HYBRID MU OPIOID RECEPTOR AND NEUROPEPTIDE FF RECEPTOR BINDING MOLECULES, THEIR METHODS OF PREPARATION AND APPLICATIONS IN THERAPEUTIC TREATMENT
20210002231 · 2021-01-07
Inventors
- Frédéric Simonin (ILLKIRCH GRAFFENSTADEN, FR)
- Armand Drieu la Rochelle (SAINT-GRÉGOIRE, FR)
- Frédéric Bihel (Fegersheim, FR)
- Steven Ballet (ITEGEM, BE)
Cpc classification
A61K38/04
HUMAN NECESSITIES
C07D223/14
CHEMISTRY; METALLURGY
International classification
Abstract
Molecules binding the mu opioid receptor (MOR) and the neuropeptide FF receptor (NPFFR), in particular molecules having a MOR agonist and NPFFR modulatory activity, and pharmaceutical compositions useful in the treatment of pain and/or hyperalgesia.
Claims
1. A molecule comprising the following structure: X1-X2-X3-X4-X5-X6-T wherein X1 has the following structure: ##STR00052## wherein R2, R.sub.3 and R.sub.4 are, independently at each occurrence H, OH, NH.sub.2, CH.sub.3, CONH.sub.2, or COCH.sub.3 R.sub.1 and R.sub.5 are, independently at each occurrence, H or Me R is H, alkyl or C(N)NH.sub.2 X is N or CRa, wherein Ra is H or Me; X2 is a natural or non-natural amino acid residue, or derivative thereof, including homologated amino acids, aza amino acids, or X1-X2 represent together the following structure: ##STR00053## R2, R.sub.3 and R4 are, independently at each occurrence H, OH, NH2, CH.sub.3, CONH2, or COCH.sub.3 R.sub.1, R.sub.5 and R.sub.6 are, independently at each occurrence, H or Me R is H or C(N)NH.sub.2 R is H or a group of atoms, including natural and non-natural amino acid side chains X is N or CRa, wherein Ra is H or Me, X3 is a natural or non-natural amino acid residue, X4 is one to five natural or non-natural amino acid residue or a derivative thereof, X5 has the following structure: ##STR00054## wherein Rx.sub.5 is a cyclic or acyclic tertiary amine group linked by the nitrogen atom of the tertiary amine group, wherein: if m=0, then n=2, 3, or 4; if m=1, or 2, then n=1 wherein X is CRa or N, wherein Ra is H or Me, wherein R is H or Alkyl (for example Me, Et); wherein each carbon atom of (CH2)n and (CH2)m can be substituted independently of each other; X6 is a natural or non-natural amino acid residue T is a chemical terminal group of atoms, ##STR00055## represents a covalent link.
2. The molecule of claim 1 wherein X1 is ##STR00056## wherein R is alkyl (methyl, ethyl), or ##STR00057## wherein R1 is NH2, CH.sub.3, CONH.sub.2, COCH.sub.3 and R is H or alkyl (methyl, ethyl), ##STR00058## wherein: R.sub.1 is Me, R2 is Et and R.sub.3 is H or R.sub.1 is Me, R2 is iPr and R.sub.3 is H or R.sub.1, R2 and R.sub.3 are Me.
3. The molecule of claim 1 wherein X2 is ##STR00059## wherein X is CRa or N and Ra is H or Me; wherein R is H or alkyl (typically Me); wherein R.sub.x2 is H, alkyl chain bearing an substituted amino group or a substituted guanidine group, wherein: if m=0, then n=2, 3, or 4; if m=1, or 2, then n=1.
4. The molecule of claim 1 wherein X2 has the following structure: ##STR00060## wherein R.sub.x2 is a cyclic or acyclic tertiary amine group linked by the nitrogen atom of the tertiary amine group or is a phenyl group optionally substituted by an alkyl group or an amino acid side chain, wherein: if m=0, then n=2, 3, or 4; if m=1, or 2, then n=1 wherein each carbon atom of (CH2)n and (CH2)m can be substituted independently of each other.
5. The peptide sequence of claim 1 wherein X3 has the following structure: ##STR00061## wherein R4 is an amino acid side chain; R1, R2 and R.sub.3 are each independently H, halogen, alkyl, alkenyl, (hetero)aryl; wherein X is CRa or N, wherein Ra is H or Me.
6. The molecule of claim 1 wherein X3-X4 together represent the following structure: ##STR00062## Wherein R is an amino acid side chain Ra is H or Me R.sub.1 to R.sub.4 and R.sub.6 are each independently H, halogen, alkyl, alkenyl, (hetero)aryl; ##STR00063## wherein X is CRa or N, wherein Ra is H or Me wherein R.sub.4 is an amino acid side chain Ra is H or Me R.sub.1 to R.sub.5 are each independently H, halogen, alkyl, alkenyl, (hetero)aryl.
7. The molecule of claim 1 wherein X4 has the following structure: -amino acids ##STR00064## wherein R is an amino acid side chain or derivative thereof wherein Ra is H or Me wherein R.sub.N is H or alkyl 3-homo-amino acids ##STR00065## wherein R is an amino acid side chain or derivative thereof wherein Ra is H or Me wherein R.sub.N is H or alkyl 2-homo-amino acids ##STR00066## wherein R is an amino acid side chain or derivative thereof wherein R.sub.N is H or alkyl aza-amino acids ##STR00067## wherein R is an amino acid side chain or derivative thereof wherein Ra is H or Me wherein R.sub.N is H or alkyl.
8. The molecule of claim 1, wherein X4 comprises one of the following group C-terminal group forming a natural or non-natural amino acid residue or a derivative thereof: ##STR00068## wherein 0m5; ##STR00069## wherein 0m5; ##STR00070## wherein 1m3; ##STR00071## wherein 0m3 and 0n3 ##STR00072## wherein 0m3 and 0n3. ##STR00073## wherein 0m5 and 0n5 ##STR00074## wherein 0m5 and
9. The molecule of claim 1 wherein R.sub.x5 is selected from the group consisting of: a cyclic or acyclic guanidine bearing substituents: ##STR00075## a cyclic or acyclic urea or thiourea bearing substituents: ##STR00076## and a cyclic or acyclic tertiary amine group: ##STR00077## wherein A is (CH.sub.2).sub.n with n=0, 1, 2, 3, O, S, or NH, wherein each carbon atom of (CH.sub.2)n is optionally substituted independently of each other, wherein R1 is Aryl or Heteroaryl, optionally substituted.
10. The molecule of claim 1, wherein X6 is selected from the group consisting of: a natural or non-natural amino acids of configuration L or D including one of the following structure: Gly, Ala, Val, Ile, Leu, Nle, cHex, Phe, Hphe, Tyr, Trp, Asn, Gln, Pro Arg, Lys, Cys, Met, Asp, Glu; and a bridged amino acids including: ##STR00078##
11. The molecule of claim 1 wherein said terminal group T is selected from the group consisting of: H, Alkyl, (CH.sub.2).sub.n-Aryl (when X6 is a bridged amino acid), and NH.sub.2, NHR or cyclic/acyclic NR.sub.1R.sub.2 wherein R, R.sub.1, R.sub.2 is H, Alkyl or (CH.sub.2).sub.n-Aryl.
12. The molecule of claim 1 wherein said molecule comprises from 6 to 10 amino acid residues or derivative thereof.
13. The molecule of claim 1 wherein X1-X2-X3-X4 represent a opioid peptide-based peptide analogue structure or a dermophin peptide based peptide analogue structure.
14. A molecule according to claim 1, wherein said molecule is binding MOR and NPFFR.
15. A molecule according to claim 1, wherein said molecule is an NPFFR1 or NPFFR2 antagonist, and in particular a NPFFR1 and NPFFR2 antagonist.
16. A method of treating an animal or human body, said method comprising administering to said animal or human body of an effective amount of at least one molecule as defined in claim 1.
17. The method according to claim 16, wherein said method is a method of treatment of pain and/or hyperalgesia.
18. The method according to claim 16, wherein said method is a method of treatment of a disease or condition associated with MOR.
19. The method according to claim 16, wherein said method is a method of treatment of a disease or condition associated with NPFFR1 and/or NPFFR2.
20. The method according to claim 16, wherein said method is method of treatment of behavioral and somatic signs of opioid withdrawal syndrome.
21. A pharmaceutical composition comprising at least one molecule according to claim 1 and one or more pharmaceutically acceptable excipients.
22. A pharmaceutical composition comprising at least one molecule according to claim 2 and one or more pharmaceutically acceptable excipients.
23. A pharmaceutical composition comprising at least one molecule according to claim 6 and one or more pharmaceutically acceptable excipients.
24. A pharmaceutical composition comprising at least one molecule according to claim 8 and one or more pharmaceutically acceptable excipients.
25. A pharmaceutical composition comprising at least one molecule according to claim 9 and one or more pharmaceutically acceptable excipients.
Description
[0247] In the figures:
[0248]
A: KGFF compounds combining the opioid ligand KGOP01 and NPFF ligand pharmacophores. From this strategy, 16 compounds were synthesized and screened in vitro for their affinity for the MOPr, NPFF1R and NPFF2R.
B: Chemical diversity at the NPFF ligand pharmacophore with Arg mimetics/Orn derivatives serving as Arg mimetics for the NPFF ligand pharmacophore.
C: Binding affinity constants (pK.sub.i values) were determined in radioligand competition binding assays with membranes from CHO cells expressing hMOPr, hNPFF1R or hNPFF2R. Values are meanSEM of at least 2 independent experiments performed in duplicate.
[0249]
A: Inhibition of forskolin-induced cAMP accumulation in HEK293-Glo20E-hMOPr cells.
B, E: hNPFF1R activation measured by [.sup.35S]-GTPS binding with membranes from CHO-hNPFF1R cells.
C, F: hNPFF2R activation measured by [.sup.35S]-GTPS binding with membranes from CHO-hNPFF2R cells.
D: eYFP-labelled -arrestin-2 translocation to Rluc-hMOPr in HEK293 cells. Agonist specific BRET1 ratio were determined by subtracting BRET1 ratio of non-activated cells, and normalized to the maximal effect of DAMGO.
(A, B, C) Potency constants (pEC.sub.50) and efficacy values (E.sub.max) are shown on left panels and representative experiments for KGOP01, KGFF03 and KGFF09 on right panels. Efficacy values (E.sub.max) are relative to DAMGO (A), RFRP3 (B) or NPFF (C) response. NPFF1R (E) and NPFF2R (F) antagonisms were assessed with RFRP3 or NPFF dose-response curve, respectively, in the presence of increasing KGFF09 concentrations. Values are meanSEM of at least 2 independent experiments performed in duplicate or triplicate.
[0250]
[0251]
A: Development of hyperalgesia and analgesic tolerance upon chronic treatment with KGOP01, KGFF03 and KGFF09. C57BL/6N mice received daily (d1 to d8) injections of KGOP01 (1.8 mol/kg, sc.), KGFF03 (1.2 mol/kg, sc.), KGFF09 (7.4 mol/kg, sc.) or saline (A, B, C).
B: Development of hyperalgesia upon chronic treatment of C57BL/6N mice with KGOP01, KGFF03 and KGFF09. Basal nociceptive values were measured for two days before drug treatment and once daily before drug administration (d1 to d8), using the tail immersion test. Each day of injection is shown with an arrowhead.
C: Development of analgesic tolerance upon chronic treatment of C57BL/6N mice with KGOP01, KGFF03 and KGFF09. Comparison between groups of area-under-the-curve (AUC) values over the 0-7 h time course is shown on the left panel and development of tolerance (%) at day 8 relative to day 1 on the right panel.
D: Effect of KGOP01, KGFF03 or KGFF09 on naltrexone-precipitated withdrawal signs after chronic treatment of C57BL/6N mice. The different signs of withdrawal were measured over 30 min immediately after naltrexone injection (1 mg/kg, sc.) and a global withdrawal score (GWS) was calculated. KGOP01 (1.8 mol/kg, sc.), KGFF03 (1.2 mol/kg, sc.) and KGFF09 (7.4 mol/kg, sc.) were administered twice daily for 7 days.
E: Effect of KGOP01, KGFF03 and KGFF09 on respiratory frequency after sc. administration to C57BL/6N mice, measured by whole body mouse plethysmography immediately after injection of KGOP01 (1.8 mol/kg, sc.), KGFF03 (1.2 mol/kg, sc.), KGFF09 (7.4 mol/kg, sc.) or saline (sc.). Comparison between groups of respiratory frequency (left panel) and area-under-the curve (AUC, right panel) values over the 100 min kinetics are shown. Data are expressed as meanSEM. Groups were compared using one-way (C right panel, and D, E right panel) or two-way (B, C left panel, and E left panel) ANOVA with Bonferroni post hoc test *p<0.05, **p<0.01, ***p<0.001 as compared to saline, ##p<0.01 as compared to KGOP01, +p<0.001 as compared to KGFF09.
[0252]
C57BL/6N mice were injected on day 1 with CFA or saline in the tail, and then daily administered with KGOP01 (1.8 mol/kg/d, sc.), KGFF03 (1.2 mol/kg/d, sc.), KGFF09 (7.4 mol/kg/d, sc.) or saline (sc., CFA/saline and saline/saline).
A: Analgesic tolerance to the thermal antinociceptive effect was measured 2 h after daily sc. administration on days 2-3-5-7 by the tail immersion test. Comparison between groups of the % MPE (left panel) and tolerance (%) at day 7 relative to day 2 (right panel) are shown.
B: Analgesic tolerance to the mechanical antinociceptive effect was measured 2 h after daily sc. injection on days 2-4-6-8 by the tail pressure test. Comparison between groups of percentage of the % MPE (left panel) and tolerance (%) at day 8 relative to day 2 (right panel) are shown.
C, D: Anti-hyperalgesic activity of KGOP01, KGFF03 and KGFF09. Basal nociceptive thresholds were measured by the tail immersion test (C) or tail pressure test (D) once daily before drug administration to visualize CFA-induced pain hypersensitivity.
Data are expressed as meanSEM, n=8-10. Statistical significance was calculated with two-way ANOVA with Bonferroni post hoc test *p<0.5, **p<0.01, ***p<0.001 vs saline mice, +p<0.5, ++p<0.01, +++p<0.001 vs KGOP01, and with one-way ANOVA with Bonferroni post hoc test ###p<0.001 vs KGFF09.
