NOVEL PEPTOIDS AND USE THEREOF FOR PREVENTING OR TREATING CHRONIC PAIN
20230374067 · 2023-11-23
Inventors
- Jérôme BUSSEROLLES (Saulzet, FR)
- Emmanuel BOURINET (SAINT MATHIEU DE TREVIERS, FR)
- Claude TAILLEFUMIER (CHIDRAC, FR)
- Olivier ROY (COURNON D'AUVERGNE, FR)
- Lionel NAUTON (CHAMPEIX, FR)
- Youssef AISSOUNI (RIOM, FR)
Cpc classification
International classification
Abstract
The invention concerns new peptidomimetic (or peptoid) molecules for the prevention and/or treatment of chronic pain, particularly that resulting from peripheral neuropathies.
Claims
1. Peptoid of the following general formula (I): ##STR00029## where R.sup.1 is independently CH.sub.3 (L-Ala), CH(CH.sub.3).sub.2 (L-Val), CH(CH.sub.3)CH.sub.2CH3 (L-Ile), CH.sub.2CH(CH.sub.3).sub.2 (L-Leu and D-Leu), CH.sub.2C(CH.sub.3).sub.3, C(CH.sub.3).sub.3, CH.sub.2(cyclobutyl); R.sup.2 and R.sup.3 are independently selected from: CH.sub.2CH(CH.sub.3).sub.2, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2CH.sub.2OH, CH.sub.2CH(OH)CH.sub.3, CH.sub.2CONH.sub.2, CH.sub.2CH.sub.2CONH.sub.2; R.sup.4 is an aliphatic or aromatic group selected from: ##STR00030## R.sup.5 is independently CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2C.sub.6H.sub.5, C.sub.6H.sub.4X (where X in the ortho, meta or para position is independently H, OMe, CF.sub.3, CH.sub.3, CH.sub.2CH.sub.3, F, Br, Cl, NO.sub.2), 3-indolyl, 3-quinolinyl.
2. Peptoid according to claim 1 of the following formula: ##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##
3. Peptoid of the following general formula (II): ##STR00036## wherein R.sup.1, R.sup.2 and R.sup.3 are as defined in claim 1; R.sup.6 is R.sup.3, H, COR.sup.5 (wherein R.sup.5 is as defined above) or R.sup.7; R.sup.7 is independently one of: ##STR00037##
4. A pharmaceutical composition comprising a peptoid of claim 1, and a pharmaceutically acceptable carrier.
5. A method comprising: administering the peptoid of claim 1 to a subject in need thereof as a medicament.
6. A method comprising: administering the peptide of claim 1 to a subject in need thereof to prevent or treat chronic pain.
7. The method according to claim 6, wherein the chronic pain is of neuropathic origin.
8. A method comprising: administering the pharmaceutical composition of claim 4 to a subject in need thereof as a medicament.
9. A method comprising: administering the pharmaceutical composition of claim 4 to a subject in need thereof to prevent or treat chronic pain.
10. The method according to claim 9, wherein the chronic pain is of neuropathic origin.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0039]
[0040]
[0041]
[0042]
[0043]
[0044]
[0045]
[0046]
[0047]
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
[0055]
[0056]
[0057]
[0058]
EXAMPLES
Example 1
Material and Methods
Animals
[0059] Experiments were performed on 20-24 g male C57BI/6JRj mice or 150-175 g male Sprague-Dawley rats provided by Janvier Laboratories (France), maintained on a 12 h light/dark cycle and fed and watered ad libitum. Behavioral experiments were performed blindly, in a quiet room, by the same experimenter for a given test, taking great care to minimize or avoid animal discomfort. All animal procedures were approved by the local animal ethics committee and experiments were performed in accordance with the guidelines provided by the European Community on the care and use of animals (Directive 2010/63/EU).
Behavioral Tests
[0060] Von Frey test: Mechanical sensitivity was assessed with calibrated Von Frey filaments of 0.07 g, 0.6 g or 1.4 g. The filaments were applied five times in order of increasing stiffness, perpendicularly to the plantar surface of the hind paw and pressed until they bent. The number of responses for a given filament force was counted.
[0061] Immersion test: The tail or paw was immersed in a water bath set at 10° C. or 46° C. until shrinkage was observed (cut-off time: 30 s). The average of two separate determinations of withdrawal latency was calculated (Janssen, Niemegeers & Dony, 1963) [14].
[0062] Paw pressure test in rats: Rats were subjected to the paw pressure test previously described by Randall and Selitto (1957) [15]. Nociceptive thresholds, expressed in grams, were measured with a Ugo Basile analgesic meter (Apelex, probe tip diameter 1 mm, weight 30 g) by applying increasing pressure to the right hind paw of the rats until a pain sign (vocalization threshold) was obtained (cut-off was 750 g). Before the treatments, the rats were habituated to the test by handling them without subjecting them to paw pressure. Then, after obtaining two consecutive stable vocalization threshold values, the treatment effects were evaluated after 15, 30, 45, 60, 90, and 120 min. Results are expressed as vocalization thresholds, in grams. To investigate the overall effects, the areas under the time course curves (AUC, g.min) of the antihyperalgesic effects were calculated from the individual scores at each time point, using the trapezoidal method. Data were analyzed by a 2-way ANOVA followed by a Bonferroni test, when the time course of the effects was studied. A one-way ANOVA followed by a Student Newman-Keuls test was used to analyze the effect of the different treatments determined by the CSAs. The level of statistical significance was set at p<0.05.
