WO2008114976A1

WO2008114976A1 - Spinorphin derivatives as potent and selective human p2x3 receptor antagonist

Info

Publication number: WO2008114976A1
Application number: PCT/KR2008/001476
Authority: WO
Inventors: Yong-Chul Kim; Kwan-Young Jeong
Original assignee: Anygen Co., Ltd.
Priority date: 2007-03-19
Filing date: 2008-03-17
Publication date: 2008-09-25
Also published as: KR100880370B1; KR20080085245A; WO2008114976A8

Abstract

The present invention relates to a spinorphin derivative useful as a potent and selective human P2X3 receptor antagonist. More particularly, the present invention provides a spinorphin derivative useful as a potent and selective human P2X3 receptor antagonist selected from peptide AVVYPWT, peptide LAVYPWT, peptide LVAYPWT, peptide LVVAPWT and/ or cyclic peptide LWYPWT. Human P2X3 receptor antagonist has been developed on the electrophysiological evaluation of spinorphin peptide derivatives, truncated peptide analogues, cyclic peptides and a retroinverso peptide at human P2X3 receptors to determine the channel blocking activity.

Description

SPINORPHIN DERIVATIVES AS POTENT AND SELECTIVE HUMAN

P2X3 RECEPTOR ANTAGONIST

TECHNICAL FIELD

The present invention relates to a spinorphin derivatives as potent and selective human P2X₃ receptor antagonist. More particularly, this invention relates to a spinorphin derivative as potent and selective human P2X₃ receptor antagonist selected from the group consisting of peptide AVVYPWT, peptide LAVYPWT, peptide LVAYPWT, peptide LVVAPWT and cyclic peptide LVVYPWT.

BACKGROUND ART

P2X₃ receptors, members of the P2 purinergic receptor family, are ligand-gated ion channels activated by extracellular ATP as an endogenous ligand. The expression of P2X₃ receptors are highly localized in the peripheral and central processes of sensory afferent neurons. To this end, extracellular ATP has been studied as a neurotransmitter at synapses in sympathetic ganglia and in the brain. The activation of P2X₃ receptors by ATP based on a pronociceptive effect has been shown to initiate the pain signaling involved in chronic inflammatory nociception and neuropathic pain due to nerve injury, implicating a possibility of a new drug target for pain control.

Among a number of P2X₃ receptor antagonists (Figure 1), compound 3 (A317491) has shown potent antagonistic activity at human P2X3 receptors, with an IC50 value of 99 nM in patch clamp assays and painkilling effects in several animal models. However, most of the known antagonists for P2X₃ receptors, including, contain anionic functional groups such as carboxylic acids and phosphates, which generally limit the bioavailability of these compounds as potential drug candidates.

Spinorphin has been isolated from bovine spinal cords as an endogenous peptide consisting of seven amino acids, LVVYPWT, and is known for its inhibitory effect of enkephalin-degrading enzymes, resulting in analgesic activity in the tail pinch assay with intracerebroventricular administration. Moreover, it has been reported that spinorphin has completely inhibited the effects of the P2X receptor agonist, 2-MeS-ATP, including agonist induced nociception, which was not blocked by morphine, an inhibitor of bradykinin-induced nociceptive response. For this reason, a detailed structure-activity relationship study of spinorphin as the first potent peptide antagonist of P2X₃ receptors, was undertaken.

In the present invention, we have measured the electrophysiological effect of seven alanine scanned spinorphin peptide derivatives, truncated peptide analogs, cyclic peptides, and a retroinverso peptide at the recombinant human P2X₃ receptors expressed in Xenopus oocytes in order to characterize the channel blocking activity and recognition of fragments of spinorphin at P2X₃ receptors.

DISCLOSURE OF INVENTION

The object of the present invention is to provide a spinorphin derivative as human P2X₃ receptor antagonist selected from the group consisting of peptide (a) AWYPWT (SEQ ID NO: I)₇ (b) peptide LAVYPWT (SEQ ID NO: 2), (c) peptide LVAYPWT (SEQ ID NO: 3), (d) peptide LVVAPWT (SEQ ID NO: 4) and (e) cyclic peptide LWYPWT (SEQ ID NO: 5).