[0253]
Agonist (A) or antagonist (B) activity of KGFF03 and KGFF09 measured by inhibition of forskoline-induced cAMP accumulation (A, B) in HEK293-Glo20F cells stably expressing NPFF1R. Data are expressed as percentage of maximal cAMP levels. Agonist (C) or antagonist (D) activity of KGFF03 and KGFF09 measured by Ca.sup.2+ release (C, D) in HEK293-Glo20F cells stably expressing NPFF2R. Data are expressed as percentage of maximal digitonine-induced Ca.sup.2+ response, relative to basal. Data are meanSEM of at least 2 independent experiments performed in duplicate.
[0254]
Agonist (A, B and E) or antagonist (C, D, F and G) activity of KGOP01, KGFF03 and KGFF09 measured by inhibition of forskoline-induced cAMP accumulation in HEK293-Glo20F cells stably expressing human DOPr (A), KOPr (B, C and D) or NOPr (E, F and G). Data are expressed as percentage of maximal cAMP levels and shown as meanSEM of at least 2 independent experiments performed in duplicate.
[0255]
The signs of withdrawal were measured over 30 min immediately after naltrexone sc. injection. Mice were treated with KGOP01 (1.8 mol/kg, sc.), KGFF03 (1.2 mol/kg, sc.), KGFF09 (7.4 mol/kg, sc.) or saline (control) twice daily over a 7-days period. Data are meanSEM, n=6-8. One way ANOVA with Bonferroni's post hoc test *p<0.05, **p<0.01, ***p<0.001 as compared to saline, +p<0.05, +++p<0.001 as compared to KGOP01 and #p<0.05, ##p<0.01, ###p<0.001 as compared to KGFF03.
[0256]
Time-dependent antinociceptive effect of KGOP01, KGFF03 and KGFF09 sc. administration in C57BL/6 mice in the tail immersion test (A) and in the tail pressure test (B). KGOP01 (1.8 mol/kg/d, sc.), KGFF03 (1.2 mol/kg/d, sc.), KGFF09 (7.4 mol/kg/d, sc.) or saline (control) were administered 24 h after CFA (or saline) injection in the tail. Data are meanSEM, n=9-10.
EXAMPLES
[0257] 1. Materials and Methods
1.1. Chemical Synthesis
1.1.1. Peptides KGFF01-KGFF07
[0258] Peptides KGFF01-KGFF07 were synthesized manually by standard Fmoc-SPPS on Rink amide AM resin. Standard couplings were performed with 3 equivalents (equiv.) of Fmoc-protected amino acid and 3 equiv. of coupling reagent (HCTU) in 0.4 NMM in DMF during 1.5 h. Fmoc-Aba--Ala-OH was coupled in only 1.5 equiv. excess of both Fmoc-dipeptide and coupling reagent for 3 h. Boc-Dmt-OH was coupled using 1.5 equiv. of amino acid and 1.5 equiv. of HOBt/DIC in DMF for 2 h, without addition of base to avoid coupling to the unprotected phenol group. For Fmoc-deprotection, the resin was treated with 20% 4-methylpiperidine in DMF for consecutively 5 and 15 min, or with DBU/Piperidine/DMF 2/2/96 for consecutively 330 s, and 7 min. Washing of the resin was performed after every coupling and after deprotection step with DMF (3), iPrOH or MeOH (3) and CH.sub.2Cl.sub.2 (3). Final cleavage and deprotection were done with the cleavage mixture (TFA/TES/H.sub.2O 95:2.5:2.5, TES can be replaced by TIS) during 3 h. After filtration and concentration of TFA, the residue was added to cold ether to precipitate the peptide. The ether phase was decanted and the peptide was dissolved in acetonitrile/H.sub.2O or water and lyophilized to obtain the crude peptides as a powder. The crude peptides were dissolved in H.sub.2O and acetonitrile was added until complete dissolving was observed. This solution was purified by preparative RP-LC (system PLC-A or PLC-B). Fractions containing the product were combined and lyophilized. The final peptides were obtained with a purity >95% as white powders. The compounds were characterized by high-resolution electrospray mass spectroscopy.
1.1.2. Peptides KGFF08-KGFF11, DP0001-DP0005, DP0032-DP0035
[0259] The couplings of these four peptides were carried out using 3 equiv. of the amino acid with 3 equiv. of DIC/HOBt for 1.5 h in DMF. Coupling of both Fmoc-Aba--Ala-OH, Fmoc-Apa-OH and Fmoc-Bpa-OH were performed with only 1.5 equiv. of amino acid and coupling reagent (DIC/HOBt) in DMF during 3 h. Boc-Dmt-OH was coupled with the same number of equivalents, but the reaction mixture was stirred only for 2 h. Further standard SPPS coupling, cleavage and purification were performed as described for peptides KGFF01-KGFF07
1.1.3 Peptide KGFF12
[0260] The dipeptide Boc-Dmt-D-Arg(Pbf)-OH was first synthesized on 2-chlorotrityl chloride resin, using the same coupling and deprotection conditions as described above. Cleavage was performed with 1% TFA in CH.sub.2Cl.sub.2 to retain the side chain protective group. The solution was then evaporated and the dipeptide (1.1 equiv.) was dissolved in CH.sub.2Cl.sub.2. Two equiv. DIPEA and 1.5 equiv. DIC/HOBt were added and the mixture was stirred for 30 min at 0 C. The free 4-amino-Aba-NH was added and stirred for another 30 min in an ice bath. The reaction was then left on stirring during 16 h at room temperature. After this coupling, the protective groups were removed with the same cleavage cocktail as described for the preparation of peptides KGFF01-KGFF07, and purification was performed analogously.
1.1.4. Peptide KGFF13
[0261] This synthesis was performed on FMPB-AM resin (4-(4-formyl-3-methoxyphenoxy)butyrylaminomethyl resin). Methylamine hydrochloride (10 equiv.) was coupled to the aldehyde resin by a reductive amination with sodium cyanoborohydride (10 equiv.) in MeOH/DMF for 2.5 h at 80 C. Complete coupling was determined by the DNPH-color test. Further standard SPPS coupling, cleavage and purification were performed as described for peptides KGFF01-KGFF07 (vide infra).
1.1.5. Peptides KGFF14-KGFF16
[0262] These 3 peptides were synthesized on MBHA-resin. The couplings (Boc-Dmt-OH, Boc-D-Arg(Tos)-OH, Boc-Phe-OH) were performed with 3 equiv. of amino acid and 3 equiv. of coupling reagent (HCTU) in 0.4 NMM in DMF for 1.5 h. Both the arginine mimetics and Boc-Dmt-OH were coupled with DIC/HOBt in DMF with respectively 1.5 (Arg mimetics) and 2 equiv. of amino acid and coupling reagent. Fmoc-Aba--Ala-OH was coupled with 1.5 equiv. of HCTU in 0.4 NMM in DMF for 3 h. This Fmoc-group was removed as described above. The Boc-deprotection was performed with 50% TFA in CH.sub.2Cl.sub.2 for 5 and 15 min and washed as previously described. The neutralization step after every Boc-deprotection was realized with a solution of 20% triethylamine in CH.sub.2Cl.sub.2 during 10 min (2 times). Final cleavage was done with liquid HF (10 mL/g resin) and anisole (0.5 mL per 50 mg resin) as scavenger for 1 h at 0 C. After HF distillation, cold ether was added to precipitate the peptides. The peptides were filtered and dissolved in acetic acid/H.sub.2O, and lyophilized. The white powders could be purified by preparative RP-HPLC. Only in case of the paramethoxybenzyl-protected benzoimidazole-containing peptide (KGFF14), an extra step had to be performed to fully cleave the protective group: the peptide was treated with 10% triflic acid (0.5 mL) in TFA (4.5 mL) for 4 h, the solvent was evaporated and the product was purified by preparative RP-HPLC.
1.1.2. Peptides DP0007 to DP0031
[0263] The couplings of these peptides were carried out using 1.5 equiv. of the amino acid with 3 equiv. of DIC and 5 equiv. of oxyma-pure for 3 h to 4 h in DMF. Further standard SPPS coupling, cleavage and purification were performed as described for peptides KGFF01-KGFF07
1.1.7. Peptide DP0032
[0264] DP0032 was synthesized using 1.5 eq of amino acid, 3 eq of DIC and HOBt in DMF for 3 to 4 h. Fmoc-Dmt-OH was coupled using 2 eq of amino acid and 3 eq of DIC/Oxyma pure in DMF assisted by micro-waves (75 C. for 30 min). N-terminal guanidylation was performed using 4 eq of N,N-di-Boc-1H-pyrazole-1-carboxamidine in DMF for 16 h (repeated two times). Further standard SPPS coupling, cleavage and purification were performed as described for peptides KGFF01-KGFF07
1.1.8. Peptide DP0035
[0265] DP0035 was synthesized using 1.5 eq of amino acid, 3 eq of DIC and HOBt for 3 to 4 h. The N-alkylated glycine residue was introduced following the sub-monomer strategy. After Fmoc-Aba-bAla-OH coupling and Fmoc removal, the N-terminal amine was bromoacylated using 6 eq of bromoacetic acid and 6 eq DIC in DMF for 30 min. The bromide derivative was then displaced using 15 eq of N-Boc-1,4-butanediamine in DMF for 1 h. Finally, Boc-Dmt-OH was coupled with the resulting secondary amine using 3 eq of amino acid and 3 eq of DIC/Oxyma pure in DMF assisted by micro-waves (75 C. for 30 min). Further standard SPPS coupling, cleavage and purification were performed as described for peptides KGFF01-KGFF07.
1.2 Peptide Characterization and Synthesis of the Arginine Mimetics and Ornithine Mimetics
1.2.1. Materials
[0266] Naltrexone hydrochloride, forskolin, 3-isobutyl-1-methylxanthine (IBMX), [D-Ala.sup.2,Me-Phe.sup.4,Gly-ol.sup.5]enkephalin (DAMGO), probenecid and Complete Freund's Adjuvant (CFA) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Glass bead were purchased from Sigma Aldrich Chemicals (St; Louis, Mo., USA). [D-Pen.sup.2, D-Pen.sup.5]enkephalin (DPDPE) and dynorphin were obtained from Abcam (Paris, France), nociception from Polypeptide (Strasbourg, France), morphine hydrochloride from Francopia (Paris, France) and the Fluo-4 acetoxymethyl ester from Molecular Probes (Invitrogen, Cergy Pontoise, France). Human RF-amide peptides were obtained from Genecust (Luxembourg; Kp-10, NPFF, QRFP26 or 26RFa, PrRP-20 and RFRP-3). [.sup.125I]-1-DMe-NPFF (2200 Ci/mmol) and [.sup.3H]-PrRP-20 (150 Ci/mmol) were obtained from Hartmann Analytic (Braunschweig, Germany). [.sup.35S]Guanosine 5-O-[g-thio] triphosphate ([.sup.35S]GTPgS; 1250 Ci/mmol), [.sup.3H]-diprenorphine (42.3 Ci/mmol), [.sup.3H]-nociceptine (114.7 Ci/mmol), [.sup.125I]-Kp-10 (2200 Ci/mmol) and [.sup.125I]-QRFP43 (2200 Ci/mmol) were purchased from Perkin Elmer Life and Analytical Sciences (Courtaboeuf, France) and Luciferin from Synchem UG & Co KG (Felsberg, Germany). All other chemicals were of analytical grade and obtained from standard commercial sources.
1.2.2. Synthesis and Compound Characterization, General
[0267] Thin-layer chromatography (TLC) was performed on glass plates precoated with silica gel 60F254 (Merck, Darmstadt, Germany) using the mentioned solvent systems. Mass Spectrometry (MS) was done on a Q-Tof spectrometer with electrospray ionisation (ESI). Data collection and spectrum analysis was done with Masslynx software. Analytical RP-HPLC was performed using system LC-A (including a Waters 717plus Autosampler, a Waters 1525 Binary HPLC Pump and a Waters 2487 Dual Absorbance Wavelength Detector (Milford, Mass.), with a Grace (Deerfield, Ill.) Vydac RP 018 column (25 cm4.6 mm5 m) using UV detection at 215 nm) or system LC-B (including a LC 1200 Agilent with a Zorbax Agilent 018-column (018, 50 mm2.1 mm; 1.8 m), using UV detection with DAD scan from 190 nm to 700 nm). For LC-A, the mobile phase is a mixture of water and acetonitrile and contains 0.1% TFA. The used gradient runs from 3 to 100% acetonitrile in 20 minutes at a flow rate of 1 mL/min. For LC-B, the mobile phase is a mixture of water and acetonitrile and contains 0.05% formic acid. The used gradient runs from 2 to 100% acetonitrile in 8 minutes at a flow rate of 0.5 mL/min. Preparative RP-HPLC purification was done on system PLC-A (including a Gilson (Middleton, Wis.) HPLC system with Gilson 322 pumps, controlled by the software package Unipoint, and a reversed phase 018 column (Discovery BIO SUPELCO Wide Pore 018 column, 25 cm2.21 cm, 5 mm) with a linear gradient of 1%/min increase of acetonitrile in water (both having 0.1% TFA)) or a system PLC_B (including a Prep Spot II from Armen, and a reversed phase 018 column (Waters XSelect CSH Prep C18 5 M 19150 mm) with a gradient of acetonitrile in water (having 0.1% TFA), running from 5% to 100% in 30 to 50 minutes). After purification, the purity of all compounds was evaluated as being more than 95% by analytical RP-HPLC. All fractions were lyophilized using a Flexy-Dry lyophilizer (FTS Systems, Warminster, Pa.) or a Lyovapor L-200 (Buchi). .sup.1H and .sup.13C NMR spectra were recorded at 500 and 125 MHz on a Bruker Avance II 500 or at 400 and 100.62 MHz on a Bruker Avance 400 (Bruker Corp, Billerica, Mass.). Tetramethylsilane (TMS) or residual solvent signals are used as internal standard. The solvent used is mentioned in all cases, and the abbreviations used are as follows: s (singlet), d (doublet), dd (double doublet), t (triplet), q (quadruplet) and m (multiplet).