[0063] Rotarod Test: For the Rotarod test (Bioseb), the animals were first habituated to remain on the stationary cylinder for 10 min before switching to another 10 min with a rotational speed of 4 rpm. The animals were given the drug immediately after training and the test was performed 30 min later. The test consisted of measuring the latency to fall of the animals as the rotation increased from 4 to 40 rpm linearly over 5 min. The average of the two closest values over 3 trials was calculated.
[0064] In vivo heart rate recording: ECG telemetry recordings were performed using TA10EA-F20 telemetry transmitters (DSI) in a subcutaneous pouch with paired wire electrodes placed on the thorax as previously published (Mesirca et al., 2014) [27].
Animal Pain Model
[0065] Oxaliplatin-induced acute neuropathy in mice: Mice were injected intraperitoneally (i.p.) with 6 mg/kg oxaliplatin dissolved in 5% glucose directly after the first behavioral assessment. At 90 h after drug administration, corresponding to the time required to reach peak hyperalgesia (Descoeur et al., 2011) [9], a second assessment of cold sensitivity and mechanical sensitivity was performed.
[0066] Acute paclitaxel-induced neuropathy in rats: Rats were injected i.p. with dimethyl sulfoxide (DMSO; solvent) or 1 mg/kg/1 mL paclitaxel (Sigma-Aldrich, Lyon, France) on days D0, D2, D4, and D7. The behavioral test was performed on the day of the first paclitaxel injection (D0) and on days 10 (D10) and 14 (D14) after paclitaxel injection.
[0067] Spinal nerve ligation induced neuropathy in rats: According to the procedure of Kim and Chung (1992) [16], rats were placed in the supine position and anesthetized with 10 mg/kg xylazine and 75 mg/kg ketamine (i.p). The fur on the left side of the spine was then shaved to expose the skin. The skin was cut parallel to the spine and the paravertebral muscles were separated. The L5 transverse apophysis was removed until the L5 spinal nerve was exposed. The L5 nerve was isolated and ligated with silk thread. Finally, the muscle and skin incisions were sutured and the wound was disinfected with iodophor and 75% ethyl alcohol. In the sham group, the rats underwent the same surgical procedure without the nerve ligation.
Ex Vivo Tests
[0068] RNA isolation and quantification: DRG pairs from lumbar segments L4 to L6 were dissected and pooled. RNA was extracted using TRIzol® reagent (Nitrogen) according to the manufacturer's instructions (Thermofisher). RNA quality was assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies), samples with RIN (RNA Integrity Number) greater than 8 were retained for further analysis. Reverse transcription was performed with SuperScript II reverse transcriptase according to the protocol provided by Invitrogen. For real-time quantitative PCR, amplification was performed using the Light Cycler Fast Start DNA kit (Roche diagnostics) and the amount of DNA in real-time was measured with a Mastercycler Realplex thermal cycler (Eppendorf). The relative amount of cDNA in each triplicate was averaged using a standard concentration curve and normalized to the GUS gene cDNA.
[0069] Amplification conditions were as follows: preincubation for 7 min at 95° C. followed by 50 cycles of 20 s at 95° C. and 20 s at the specific hybridization temperature of the primer pair followed by 20 s at 72° C.
[0070] RNA sequencing: We used RNA sequencing analysis on L4-L5-L6 DRGs from mice treated with oxaliplatin (3 mg/kg, i.p.) or vehicle twice a week for 3 weeks. At the end of the experiment (D21), mice were anesthetized and killed, and L4-L5-L6 DRGs were rapidly harvested, frozen in liquid nitrogen and stored at −80° C. until use. Total RNAs from L4-L5-L6 DRGs were extracted using the RNeasy Micro kit (Qiagen) according to the manufacturer's protocol and Ribosomal RNA removed using Ribo-Zero Plus rRNA depletion kit (Illumina). RNA concentration was determined using the Take3 Nucleic Acid Quantification Plate (Epoch, BioTek, USA). RNA integrity was analyzed by a capillary electrophoresis instrument (Fragment Analyser, Agilent®) and sent to Fasteris (https://www.fasteris.com) for RNA sequencing. Libraries were prepared using the Illumina TruSeq protocol and SBS-based sequencing was performed using a HiSeq 2500 platform (Illumina). The analysis was performed in different steps: splice junction mapping (TopHat2), counting (HTSeq-count), filtering, normalization (edgeR, DESeq and DESeq2) and differential analysis (edgeR, DESeq and DESeq2) performed by Benjamin Bertin and Yoan Renaud (GreD, Clermont-Ferrand).