Further, the present invention provide a P2X3 receptor antagonist pharmaceutical composition comprising spinorphin derivative of claim 1 as active ingredient and pharmaceutically acceptable carriers.

Further, the present invention provide a process for preparing spinorphin derivatives using a stepwise fluoren-9-yl-methoxycarbony (Fmoc) solid phase synthesis method comprising the steps of: i) storing Fmoc amino acids as 0.5 M of N-methyl-2-pyrrolidone (NMP) solution; ii) pre-dissolving the coupling reagents in 0.5 M of NMP solution; iii) reacting 10 equivalents of amino acids with coupling agent; iv) performing Fmoc deprotection with 20% of piperidine in NMP solution; v) cleavaging peptide from the resin of amino acid side chains with TFA/H2O (95:5=v/v); and vi) precipitating peptide product with diethylether and lyophilizing the obtained peptide product after centrifuging it.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows structures and biological activities of the P2X₃ receptor antagonists.

FIG. 2(A) shows inhibition of human P2X₃ receptor-mediated currents by spinorphin. Representative inward current elicited by 2 μ M ATP in control and in the presence of three concentrations of spinorphin, 5, in Xenopus oocytes expressing human P2X3 receptor subtypes.

FIG. 2(B) shows the concentration-inhibitory curves of peptide 5 (■), 6 (#), 7 (A), 13(V), and iso-PPADS, 4 (♦) for human P2X₃ receptors. The continuous line for ATP is fit to the data using the equation: I = I_max/(1 + ICso/L)^nH), where 'Y is the actual current for a ligand concentration (⁷L'), 'nH' is the Hill coefficient and 'Im_3x' the maximal current. The IC₅₀ values were 8.3 ± 2.2 pM (5); 14.3 ± 5.1 nM (6); 32.4 ± 8.7 nM (7); 82.4 ± 17.0 nM (13); 108.2 ± 11.4 nM (4). The Hill coefficients were -0.38 ± 0.10 (5); -1.08 ± 0.13 (6); -1.27 ± 0.15 (7); -0.60 ± 0.05 (13); -0.92 ± 0.08 (4). Each value is the mean ± SEM of 4 observations. The recordings were carried out at a holding potential of -90 mV with 2 μ M ATP.

FIG. 3 shows the figure shows the concentration-response curves for ATP in the presence of increasing concentrations of spinorphin (peptide 5) and peptide 6 of present invention as indicated. The data correspond to oocytes expressing human P2X3 subunits. The EC50 values and Hill coefficients for ATP ( μ M) are: (A) control (■) 1.66 ± 0.23, 0.84 ± 0.09, 100 nM 5 (•) 8.45 ± 2.00, 0.73 ± 0.11, 500 nM 5 (A) 27.66 ± 1.89, 0.68 ± 0.03, and 1 μ M 5 (T) 54.31 ± 15.90, 0.57 ± 0.08. (B) control (■) 1.66 ± 0.23, 0.84 ± 0.09, 500 nM 6 (•) 9.76 ± 1.26, 0.66 ± 0.05, and 1 μ M 6 (A) 17.31 ± 4.16, 0.63 ± 0.07. Each value is the mean ± SEM of 3 to 7 oocytes.

BEST MODE FOR CARRYING OUT THE INVENTION Spinorphin, an endogenous antinociceptive peptide (LVVYPWT), showed potent antagonistic properties at the human P2X3 receptor, with an IC50 value of 8.3 pM in a two-electrode voltage clamp (TEVC) assay with recombinant human P2X3 receptors expressed in Xenopus oocytes.

Other P2X receptor subtypes, mouse P2Xi and human P2X7, were not affected by spinorphin and its derivatives. Several peptide derivatives including alanine substituted, truncated, cyclic peptides, and a retro-inverso peptide of spinorphin are evaluated for a structure-activity relationship study.

It was determined that single alanine substituted peptides 6 to 9, sustained antagonistic properties at the human P2X3 receptors with a nanomolar range of IC50 values, whereas the threonine to alanine substitution (peptide 12) resulted in an enhancing effect of agonist activity. The cyclic form of the LVVYPWT sequence (peptide 13) also displayed an antagonistic effect at the human P2X₃ receptor, with an IC50 value of 82.4 nM.