1.2.3. Peptide characterization
H-Dmt-D-Arg-Aba-Gly-Arg-Phe-NH.sub.2 (KGFF01). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 15%). HPLC (LC-A): t.sub.R=9.9 min. TLC Rf 0.69 (EBAW). HRMS (ESP.sup.+) found m/z 884.4915 [M+H].sup.+, [C.sub.44H.sub.61N.sub.13O.sub.7+H.sup.+] required 884.4890.
H-Dmt-D-Arg-Aba-Gly-Arg-Phe-OH (KGFF02). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 20%). HPLC (LC-A): t.sub.R=10.4 min. TLC Rf 0.65 (EBAW). HRMS (ESP.sup.+) found m/z 885.4764 [M+H].sup.+, [C.sub.44H.sub.60N.sub.12O.sub.8+H.sup.+] required 885.4730.
H-Dmt-D-Arg-Aba-b-Ala-Arg-Phe-NH.sub.2 (KGFF03). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 18%). HPLC (LC-A): t.sub.R=10.0 min. TLC Rf 0.67 (EBAW). HRMS (ESP.sup.+) found m/z 898.5076 [M+H.sup.+], [C.sub.45H.sub.63N.sub.13O.sub.7+H.sup.+] required 898.5046.
H-Dmt-D-Arg-Aba-Gly-Orn-Phe-NH.sub.2 (KGFF04). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 17%). HPLC (LC-A): t.sub.R=9.9 min. TLC Rf 0.67 (EBAW). HRMS(ESP.sup.+) found m/z 842.4694 [M+H].sup.+, [C.sub.43H.sub.59N.sub.11O.sub.7+H.sup.+] required 842.4672.
H-Dmt-D-Arg-Phe-Orn-Phe-NH.sub.2 (KGFF05). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 27%). HPLC (LC-A): t.sub.R=10.1 min. TLC Rf 0.67 (EBAW). HRMS (ESP.sup.+) found m/z 773.4477 [M+H].sup.+, [C.sub.40H.sub.56N.sub.10O.sub.6+H.sup.+] required 773.4457.
H-Dmt-Arg-Phe-NH.sub.2 (KGFF06). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 44%). HPLC (LC-A): t.sub.R=10.1 min. TLC Rf 0.68 (EBAW). HRMS (ESP.sup.+) found m/z 512.2994 [M+H.sup.+], [C.sub.26H.sub.37N.sub.7O.sub.4+H.sup.+] required 512.2980.
H-Dmt-D-Arg-Phe-NH.sub.2 (KGFF07). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 63%). HPLC (LC-A): t.sub.R=8.9 min. TLC Rf 0.71 (EBAW). HRMS (ESP.sup.+) found m/z 512.2977 [M+H.sup.+], [C.sub.26H.sub.37N.sub.7O.sub.4+H.sup.+] required 512.2980.
H-Dmt-D-Arg-Aba-b-Ala-Apa-Phe-NH.sub.2 (KGFF08). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 20%). HPLC (LC-A): t.sub.R=10.2 min. TLC Rf 0.46 (EBAW). HRMS (ESP.sup.+) found m/z 924.5476 [M+H.sup.+], [C.sub.49H.sub.69N.sub.11O.sub.7+H.sup.+] required 924.5454.
H-Dmt-D-Arg-Aba-b-Ala-Bpa-Phe-NH.sub.2 (KGFF09). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 17%). HPLC (LC-A): t.sub.R=12.0 min. TLC Rf 0.67 (EBAW). HRMS (ESP.sup.+) found m/z 1014.5931 [M+H.sup.+], [C.sub.56H.sub.75N.sub.11O.sub.7+H.sup.+] required 1014.5923.
H-Dmt-Apa-Phe-NH.sub.2 (KGFF10). Preparative RP-HPLC (PLC-A) yielded the desired powder, (25%). HPLC (LC-A): t.sub.R=10.0 min. TLC Rf 0.43 (EBAW). HRMS (ESP.sup.+) found m/z [C.sub.30H.sub.43N.sub.5O.sub.4+H.sup.+] required 538.3388.
H-Dmt-Bpa-Phe-NH.sub.2 (KGFF11). Preparative RP-HPLC (PLC-A) yielded the desired powder, (9%). HPLC (LC-A): t.sub.R=12.0 min. TLC Rf 0.70 (EBAW). HRMS (ESP.sup.+) found m/z [C.sub.37H.sub.49N.sub.5O.sub.4+H.sup.+] required 628.3857.
H-Dmt-D-Arg-Aba-NH (KGFF12). Preparative RP-HPLC (PLC-A) yielded the desired powder, (15%). HPLC (LC-A): t.sub.R=9.0 min. TLC Rf 0.66 (EBAW). HRMS (ESP.sup.+) found m/z [C.sub.27H.sub.37N.sub.7O.sub.4+H.sup.+] required 524.2980.
H-Dmt-D-Arg-Phe-NHMe (KGFF13). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 8%). HPLC (LC-A): t.sub.R=9.0 min. TLC Rf 0.66 (EBAW). HRMS (ESP.sup.+) found m/z 526.3140 [M+H.sup.+], [C.sub.31H.sub.39F.sub.6N.sub.7O.sub.6+H.sup.+] required 526.3136.
H-Dmt-D-Arg-Aba-b-Ala-Lys(Bim)-Phe-NH.sub.2 (KGFF14). (PLC-A) Preparative RP-HPLC yielded the desired compound (white powder, 22%). HPLC (LC-A): t.sub.R=11.2 min. TLC Rf 0.69 (EBAW). HRMS (ESP.sup.+) found m/z 986.5317 [M+H.sup.+], [C.sub.52H.sub.67N.sub.13O.sub.7+H.sup.+] required 986.5359.
H-Dmt-D-Arg-Aba-b-Ala-Lys(Box)-Phe-NH.sub.2 (KGFF15). (PLC-A) Preparative RP-HPLC yielded the desired compound (white powder, 43%). HPLC (LC-A): t.sub.R=11.3 min. TLC Rf 0.74 (EBAW). HRMS (ESP.sup.+) found m/z 987.5257 [M+H.sup.+], [C.sub.52H.sub.66N.sub.12O.sub.8+H.sup.+] required 987.5200.
H-Dmt-D-Arg-Aba-b-Ala-Lys(Bth)-Phe-NH.sub.2 (KGFF16). (PLC-A) Preparative RP-HPLC yielded the desired compound (white powder, 39%). HPLC (LC-A): t.sub.R=11.4 min. TLC Rf 0.70 (EBAW). HRMS (ESP.sup.+) found m/z 1003.4985 [M+H.sup.+], [C.sub.52H.sub.66N.sub.12O.sub.7S+H.sup.+] required 1003.4970.
H-Dmt-D-Arg-Aba--Ala-Bpa-Val-NH.sub.2 (DP0001). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 8%). HPLC (LC-B): t.sub.R=4.16 min. HRMS (ESP.sup.+) found m/z 965.5859 [M+H.sup.+], [C.sub.52H.sub.75N.sub.11O.sub.7+H.sup.+] required 965.5851.
H-Dmt-D-Arg-Aba--Ala-Bpa-Ile-NH.sub.2 (DP0002). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 15%). HPLC (LC-B): t.sub.R=4.29 min. HRMS (ESP.sup.+) found m/z 979.6017 [M+H.sup.+], [C.sub.53H.sub.77N.sub.11O.sub.7+H.sup.+] required 979.6007.
H-Dmt-D-Arg-Aba--Ala-Bpa-Leu-NH.sub.2 (DP0003). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 24%). HPLC (LC-B): t.sub.R=4.32 min. HRMS (ESP.sup.+) found m/z 979.6019 [M+H.sup.+], [C.sub.53H.sub.77N.sub.11O.sub.7+H.sup.+] required 979.6007.
H-Dmt-D-Arg-Aba--Ala-Bpa-Tyr-NH.sub.2 (DP0004). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 28%). HPLC (LC-B): t.sub.R=4.18 min. HRMS (ESP.sup.+) found m/z 1029.5819 [M+H.sup.+], [C.sub.56H.sub.75N.sub.11O.sub.7+H.sup.+] required 1029.5800.
H-Dmt-D-Arg-Aba--Ala-Bpa-Trp-NH.sub.2 (DP0005). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 15%). HPLC (LC-B): t.sub.R=4.49 min. HRMS (ESP.sup.+) found m/z 1052.5938 [M+H.sup.+], [C.sub.58H.sub.76N.sub.12O.sub.7+H.sup.+] required 1052.5960.
H-Dmt-N(Me)-D-Ala-Aba--Ala-Bpa-Phe-NH.sub.2 (DP0007). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 2%). HPLC (LC-B): t.sub.R=6.12 min. HRMS (ESP.sup.+) found m/z 942.5409 [M+H.sup.+], [C.sub.54H.sub.70N.sub.8O.sub.7+H.sup.+] required 942.5367.
H-Dmt-D-Pro-Aba--Ala-Bpa-Phe-NH.sub.2 (DP0008). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 9%). HPLC (LC-B): t.sub.R=5.80 min. HRMS (ESP.sup.+) found m/z 954.5410 [M+H.sup.+], [C.sub.55H.sub.70N.sub.8O.sub.7+H.sup.+] required 954.5367.
H-Dmt-D-Bpa-Aba--Ala-Bpa-Phe-NH.sub.2 (DP0009). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 4%). HPLC (LC-B): t.sub.R=5.94 min. HRMS (ESP.sup.+) found m/z 1129.6759 [M+H.sup.+], [C.sub.67H.sub.87N.sub.9O.sub.7+H.sup.+] required 1129.6728.
H-Dmt-D-Arg-1AnaGly-Bpa-Phe-NH.sub.2 (DP0012). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 1%). HPLC (LC-B): t.sub.R=4.58 min. HRMS (ESP.sup.+) found m/z 1049.5877 [M+H.sup.+], [C.sub.59H.sub.75N.sub.11O.sub.7+H.sup.+] required 1049.5851.
H-Dmt-D-Arg-Phe-N(Me)--Ala-Bpa-Phe-NH.sub.2 (DP0013). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 5%). HPLC (LC-BA): t.sub.R=4.34 min. HRMS (ESP.sup.+) found m/z 1015.6013 [M+H.sup.+], [C.sub.56H.sub.77N.sub.11O.sub.7+H.sup.+] required 1015.6007.
H-Dmt-N(Me)-D-Ala-1AnaGly-Bpa-Phe-NH.sub.2 (DP0014). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 3%). HPLC (LC-B): t.sub.R=5.34 min. HRMS (ESP.sup.+) found m/z 978.5375 [M+H.sup.+], [C.sub.57H.sub.70N.sub.8O.sub.7+H.sup.+] required 978.5367.
H-Dmt-D-Arg-Aba--Ala-Bpa-D-Phe-NH.sub.2 (DP0015). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 1%). HPLC (LC-B): t.sub.R=4.33 min. HRMS (ESP.sup.+) found m/z 1013.5872 [M+H.sup.+], [C.sub.56H.sub.75N.sub.11O.sub.7+H.sup.+] required 1013.5851.
H-Dmt-D-Arg-Aba--Ala-Bpa-Phe--Ala-NH.sub.2 (DP0016). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 1%). HPLC (LC-B): t.sub.R=4.26 min. HRMS (ESP.sup.+) found m/z 1084.6235 [M+H.sup.+], [C.sub.59H.sub.80N.sub.12O.sub.7+H.sup.+] required 1084.6222.
H-Dmt-N(Me)-D-Ala-AbaGABA-Bpa-Phe-NH.sub.2 (DP0017). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 6%). HPLC (LC-B): t.sub.R=5.03 min. HRMS (ESP.sup.+) found m/z 956.5498 [M+H.sup.+], [C.sub.55H.sub.72N.sub.8O.sub.7+H.sup.+] required 956.5524.
H-Dmt-D-Arg-AbaGABA-Bpa-Phe-NH.sub.2 (DP0018). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 2%). HPLC (LC-B): t.sub.R=4.36 min. HRMS (ESP.sup.+) found m/z 1027.5985 [M+H.sup.+], [C.sub.55H.sub.77N.sub.11O.sub.7+H.sup.+] required 1027.6007.
H-Dmt-D-Arg-Phe--Ala-Bpa-Phe-NH.sub.2 (DP0019). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 2%). HPLC (LC-B): t.sub.R=4.30 min. HRMS (ESP.sup.+) found m/z 1001.5834 [M+H.sup.+], [C.sub.55H.sub.75N.sub.11O.sub.7+H.sup.+] required 1001.5851.
H-Dmt-D-Arg-AbaGABA-Bpa-Val-NH.sub.2 (DP0020). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 3%). HPLC (LC-B): t.sub.R=4.12 min. HRMS (ESP.sup.+) found m/z 979.5993 [M+H.sup.+], [C.sub.53H.sub.77N.sub.11O.sub.7+H.sup.+] required 979.6007.
H-Dmt-D-Arg-1AnaGly-Bpa-Val-NH.sub.2 (DP0021). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 4%). HPLC (LC-B): t.sub.R=4.41 min. HRMS (ESP.sup.+) found m/z 1001.5853 [M+H.sup.+], [C.sub.55H.sub.75N.sub.11O.sub.7+H.sup.+] required 1001.5851.
H-Dmt-D-Arg-AbaGABA-Bpa-Trp-NH.sub.2 (DP0022). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 1%). HPLC (LC-B): t.sub.R=4.42 min. HRMS (ESP.sup.+) found m/z 1066.6101 [M+H.sup.+], [C.sub.59H.sub.78N.sub.12O.sub.7+H.sup.+] required 1066.6116.
H-Dmt-D-Arg-1AnaGly-Bpa-Trp-NH.sub.2 (DP0023). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 1%). HPLC (LC-B): t.sub.R=4.65 min. HRMS (ESP.sup.+) found m/z 1088.5926 [M+H.sup.+], [C.sub.61H.sub.76N.sub.11O.sub.7+H.sup.+] required 1088.5960.
H-Dmt-D-Arg-Phe-N(Me)Gly-Bpa-Phe-NH.sub.2 (DP0024). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 27%). HPLC (LC-B): t.sub.R=4.37 min. HRMS (ESP.sup.+) found m/z 1001.5822 [M+H.sup.+], [C.sub.55H.sub.75N.sub.11O.sub.7+H.sup.+] required 1001.5851.