[0071] Membrane Fractionation:Tissues were homogenized in HEPES buffer (20 mM HEPES pH 7.4, 320 mM sucrose, 5 mM EDTA, 5 mM EGTA) containing protease inhibitors at 4° C., sonicated and centrifuged at 1000 g for 15 min at 4° C. The pellets were discarded. For organelle spin, supernatants were centrifuged at 15,000 g for 10 min at 4° C. and organelle pellets were retained. For cytosolic spin, supernatants were centrifuged at 100,000 g for 1 h at 4° C. The cytosolic fraction was in the supernatants and the membrane fraction in the pellets. The membrane pellets were then resuspended in SB buffer (20 mM HEPES pH 7.4, 1M Kl, 5 mM EDTA, 5 mM EGTA at 4 degrees) and centrifuged at 100,000 g for 1 hour at 4° C. to remove weakly bound membrane proteins. The membrane fractions were then resuspended in RB buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 5 mM EDTA, 5 mM EGTA) containing Triton (1%) and protease inhibitors. After sonication in an ice bath, the membrane fractions were subsequently subjected to gentle agitation for 2 hours at 4° C. The solubilized membrane fractions were centrifuged at 100,000 g for 1 hour at 4° C. The membrane fraction was in the supernatant.
[0072] Western blot: L4 to L6 DRGs from three mice were pooled and homogenized in lysis buffer containing triton (1%) and protease inhibitors. Homogenates were centrifuged at 18,000 g 20 min at 4° C. and supernatants collected. 50 μg of total protein was separated by SDS-PAGE on a 7.5% gel. After transfer to nitrocellulose membrane and saturation with BSA (5%), primary antibodies were used at a dilution of 1/500 for HCN1 and HCN2 (Neuromab), 1/1000 for TRIP8b (Neuromab), and 1/5000 for Actin□ (Sigma-Aldrich). After 3 successive washes with TBST, the corresponding HRP-coupled secondary antibodies (ThermoFisher) were incubated at a 1:10,000 dilution and detected with the chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate, Thermofisher).
[0073] Patch-clamp recording: The electrophysiology experiments were performed on isolated pacemaker heart cells as previously described (Marger et al., 2011) [28]. The whole-cell patch-clamp technique was used to record If currents generated by HCN channels, employing an Axopatch 200A amplifier (Axon Instruments Inc., Foster USA).
[0074] Recording electrodes of ˜5 m MΩ resistance were filled with an intracellular solution containing (mM/L): K+-aspartate, 130; NaCl, 10; ATP-Na+ salt, 2; creatine phosphate, 6.6; GTP-Mg2+, 0.1; CaCl2, 0.04 (pCa=7); Hepes-KOH, 10; (pH=7.2 with KOH). A Tyrode extracellular solution containing 5 mM BaCl2 to block IK1 currents was used. The effect of NUCC5953 (5 μM) on If current amplitude and kinetics was compared with that of ivabradine (3 μM), both diluted in the extracellular solution. Data acquisition was performed using pClamp software (ver. 9, Axon Instruments Inc.).
In Vitro Assays
[0075] Fluorescence polarization assay: a fluorescence polarization (FP) based assay was used to identify compounds capable of disrupting the interaction between HCN and the TRIP8b tail, as published (Han et al., 2015) [13].
Example 2
Results
[0076] For the first time the presence of TRIP8b mRNA (RNAseq data obtained from DRG mice showed the presence (FPKM>1) of splice variants 2-5: NM_01163516, NM_01163517, NM_01289505, NM_01310460) and protein in DRG and TG neurons in mice was observed. In addition, transcript and protein expression of HCN1-2 channels and TRIP8b were significantly increased in peripheral neurons of oxaliplatin-treated animals compared with controls, suggesting the involvement of the 2 proteins in the development of OIPN (oxaliplatin-induced neuropathy model mouse) (
[0077] Using NUCC5953, a molecule capable of disrupting the TRIP8b-HCN interaction (Han et al., 2015) [13], it was shown that oxaliplatin-induced acute cold hypersensitivity can be reversed in a dose-dependent manner either i.t. (0.2; 1; 5 μg/mouse;
[0078] No alteration in locomotor activity was observed in the Rotarod test when mice were treated with NUCC5953 (4 mg/kg) administered subcutaneously (
[0079] At the same time, preliminary data also suggest that administration of NUC5953 reduces the overexpression of HCN1 and 2 and TRIP8b proteins in the membrane fraction of DRG neurons from OIPN animals (
[0080] Patch-clamp experiments on isolated cardiac pacemaker cells showed no effect of NUCC5953 on If current amplitude, in contrast to the pan-HCN blocker Ivabradine (
[0081] NUCC5953 does not affect heart rate in vivo when administered at an analgesic dose (4 mg/kg, i.p.) (
Example 3
Design of Peptidomimetics
[0082] Design of peptidomimetics
[0083] A—Tetrameric Peptidomimetics
[0084] Peptidomimetic compounds mimicking the sequence of the carboxy-terminus of the HCN2 channel were designed and synthesized. The design of these peptidomimetics was based on an extensive X-ray structure study of a co-crystal of TRIP8b with the C-terminal region of HCN2, available in the protein database (pdb: 4EQF). Preparatory modeling work to complete and improve this structure was performed. The missing parts were modeled by sequence homology from different PEX5 structures (PDB: 1FCH, 3R9A, 4KXK, 4KYO, 2J9Q, 2COM and 2COL) showing sequence identity ranging from 30 to 60% with TRIP8b. The resulting model identified the contacts between residues responsible for the formation of the HCN-TRIP8b complex. In particular, this study identified two hydrophobic pockets in the TPR domain of TRIP8b, which are essential for good affinity of ligands to this protein domain (
[0085] The hydrophobic pocket 1 of TRIP8b provides side-chain anchoring of the C-terminal residue of the ligands. Effective interaction of the ligands with hydrophobic pocket 2, located near the central region of TRIP8b, identified as a binding site for the CNBD domain of HCN, also enhances the affinity of the inhibitors. Several 4-residue peptide-peptoid hybrid oligomers bearing various side chains capable of targeting these hydrophobic pockets were designed and evaluated by molecular dynamics (MD).