The truncated and retro-inverso peptide analogs showed complete loss of antagonistic activity at the human P2X₃ receptors. These results suggest that the high potency of spinorphin and its derivatives at the P2X3 receptor is consistent with its antinociceptive properties.

Spinorphin (peptide 5) and other peptide derivatives were synthesized using solid phase peptide synthesis on a Wang resin loaded with Fmoc-protected amino acids. Here, HOBt and DCC were used for the loading and subsequent coupling of each amino acid on the resin after deprotection of the Fmoc group. The cleavage of the peptides from the resin was carried out for 3 h with TFA/H2O (95:5 v/v). Note that the representative procedure for the synthesis of spinorphin is depicted in Scheme 1.

[Scheme 1]

The synthesized peptides were then tested in a functional ion channel assay to measure the ATP-induced current at the recombinant mouse P2Xi and human P2X₃ receptors, expressed in Xenopus oocytes, using the two-electrode voltage clamping (TEVC) technique.

Since purinergic Pl and P2 receptors are present on the follicle cell layer of Xenopus oocytes, the TEVC experiment was carried out on defolliculated oocytes. Control experiments were repeated over five times with a delay of 20 min for the human P2X₃ receptor to confirm the reproducible magnitude of peak currents in response to 2 μ M ATP, and the antagonistic effect of each peptide was measured based on the difference of the currents. The effects of spinorphin derivatives at the human P2X7 receptors stably expressed in the HEK293 cells were determined with an ethidium accumulation assay using BzATP as a selective agonist.

The antagonistic activity of spinorphin (peptide 5) and its derivatives at various P2X receptor subtypes are listed in Table 1. A P2X receptor antagonist, iso-PPADS (pyridoxal- a 5-phosphate-6-zaophenyl-2',5'- disulfonic acid) (peptide 4) was used as a positive control, which displayed 64%, 48%, and 97% inhibition in 100 nM at the mouse P2X_lx human P2X₃, and in 10 μ M at the human P2X7 receptor, respectively. Spinorphin (peptide 5) and its derivatives with single alanine substitutions of the 1st to 4th amino acids from the N-terminus of spinorphin showed higher (peptide 5 and peptide 6) or equal (peptide 7 to 9) antagonistic potency compared with iso-PPADS at the human P2X₃ receptor.

Table 1. Antagonistic effects of peptide analogues at mouse P2Xi, human P2X₃, and human P2X7 receptors % inhibition or normalized current (I/Imax) no. sequence

4

64.2±3.1%^c 48.3 ± 5.6% 97.6 ± 4.7%

(isoPPADS)