H-Dmt-D-Arg-Phe-N(Me)Gly-Bpa-Val-NH.sub.2 (DP0025). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 22%). HPLC (LC-B): t.sub.R=4.09 min. HRMS (ESP.sup.+) found m/z 953.5872 [M+H.sup.+], [C.sub.51H.sub.75N.sub.11O.sub.7+H.sup.+] required 953.5851.
H-Dmt-D-Arg-Aba--Ala-THIQ-Phe-NH.sub.2 (DP0026). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 4%). HPLC (LC-B): t.sub.R=3.97 min. HRMS (ESP.sup.+) found m/z 971.5374 [M+H.sup.+], [C.sub.53H.sub.69N.sub.11O.sub.7+H.sup.+] required 971.5381.
H-Dmt-D-Arg-Aba--Ala-D-Bpa-Phe-NH.sub.2 (DP0027). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 2%). HPLC (LC-B): t.sub.R=4.37 min. HRMS (ESP.sup.+) found m/z 1013.5862 [M+H.sup.+], [C.sub.56H.sub.75N.sub.11O.sub.7+H.sup.+] required 1013.5851.
H-Dmt-D-Orn-Aba--Ala-Bpa-Phe-NH.sub.2 (DP0028). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 11%). HPLC (LC-B): t.sub.R=4.27 min. HRMS (ESP.sup.+) found m/z 971.5647 [M+H.sup.+], [C.sub.55H.sub.73N.sub.9O.sub.7+H.sup.+] required 971.5633.
H-Dmt-D-Lys-Aba--Ala-Bpa-Phe-NH.sub.2 (DP0029). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 9%). HPLC (LC-B): t.sub.R=4.30 min. HRMS (ESP.sup.+) found m/z 985.5805 [M+H.sup.+], [C.sub.56H.sub.75N.sub.9O.sub.7+H.sup.+] required 985.5789.
H-Dmt-D-Arg-Phe-N(Me)-D-Ala-Bpa-Phe-NH.sub.2 (DP0030). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 11%). HPLC (LC-B): t.sub.R=4.51 min. HRMS (ESP.sup.+) found m/z 1015.6033 [M+H.sup.+], [C.sub.56H.sub.77N.sub.11O.sub.7+H.sup.+] required 1015.6007.
7-OH-Tic-D-Arg-Aba--Ala-Bpa-Phe-NH.sub.2 (DP0031). Preparative RP-HPLC (PLC-B) yielded the desired compound (white powder, 1%). HPLC (LC-B): t.sub.R=4.35 min. HRMS (ESP.sup.+) found m/z 997.5539 [M+H.sup.+], [C.sub.55H.sub.71N.sub.11O.sub.7+H.sup.+] required 997.5538.
Guanidyl-Dmt-D-Arg-Aba--Ala-Bpa-Phe-NH.sub.2 (DP0032). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 10%). HPLC (LC-B): t.sub.R=4.51 min. HRMS (ESP.sup.+) found m/z 1055.6045 [M+H.sup.+], [C.sub.57H.sub.77N.sub.13O.sub.7+H.sup.+] required 1055.6069.
H-Dmt-D-hArg-Aba--Ala-Bpa-Phe-NH.sub.2 (DP0033). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 14%). HPLC (LC-B): t.sub.R=4.48 min. HRMS (ESP.sup.+) found m/z 1027.6010 [M+H.sup.+], [C.sub.57H.sub.77N.sub.11O.sub.7+H.sup.+] required 1027.6007.
H-Dmt-D-Lys(Nic)-Aba--Ala-Bpa-Phe-NH.sub.2(DP0034). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 21%). HPLC (LC-B): t.sub.R=5.05 min. HRMS (ESP.sup.+) found m/z 1090.6024 [M+H.sup.+], [C.sub.62H.sub.78N.sub.10O.sub.8+H.sup.+] required 1090.6004.
H-Dmt-Nlys-Aba--Ala-Bpa-Phe-NH.sub.2 (DP0035). Preparative RP-HPLC (PLC-A) yielded the desired compound (white powder, 19%). HPLC (LC-B): t.sub.R=4.46 min. HRMS (ESP.sup.+) found m/z 985.5801 [M+H.sup.+], [C.sub.56H.sub.75N.sub.9O.sub.7+H.sup.+] required 985.5789.
1.2.4. Synthesis of the Arginine Mimetics
[0268] ##STR00051##
1.2.5. Synthesis of Boc-Lys(NC)-OMe
[0269] Boc-Lys-OMe hydrochloride (3.00 g, 10.1 mmol, 1.0 equiv.) was dissolved in ethylformate (28.8 mL, 35.5 mmol, 35 equiv.). To this solution, triethylamine (1.4 mL, 10.1 mmol, 1.0 equiv.) was added and stirred for 4 h at 80 C. After cooling down to room temperature, the mixture was evaporated in vacuo. The crude formamide was obtained as a white solid and used without further purification. The formamide was dissolved in dry CH2Cl2 (20 mL) and triethylamine (7.0 mL, 50.5 mmol, 5.0 equiv.) was added to this solution. The solution was flushed with argon and cooled to 0 C. Subsequently, phosphoryl chloride (1.4 mL, 15.2 mmol, 1.5 equiv.) was added dropwise while stirring. After the addition, the ice bath was removed and the mixture was stirred for an additional 2.5 h at room temperature. The mixture was poured in cold water (30 mL) and extracted with CH2Cl2 (330 mL). The combined organic layers were washed with water and brine (230 mL) and dried over MgSO4. After concentration, the product was purified by flash chromatography using heptane/EtOAc (1/1) as eluent. (S)-methyl-2-((tert-butoxycarbonyl)amino)-6-isocyanohexanoate was obtained in 80% yield. Yield: 80% (yellow oil, 3.64 g); Formula: C.sub.13H.sub.22N.sub.2O.sub.4; MW=270.32 g/mol; TLC Rf: =0.59 (EtOAc/heptane 1/1); 1H-NMR (CDCl3, 400 MHz) in ppm d: 1.44 (s, 9H), 1.48-1.55 (m, 2H), 1.63-1.72 (m, 3H), 1.82-1.89 (m, 1H), 3.39 (t, J=6.0 Hz, 2H), 3.75 (s, 3H), 4.31 (br s, 1H), 5.04 (br s, 1H); 13C-NMR (CDCl3, 100 MHz) in ppm d: 22.1, 28.3, 28.5, 32.0, 41.3 (t, J=6.4 Hz), 52.4, 53.0, 80.1, 155.4, 156.4, 172.9.
1.2.6. Synthesis of Boc-Lys(Bim-PMB)-OMe
[0270] A 25 mL Kjeldahl flask was flame dried under vacuum and refilled with argon. Subsequently, the vial was charged with Pd(OAc).sub.2 (11 mg, 0.05 mmol, 0.05 equiv.), N-(p-methoxybenzyl)-o-phenylenediamine (228 mg, 1.00 mmol, 1.0 equiv.) and 4 molecular sieves (300 mg). The flask was equipped with a reflux condenser, evacuated and back filled with 02 (three times). (S)-methyl-2-((tert-butoxycarbonyl)amino)-6-isocyanohexanoate (324 mg, 1.20 mmol, 1.2 equiv.) was dissolved in 2-MeTHF (1 mL) in a separate vial under argon. This mixture was added to the Kjeldahl flask followed by 2-MeTHF (1.5 mL). The reaction mixture was stirred at 75 C. under 02 atmosphere for 21 h. After cooling down to room temperature, the solution was filtered through celite using EtOAc (50 mL) and concentrated in vacuo. The product was purified by automated flash chromatography applying a heptane/EtOAc gradient (from 100% heptane to 10% EtOAc, 25 mL/min). Methyl-2-((tert-butoxycarbonyl)amino)-6-((1-(4-methoxybenzyl)-1H-benzoimidazol-2-yl)amino)hexanoate was obtained in 86% (425 mg) yield. Yield: 86% (brown oil, 425 mg); Formula: C.sub.27H.sub.36N.sub.4O.sub.5; MW=496.60 g/mol; TLC Rf=0.61 (acetone/heptane 1/4); HPLC: t.sub.R=14.8 min; HRMS (ES.sup.+): found 497.2744, calculated 497.2758 [M+H].sup.+; 1H-NMR (CDCl.sub.3, 400 MHz) in ppm 1.23-1.40 (m, 4H), 1.43 (s, 9H), 1.57-1.67 (m, 4H), 1.75-1.81 (m, 2H), 3.47 (q, J=6.5 Hz, 2H), 3.70 (s, 3H), 3.78 (s, 3H), 4.01 (br s, 1H), 4.25 (br s, 1H), 5.02 (br s, 3H), 6.85-6.88 (m, 2H), 7.01-7.14 (m, 5H), 7.51 (d, J=7.8 Hz, 1H); 13C-NMR (CDCl.sub.3, 100 MHz) in ppm : 22.5, 28.2, 29.1, 32.2, 42.9, 45.0, 52.0, 53.2, 55.1, 79.7, 107.1, 114.4, 116.2, 119.5, 121.1, 127.3, 127.7, 134.6, 142.1, 154.2, 155.3, 159.3, 173.1.
1.2.7. Synthesis of Boc-Lys(Box)-OMe
[0271] A 25 mL Kjeldahl flask was flame dried under vacuum and refilled with argon. Subsequently, the vial was charged with Pd(OAc).sub.2 (11 mg, 0.05 mmol, 0.05 equiv.), 2-aminophenol (109 mg, 1.0 mmol, 1.0 equiv.) and 4 molecular sieves (300 mg). The flask was equipped with a reflux condenser, evacuated and back filled with 02 (three times). (S)-methyl-2-((tert-butoxycarbonyl)amino)-6-isocyanohexanoate (324 mg, 1.20 mmol, 1.2 equiv.) was dissolved in 2-MeTHF (1 mL) in a separate vial under argon. This mixture was added to the Kjeldahl flask followed by 2-MeTHF (1.5 mL). The reaction mixture was stirred at 75 C. under 02 atmosphere for 21 h. After cooling down to room temperature, the solution was filtered through celite using EtOAc (50 mL) and concentrated in vacuo. The product was purified by an automated flash chromatography applying a heptane/EtOAc gradient (from 100% heptane to 10% EtOAc, 25 mL/min). Tert-butyl 6-(benzoxazol-2-ylamino)-2-((methoxycarbonyl)amino)hexanoate was obtained in 72% (270 mg) yield. Yield: 72% (brown solid, 270 mg); Formula: C.sub.19H.sub.27N.sub.3O.sub.5; MW=377.43 g/mol; TLC Rf=0.32 (EtOAc/heptane 1/1); HPLC t.sub.R=13.0 min; HRMS (ES.sup.+): found 378.2000, calculated 378.2023 [M+H].sup.+; 1H-NMR (CDCl3,400 MHz) in ppm =1.44-1.51 (s, 11H), 1.64-1.90 (m, 4H), 3.48 (m, 2H), 4.73 (s, 3H), 4.32 (br s, 1H), 5.08 (s, 2H), 7.02 (td, J=7.8, 1.0 Hz, 1H), 7.15 (td, J=7.7, 0.8 Hz, 1H), 7.22 (d, J=7.9 Hz, 1H), 7.36 (d, J=7.7 Hz, 1H); 13C-NMR (CDCl3, 100 MHz) in ppm 22.6, 28.5, 29.2, 32.8, 43.0, 52.5, 53.2, 80.2, 108.8, 116.5, 121.0, 124.0, 143.2, 148.7, 155.7, 162.2, 173.3.
1.2.7. Synthesis of Boc-Lys(Bth)-OMe
[0272] A 25 mL Kjeldahl flask was flame dried under vacuum and refilled with argon. Subsequently, the vial was charged with Pd(OAc).sub.2 (11 mg, 0.05 mmol, 0.05 equiv.), 2-aminothiophenol (1070, 1.00 mmol, 1.0 equiv.) and 4 molecular sieves (300 mg). The flask was equipped with a reflux condenser, evacuated and back filled with 02 (three times). (S)-methyl-2-((tert-butoxycarbonyl)amino)-6-isocyanohexanoate (324 mg, 1.20 mmol, 1.2 equiv.) was dissolved in 2-MeTHF (1 mL) in a separate vial under argon. This mixture was added to the Kjeldahl flask followed by 2-MeTHF (1.5 mL). The reaction mixture was stirred at 75 C. under 02 atmosphere for 21 h. After cooling down to room temperature, the solution was filtered through celite using EtOAc (50 mL) and concentrated in vacuo. The product was purified by an automated flash chromatography applying a heptane/EtOAc gradient (from 100% heptane to 10% EtOAc, 25 mL/min). Tert-butyl 6-(benzothiazol-2-ylamino)-2-((methoxycarbonyl)amino)hexanoate was obtained in 80% yield. Yield: 80% (white solid, 316 mg); Formula: C.sub.19H.sub.27N.sub.3O.sub.4S; MW: 393.50 g/mol; TLC Rf: =0.61 (EtOAc/heptane 1/2); HPLC: t.sub.R=13.1 min; HRMS (ES.sup.+): found 394.1779, calculated 394.1795 [M+H].sup.+; 1H-NMR (CDCl.sub.3, 400 MHz) in ppm 1.44-1.48 (m, 10H), 1.50-1.67 (m, 3H), 1.84-1.89 (m, 1H), 3.44 (t, J=6.8 Hz, 2H), 3.73 (s, 3H), 4.32 (br s, 1H), 5.07 (br s, 1H), 5.41 (br s, 1H), 7.07 (td, J=7.6, 0.9 Hz, 1H), 7.26-7.30 (m, 1H), 7.53 (d, J=7.8 Hz, 1H), 7.57 (d, J=7.8 Hz, 1H); 13C-NMR (CDCl.sub.3, 100 MHz) in ppm : 22.8, 28.5, 29.1, 31.0, 32.8, 45.3, 52.5, 53.2, 80.2, 119.1, 120.9, 121.7, 126.1, 130.6, 152.7, 167.4, 173.3.