[0086] The structure-based design identified peptidomimetic ligands interacting at the SNL peptide binding site (TPR domains) of TRIP8b. These ligands have a formula that is represented by the above LF108 formula or the following general formula (I):
##STR00015##
where
R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined above.
[0087] The compounds are peptide-peptoid hybrid oligomers (peptomers) (Ostergaard et al., 1997) [17]. They comprise four residues including an α-amino acid positioned at the C-terminus of the sequence, and three consecutive peptoid residues. The third residue (from the N-terminus) is either a β-peptoid unit or an α-peptoid unit. The α-peptoids generally referred to as “peptoids” refer to N-substituted glycine oligomers (Simon et al., 1992) [18]. Compared to peptides, the side chains are attached to the nitrogen atoms of the amide bonds rather than to the α-carbon atoms. The β-peptoids are oligomers composed of N-substituted β-aminopropionic acid units (Hamper et al., 1998) [19]. Thus, a β-peptoid residue contains an additional methylene group in the backbone compared to an α-peptoid residue. Peptoids have advantageous characteristics for the development of bioactive compounds. They are amenable to solid-phase synthesis, using an iterative “submonomer” approach in which each new unit is constructed in two steps (Zuckermann et al., 1992) [20]. Peptoids can incorporate a wide variety of side chains, both proteinogenic and non-natural side chains (Cuff et al., 2010) [21]. “Sub-monomer” syntheses use simple starting molecules such as bromoacetic acid and primary amines for the construction of α-units and acryloyl chloride and primary amines for the construction of β-peptoid units. The primary amines, from which the side chains are incorporated, are commercially available by the hundreds, or can be prepared prior to their use where appropriate. The peptoid backbone based on N,N-disubstituted amides (tertiary amides) has been shown to be resistant to proteases (Miller et al., 1995) [22]. The presence of tertiary amide linkages also gives them better cell permeability (Kwon et al., 2007; Vollrath et al., 2013) [23, 24], and a few studies tend to show that peptoids are less immunogenic than peptides (Li et al., 2010; Chongsiriwatana et al., 2008) [25, 26].
[0088] The syntheses were performed on 2-chlorotrityl chloride (2-CTC) resin leading to a carboxylic acid at the C-terminus of the peptomer after cleavage. The first residue immobilized on the support, an α-amino acid, was selected primarily from the group of hydrophobic proteinogenic amino acids, including L-leucine which corresponds to the C-terminus of the SNL peptide. The α-amino acids were immobilized on the resin in a protected N-Fmoc form. After removal of the Fmoc group, chain extension by peptoid residues was achieved by “submonomer chemistry” approaches. Known protocols were implemented but some technical adjustments were required for the synthesis of the β-peptoid monomer. The N-terminus was acylated prior to cleavage under acidic conditions with hexafluoroisopropanol (HFIP) and deprotection of side chains with trifluoroacetic acid (TFA) in dichloromethane.
[0089] The crude peptomeric compounds were analyzed by LC-MS and then purified on C18 flash chromatography cartridges with UV detection at 214 and 220 nm. The pure fractions were collected and lyophilized. Pure compounds were analyzed by reverse phase HPLC on Nucleodur® C4 column (5 μm, 300 Å, 250 mm×4.6 mm). Table 2 below shows the HRMS and HPLC data of the compounds.
