz, 1 H), 4.17 (q, J = 7.1 Hz, 2 H), 4.50 (m, 1 H); 13C
RMN (100 MHz, CD3OD) δ 14.4, 21.9, 23.4, 25.9, 26.4, 33.3, 42.0, 42.3, 53.1, 62.2, 72.1 (q, J = 26.8 Hz),
127.4 (q, J = 283.7 Hz), 170.9, 171.3, 174.6; 19F RMN (376 MHz, CD3OD) δ −79.14 (s, CF3); LCMS
(ESI+) rt = 4.43 min, m/z = 382 [M + H]+ (100); ELSD pur. 99%, UV pur. 100%.
4.1.3.2. (S)-(α−Tfm)-Pro-Leu-Gly-NH2 (2)
The product was prepared following the corresponding general procedure starting from HCl.Leu-Gly-
NH2 4b (200 mg, 0.9 mmol, 1.6 equiv) in DMF (6 mL), TEA (260 µL, 1.9 mmol, 3.4 equiv), HOBt (110
mg, 0.8 mmol, 1.5 equiv), EDCI (157 mg, 0.8 mmol, 1.5 equiv), (S)-(αTfm)-Proline (100 mg, 1 equiv).
Purification by reverse phase chromatography (100% H2O then 5% gradient H2O/CH3CN) gave 170 mg
(89%) of tripeptide 2. Colorless hygroscopic oil; IR (neat) 3305, 3080, 3200, 2958, 2928, 2872, 1654, 1514,
1283, 1158 cm−1; [α]24D −30.0 (c 0.5, MeOH); 1H RMN (400 MHz, CD3OD) δ 0.93 (d, J = 6.0 Hz, 3 H),
0.97 (d, J = 5.7 Hz, 3 H), 1.58-1.80 (m, 4 H), 1.89 (m, 1 H), 2.21-2.29 (m, 2 H), 2.99-3.09 (m, 2 H), 3.77 (d,
J = 17.0 Hz, 1 H), 3.90 (d, J = 17.0 Hz, 1 H), 4.40 (m, 1 H); 13C RMN (100 MHz, CD3OD) δ 21.9, 23.4,
26.0, 26.4, 33.3, 41.6, 43.1, 48.2, 53.9, 72.1 (q, J = 26.2 Hz), 127.4 (q, J = 289.0 Hz), 171.9, 174.0, 174.8;
19F RMN (376 MHz, CD3OD) δ −79.1 (s, CF3); LCMS (ESI+) rt = 2.41 min, m/z = 353 [M + H]+ (100);
ELSD pur. 99%, UV pur. 100%.
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4.2. Biology
4.2.1. Animals
The experiments were carried out on male Wistar rats (180–200 g), housed at 12 h light/dark cycle. Food
and water were available ad libitum. All experiments were carried out between 09.00 a.m. and 12.00 p.m.
Each group included five rats for nociceptive tests.
4.2.2. Nociceptive tests
Paw-pressure test (Randall-Selitto test): The changes in the mechanical nociceptive threshold of the rats
were measured using an analgesimeter (Ugo Basile). The pressure was applied to the hind-paw and the
pressure (g) required eliciting nociceptive responses, such as squeak, and struggle was taken as the
mechanical nociceptive threshold. A cut-off value of 500 g was used to prevent damage of the paw.
Hot plate test: The latency of response to pain was measured from the moment of placing an animal on a
metal plate (heated to 55 ± 0.5oC) to the first signs of pain (paw licking, jump). The cut-off time was 30 s.
4.2.3. Drugs and treatment
MIF-1, a non-specific opioid receptor antagonist naloxone (Nal), L-Arginine (L-Arg, the natural precursor
of NO) (all at a dose of 1 mg/kg) and NO synthase inhibitor L-NG-nitroarginine ester (L-NAME) (10 mg/kg),
were obtained from Tocris. All drugs were dissolved in a sterile saline (0.9% NaCl) solution and injected
intraperitoneally (i.p.).
The experimental procedures were carried out in accordance with the institutional guidance and general
recommendations on the use of animals for scientific purposes.