5 LVVYPWT 7.4 ±1.3% 67.2 ±3.1% 8.3 ± 0.9%

6 AVVYPWT inactive 82.5 ± 7.5% inactive

7 LAVYPWT 8.1 ±0.9% 48.4 ± 6.3% 7.6 ± 0.3%

8 LVAYPWT inactive 53.4 ±1.2% inactive

9 LVVAPWT inactive 52.3 ± 8.9% inactive

10 LVVYAWT inactive 12.9 ±1.0% inactive

11 LVVYPAT 4.2 ±1.2% 17.0 ±6.2% 16.7 ±1.4%

12 LVVYPWA 9.4 ±1.5% 2.5±0.1^d 6.7 ±1.2%

13 cyclic LVVYPWT 11.5 ±1.7% 36.3 ±1.6% 8.8 ±1.0%

14 cyclic AVVYPWT 6.4 ± 0.8% 11.4 ±2.3% inactive

15 (D)-TWPYVVL 15.5 ±2.4% inactive inactive

16 VYP n.d. 16.2±2.9%^e n.d.

17 VYPW n.d. inactive ^e n.d.

18 VYPWT n.d. inactive ^e n.d.

19 YPW n.d. 15.6 ± 5.4% ^e n.d.

20 YPWT n.d. 13.3 ± 3.5% ^e n.d.

21 PWT n.d. 32.7±6.8%^e n.d.

22 VVYPWT n.d. 29.3 ±1.8% ^e n.d.

^aThe ion current was induced by 2 μM ATP at the recombinant P2X receptors expressed in Xenopus oocytes and % inhibition of the ion current by 10 μM and 100 nM compounds was measured for mouse P2Xi and human P2X₃ receptors, respectively (mean ± SEM, n = 4). bThe accumulation of ethidium⁺ was induced by 6 μM BzATP at the human P2X7 receptors expressed in HEK293 cells and % inhibition of the accumulation by 10 μM compounds was measured (mean ± SEM, n = 3). ^c100 nM compound 4, isoPPADS, was used. Normalized ion current evoked by peptide 12 in the presence of 2 μM ATP. ^e10 μM compounds were tested. In terms of subtype selectivity, peptide analogs displaying antagonistic effects at the P2X3 receptor are all highly selective, showing very weak or inactive activity at P2Xi,7 receptors. The alanine substitutions of the 5th and 6th amino acids of spinorphin (peptide 10 and 11) significantly reduced the antagonistic activity, displaying only 10 to 20% inhibition at 100 nM (Table 1). Interestingly, the substitution of threonine of spinorphin to alanine (peptide 12) resulted in a 2.5-fold increase of ATP induced ion current, showing a dramatic reverse of the antagonistic character of spinorphin into a strong potentiator of ATP effect at the human P2X3 receptor.

Thus, the 5th to 7th amino acids in the sequence of spinorphin seem to be important in recognizing the human P2X₃ receptor as an antagonist. Peptides 13 and 14, as the cyclic forms of peptides 5 and 6, at a concentration of 100 nM were found to have relatively weak antagonism, showing 36% and 11 % inhibition against the ATP induced ion current at the human P2X₃ receptor, respectively. A retro-in verso peptide (peptide 15) an isomer with reverse sequence and inverted chirality, was inactive in 10 μ M, suggesting that terminal functions such as primary amine and carboxylic acid units may need to be appropriately arranged.

Figure 2(A) shows the dose dependent inhibition of spinorphin (peptide 5) against the ATP-induced ion current in defolliculated Xenopus oocytes expressing the human P2X₃ receptor.

As can be seen in the figure, the antagonistic effects of the peptide analogs were completely reversed after wash-out. Additionally, the full dose-response curves of iso-PPADS (peptide 4), spinorphin (peptide 5), alanine substituted peptides (peptide 6, 7), and a cyclic form of spinorphin (peptide 13) are depicted in Figure 2(B). As can be seen, spinorphin showed the most potent antagonistic effect against the inward current elicited by ATP, with an IC50 value of 8.3 pM and a Hill coefficient of -0.38, suggesting the possibility of a binding mode with negative cooperative antagonism.

The alanine substituted peptides 6 and 7 displayed decreased antagonistic activity, with IC50 values of 14.3 nM and 32.4 nM and Hill coefficients of -1.08 and -1.27, respectively, which implies a changed binding mode from negative to positive cooperative antagonism due to the alanine substitutions. The cyclic form of the spinorphin sequence (LWYPWT) also has an appreciable antagonistic effect at human P2X3 receptor, with an IC50 value of 82.4 nM and Hill coefficient of -0.60.

In an effort to search the active fragments of spinorphin (peptide 5) that maintain antagonistic properties at the human P2X₃ receptor, a number of truncated peptide analogues were evaluated. However, removal of certain amino acid sequences from the original peptide resulted in a significant loss of antagonistic activity, showing 10 to 30% inhibition even at 10 μ M concentration, as presented in Table 1. In addition, the leucine deleted peptide (peptide 22) was also less active than spinorphin, showing only 30% inhibition at 10 μ M concentration. Thus, the data suggests that most of the amino acid residues of spinorphin are important in conferring the antagonistic activity for the human P2X3 receptor subtype.

To investigate whether the mechanism of the interaction of peptide 5 with human P2X₃ receptors is competitive, the ATP-induced currents in the presence of three concentrations of peptide 5 were measured, and the dose-response curves for ATP were compared (Figure 3(A)). Each peak current was normalized to the current value obtained from the treatment with 200 μ M ATP, a concentration that showed the maximum ATP-evoked current; the EC50 value from the dose-response curve of ATP in the absence of antagonist (control) was determined to be 1.66 μ M.