TABLE-US-00001 TABLE 2 retention time (min) chromatograms HPLC Molecule expected LCMSª purity reference mass observed mass/formula or HPLC.sup.b (%).sup.c YC55 564.3159 565.3227 [M + H].sup.+/ 22.02.sup.b 97 C.sub.28H.sub.45N.sub.4O.sub.8 YC60 564.3159 565.3229 [M + H].sup.+/ 3.74ª n.d. C.sub.28H.sub.45N.sub.4O.sub.8 MP208 598.3003 599.3075 [M + H].sup.+/ 22.86.sup.b 92 C.sub.31H.sub.43N.sub.4O.sub.8 MP281 536.2846 537.2919 [M + H].sup.+/ 20.69.sup.b 96 C.sub.26H.sub.41N.sub.4O.sub.8 MP341 578.3316 579.3388 [M + H].sup.+/ 4.05ª n.d. C.sub.29H.sub.47N.sub.4O.sub.8 MP354 578.3316 579.3388 [M + H].sup.+/ 3.81ª n.d. C.sub.29H.sub.47N.sub.4O.sub.8 MP376 578.3316 579.3388 [M + H].sup.+/ 3.76ª n.d. C.sub.29H.sub.47N.sub.4O.sub.8 MP377 592.3472 593.3545 [M + H].sup.+/ 3.81ª n.d. C.sub.30H.sub.49N.sub.4O.sub.8 MP405 550.3003 551.3075 [M + H].sup.+/ 21.59.sup.b 85 C.sub.27H.sub.43N.sub.4O.sub.8 LF40 563.2955 564.3021 [M + H].sup.+/ 21.66.sup.b 91 C.sub.27H.sub.42N.sub.5O.sub.8 LF96 594.3265 595.3337 [M + H].sup.+/ 22.06.sup.b 92 C.sub.29H.sub.47N.sub.4O.sub.8 LF108 609.3010 608.2939 [M + H].sup.−/ 22.07.sup.b 92 C.sub.28H.sub.42N.sub.5O.sub.10 LF176 577.3112 578.1388 [M + H].sup.+/ 21.53.sup.b 97 C.sub.28H.sub.44N.sub.5O.sub.8 LF126 578.3316 579.3381 [M + H].sup.+/ 24.53.sup.b 89 C.sub.29H.sub.47N.sub.4O.sub.8 LF188 632.3033 633.3099 [M + H].sup.+/ 23.30.sup.b 98 C.sub.29H.sub.44F.sub.3N.sub.4O.sub.8 LF222 564.3159 565.3229 [M + H].sup.+/ 21.55.sup.b 94 C.sub.28H.sub.45N.sub.4O.sub.8 LF239 564.3159 565.3228 [M + H].sup.+/ 21.12.sup.b 95 C.sub.28H.sub.45N.sub.4O.sub.8 LF261 582.3065 583.3138 [M + H].sup.+/ 21.34.sup.b 98 C.sub.28H.sub.44FN.sub.4O.sub.8 LF275 563.3319 564.3395 [M + H].sup.+/ 30.79.sup.b 94 C.sub.28H.sub.46N.sub.5O.sub.7 LF295 550.3003 551.3068 [M + H].sup.+/ 20.36.sup.b 86 C.sub.27H.sub.43N.sub.4O.sub.8 LF306 578.3316 579.3387 [M + H].sup.+/ 21.32.sup.b 95 C.sub.29H.sub.47N.sub.4O.sub.8 LF329 563.3319 564.3383 [M + H].sup.+/ 20.90.sup.b 95 C.sub.28H.sub.46N.sub.5O.sub.7 LF400 578.3316 579.3381 [M + H].sup.+/ 21.78.sup.b 91 C.sub.29H.sub.47N.sub.4O.sub.8 LF369 389.4490 390.2231 [M + H].sup.+ 18.32.sup.b 90 C.sub.17H.sub.32N.sub.3O.sub.7 .sup.aLCMS. UHPLC Ultimate 3000 RSLC chain (ThermoScientific), Kinetex EVO C18 column (100 × 2.1 mm; 1.7 μm) (Phenomenex), flow rate: 0.45 mL/min, column oven: 30° C., H.sub.2O gradient (0.1% formic acid/ACN (0.1% formic acid): t = 0 min 95:5; t = 7.5 min 1:99, t = 8.5 min 1:99; t = 9 min 95:5; t = 11 min 95:5. .sup.bHPLC. Series 1100 Agilent chain, Nucleodur ® C4 column (5 μm, 300 Å, 250 mm × 4.6 mm), flow rate 0.5 mL/min. H2O/ACN gradient (0.1% TFA): t = 0 min 95:5; t = 5 min 95:5; t = 25 min 5:95; t = 35 min 5:95, t = 40 min 95:5, t = 50 min 95:5. .sup.cUV 214 nm detection.
Side Chains
[0090] R.sup.1 is independently CH.sub.3 (L-Ala), CH(CH.sub.3).sub.2 (L-Val), CH(CH.sub.3)CH.sub.2CH.sub.3 (L-Ile), CH.sub.2CH(CH.sub.3).sub.2 (L-Leu and D-Leu), CH.sub.2C(CH.sub.3).sub.3, C(CH.sub.3).sub.3, CH.sub.2(cyclobutyl);
R.sup.2, R.sup.3, and R.sup.4 are the side chains of the peptoid units incorporated sequentially during the synthesis from primary amines;
R.sup.2 and R.sup.3 are independently selected from: CH.sub.2CH(CH.sub.3).sub.2, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2CH.sub.2OH, CH.sub.2CH(OH)CH.sub.3, CH.sub.2CONH.sub.2, CH.sub.2CH.sub.2CONH.sub.2. The side chains with an alcohol or amine function have been incorporated into the oligomers in protected form: silyl ether for the alcohol function and tert-butyl carbamate (Boc) for the amine function;
R.sup.4 is an aliphatic or aromatic group chosen from:
##STR00016##
R.sup.5 is independently CH3, CH(CH3)2, CH2C6H5, C6H4X (where X in the ortho, meta or para position is independently H, OMe, CF3, CH3, CH2CH3, F, Br, Cl, NO2), 3-indolyl, 3-quinolinyl.
Representative Synthetic Scheme on 2-chlorotrityl Chloride Resin for the Synthesis of N-acylated Peptomers Incorporating an α-amino acid at the C-Terminus, Followed by a β- and two α-peptoid Residues.
##STR00017##
Synthesized molecules
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
Synthesis
[0091] Compounds corresponding to the general formula (I) were synthesized on 2-chlorotrityl chloride resin (Novabiochem 100-200 mesh) in 5 mL plastic syringes equipped with a sinter and stopcock. The syringes were placed on an orbital shaker heating platform during the reactions.