4.2.4. Statistical analysis
The results were statistically assessed by the analysis of variance ANOVA. Values are mean ± S.E.M.
Values of P < 0.05 were considered to indicate statistical significance.
Aknowledgments
The authors benefit of financial support from ‘RILA’ program for French-Bulgarian Partnership
(PROJET PHC RILA 2011 N° 25325VG).
French partner is grateful to Central Glass Company for their generous gift of ethyl trifluoropyruvate and
for financial support.
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List of captions
Fig 1. Chemical structure of (MIF-1) 1 and CF3-(MIF-1) 2
Fig 2. Retrosynthetic pathway of CF3-(MIF-1) 2
Scheme 1. Reagents and conditions : (a) AllylTMS, BF3.OEt2, CH2Cl2, 72 h, 0 °C ꢀ r.t.; (b) PTSA cat.,
Toluene, reflux, 24 h; (c) I2, Toluene, 95°C, 48 h; (d) H2 (5 bars), Pd/C, AcONa, EtOH, 24 h; (e)
Chromatographic separation of both diastereomer; (f) H2 (5 bars), Pd(OH)2, EtOH, 48 h.
Scheme 2. Reagents and conditions : (a) HOBt, EDCI, Et3N, DMF, 16 h, 0 °C ꢀ r.t.; (b) BOPCl, Et3N,
CH2Cl2, 12 h; (c) HClg, 1,4-dioxane; (d) (S)-3, HOBt, EDCI, Et3N, DMF, 72 h, 0 °C ꢀ r.t.; (e) NH3, EtOH,
2 h.
Fig.3. Effects of MIF-1, its analogue CF3-MIF-1 and combination of CF3-MIF-1 with Nal (all at a dose of 1
mg/kg, i.p.) estimated by PP test in male Wistar rats. Data are presented as mean ±S.E.M.; *P<0.05, **
P<0.01 vs. control; +P<0.05, ++ P<0.01 vs. MIF-1.
Fig.4. Effects of MIF-1, its analogue CF3-MIF-1 and combination of CF3-MIF-1 with Nal (all at a dose of 1
mg/kg, i.p.) estimated by HP test in male Wistar rats. Data are presented as mean ±S.E.M.; *P<0.05, **
P<0.01 vs. control; +P<0.05, ++ P<0.01 vs. MIF-1.
Fig.5. Effects of MIF-1, its analogue CF3-MIF-1 (both at a dose of 1 mg/kg, i.p.) and their combination with
L-NAME (10 mg/kg, i.p.) estimated by PP test in male Wistar rats. Data are presented as mean ±S.E.M.; **
P<0.01 vs. control; ++ P<0.01 vs. L-NAME.
Fig.6. Effects of MIF-1, its analogue CF3-MIF-1 (both at a dose of 1 mg/kg, i.p.) and their combination with
L-NAME (10 mg/kg, i.p.) estimated by HP test in male Wistar rats. Data are presented as mean ±S.E.M.;
*P<0.05, ** P<0.01 vs. control; ++ P<0.01 vs. L-NAME.
Fig.7. Effects of MIF-1, its analogue CF3-MIF-1 and their combination with L-Arg (all at a dose of 1 mg/kg,
i.p.) estimated by PP test in male Wistar rats. Data are presented as mean ±S.E.M.; *P<0.05, ** P<0.01 vs.
control; ++ P<0.01 vs. L-Arg.
Fig.8. Effects of MIF-1, its analogue CF3-MIF-1 and their combination with L-Arg (all at a dose of 1 mg/kg,
i.p.) estimated by HP test in male Wistar rats. Data are presented as mean ±S.E.M.; *P<0.05, ** P<0.01 vs.
control; ++ P<0.01 vs. L-Arg.
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Highlights
- A MIF-1 analogue containing enantiopure (S)-α-trifluoromethyl proline was synthesized
- The nociception during acute pain of the CF3-MIF-1 was evaluated in rat model
- Significant analgesic effect in paw pressure test compared to the native peptide

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