Increasing concentrations of peptide 5 shifted the dose-response curve of ATP to the right and at the same time reduced the maximal current, resulting in EC50 values of 8.45 μ M, 27.66 μ M, and 54.31 μ M for 100 nM, 500 nM, and 1 μ M of peptide 5, respectively. Hill coefficient values, nH, of each dose-response curves also changed (0.84, 0.73, 0.68, and 0.57 for control, 100 nM, 500 nM, and 1 μ M of peptide 5, respectively), indicating that the mode of antagonism of peptide 5 is noncompetitive. Additionally, an alanine substituted peptide of the second amino acid, valine, was also found to be a noncompetitive antagonist at the human P2X3 receptor, showing increasing EC50 values and decreasing Hill coefficient values (Figure 3(B)).

In summary, we established potent antagonism and structure-activity relationships of a series of spinorphin derivatives by electrophysiological evaluation with P2Xi,3 and 7 receptors expressed in oocyte or mammalian cell systems. Although the mode of antagonism was found to be noncompetitive, the peptide derivatives showed great potency and specificity for the human P2X3 receptors in comparison to the mouse P2Xi and human P2X7 receptors.

The peptide antagonists discovered in this invention will be valuable for future study regarding the structure and function of P2X receptors, and the information collected on the peptide sequences for antagonism can be used for further design of non-peptide antagonists.

The present invention will be more specifically explained by the following examples. However, it should be understood that the examples are intended to illustrate but not in any manner to limit the scope of the present invention.

EXAMPLES

Synthesis, Materials and Analytical Methods

Peptides 5 to 22 were prepared as described by a solid phase synthesis method. Pyridoxal 5' -phosphate monohydrate and aniline 2,4-disulfonic acid used in azo coupling reaction to prepare compound 4 were purchased from Aldrich (St. Louis, MO) and TCI (Tokyo). All amino acids used the L-configuration unless otherwise indicated. Fmoc-protected amino acids were purchased from Novabiochem (Darmstadt, Germany). All other reagents and solvents were of analytical or peptide synthesis grade, and purchased from Merck (Darmstadt, Germany), B&J Bioscience.

Proton nuclear magnetic resonance spectroscopy was performed on a JEOL JNM-LA 300WB spectrometer, and spectra were taken in Unless otherwise noted, chemical shifts are expressed as ppm downfield from internal tetramethylsilane, or relative ppm from DMSO (2.5 ppm). Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; b, broad; app.: apparent), coupling constants, and integration. Mass spectroscopy was carried out on MALDI-TOF and FAB instruments. High-resolution mass spectra (m/z) were recorded on a FAB instrument. [FAB source: JEOL FAB source and ion gun (Cs ion beam, 30 kV acceleration)]. High-resolution mass analysis was performed at the Seoul Branch Analytical Laboratory of the Korea Basic Science Institute.

The determination of purity was performed on a Shimadzu SCL-IOA VP HPLC system using a Shimadzu Shim-pack C18 analytical column (250 mm X 4.6 mm, 5 μ m, 100 A) in linear gradient solvent systems. One solvent system (A) was 0.1% TFA in H₂O:0.1% TFA in CH3CN = 95:5 to 35:65 in 30 min with flow rate = 1 mL/min. The other (B) was 0.1% TFA in H₂O:0.1% TFA in MeOH = 95:5 to 35:65 in 30 min with flow rate = 1 mL/min. Peaks were detected by UV absorption using a diode array detector.

The cRNA of human P2X3 receptor was obtained by reverse transcription of the cDNA of human P2X₃ receptor, which was generously provided by Dr. W. Stuhmer and Dr. F. Soto of the Max-Plank Institute. The EST clones containing full-length cDNAs of mouse P2Xi (clone ID: 4189541) and human P2X₇ receptor (clone ID: 5286944) were purchased (Invitrogen, CA, U.S.A.), and their sequences were confirmed by DNA sequencing.

(Preparation Example 1) Peptide synthesis

The peptides were synthesized by the stepwise fluoren-9-yl methoxycarbonyl (Fmoc) solid-phase method. Fmoc amino acids were stored as 0.5 M N-methyl-2-pyrrolidinone (NMP) solutions. Coupling reagents were pre-dissolved in NMP (0.5 M solutions), which the activators EDCI, HOBt as 2 M solutions. All cases utilized 10 equivalents of amino acids and coupling reagents, at a single 2 h coupling time. Fmoc deprotections were performed with 20% piperidine in NMP. Peptide cleavage from the resin of the amino acids side chains was carried out for 3 h with TFA/H₂O (95:5=v/v). The resins were washed with TFA and the filtrates partially evaporated. The crude products were precipitated with diethyl ether, collected by centrifugation, dissolved in H₂O, and lyophilized.