[0092] In a typical synthesis described with L-leucine at the C-terminus and a β-peptoid residue preceding this amino acid, 2-chlorotrityl chloride resin (1.33 mmol g−1, 0.100 g, 0.133 mmol) was swollen in 2 mL of dry CH.sub.2Cl.sub.2 (2×20 min). After filtration, 0.12 mL of a 2.2 M solution of Fmoc-Leu-OH in CH.sub.2Cl.sub.2 (2.0 equivalents) was added, followed by 2.0 mL of a 0.4 M solution of DIEA in CH.sub.2Cl.sub.2 (6.0 equivalents). The mixture was stirred for 40 min at 25° C. The resin was drained, washed with CH.sub.2Cl.sub.2 (2×2 mL), and the binding of Fmoc-Leucine to the resin was repeated under the same conditions. The resin was washed with CH.sub.2Cl.sub.2 (5×2 mL). The Fmoc group was removed by adding 2.0 mL of a 20% piperidine solution in DMF and stirring for 15 min at room temperature. The operation was repeated under the same conditions, after which the resin was washed with DMF (5×2 mL) and drained.
[0093] Introduction of the β-peptoid residue. The resin was cooled by two washes with CH.sub.2Cl.sub.2 at −20° C. 1.21 mL of a 0.22 M solution of acryloyl chloride (2.0 equivalents) in CH.sub.2Cl.sub.2 cooled to −20° C. was added, followed by 0.8 mL of a 0.5 M solution of Et3N (3.0 equivalents) in cooled CH.sub.2Cl.sub.2 (−20° C.). The resin was stirred for 1 h at 25° C., drained, and then treated again with acryloyl chloride under the same conditions. The resin was filtered and washed with CH.sub.2Cl.sub.2 (5×2 mL) and drained. The resulting acrylamide was treated with a 2.0 M solution of the amine R.sup.2NH.sub.2 in isopropanol (12.0 equivalents) for 24 h at 50° C. After filtration of the resin, the aza-Michael reaction was repeated under the same conditions. The resin was washed with isopropanol (5×2 mL) before submonomer synthesis of two α-peptoid residues.
[0094] Synthesis of α-peptoid residues. To the resin were added 2.0 mL of a 0.4 M solution of bromoacetic acid in DMF (6.0 equivalents), followed by 2.13 mL of a 0.5 M solution of CID in DMF (8.0 equivalents). After stirring for 5 min at 25° C., the resin was drained and washed with DMF (5×2 mL). The resulting bromoacetamide was then treated with a 2.0 M solution of the amine R.sup.3NH.sub.2 (20.0 equiv) in DMF and the mixture was left on the stirring platform for 1 h at 40° C. The resin was filtered and washed with DMF (5×2 mL). The last two bromoacetylation and substitution steps were repeated under the same conditions to construct the fourth residue bearing the desired R.sup.4 side chain. The N-terminus of the peptomer was then acylated according to one of the general procedures described below, prior to its removal from the resin.
[0095] For cleavage, the resin was treated with 1.5 mL of a 2.4 M solution of HFIP in CH.sub.2Cl.sub.2. After stirring for 40 min at 25° C., the resin was filtered and washed with CH.sub.2Cl.sub.2 (5×2 mL). The solvent was removed under reduced pressure. Finally, the silylated or Boc protecting groups of the side chains were removed by treating the peptomer with 2 mL of 25% (v/v) TFA in CH.sub.2Cl.sub.2 for 10 min at room temperature. The reaction mixture containing the final product was diluted with CH.sub.2Cl.sub.2 (5 mL) before complete evaporation in vacuo. In order to remove all traces of TFA, the product dissolved in CH2Cl2 was evaporated under vacuum, repeating this operation 5 times.
General Procedures for Acylation of the N-Terminus of the Resin-Bound Peptomer
[0096] Acetylation: To the resin was added 1.56 mL of a 1.7 M solution of acetic anhydride in DMF (20.0 equivalents), followed by 0.8 mL of a 2.0 M solution of DIEA in DMF (12.0 equivalents). After stirring for 90 min at 25° C., the resin was filtered and washed with DMF (5×2 mL).
[0097] Benzoylation: To the resin was added 0.8 mL of a 1.0 M solution of benzoyl chloride in CH.sub.2Cl.sub.2 (6.0 equiv.), followed by 0.8 mL of a 2.0 M solution of DIEA in CH.sub.2Cl.sub.2 (12.0 equiv.,). After stirring for 40 min at 25° C., the resin was drained and washed with CH.sub.2Cl.sub.2 (5×2 mL).
[0098] Acylation from carboxylic acids: In a typical coupling reaction described here from phenylacetic acid, 2.0 mL of a 0.4 M solution of phenylacetic acid in DMF (6.0 equiv.) and 2.13 mL of a 0.5 M solution of DIC (8.0 equiv.) in DMF were added successively to the resin. After stirring for 1 h at 25° C., the resin was drained and washed with DMF (5×2 mL).
[0099] Of the compounds (YC55, YC-60) that were synthesized, YC55 demonstrated equal potency to NUCC5953 in disrupting the TRIP8b-HCN interaction (Table 1: IC50 of peptoids).
TABLE-US-00002 TABLE 1 YC55 YC60 Acox3 IC50 8.862 12.71 0.6029
[0100] The analgesic effect of novel peptidomimetic compounds injected either intrathecally or systemically has been demonstrated in oxaliplatin-induced acute neuropathic pain in mice.