1.2.8. Boc-Lys(Bim-PMB)-OH
[0273] Boc-Lys(Bim,PMB)-OMe (250 mg, 0.50 mmol) was dissolved in a mixture of THF/H.sub.2O (7:1, total 3.2 mL). Lithium hydroxide monohydrate (148 mg, 3.52 mmol, 7 equiv.) was added and continuously stirred for 16 h at room temperature. The reaction mixture was concentrated by evaporation and resuspended in H.sub.2O (10 mL). The aqueous phase was washed with CH2Cl2 (25 mL) and carefully acidified to pH=3, as indicated by pH paper, with 1N HCl. The aqueous layer was extracted with CH2Cl2 (410 mL). The combined organic layers were collected, washed with brine (120 mL) and dried over MgSO4, filtered and concentrated to obtain the corresponding carboxylic acid as a pink solid in 87% yield. The building block was used in Boc-based SPPS without further purification. Yield: 87% (211 mg); HPLC: t.sub.R=14.0 min; HRMS: (ES.sup.+): found 483.2580, calculated 493.2602 [M+H].sup.+.
1.2.9. Boc-Lys(Box)-OH
[0274] Boc-Lys(Box)-OMe (190 mg, 0.50 mmol) was dissolved in a mixture of THF/H.sub.2O (7:1, total 3.2 mL). Lithium hydroxide monohydrate (148 mg, 3.52 mmol, 7 equiv.) was added and continuously stirred for 16 h at room temperature. The reaction mixture was concentrated by evaporation and resuspended in H.sub.2O (10 mL). The aqueous phase was washed with CH2Cl2 (25 mL) and carefully acidified to pH=3, as indicated by pH paper, with 1N HCl. The aqueous layer was extracted with CH2Cl2 (410 mL). The combined organic layers were collected and washed with brine (120 mL) and dried over MgSO4, filtered and concentrated to obtain the corresponding carboxylic acid as a pink solid in 74% yield (135 mg). The building block was used in Boc-based SPPS without further purification. Yield: 74% (135 mg); HPLC: t.sub.R=11.8 min; HRMS (ES.sup.+): found 364.1867, calculated 364.1867 [M+H].sup.+.
1.2.10. Boc-Lys(Bth)-OH
[0275] Boc-Lys(Bth)-OMe (198 mg, 0.50 mmol) was dissolved in a mixture of THF/H.sub.2O (7:1, total 3.2 mL). Lithium hydroxide monohydrate (148 mg, 3.52 mmol, 7 equiv.) was added and continuously stirred for 16 h at room temperature. The reaction mixture was concentrated by evaporation and resuspended in H.sub.2O (10 mL). The aqueous phase was washed with CH2Cl2 (25 mL) and carefully acidified to pH=3, as indicated by pH paper, with 1N HCl. The aqueous layer was extracted with CH2Cl2 (410 mL). The combined organic layers were collected and washed with brine (120 mL) and dried over MgSO4, filtered and concentrated to obtain the corresponding carboxylic acid as a pink solid in 72% yield. The building block was used in Boc-based SPPS without further purification. Yield: 72% (13 8 mg); HPLC: t.sub.R=12.1 min; HRMS (ES.sup.+): found 380.1617, calculated 380.1638 [M+H].sup.+.
1.2.11. Chiral Derivatization of Boc-Lys(Bim,PMB)-OH, Boc-Lys(Box)-OH and Boc-Lys(Bth)-OH with Marfey's Reagent (FDAA)
The chiral derivatization was performed starting from the N-Boc-deprotected substrates. Compounds (10 mg) are treated with a cocktail of TFA/CH.sub.2Cl.sub.2 (1:1) for 1 h and concentrated. The crude was redissolved in H.sub.2O with a minimal amount of AcN and lyophilized to obtain the deprotected mimetics as white to off-white solids in quantitative yields. The samples were analyzed via LC/MS and used without further purification.
Subsequently, the enantiomeric purity was checked via chiral derivatization with Marfey's reagent (FDAA). For this, a stock solution of 20.0 mM FDAA in acetone was prepared (5.44 mg FDAA/1 mL acetone). The analyte (1 mg) was dissolved in 1 mL 1 M NaHCO.sub.3. Two equiv. of the stock solution were added to 100 L of the analyte solution and the mixture was incubated at 40 C. overnight. After quenching with 100 L of a 1 M HCl solution, the sample was diluted to 1 mL with water and analyzed by LC-MS. Integration of the peak area (340 nm) gave an estimate of the enantiomeric excess. The derivatized product was obtained as a single peak, indicating that no epimerization took place during the synthesis.
Further analysis of the enantiomeric excess was performed by coupling of Boc-Lys(Bim-PMB)-OH to HCl.H-Ala-OMe by use of EDC/HOAt (1.6 equiv.) to yield the dipeptide. Analysis via HPLC showed only one single peak and NMR did not show any trace of the other diastereomer.
1.2.12. Synthesis of 4-amino-tetrahydro-aminobenzazepinon (Aba)-NH
N-Boc-ortho-aminomethyl-L-Phe (507 mg, 1.72 mmol, 1 equiv.), was submitted to an intramolecular cyclization [1]. The product was dissolved in 172 mL of CH2Cl2 (10 mM) and EDC.HCl (495 mg, 2.58 mmol, 1.5 equiv.), Et.sub.3N (601 L, 4.31 mmol, 2.5 equiv.) and HOBt.H.sub.2O (396 mg, 2.58 mmol, 1.5 equiv.) were added. The reaction was stirred for 16 h and then the organic phase was washed with 20% citric acid and sat. NaHCO.sub.3-solution. The solvent was dried with MgSO4 and evaporated. The product was purified by flash chromatography using ethyl acetate/petroleum ether (4/6) to give a white solid in 19% yield. Yield: 19% (92.4 mg); Formula: C.sub.15H.sub.20N.sub.2O.sub.3; MW=276.15 g/mol; TLC Rf=0.34 (EtOAc/petroleum ether4/6); HPLC t.sub.R=13.9 min; MS (ES.sup.+): 277 [M+H].sup.+, 299 [M+Na].sup.+, 177 [M-Boc].sup.+; Melting interval 149.0-150.5 C.; 1H-NMR (CDCl.sub.3, 500 MHz) in ppm : 1.46 (9H, s, Boc), 2.97 (1H, dd, .sup.2J=16.7 Hz, .sup.3J=13.0 Hz, H.sub.), 3.41 (1H, dd, .sup.2J=16.9 Hz, .sup.3J=3.2 Hz, H.sub.) 3.96 (1H, dd, .sup.2J=16.7 Hz, .sup.3J=7.1 Hz, H.sub.), 4.85 (1H, dd, .sup.2J=16.5 Hz, .sup.3J=3.8 Hz, H.sub.), 5.86 (1H, d, .sup.2J=5.8 Hz, NH), 7.01 (1H, d, .sup.3J=7.5 Hz, CH arom.), 7.10 (2H, m, CH arom.), 7.19 (1H, m, CH arom.); 13C-NMR (CDCl3, 125 MHz) in ppm : 28.6 (Boc), 37.2 (-CH2), 46.0 (-CH2), 49.4 (-CH), 79.9 (Cq Boc), 126.5 (CH arom.), 128.0 (CH arom.), 128.4 (CH arom.), 131.3 (CH arom.), 134.2 (Cq arom.), 135.7 (Cq arom.), 155.4 (CO Boc), 174.4 (CO Aba).
The product could be deprotected with 50% TFA in CH2Cl2 for 2 h and directly coupled to the dipeptide.
1.2.13. Synthesis of Fmoc-1-AnaGly-OH
[0276] Synthesis of Fmoc-1-AnaGly-OH was performed using the procedure described in the literature (Van der Poorten et al. ACS Med. Chem. Lett. (2017) 8, 11, 1177-82)
1.2.14. Synthesis of AbaGABA
[0277] Synthesis of Phth-Phe-OH
[0278] To a 250 mL round bottom flask, L-phenylalanine (7.72 g, 46.7 mmol) and phthalic anhydride (6.92 g, 46.7 mmol) were added. The solids were then carefully heated up to 145 C. (not above to avoid racemization) and mechanically stirred. Upon heating, the solids started to melt and a brown solid was formed after 1 h indicating completion. The residue was dissolved in hot MeOH (70 mL) stirred and filtered. Cold water (50 mL) was then slowly added to allow crystallization. Pure Phth-Phe-OH was obtained as white crystals after filtration and drying (12.54 g, 91%).
[0279] Formula: C17H13NO4; MW: 295.29 g/mol; TLC: Rf=0.43 (CH2Cl2/MeOH 95:5+1% AcOH); HPLC: t.sub.R=2.5 min; LC-MS (ES+): [MCOOH].sup.+=249.91 Da, [M+H].sup.+=295.90 Da; 1H NMR: (500 MHz, 298 K, CDCl3): (ppm) 7.75 (m, 2H, arom. H Phth), 7.70 (m, 2H, arom. H Phth), 7.13 (m, 5H, arom. H Phe), 5.22 (t, 1H, H.sub., J=8.11), 3.61 (dd, 2H, CH.sub., J=8.60 Hz, J=1.21 Hz).
[0280] Synthesis of Phth-Phe-GABA-OEt
[0281] Phth-Phe-OH (3.02 g, 10.2 mmol), ethyl 4-aminobutanoate (1.88 g, 11.2 mmol), TBTU (9.75 g, 30.6 mmol) and DCM (50 mL) were added to a 100 mL round bottom flask. TEA (4.25 mL, 30.6 mmol) was then added giving a yellow solution. The pH was monitored and adjusted to 9 until reaction completion by means of TEA addition. After 3 h, the reaction mixture was concentrated and the residue dissolved in ethyl acetate, washed with 1M HCl (3 times), saturated NaHCO.sub.3 solution (3 times) and brine (2 times). The resulting organic phase was dried with MgSO.sub.4, filtered and concentrated under reduced pressure. The crude compound was finally purified by column chromatography using ethyl acetate/petroleum ether as eluent yielding the desired dipeptide (3.02 g, 73%).
[0282] Formula: C23H24N2O5; MW: 408.45 g/mol; HPLC: t.sub.R=2.7 min; LC-MS (ES+): [M+H]+=408.90, [M+Na].sup.+=430.87 Da; HRMS: Calculated [M+H]+=409.1763, mass found [M+H]+=409.1755; TLC: Rf=0.39 (PE/EtOAc 1:1); 1H NMR (500 MHz, 298 K, CDCl3): (ppm) 7.79 (m, 2H, arom. H), 7.70 (m, 2H, arom. H), 7.13 (m, 5H, arom. H Phe), 5.10 (dd, 1H, H.sub. Phe, J=11.54 Hz, J=5.40 Hz), 3.98 (m, 2H, OCH.sub.2CH.sub.3, J=7.16 Hz, J=0.96 Hz), 3.60 (dd, 1H, H.sub. Phe, J=14.2 Hz, J=11.2 Hz)), 3.52 (dd, 1H, H.sub. Phe, J=14.2 Hz, J=5.6 Hz), 3.35 (m, 2H, 2H.sub. GABA), 2.37 (t, 2H, 2H.sub. GABA, J=6.80 Hz), 1.81 (q, 2H, 2H.sub. GABA, J=6.57 Hz), 1.18 (t, 3H, OCH.sub.2CH.sub.3, J=7.16 Hz); 13C NMR (126 MHz, 295 K, CDCl3): (ppm) 174.04 (CO ester), 168.67 (CO amide bond), 168.15 (CO amide Phth), 137.13 (CH arom. Phe), 134.32 (CH arom. phth), 131.74 (Cq arom. Phth), 129.02 (CH arom. Phe), 128.77 (CH arom. Phe), 127.04 (CH arom. Phe), 123.59 (CH arom. Phth), 60.74 (OCH.sub.2CH.sub.3), 55.96 (CH, Phe), 39.84 (CH.sub.2 GABA), 34.82 (CH.sub.2 Phe), 32.11 (CH.sub.2 GABA), 24.71 (CH.sub.2 GABA) and 14.33 (OCH.sub.2CH.sub.3).
[0283] Synthesis of Phth-Aba-GABA-OEt
[0284] To a 250 mL two-necked round bottom flask equipped with a Dean-Stark apparatus, phosphorus pentoxide (12 g, 42.4 mmol) and 85% phosphoric acid (2.73 mL, 40.5 mmol) in acetic acid (60 mL) and benzene (90 mL) were added to give a yellow suspension. The mixture was refluxed for 1 h at 115 C. under stirring and Ar atmosphere. Then Phth-Phe-GABA-OEt (1.5 g, 3.67 mmol) and 1,3,5-trioxane (2.26 g, 25.1 mmol) were added. The mixture was refluxed at 115 C. for 3 h while 1,3,5-trioxane (2.26 g, 25.1 mmol) was added every 30 min. The reaction mixture was cooled to rt and concentrated under reduced pressure giving an orange oil. The residue was dissolved in ethyl acetate and washed with 1M HCl (3 times), saturated NaHCO.sub.3 solution (six times), and once with brine. The resulting organic phase was dried with MgSO4, filtered and concentrated under reduced pressure giving an orange oil. The crude was finally purified by flash chromatography using a Interchim 80 g silica column and petroleum ether/ethyl acetate as eluent (gradient from 20 to 60% ethyl acetate in 25 min) yielding the desired product (0.722 g, 47%).
[0285] Formula: C24H24N2O5; MW: 420.47 g/mol; HPLC: t.sub.R=2.9 min; LC-MS (ES+): [M+Na]+=442.94 Da, [M+H]+=420.27 Da; HRMS: Calculated [M+H]+=421.1758 Da, mass found [M+H]+=421.1732 Da; TLC: Rf=0.26, EtOAc/PE (1:1); 1H NMR (500 MHz, 298 K, CDCl3): (ppm) 7.88 (m, 2H, arom. Phth), 7.75 (m, 2H, arom. Phth), 7.29 (m, 4H, arom. Aba), 5.40 (dd, 1H, H.sub. Aba, J=13.04 Hz, J=4.70 Hz), 4.73 (d, 1H, H.sub. Aba, J=15.60 Hz), 4.60 (d, 1H, H.sub. Aba, J=15.81 Hz, 65.21 Hz), 4.12 (q, 2H, OCH.sub.2CH.sub.3, J=7.05 Hz), 3.58 (m, 2H, 2H.sub. GABA), 3.15 (dd, 2H, 2H.sub. Aba, J=15.71 Hz, J=4.59 Hz), 2.28 (t, 2H, 2H.sub. GABA, J=7.07 Hz), 1.91 (m, 2H, 2H.sub. GABA), 1.24 (t, 3H, OCH.sub.2CH.sub.3, J=7.05); 13C NMR (126 MHz, 298 K, CDCl3): (ppm) 173.34 (CO ester), 168.54 (CO amide bond Aba), 168.22 (CO Phth), 136.16 (Cq arom. Aba), 135.68 (Cq arom. Aba), 134.33 (CH arom. Phth), 132.27 (Cq arom. Phth), 130.30 (CH arom. Aba), 129.30 (CH arom. Aba), 128.78 (CH arom. Aba), 128.44 (CH arom. Aba), 126.97 (CH arom. Aba), 123.70 (CH arom. Phth), 60.60 (OCH.sub.2CH.sub.3), 51.48 (CH.sub. Aba), 51.37 (CH.sub.2 Aba), 49.02 (CH.sub.2 GABA), 34.30 (CH.sub.2 Aba), 31.42 (CH.sub.2 GABA), 23.26 (CH.sub.2 GABA), 14.41 (OCH.sub.2CH.sub.3).