Peptide 5 (Spinorphin; LWYPWT)

¹H-NMR (DMSO- ₆, 300 MHz) δ 10.74 (s, IH, Trp); 9.15 (s, IH, Tyr); 8.46 (d, / = 8.4 Hz, IH, Tyr); 8.06 (d, / = 7.5 Hz, IH, Trp); 7.95 (m, 2H, Leu); 7.86 (d, / = 9.6 Hz, IH, Thr); 7.79 (d, / = 7.8 Hz, IH, VaI); 7.73 (d, / =8.4 Hz, IH, VaI); 7.55 (d, / =7.5 Hz, IH, Trp); 7.28 (d, / = 7.8 Hz, IH, Trp); 7.13 (s, IH, Trp); 7.03 (d, / = 8.4 Hz, 2H, Tyr); 7.02-7.09 (m, IH, Trp); 6.95 (dd, / = 8.4, 7.5 Hz, IH, Trp); 6.60 (d, / = 8.4 Hz, 2H, Tyr); 4.64 (m, IH, Tyr); 4.55-4.61 (m, IH, Trp); 4.28-4.36 (m, IH, Leu); 4.21-4.27 (m, 2H, 2H of each VaI); 4.10-4.19 (m, 2H₇ each IH of Thr); 3.84 (t, J = 6.9 Hz, IH, Pro); 3.51-3.63 (m, 2H, Pro); 3.19-3.24 (m, IH, Trp); 2.97-3.05 (m, IH, Trp); 2.64-2.77 (tt, / = 10.2, 9.8 Hz, 2H, Tyr); 1.83-1.92 (m, 3H, 2H of each VaI + IH of Pro); 1.72 (m, 2H, Pro); 1.56 (m, IH, Leu); 1.46 (dd, / = 6.3, 6.9 Hz, 2H, Leu); 1.03 (d, / = 6.3 Hz, 3H, Thr); 0.78-0.89 (m, 12H, 12H of each VaI); 0.68-0.74 (d, / = 6.9 Hz, 6H, Leu). HRMS (FAB) Calcd. For C₄₅H₆₅N₈Oi₀ : 877.4824. Observed m/z 877.4829 HPLC (tR, min) system A: 21.6, system B: 25.6

(Preparation Example 2) Synthesis of Peptide 6 (AWYPWT) ¹H-NMR (OMSO-dβ, 300 MHz) δ 12.59 (s, IH, Thr); 10.79 (s, IH, Trp); 9.15 (s, IH, Tyr); 8.36 (d, / = 8.4 Hz, IH, Tyr); 8.08 (d, / = 7.2 Hz, IH, Trp); 8.03 (m, 2H, Ala); 7.81 (d, / = 9.0 Hz, IH, Thr); 7.79 (d, / = 9.0 Hz, IH, VaI); 7.76 (d, / = 9.0 Hz, IH, VaI); 7.55 (d, / = 8.1 Hz, IH, Trp); 7.28 (d, / = 8.1 Hz, IH, Trp); 7.13 (s, IH, Trp); 7.04 (d, / = 8.7 Hz, 2H, Tyr); 7.02-7.04 (m, IH, Trp); 6.95 (dd, / = 9.0, 8.1 Hz, IH, Trp); 6.60 (d, / = 8.7 Hz, 2H, Tyr); 4.87 (d, / = 4.5 Hz, IH, Thr); 4.64 (m, IH, Tyr); 4.55 (m, IH, Trp); 4.28-4.31(m, IH, Ala); 4.24-4.27 (m, IH, VaI); 4.22-4.24 (m, IH, VaI); 4.11-4.18 (m, IH, Thr); 4.11-4.15 (m, IH, Thr); 3.90 (m, IH, Pro); 3.53 (m, IH, Pro); 3.16-3.27 (m, IH, Trp); 2.95-3.05 (m, IH, Trp); 2.64-2.77 (tt, / = 10.2, 9.8 Hz, 2H, Tyr); 1.83-1.96 (m, 3H, 2H of each VaI + IH of Pro); 1.67-1.75 (m, 2H, Pro); 1.03 (d, / = 6.9 Hz, 3H, Ala); 0.70-0.89 (m, 12H, 12H of each VaI). HRMS (FAB) Calcd. For C₄₂H₅₉N₈O₁₀ : 835.4354. Observed m/z 835.4351 HPLC (tR, min) system A: 19.8 system B: 21.7