[0101] Thus, the lead compound YC55 demonstrated a dose-dependent analgesic effect in OIPN mice when administered i.t. (
[0102] Compound YC55 also demonstrated a dose-dependent analgesic effect in OIPN mice when administered s.c. (
[0103] No alteration in locomotor activity was observed in the Rotarod test when mice are treated with YC55 administered subcutaneously (
[0104] YC55 also demonstrated a dose-dependent analgesic effect in a rat model of paclitaxel-induced neuropathy when administered i.t. (
[0105] Intrathecal administration of YC55 showed an antihyperalgesic effect in a model of neuropathy induced by traumatic sciatic nerve injury (
[0106] Compounds MP208, MP405 (5 μg, i.t.) and MP341, MP354, MP376 (10 mg/kg, s.c.) demonstrated an analgesic effect in an oxaliplatin-induced neuropathy model in mice (
[0107] Similarly, compounds LF108, LF306, LF188 (
B—Trimeric Peptomimetics
[0108] The trimeric peptomeric compounds are also mimics of the carboxy-terminal sequence of the HCN2 channel. They were designed based on the extensive X-ray structure study of a co-crystal of TRIP8b with the C-terminal region of HCN2 (bp: 4EQF).
[0109] These ligands have a formula which is represented by the formula LF369 below or the following general formula (II):
##STR00023##
where
R.sup.1, R.sup.2 and R.sup.3 are as previously defined;
R.sup.6 is H, R.sup.3, CO R.sup.5 (where R.sup.5 is as defined above), or R.sup.7;
R.sup.7 is independently one of:
##STR00024##
[0110] The compounds are peptide-peptoid hybrid oligomers (peptomers) (Ostergaard et al., 1997) [17]. They comprise three units including an α-amino acid positioned at the C-terminus of the sequence, followed by two peptoid residues.
[0111] The syntheses were performed in solution from an α-amino acid protected as a tert-butyl ester (Xaa-tBu, the L-Leu-tBu in the case of the synthesis of LF369). These syntheses can also be performed on resin as previously described for tetrameric peptidomimetics. From this unit, the elongation was performed as for the molecules corresponding to the general formula (I), by “submonomer chemistry” approaches. When the core unit is of the β-peptoid type, the two steps necessary for its construction are the reaction of Xaa-tBu with acryloyl chloride, followed by an aza-Michael reaction to introduce the desired side chain (R.sup.2) to the backbone. The third α-peptoid unit is constructed by acylation of the dimer with bromoacetic acid bromide followed by substitution of the bromine atom with an amine. In the case where R.sup.6 is H, the amine used is a primary amine R.sup.3NH.sub.2. When R.sup.6 is COR.sup.5, the secondary amine obtained after treatment with R.sup.3NH.sub.2 is acylated with an acid chloride, acid anhydride or carboxylic acid under coupling conditions. Compounds in which R.sup.6=R.sup.7 are obtained by treating the N-terminally bromoacetylated dimers with a secondary amine R.sup.3R.sup.7NH. They can also be obtained by reductive amination of compounds with NHR.sup.3 at the N-terminus. The final step is to deprotect the side chains and the C-terminus by treatment with TFA.
The Precise Experimental Protocol for the Synthesis of LF369 is as Follows:
[0112] In a flask, L-leu-tBu (1.74 g, 7.8 mmol) is dissolved in 45 mL of anhydrous THF. The flask under an inert argon atmosphere is cooled to 0° C. Triethylamine (2.3 mL, 31.4 mmol, 4 equiv) is added, followed by acryloyl chloride (0.76 mL, 9.4 mmol, 1.2 equiv). After 3 h stirring at 0° C. a thin layer chromatography (50/50 cyclohexane/AcOEt) indicates full conversion. The salts formed are removed by filtration, the filtrate is evaporated under vacuum and the product obtained is purified by silica gel chromatography (50/50 cyclohexane/AcOEt). The acrylamide is isolated in 93% yield (1.76 g, 7.27 mmol) as a white solid.
[0113] Aza-Michael reaction. The acrylamide formed previously (1.76 g, 7.27 mmol) is dissolved in 20 mL of ethanol. Ethanolamine with the alcohol function protected as tertbutyldimethylsilyl ether (2.68 g, 15.28 mmol, 2.1 equiv.) is added and the reaction mixture is heated to reflux for 60 h. The solvent is then evaporated and the reaction crude purified by silica gel chromatography (90/10/0.1 AcOEt/MeOH/Et3N) affording the dimer in 90% yield (2.74 g, 6.58 mmol) as a yellowish oil.
[0114] Acylation reaction of the dimer. The dimer (2.74 g, 6.58 mmol) is dissolved in 10 mL THF, the reactor is inerted with argon and then cooled to −20° C. Triethylamine (1.1 mL, 7.89 mmol, 1.2 equiv.) is added to the solution, followed by the addition of 2-bromoacetyl bromide (0.62 mL, 7.89 mmol, 1.2 equiv.). After 3 h of stirring at −20° C., the reaction mixture is filtered. The resulting filtrate is evaporated and the reaction crude is chromatographed on silica gel (60/40/0.1 Cyclohexane/AcOEt/Et.sub.3N). The bromoacetylated dimer is isolated in 67% yield (2.36 g, 4.4 mmol) as a yellowish oil.