[0286] Synthesis of Phth-Aba-GA BA-OH
[0287] To a 100 mL round bottom flask, Phth-Aba-GABA-OEt (0.722 g, 1.72 mmol), 1M HCl (10 mL) and acetone (10 mL) were added. The reaction mixture was heated at reflux (90 C.) for 16 h under Ar atmosphere. The reaction mixture was concentrated under reduced pressure yielding the desired product as a yellow solid (0.692 g, quantitative) which was used without further purification.
[0288] Formula: C22H20N2O5; MW: 392.14 g/mol; HPLC: t.sub.R=2.4 min; LC-MS (ES+): [M+H]+=392.80 Da; HRMS: Calculated [M+Na]+=415.1264 Da, mass found [M+Na]+=415.1274 Da; 1H NMR (500 MHz, 298 K, CDCl3): (ppm) 7.93 (m, 2H, arom. Phth), 7.89 (m, 2H, arom. Phth), 7.36 (m, 1H, arom. Aba), 7.28 (m, 3H, arom. Aba), 5.28 (dd, 1H, H.sub. Aba, J=12.38, Hz, J=5.27 Hz), 4.84 (d, 1H, H.sub. Aba, J=15.90 Hz), 4.52 (d, 1H, H.sub. Aba, J=15.81 Hz), 3.94 (dd, 1H, H.sub. GABA, J=15.92 Hz, J=12.50 Hz), 3.50 (m, 1H, H.sub. GABA), 3.28 (dd, 2H, 2H.sub. Aba, J=16.00 Hz, J=5.25 Hz), 2.10 (t, 2H, 2H.sub. GABA, J=7.70 Hz), 1.69 (m, 2H, 2H.sub. GABA); 13C NMR (126 MHz, 298 K, CDCl3): (ppm) 207.24 (COCOOH), 174.62 (CO amide bond), 168.30 (CO Phth), 136.87 (Cq arom. Aba), 135.44 (CH arom. Phth), 132.04 (Cq arom. Phth), 130.43 (CH arom. Aba), 128.95 (CH arom. Aba), 127.36 (CH arom. Aba), 123.95 (CH arom. Phth), 52.35 (CH.sub. Aba), 51.03 (CH.sub.2 Aba), 48.37 (CH.sub.2 GABA), 33.57 (CH.sub.2 Aba), 31.41 (CH.sub.2 GABA), 23.61 (CH.sub.2 GABA).
[0289] Synthesis of Fmoc-Aba-GABA-OH
[0290] To a 100 mL round bottom flask, Phth-Aba-GABA-OH (0.674 g, 1.72 mmol), hydrazine monohydrate (543 L, 11.2 mmol) and EtOH (20 mL) are added. The reaction mixture was refluxed at 90 C. for 2 h then evaporated and dried under vacuum for 2 h. The residue was dissolved in 10 mL water then pH was adjusted from 8 to 4 by acetic acid addition. The resulting suspension was stirred at rt for 30 min then filtered and concentrated to give the Phth-deprotected intermediate used without further purification.
[0291] The residue was dissolved in acetone (10 mL) and water (10 mL). Then a solution of Fmoc-OSu (0.608 g, 1.80 mmol) and Na.sub.2CO.sub.3 (0.210 g, 1.98 mmol) in water (10 mL) and acetone (10 mL) was added. The reaction mixture was stirred at rt overnight under Ar atmosphere. Acetone was then evaporated and the resulting aqueous solution was acidified to pH 2 using a 6M HCl solution. The suspension was extracted with ethyl acetate (4 times) and the resulting organic phases were combined, washed once with brine, dried with MgSO.sub.4, filtered and concentrated. The crude was finally purified by column chromatography using DCM/MeOH 98/2 with 1% acetic acid as eluent yielding the desired compound (0.564 g, 68%).
[0292] Formula: C.sub.29H.sub.28N.sub.2O.sub.5; MW: 484.20 g/mol; HPLC: t.sub.R=2.82 min; LC-MS (ES+): [M+H]+=485.19 Da; .sup.1H NMR (251 MHz, CDCl.sub.3) (ppm) 7.68 (m, 4H, H arom.), 7.36 (m, 4H, H arom.), 7.24-7.01 (m, 4H, H arom.), 6.36 (d, J=6.6 Hz, 1H), 5.41-5.02 (m, 2H, H.sub. and H.sub. Aba), 4.39 (d, J=7.2 Hz, 2H, CH2 Fmoc), 4.24 (t, J=7.2 Hz, 1H, CH Fmoc), 3.89 (d, J=16.7 Hz, 1H, H.sub.), 3.65-3.37 (m, 3H, H.sub. Aba and 2H.sub. GABA), 2.98 (dd, J=16.9, 13.1 Hz, 1H, H.sub.), 2.41-2.11 (m, 2H, 2H.sub. GABA), 1.84 (m, 2H, 2H.sub. GABA), 13C NMR (63 MHz, CDCl3) (ppm) 177.2 (COCOOH), 171.8 (CO amide), 155.9 (CO Fmoc) 144.1 (Cq arom. Fmoc), 141.4 (Cq arom. Fmoc), 135.6 (Cq arom. Aba), 133.2 (Cq arom. Aba), 131.1 (CH arom. Fmoc) 128.7 (CH arom. Aba), 128.3 (CH arom. Fmoc), 127.8 (CH arom. Aba), 127.2 (CH arom. Aba), 126.5 (CH arom. Aba), 125.4 (CH arom. Fmoc), 120.2 (CH arom. Fmoc), 67.3 (CH.sub.2 Fmoc), 52.4 (CH.sub.2 Aba), 50.0 (CH.sub. Aba), 47.3 (CH Fmoc and CH.sub.2 GABA), 37.0 (CH.sub.2 Aba), 30.9 (CH.sub.2 GABA), 23.2 (CH.sub.2 GABA).
1.2.13. Synthesis of Fmoc-Apa-OH, Fmoc-Bpa-OH, Fmoc-D-Bpa-OH, Fmoc-THIQ-OH
[0293] N-protected amino acids N-Fmoc-Apa-OH, N-Fmoc-Bpa-OH, N-Fmoc-D-Bpa-OH and N-Fmoc-THIQ-OH were prepared as described by Schneider and al. [46]
1.3. Receptor cDNA Constructs, Cell Expression and Membrane Preparations
Human embrionic kidney 293 (HEK293) cells expressing Glo-sensor 20F were a gift from M. Hanson (GIGA, Lige, Belgium). Human MOPr, DOPr, KOPr, NOPr, NPFF1R, NPFF2R and GPR54 cDNAs were subcloned into the pCDNA3.1 expression vector (Invitrogen, Cergy Pontoise, France) and transfected into Chinese Hamster ovary (CHO) cells or HEK293-Glo-20F cells before selection for stable expression, as reported [13]. CHO cells expressing human GPR10 and GPR103 were a gift from M. Parmentier (IRIBHM, Brussels, Belgium). All cell membranes were prepared as described [13] and stored at 80 C. as aliquots (1 mg prot/mL) until use.
1.4. Radioligand binding assays
Binding assay conditions were essentially performed as described [13]. Briefly, membranes from CHO cells stably expressing human GPR10, GPR54, GPR103, NPFF1R or NPFF2R were incubated with 0.6 nM [.sup.3H]-PrRP-20, 0.05 nM [.sup.125I]-Kp-10, 0.03 nM [.sup.125I]-43RFa, or 0.015 nM [.sup.125I]-1-DMe-NPFF (for NPFF1R and NPFF2R), respectively. Membranes from HEK293 cells stably expressing human MOP, DOP and KOP receptors were incubated with 1 nM [.sup.3H]-diprenorphine. Membranes from HEK293 cells transiently expressing human NOP receptors were incubated with 0.2 nM [.sup.3H]-nociceptin. Competition binding experiments were performed at 25 C., under equilibrium conditions (60 min, 0.25 mL final volume), in the presence of increasing concentrations of unlabeled peptides or test compounds. Membrane-bound radioactivity was separated from free radioligand by rapid filtration through a 96-well GF/B unifilter apparatus (Perkin Elmer Life and Analytical Sciences, Courtaboeuf, France) and quantified using a TopCount scintillation counter (Perkin Elmer).
1.5. [.SUP.35.S]-GTPS Binding Assay
[0294] Stimulation by endogenous RF-amide peptides or test compounds for [.sup.35S]-GTPS binding to membranes from CHO cells expressing human NPFF1R or NPFF2R, were examined as reported [48].
1.6. Glo-Sensor cAMP Assay
cAMP accumulation assay was essentially done as described [45] with the following modifications: HEK293 cells stably expressing cAMP Glosensor-20F were used, with or without additional stable expression of each individual opioid receptor or NPFF1R. The cAMP responses were measured in presence of D-luciferin (1 mM). All peptides and test compounds were incubated in the presence of IBMX for 15 min before inducing cAMP production by forskolin. IBMX and forskolin concentrations were optimized for each receptor cell line: NPFF1R responses were recorded in presence of 0.1 mM IBMX and 0.4 M forskolin, NPFF2R responses with 0.5 mM IBMX and 0.3 M forskolin, MOPr responses with 0.5 mM IBMX and 0.125 M forskolin, DOPr responses with 0.1 mM IBMX and 0.125 M forskolin; KOPr and NOPr with 0.5 mM IBMX and 1.5 M forskolin. The antagonist activity of the derivatives on NPFF1R and NPFF2R was evaluated at three different concentrations (0.5, 5 and 50 M) in the presence of 50 nM RFRP3 and 200 nM NPFF, respectively.
1.7. Calcium Mobilization Assay
[0295] CHO cells expressing human NPFF2R were loaded with 2.5 M Fluo-4 AM in the presence of 2.5 mM probenecid, as described previously [13]. Agonist-evoked increases in intracellular calcium were recorded over time (5 sec intervals over 220 sec) at 37 C. through fluorescence emission at 520 nm (excitation at 485 nm). Peak response amplitudes were normalized to basal and maximal (cells permeabilized with 20 M digitonin) fluorescence levels.
1.8. -Arrestin-2 recruitment assay
The -arrestin-2 recruitment assay was performed as described [34; 45] with minor modifications. Briefly, two days prior the experiment, HEK293 cells stably expressing eYFP-tagged -arrestin-2 were transfected with the plasmid encoding Rluc8-MOP receptor. -Arrestin-2 recruitment was measured at 37 C. in presence of 5 M Coelenterazine H, 5 min after agonist addition. A BRET ratio corresponding to the signal in the acceptor channel (band-pass filter 510-560 nm) divided by the signal in the donor channel (band-pass filter 435-485 nm) was calculated. Drug-induced BRET was determined (BRET1 ratio of drug-activated cells minus BRET1 ratio of buffer-treated cells) and normalized to the maximum of DAMGO-induced BRET, defined as 100%.
1.9. In Vivo Experiments
[0296] All experiments were carried out in accordance with the European guidelines for the care of laboratory animals (European Communities Council Directive 2010/63/EU) and were approved by the local ethical committee and authorized by the French Ministry for Research and the Committee of Animal Care of the Austrian Federal Ministry of Science and Research. All efforts were made to minimize animal discomfort and to reduce the number of animals used.
1.10. Animals
[0297] Animal experiments were performed on adult male C57BL/6N male mice (25-30 g weight; Janvier labs, France). Animals were housed in groups of three to five per cage and kept under a 12 h/12 h light/dark cycle at 211 C. with ad libitum access to food and water. Experiments were performed during the light-on phase of the cycle. Mice were habituated to the testing room and equipment before starting behavioral experiments. Control and treated group assignment as well as pain response measurements were performed in a blinded manner. Every animal was used only once.
1.11. Drug administration
All drugs were dissolved in physiological saline (0.9%) and administered subcutaneously (sc.) at 10 mL/kg (volume/body weight).
1.12. Assessment of thermal nociception
The nociceptive sensitivity to thermal stimulation was determined in mice using the warm-water tail immersion tests as previously described [12; 49]. In the tail immersion test, C57BN/6N mice were restrained in a grid pocket and their tail was immersed in a thermostated water bath. The latency (in sec) for tail withdrawal from hot water (47.50.5 C.) was taken as a measure of the nociceptive response. In the absence of any nociceptive reaction, a cut-off value of 25 sec was set to avoid tissue damage.
Experiments for chronic drug effects were designed according to a protocol enabling the evaluation of time-course of opioid-induced hyperalgesia and development of analgesic tolerance using the tail immersion test. C57BL/6N mice were sc. treated daily with 1.8 mol/kg/d KGOP01, 1.2 mol/kg/d KGFF03, 7.4 mol/kg/d KGFF09 or saline (controls) for 8 days. For analgesic tolerance evaluation, nociceptive latencies were measured on day 1 and 8 according to the acute effect protocol. For the assessment of hyperalgesia, basal nociceptive latencies were measured every day, 30 min before drug or saline injection. Responses are expressed as latency times (in sec) of tail withdrawal from the hot water.