(Preparation Example 3) Synthesis of Peptide 7 (LAVYPWT)

¹H-NMR (DMSO-4, 300 MHz) δ 12.60 (bs, IH, Thr); 10.78 (s, IH, Trp); 9.16 (s, IH, Tyr); 8.61 (d, / = 7.8 Hz, IH, Tyr); 8.08 (d, / = 7.8 Hz, IH, Trp); 8.04 (m, 2H, Leu); 7.80 (m, IH, Thr); 7.75 (m, IH, VaI); 7.74 (m, IH, VaI); 7.56 (d, / = 7.2 Hz, IH, Trp); 7.28 (d, / = 8.1 Hz, IH, Trp); 7.13 (s, IH, Trp); 7.05 (d, / = 8.4 Hz, 2H, Tyr); 7.01 (m, IH, Trp); 6.92 (dd, / = 8.4, 7.2 Hz, IH, Trp); 6.61 (d, / = 8.4 Hz, 2H, Tyr); 4.90 (m, IH, Thr); 4.64 (m, IH, Tyr); 4.53 (m, IH, Trp); 4.45 (m, IH, VaI); 4.30 (m, IH, Leu); 4.21 (m, IH, Ala); 4.14-4.17 (m, IH, Thr); 4.12-4.14 (m, IH, Thr); 3.75 (m, IH, Pro); 3.55 (m, IH, Pro); 3.16-3.21 (m, IH, Trp); 2.97-3.05 (m, IH, Trp); 2.60-2.80 (m, 2H, Tyr); 1.87-1.91 (m, 2H, IH of VaI + IH of Pro); 1.67-1.79 (m, 2H, Pro); 1.55-1.65 (m, IH, Leu); 1.45-1.55 (m, 2H, Leu); 1.20 (d, / = 6.9 Hz, 3H, Ala); 1.01 (d, / = 6.0 Hz, 3H, Tyr); 0.85 (dd, / = 7.2, 6.6 Hz, 6H, VaI); 0.71 (t, / = 6.9 Hz, 6H, Leu). HRMS (FAB) Calcd. For C₄₃H₆₁N₈O₁₀ : 849.4511. Observed m/z 849.4511 HPLC (t_R, min) system A:21.1 system B: 24.0

(Preparation Example 4) Synthesis of Peptide 8 (LVAYPWT)

¹H-NMR (DMSO-rf_6/ 300 MHz) δ 10.80 (s, IH, Trp); 9.15 (s, IH, Tyr); 8.40 (d, / = 8.4 Hz, IH, Tyr); 8.05 (d, / = 7.5 Hz, IH, Trp); 8.00 (m, 2H, Leu); 7.85 (d, / = 7.2 Hz, IH, Thr); 7.70 (d, / = 9.2 Hz, IH, VaI); 7.55 (d, / = 7.5 Hz, IH, Trp); 7.28 (d, / = 7.5 Hz, IH, Trp); 7.15 (s, IH, Trp); 7.05 (d, / = 8.7 Hz, 2H, Tyr); 7.00-7.05 (m, IH, Trp); 6.95 (dd, / = 8.7, 7.5 Hz, IH, Trp); 6.60 (d, / = 8.7Hz, 2H, Tyr); 4.60-4.67 (m, IH, Thr); 4.45-4.55 (m, IH, Trp); 4.25-4.35 (m, 2H, IH of Leu + IH of VaI); 4.17-4.25 (m, 2H, IH of Ala + IH of Thr); 4.07-4.15 (m, IH, Thr); 3.65-3.85 (m, IH, Pro); 3.45-3.55 (m, IH, Pro); 3.13-3.25 (m, IH, Trp); 2.98-3.05 (m, IH, Trp); 2.60-2.85 (m, 2H, Tyr); 1.85-1.98 (m, 2H, IH of VaI + IH of Pro); 1.70-1.80 (m, 2H, Pro); 1.55-1.65 (m, IH, Leu); 1.45-1.52 (m, 2H, Leu); 1.10 (d, / = 6.9 Hz, 3H, Ala); 1.01 (d, / = 6.0 Hz, 3H, Thr); 0.81-0.95 (m, 12H, 6H of VaI + 6H of Leu). HRMS (FAB) Calcd. For C₄₃H₆₁N₈O₁₀ : 849.4511. Observed m/z 849.4514 HPLC (tR, min) system A: 20.0 system B: 22.5