[0115] Substitution reaction. The previously obtained product (2.36 g, 4.4 mmol) is dissolved in 10 mL THF. Triethylamine (1.2 mL, 8.8 mmol, 2 equiv.) and then protected ethanolamine H.sub.2NCH.sub.2CH.sub.2OTBDMS (2.31 g, 13.2 mmol, 3 equiv.) are added to the solution. The reaction medium under argon atmosphere is stirred overnight at room temperature. The reaction mixture is then filtered and the filtrate obtained is evaporated. The crude product is purified by silica gel chromatography (95/5/0.1 AcOEt/MeOH/Et.sub.3N) leading to the trimer in 83% yield (2.30 g, 3.64 mmol) as an oil.
[0116] Acetylation of the trimer. The resulting amine (2.3 g, 3.64 mmol) is dissolved in 20 mL of ethyl acetate. Triethylamine (2.02 mL, 14.5 mmol, 4 equiv.) and then acetic anhydride (2.75 mL, 29 mmol, 8 equiv.) are added to the solution. The reaction is placed under argon at room temperature for 60 h. After evaporation of the solvent the crude obtained is chromatographed on silica gel (90/10/0.1 AcOEt/cyclohexane/Et3N) leading to the N-acetylated trimer in 92% yield (2.27 g, 3.36 mmol) as a colorless oil.
[0117] Deprotection of the side chains and C-terminus. The trimeric compound (502 mg, 0.776 mmol, 1 equiv.) is dissolved in 4 mL of a TFA/CH2Cl2/H2O (47.5:47.5:5) mixture. After 2 h of stirring at room temperature, 10 mL of dichloromethane is added. The reaction medium is evaporated under vacuum and then 5 co-evaporations with dichloromethane are performed. The product is then redissolved in 5 mL of distilled water and left under stirring at room temperature for 2 h. The solvent is evaporated and then five co-evaporations with toluene are performed leading to LF369 (362 mg) as an oil.
Side Chains
[0118] R.sup.1, R.sup.2 and R.sup.3 are as previously defined;
R.sup.6 is R.sup.3, H, COR.sup.5 (where R.sup.5 is as previously defined), or R.sup.7;
R.sup.7 is independently one of:
##STR00025##
Representative Synthetic Scheme for the Solution Synthesis of Peptoids of General Formula (II), e.g. LF369
##STR00026##
Representative Synthetic Scheme for the Supported Synthesis of Peptoids of General Formula (II), e.g. LF369
##STR00027##
Synthesized Molecule
[0119] ##STR00028##
Synthesis
[0120] The compound LF369 (5 μg, i.t.) demonstrated an analgesic effect in a mouse model of oxaliplatin-induced neuropathy (
LIST OF REFERENCES
[0121] 1. Finnerup et al., Lancet Neurol., 14(2): 162-173, 2015 [0122] 2. von Hehn et al., Neuron, 73(4): 638-652, 2012 [0123] 3. Hershman et al., J. Clin. Oncol., 32(18): 1941-1967, 2014 [0124] 4. Ludwig et al., Nature, 393(6685): 587-591, 1998 [0125] 5. Santoro et al., Cell, 93(5): 717-729, 1998 [0126] 6. Seifert et al., Proc. Natl. Acad. Sci. USA, 96(16): 9391-9396, 1999 [0127] 7. Dunlop et al., Curr. Pharm. Des., 15(15): 1767-1772, 2009 [0128] 8. Lewis et al., Mol. Cell Neurosci., 46(2): 357-367, 2011 [0129] 9. Descoeur et al., EMBO Mol. Med., 3(5): 266-278, 2011 [0130] 10. Young et al., Pain, 155(9): 1708-1719, 2014 [0131] 11. Lewis et al., J. Neurosci., 29(19): 6250-6265, 2009 [0132] 12. Bankston et al., Proc. Natl. Acad. Sci. USA, 109: 7899, 2012 [0133] 13. Han et al., J. Biomol. Screen, 20(9): 1-8, 2015 [0134] 14. Janssen et al., Arzneimittelforschung, 13: 502-507, 1963 [0135] 15. Randall et Selitto, Arch. Int. Pharmacodyn. Ther., 111(4): 409-419, 1957 [0136] 16. Kim et Chung, Pain, 50(3): 355-363, 1992 [0137] 17. Ostergaard et al., Molecular Diversity, 3: 17-27, 1997( [0138] 18. Simon et al., Proc. Natl. Acad. Sci USA, 89: 9367-9371, 1992 [0139] 19. Hamper et al., J. Org. Chem., 63: 708-718, 1998 [0140] 20. Zuckermann et al., J. Am. Chem. Soc., 114: 10646-10647, 1992 [0141] 21. Culf et al., Molecules, 15: 5282-5335, 2010 [0142] 22. Miller et al., Drug Dev. Res., 35: 20-32, 1995 [0143] 23. Kwon et al., J. Am. Chem. Soc., 129: 1508-1509, 2007 [0144] 24. Vollrath et al., Organic & Biomolecular Chemistry, 11: 8497-8201, 2013 [0145] 25. Li et al., Cellular & Molecular Immunol., 7: 133-142, 2010 [0146] 26. Chongsiriwatana et al., Proc. Natl. Acad Sci. USA, 105: 2794-2799, 2008 [0147] 27. Mesirca et al., Nat. Commun., 5: 4664.doi:10.1038/ncomms5664, 2014 [0148] 28. Marger et al., Channels (Austin), 5(3): 241-250, 2011