1.13. CFA-Induced Inflammatory Pain Model
[0298] Tail inflammation in C57BL/6N mice was induced by injecting subcutaneously 20 l of a Complete Freund's Adjuvant (CFA) solution or saline (control mice) 3 cm from the tip of the tail [41]. Twenty-four hours after CFA injection (day 1), inflammation was confirmed by measuring thermal and mechanical hyperalgesia. Mice were then treated sc. daily for 7 days (from day 1 to day 7) with the test compounds or saline (control). Nociceptive threshold to heat stimulation was measured by tail immersion test (47.50.5 C.) and tail pressure test [12] each hour for 5 h after the first drug administration in order to determine the peak of the anti-hyperalgesic response. During the following days, basal nociceptive thresholds were evaluated before injection and 2 h after drug injection. Antinociceptive response was calculated as percent of Maximum Possible Effect (% MPE) according to the formula=[(test latencyCFA/saline mice latency)/(cut-off timeCFA/saline mice latency)]100. In order to limit behavioral sensitization, thermal nociception was evaluated on days 1-2-4-6 and mechanical nociception on days 1-3-5-7.
1.14. Naltrexone-Precipitated Withdrawal Syndrome
[0299] Opioid physical dependence was induced in C57BL/6N mice by sc. administration of test compounds twice daily, 1.8 mol/kg KGOP01, 1.2 mol/kg KGFF03, 7.4 mol/kg KGFF09 or saline (control) over a 7-days period. On day 7, two hours after the last drug injection, the withdrawal syndrome was precipitated by administration of naltrexone (5 mg/kg, sc.) and evaluated over 30 min. Jumping, paw tremors and wet dog shakes were recorded as number of events occurring during the total test time. Diarrhea was checked for 30 min with one point given for any signs of it during each 5 min period (maximum score: 6). Body weight was measured immediately before and after each 30 min test session, and percentage of body weight lost during the test was calculated. For each mouse, a global opiate withdrawal score was also calculated by summing the values obtained for each sign. For this purpose one point was assigned to every 3 jumps and 5 paw tremors, respectively, whereas all other signs were given the absolute values recorded during the test [39].
1.15. Respiratory Depression Measurement Using Whole Body Plethysmography
[0300] Ventilatory parameters were recorded in conscious C57BL/6N mice by whole body barometric plethysmography (Emka Technologies, Paris, France). Mice were acclimatized with the plethysmograph chamber for 30 min until a stable baseline was obtained. Then, the animal was gently removed from the chamber for sc. injection of the tested drug at TO and replaced in the chamber for the remaining measurements. Respiratory frequency (f) was recorded for 100 min and used as the index of respiratory depression [31].
1.16. Statistics
[0301] For in vitro binding and functional experiments, two to four independent experiments were performed in duplicates and data were analyzed using Prism (GraphPad Software, San Diego, Calif., USA). In vivo data are expressed as mean valuesSEM for 6 to 12 mice per group. Antinociception was quantified as the area under the curve (AUC) calculated by the trapezoidal method [8]. Data were analyzed using one-way or two-way analysis of variance (ANOVA). Post-hoc analyses were performed with Bonferroni tests. The level of significance was set at p<0.05. All statistical analyses were carried out using the StatView or GraphPad Prism softwares.
[0302] 2. Results
2.1. Design Strategy and Biological Screening of MOPr/NPFFR Peptidomimetic Ligands
[0303] In this study, the inventors designed a series of bifunctional ligands that were based on a combination of the recently described MOPr peptidomimetic KGOP01 [20], found to display potent analgesic activity following systemic administration, and standard but also more evolved NPFF pharmacophores (
TABLE-US-00003 TABLE 1 compound Sequence position X1 X2 X3 X4 X5 X6-T KGFF01 H-Dmt D-Arg Aba Gly Arg Phe-NH.sub.2 KGFF02 H-Dmt D-Arg Aba Gly Arg Phe-OH KGFF03 H-Dmt D-Arg Aba b-Ala Arg Phe-NH.sub.2 KGFF04 H-Dmt D-Arg Aba Gly Orn Phe-NH.sub.2 KGFF05 H-Dmt D-Arg Phe Orn Phe-NH.sub.2 KGFF06 H-Dmt L-Arg Phe-NH.sub.2 KGFF07 H-Dmt D-Arg Phe-NH.sub.2 KGFF08 H-Dmt D-Arg Aba b-Ala Apa Phe-NH.sub.2 KGFF09 H-Dmt D-Arg Aba b-Ala Bpa Phe-NH.sub.2 KGFF10 H-Dmt Apa Phe-NH.sub.2 KGFF11 H-Dmt Bpa Phe-NH.sub.2 KGFF12 H-Dmt D-Arg Aba-NH KGFF13 H-Dmt D-Arg Phe-NHCH.sub.3 KGFF14 H-Dmt D-Arg Aba b-Ala Lys(Bim) Phe-NH.sub.2 KGFF15 H-Dmt D-Arg Aba b-Ala Lys(Box) Phe-NH.sub.2 KGFF16 H-Dmt D-Arg Aba b-Ala Lys(Bth) Phe-NH.sub.2 KGOP-01 H-Dmt D-Arg Aba b-Ala-NH.sub.2
As a standard NPFF pharmacophore, the C-terminal RF-NH2 dipeptide segment of NPFF served as a minimal recognition motif (structure 2,
Further truncation of KGFF01 gave way to tripeptides KGFF06 and KGFF07 with complete overlap of the opioid and NPFF pharmacophores, as the 2,6-dimethyl tyrosine (Dmt) side chain could efficiently serve as the apolar group. In this case, the opioid segment is cut down to three amino acids instead of four, with well-conserved MOPr binding affinity for KGFF07. In addition to the detrimental consequence on MOPr affinity, the stereochemistry of Arg (L-Arg for KGFF06 and D-Arg for KGFF07) seems to drive a selectivity switch between NPFF1R and NPFF2R (
Following initial in vitro biological evaluation of the described hybrid peptidomimetics, the two peptide analogues, KGFF03 and KGFF07, with the most promising binding data on MOPr and NPFFR were used to design a second set of bifunctional peptides (Table 1). Within this second set, the Arg residues were replaced by both reported Orn-derivatives (
It has been recently shown that the guanidine moiety of Arg can be advantageously replaced by tertiary amines in the sequence RF-NH2, leading to new peptidomimetic ligands of NPFFRs [5]. Based on the promising biological data of KGFF01 and KGFF03, and in light of the previous work, the Arg5 residue was replaced by piperidine- and benzylpiperidine-bearing residues 3 and 4, respectively (
The Table 2b summarizes 1050 values of DP compounds for NPFF1R and NPFF2R. These compounds displayed nanomolar affinity for NPFF1/2R,
TABLE-US-00004 TABLE 2a Binding affinity constant (K.sub.i) values of KGFF compounds for human MOPr, NPFF1R and NPFF2R. Ki SEM (nM) compound MOPr NPFF1R NPFF2R DAMGO 14.6 3.8 nd nd RFRP3 nd 0.053 0.003 nd NPFF nd nd 0.28 0.16 KGOP01 0.12 0.02 4,600 1,300 4,500 1,800 KGFF01 0.59 0.21 57 10 1.2 0.5 KGFF02 0.59 0.15 2,540 460.sup. 251 25 KGFF03 0.24 0.03 2.7 0.3 0.077 0.01 KGFF04 0.67 0.29 780 90 45 8 KGFF05 3.2 1.1 3,100 1,100 360 50 KGFF06 46 22 147 20 15.9 1.8 KGFF07 0.29 0.13 11.8 3.6 84 26 KGFF08 0.94 0.19 136 26 3.9 0.1 KGFF09 2.43 0.18 83 21 3.2 0.7 KGFF10 68 48 >10,000 2,100 600.sup. KGFF11 >10,000 2,720 760.sup. 406 95 KGFF12 0.17 0.05 2,490 1,170 375 108 KGFF13 0.11 0.05 147 16 206 47 KGFF14 1.6 0.3 4.54 0.01 4.15 1.35 KGFF15 1.8 0.9 88 8 9.6 0.6 KGFF16 2.6 1.3 80 13 10.4 2.3 Data are mean SEM of at least 2 independent experiments performed in duplicate. K.sub.i values were determined from competition binding curves using [.sup.3H]-diprenorphine for MOPr, and [.sup.125I]-1-DMe-NPFF for NPFF1R and NPFF2R.
TABLE-US-00005 TABLE 2b IC.sub.50 values of DP compounds on NPFF1R and NPFF2R. IC50 (nM) compound NPFF1R NPFF2R DP0001 500 <50 DP0002 50-500 <50 DP0003 50-500 <50 DP0004 50-500 <50 DP0005 50 <50 DP0007 nd nd DP0008 nd nd DP0009 nd nd DP0012 <40 <40 DP0013 <40 40 DP0014 <40 40 DP0015 <40 <40 DP0016 nd nd DP0017 nd nd DP0018 40-400 <40 DP0019 40-400 40-400 DP0020 40-400 40-400 DP0021 40-400 <40 DP0022 40 <40 DP0023 nd nd DP0024 nd nd DP0025 nd nd DP0026 nd nd DP0027 nd nd DP0028 nd nd DP0029 nd nd DP0030 nd nd DP0031 nd nd DP0032 nd nd DP0033 nd nd DP0034 nd nd DP0035 nd nd IC50 values were estimated from a single competition binding experiment performed in duplicates in the presence of [.sup.125I]-1-DMe-NPFF for NPFF1R and NPFF2R and three concentrations of each compounds: 0.05, 0.5 and 5 M for DP0001 to DP0005 and 0.04, 0.4 and 4 M for DP0012 to DP0015 and DP0018 to DP0022. nd: not determined
2.2. Identification of an MOPr/NPFFR Agonist (KGFF03) and a Mixed MOPr Agonist/NPFFR Antagonist (KGFF09)
[0304] The capacity of the selected compounds to inhibit forskolin-induced cAMP production from MOPr overexpressing HEK cells was first evaluated. All ligands displayed full agonist activity at the MOPr (EC.sub.50 values ranging from 1.5 to 18.2 nM, as compared to DAMGO, EC.sub.50=80 nM,
Overall, these data allowed us to identify at least one molecule, KGFF03, that displays potent MOPr and NPFFR agonist activities, and at least a second ligand, KGFF09, that shows mixed MOPr agonist and NPFF1/2R antagonist activities. These two ligands underwent further in vitro and in vivo characterization, and their profiles were compared to the parent opioid ligand KGOP01.
Table 3b summarize agonist and antagonist activity constant values of DP compounds on MOP (agonist) and NPFFR1/2 (Agonist and antagonist). These compounds display mixed MOPr agonist and NPFF1/2R antagonist (and potentially partial agonist for DP0001, 0002 and 0003) activities.
TABLE-US-00006 TABLE 3a Agonist activity constant (EC.sub.50 and E.sub.max) values of KGFF compounds for human MOPr, NPFF1R and NPFF2R. MOPr MOPr NPFF1R NPFF2R (cAMP) (-arrestin-2) (GTPS) (GTPyS) EC.sub.50 E.sub.max EC.sub.50 E.sub.max EC.sub.50 E.sub.max EC.sub.50 E.sub.max compound (nM) (%) (nM) (%) (nM) (%) (nM) (%) DAMGO 80.4 23.2 100 4 240 65 100 2 nd nd nd RFRP3 nd 10.1 2.8 100 10 nd nd NPFF nd nd nd 18.2 6.4 100 4 KGOP01 0.204 0.05 89 2 1.6 0.1 103 9 >10,000 nd >10,000 nd KGFF01 2.4 1.0 101 7 155 25 44 8 128 23 95 6 KGFF02 1.5 0.7 89 2 nd nd nd nd KGFF03 12.0 2 92 1 33.9 8.8 41 11 84.8 22.2 88 7 11 2 111 17 KGFF04 9.5 1.5 70 4 >10,000 25 4 963 168 67 3 KGFF05 59.3 24.5 94 2 nd nd nd nd KGFF06 2,300 300.sup. nd nd nd nd nd KGFF07 1.7 0.4 102 2 87 7 84 8 1,930 66.sup. 87 2 KGFF08 9.9 2.0 102 3 >10,000 23 1 574 129 97 2 KGFF09 18.2 6.1 85 2 56.4 26.5 42 2 176 66 10 2 157 49 38 14 KGFF10 598 35 91 10 nd nd nd nd KGFF11 >10,000 nd nd nd nd nd KGFF12 2.7 1.5 103 4 >10,000 13 3 >10,000 32 17 KGFF13 1.0 0.4 97 2 >10,000 39 2 >10,000 68 8 KGFF14 5.9 2.5 99 4 76 26 74 1 89 7 73 18 KGFF15 4.1 1.7 92 13 1,020 610 51 5 930 210 105 15 KGFF16 6.2 2.0 93 6 273 115 51 7 526 12 88 12 Efficacy (E.sub.max) is expressed as the percentage relative to the reference compound (DAMGO, RFRP3 and NPFF for MOPr, NPFF1R and NPFF2R, respectively). Values are mean SEM of at least 2 independent experiments performed in duplicate. nd, not determined.
TABLE-US-00007 TABLE 3B Agonist activity (EC50 and Emax) of DP compounds for human MOPr and agonist and/or antagonist activity (IC50) for NPFF1R and NPFF2R. NPFF1R NPFF2R MOPr MOPr (cAMP) (cAMP) (cAMP) (-arrestin-2) Antagonist Antagonist EC.sub.50 E.sub.max EC.sub.50 E.sub.max Agonist mode Agonist mode compound (nM) (%) (nM) (%) mode IC50 (M) mode IC50 (M) DAMGO 80.4 23.2 100 4 240 65 100 2 nd nd nd nd KGFF09 18.2 6.1 85 2 56.4 26.5 42 2 5 5 DP0001 16.9 8.3 124 101 52 5 Partial 50 DP0002 8.9 2.6 110 45 49 5 Partial 50 DP0003 20.1 10 70 37 38 5 Partial 50 DP0004 4.2 0.7 88 4 126 64 <5 5 DP0005 7.9 0.5 81 0.6 98 42 <5 5 DP0007 40 20 64 6 214 12 <5 5 DP0008 10.6 1.3 53 0.7 <5 5 DP0009 800 35 nd nd 5 5 DP0012 15.1 0.4 90 2 650 22 >5 <5 DP0013 0.53 0.004 115 4 20.5 101 >5 5 DP0014 5.3 76 >5 5 DP0015 5.9 0.3 109 0.4 80 59 5 >5 DP0016 19 95 399 31 nd >5 nd >5 DP0017 14.1 70 nd >5 nd 5 DP0018 5.5 108 95.8 31 5 >5 DP0019 4 0.4 101 1 162 53 >5 >5 DP0020 6.3 101 149 53 >5 >5 DP0021 2.8 0.4 102 3 109 57 22 5 <5 DP0022 5 90 617 205 30 0.7 5 5 DP0023 9.1 61 5 <5 DP0024 9.9 97 50 95 nd >5 nd 5 DP0025 7.1 110 50 114 nd nd 50 DP0026 2.4 98 50 50 nd >5 nd 50 DP0027 13.6 95 180 68 nd >5 nd 5 DP0028 40.4 75 nd >5 nd 5 DP0029 25.7 72 nd >5 nd >50 DP0030 27.7 111 150 85 nd >5 nd >50 DP0031 3600 46 >500 40 nd 50 nd 5 DP0032 12.6 63 nd <5 nd <5 DP0033 24.9 41 nd 5 nd <5 DP0034 3.9 92 5-50 18 nd >5 nd <5 DP0035 48.4 15 nd nd nd nd For MOR: Efficacy (E.sub.max) is expressed as the percentage relative to the reference compound DAMGO. Agonist activity at NPFF1R and NPFF2R of each compound was evaluated et 2 concentrations 0.5 and 5 M. Antagonist activity at NPFF1R and NPFF2R of each compound was evaluated in the presence of 50 nM RFRP3 and 200 nM NPFF (respectively) and three concentrations (0.5, 5 and 50 M) of test compound. nd: not determined.
2.3. KGFF03 and KGFF09 are G Protein-Biased MOPr Agonists
[0305] While opioid-induced analgesia is attributed to MOPr signaling through the G protein G.sub.i, -arrestin-2 recruitment upon MOPr activation is suggested to be responsible for many acute side effects including respiratory depression and constipation [10; 31; 43]. To examine MOPr biased agonism of KGOP01, KGFF03 and KGFF09 towards activation of G protein-over -arrestin-2-mediated signaling, their functional activity, i.e. potency and efficacy, was compared across two cell-based assays that measure G protein coupling (the cAMP accumulation assay) and -arrestin-2 translocation (the BRET1 -arrestin-2 recruitment assay) at the human MOPr (
2.4. Selectivity of KGFF03 and KGFF09 at Opioid and RF-Amide Receptors
[0306] The binding affinity and functional activity of KGFF03, KGFF09 and KGOP01 at other opioid receptor types were further investigated (Table 4, which summarize binding affinity constant (K) values and agonist activity constant (EC.sub.50 and E.sub.max) values of KGFF compounds for human DOPr, KOPr, and NOPr and
[0307] KGOP01, KGFF03 and KGFF09 on opioid receptors). KGFF03 showed good affinity at the DOPr, and lower affinity for KOPr and NOPr. It displayed potent agonist activity at DOPr (EC.sub.50=0.34 nM) and lower agonist activity at KOPr (EC.sub.50=95 nM), as well as very weak antagonist activity at NOPr (pA.sub.2=5.30.34). KGFF09 showed lower affinity at the DOPr and relatively higher affinity for the KOPr, in comparison with KGOP01 and KGFF03. Its affinity for the NOPr was similar to KGFF03. Alike KGOP01 and KGFF03, KGFF09 displayed potent agonist activity at DOPr (EC.sub.50=0.78 nM). Moreover, KGFF09 displayed potent antagonist activity at the KOPr (pA.sub.2=8.160.13), but low antagonist activity at the NOPr (pA.sub.2=5.940.12).
Because NPFFRs belong to the family of RF-amide receptors, which include GPR10, GPR54 and GPR103 [13], the selectivity of the compounds for these receptors was also evaluated. KGOP01, KGFF03 or KGFF09 displayed no or low affinity for GPR10, GPR54 and GPR103 (Table 5, which summarize affinity constant (K) values of KGFF compounds for GPR10, GPR54 and GPR103).
TABLE-US-00008 TABLE 4 Binding affinity constant (K.sub.i) values and activity agonist constant (EC.sub.50 and E.sub.max) values of KGFF compounds for human DOPr, KOPr and NOPr. DOPr (cAMP) KOPr (cAMP) NOPr (cAMP) K.sub.i EC.sub.50 E.sub.max K.sub.i EC.sub.50 E.sub.max K.sub.i EC.sub.50 E.sub.max compound (nM) (nM) (%) (nM) (nM) (%) (nM) (nM) (%) Naloxone 15.9 2.3 8.4 4.3 DPDPE 57.1 18.8 0.8 0.3 100 11 nd nd nd nd nd nd Dynorphin A nd nd nd nd 1.0 0.4 100 14 nd nd nd Nociceptin nd nd nd nd nd nd 0.0013 0.0002 0.568 0.004 100 3 KGOP01 5.1 0.5 0.16 0.01 91 2 37.3 9.6 >10,000 18 1 >10,000 nd nd KGFF03 9.1 0.7 0.34 0.12 89 6 108 28 95 3 34 11 289 3 >10,000 15 2 KGFF09 186 77 0.78 0.01 90 3 3.2 0.8 >10,000 15 1 209 2 >10,000 7 3 K.sub.i values were determined from competition binding curves using [.sup.3H]-diprenorphine for DOP and KOP receptor and [.sup.3H]-nociceptin for NOP receptor. Efficacy (E.sub.max) is expressed as the percentage relative to the referent compound (DPDPE, dynorphin A and nociceptin respectively for DOP, KOP and NOP receptors). Data are mean SEM of at least two independent experiments performed in duplicate. nd, not determined.
TABLE-US-00009 TABLE 5 Binding affinity constant (K.sub.i) values of KGFF compounds for GPR10, GPR54 and GPR103. K.sub.i SEM (nM) compound GPR10 GPR54 GPR103 PrRP20 2.1 0.4 nd nd Kp10 nd .sup.0.062 0.009 nd 26RFa nd nd .sup.2.04 0.58 KGOP01 >10,000 >10,000 >10,000 KGFF03 >10,000 4,370 960 >10,000 KGFF09 >10,000 2,000 300 1,200 340 Data are mean SEM of at least two independent experiments performed in duplicate. K.sub.i values were determined from competition binding curves using [.sup.3H]-PrRP-20, [.sup.125I]-Kp-10 and [.sup.125I]-43RFa for GPR10, GPR54 and GPR103, respectively. nd, not determined.
2.5. Acute Subcutaneous Administration of KGFF03 and KGFF09 Produces Dose-Dependent, Long-Lasting Antinociception in Mice
[0308] The acute antinociceptive activity of the new MOPr/NPFFR hybrid structures, KGFF03 and KGFF09 was then evaluated, in two mouse models of thermal acute nociception after sc. administration and compared them to the parent opioid, KGOP01. All three peptides produced time- and dose-dependent increase in tail withdrawal latencies in the tail immersion test (
2.6. Chronic Subcutaneous Administration of KGFF09 does not Induce Hyperalgesia Nor Analgesic Tolerance in Nave Mice
To evaluate if the blockade of the NPFF system prevents the development of hyperalgesia and analgesic tolerance, mice were chronically administered with equianalgesic doses of either KGOP01, KGFF03 or KGFF09, as shown in the time course of analgesia on day 1 of the chronic administration scheme (
2.7. KGFF09 Displays Reduced Withdrawal Syndrome and Respiratory Depression
[0309] The development of naloxone-precipitated withdrawal syndrome after chronic sc. administration to mice of KGOP01, KGFF03 and KGFF09 was further studied. Mice were treated twice a day over a 7 days period with the same doses used in previous experiments. Administration of naltrexone (1 mg/kg, sc.), 2 h after the last injection of KGOP01, induced high scores on several somatic and vegetative signs in the drug-dependent mice, as compared to the control saline-treated animals (
Respiratory depression and constipation, two of the main side effects that occur upon acute opiate administration were next studied. As shown by the measurement of respiratory frequency (
2.8. KGFF09 Chronic Administration Efficiently Reverses CFA-Induced Hyperalgesia
[0310] Additionally, the analgesic profile of the compounds was characterized in a mouse model of persistent inflammatory pain induced by sc. injection of CFA in the mouse tail on day 1. Animals were then daily sc. administered with equianalgesic doses of KGOP01 (1.8 mol/kg/d), KGFF03 (1.2 mol/kg/d) or KGFF09 (7.4 mol/kg/d) from day 2 to day 8, and their antinociceptive activity upon thermal or mechanical nociceptive stimulation was measured on days 2, 3, 5 and 7 for the thermal stimulus, and on days 2, 4, 6, 8 for the mechanical stimulus. Measurements were performed 2 h after each daily drug injection, when analgesia was maximal (
[0311] 3. Discussion
The driving force over the years in the opioid field has been the search for an alternative to morphine that would produce effective analgesia and would be free of undesirable side effects. New chemical approaches including the design of G protein-biased MOPr agonists and/or multifunctional ligands with mixed opioid and non-opioid activities for creating analgesics with fewer adverse effects are sought, and such drugs with improved benefit/risk profile are likely to have a significant impact [21; 24; 30].
The successful design and a thorough in vitro and in vivo characterization of G protein-biased MOPr agonists, for example KGFF03 and KGFF09, possessing additional agonist and antagonist activities at NPFF1/2Rs, respectively, were reported according to the present invention. In vivo, they display potent antinociception with reduced respiratory depression after acute systemic (sc.) administration. The major finding of this study is that following chronic administration, KGFF09 but not KGFF03 produces effective antinociception with limited OIH and analgesic tolerance, as well as a reduced withdrawal syndrome, thus demonstrating the benefits of NPFF system blockade towards MOPr agonists to limit the development of tolerance and dependence, the two major adverse effects associated with chronic administration of classical opiates.
Multitarget pharmacology, or polypharmacology, is defined as the specific binding of a compound to two or more molecular targets and relies on the observation that some biological networks are resilient to single-point perturbations, with redundant functions or compensatory mechanisms leading to the attenuation of the repeated perturbation (i.e. stimulation of the MOPr; [22; 53]. Here, the strategy aimed at developing a dual acting drug combining the analgesic efficacy of opioid agonists, while blocking the NPFF system. The latter system has previously been shown to be critically involved in neuroadaptive responses of the organism to repeated exposure to opiates, resulting in OIH and analgesic tolerance [14; 48]. To determine whether the NPFFR antagonist activity was responsible for the improved profile of KGFF09, this MOPr-NPFFR hybrid peptidomimetic was compared with its parent opioid agonist KGOP01, devoid of an NPFF pharmacophore (
The beneficial effect of NPFFRs inhibition on the development of analgesic tolerance and long-lasting inflammatory hyperalgesia were further demonstrated in a model of inflammatory pain, suggesting that the endogenous NPFF system could be activated upon administration of inflammatory agents, such as CFA. This result is in agreement with a recent report showing an upregulation of NPFF and NPFF2R mRNA in the spinal cord of mice treated with CFA or carrageenan [28]. Overall, the current data therefore indicate that activation of the NPFF system might represent a common feature in the development of hyperalgesia, whether induced by inflammation or repeated opioid stimulation.
Detailed in vitro characterization of the newly designed MOP-NPFF hybrids revealed that the addition of either -Arg-Phe-NH2 (KGFF03) or -Bpa-Phe-NH2 (KGFF09) to the C-terminus of the parent opioid structure KGOP01 not only conferred the expected NPFF1/2R affinity to the designed compounds, but also shows an advantageous switch of MOPr activity towards the G protein signaling over -arrestin-2 recruitment. Studies on -arrestin-2 knockout mice (-arrestin-2 KO) report less opioid-associated adverse effects, such as respiratory depression, constipation, analgesic tolerance and physical dependence, as well as higher opioid antinociceptive effects [7]. Although KGFF03 and KGFF09 are not completely devoid of the -arrestin-2 recruitment activity, their bias appeared sufficient for alleviating respiratory depression, compared to the unbiased MOPr agonist KGOP01. This result is in agreement with previous observations made with other G protein-biased MOPr agonists [10; 31]. The lower efficacy of KGFF09 to promote MOP-induced -arrestin-2 recruitment over G protein activation could also be responsible for less analgesic tolerance and physical dependence. However, reports on the development of analgesic tolerance induced by opioids in -arrestin-2 KO mice or following chronic treatments with G protein-biased MOPr agonists are conflicting [1; 6; 25]. Concerning physical dependence, the severity of antagonist-precipitated withdrawal response in -arrestin-2 KO mice was reduced only when animals were chronically treated with low doses of morphine [43] and the biased MOPr agonist TRV130 was reported to induce similar withdrawal symptoms than morphine [50]. In this study, it was shown that KGFF03, having a biased activity on the MOPr, produces similar analgesic tolerance and physical dependence to the unbiased KGOP01 parent opioid agonist, suggesting that -arrestin-2 bias is not critical for the development of these side effects. Similarly to other biased MOPr agonists [10; 31], KGFF09 also binds to DOPr and KOPr, with DOPr agonist and KOPr antagonist activities. DOP agonists have been described to play no or limited analgesic activity in naive animals, but display potent anti-hyperalgesic activity in neuropathic and inflammatory pain models in rodents [17]. Although DOPr agonist activity is an interesting characteristic to consider when developing multitarget analgesic drugs, tolerance to DOPr-mediated analgesia has a very fast onset [42], which could limit the utility of this activity in chronic treatment. It was also found that KGFF09 displays potent KOPr antagonist activity, a property also shared by PZM21, the recently described biased MOP agonist [31]. The KOPr has been shown to display anti-MOPr activity [3; 38], and its blockade could present synergism with MOP and DOP agonist activity, leading to the observed analgesic potency of KGFF09. Moreover, as DOPr agonists and KOPr antagonists have been shown to have an antidepressant potential [29], KGFF09 may also have beneficial effects on the affective component of chronic pain syndromes.
In summary, for the first time a dual acting, G protein biased MOPr agonistNPFFRs antagonist molecule, in particular KGFF09, was reported. The association of both properties within a single molecule gathers the beneficial effects of biased MOPr agonists on acute side effects (respiratory depression) and those of NPFFRs antagonists on chronic side effects (OIH, tolerance, withdrawal syndrome), altogether leading to a potent analgesic with an improved safety profile. Hence, the present invention supports therapeutic strategies for potent antinociceptive drugs with limited side effects upon both acute and chronic use.
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