(Preparation Example 5) Synthesis of Peptide 9 (LVVAPWT)

¹H-NMR (OMSO-de, 300 MHz) δ 10.80 (s, IH, Trp); 8.43 (d, / = 8.1 Hz, IH, Ala); 8.00 (d, / = 7.2 Hz, IH, Trp); 7.93 (d, / = 9.0 Hz, IH, Thr); 7.82 (d, / = 7.5 Hz, IH, VaI); 7.66 (d, / = 8.1 Hz, IH, VaI); 7.55 (d, / = 7.8 Hz, IH₇ Trp); 7.30 (d, / = 7.8 Hz, IH, Trp); 7.13 (s, IH, Trp); 7.02 (dd, / = 6.9, 7.8 Hz, IH, Trp); 6.93 (dd, / = 6.9, 7.8 Hz, IH, Trp); 4.50-4.55 (m, IH, Ala); 4.40-4.48 (m, IH, Trp); 4.26-4.30 (m, 2H, 2H of each VaI); 4.12-4.15 (m, 2H, IH of Leu + IH of Thr); 4.05-4.10 (m, IH, Thr); 3.72-3.80 (m, IH, Pro); 3.16-3.23 (m, IH, Trp); 2.97-3.05 (m, IH, Trp); 1.85-2.00 (m, 3H, IH of Pro + 2H of each VaI); 1.70-1.80 (m, 2H, Pro); 1.55-1.65 (m, IH, Leu); 1.40-1.50 (m, 2H, Leu); 1.10 (d, / = 7.2 Hz, 3H, Ala); 1.00 (d, / = 6.0 Hz, 3H, Thr); 0.75-0.88 (m, 18H, 6H of Leu + 12H of each VaI). HRMS (FAB) Calcd. For C₃₉H₆IN₈O₉ : 785.4562. Observed m/z 785.4579 HPLC (t_R, min) system A: 32.4 system B: 23.9

(Preparation Example 6) Synthesis of Peptide 10 (LVVYAWT)

¹H-NMR (OMSO-de, 300 MHz) δ 10.77 (s, IH, Trp); 9.08 (s, IH, Tyr); 8.40 (m, IH, Ala); 8.07 (d, / = 7.8 Hz, IH, Tyr); 7.91-7.96 (app.m, 3H, 2H of each VaI + IH of Trp); 7.68 (d, / = 6.9 Hz, IH, Thr); 7.55 (d, / = 7.8 Hz, IH, Trp); 7.28 (d, / = 8.1 Hz, IH, Trp); 7.13 (s, IH, Trp); 7.03 (dd, / = 6.9, 6.6 Hz, IH, Trp); 6.98 (d, / = 8.4 Hz, 2H, Tyr); 6.90-7.00 (m, IH, Tyr); 6.58 (d, / = 8.4 Hz, 2H, Tyr); 4.55-4.65 (m, IH, Tyr); 4.39-4.50 (m, IH, Trp); 4.20-4.30 (m, 2H, 2H of each VaI); 4.05-4.15 (m, 3H, IH of Leu + 2H of Thr); 3.70-3.80 (m, IH, Ala); 3.16-3.20 (m, IH, Trp); 2.92-3.02 (m, IH, Trp); 2.60-2.85 (m, 2H, Tyr); 1.80-1.95 (m, 2H, 2H of each VaI); 1.50-1.65 (m, IH, Leu); 1.37-1.50 (m, 2H, Leu); 1.15 (d, / = 6.9 Hz, 3H, Ala); 0.97 (d, / = 6.3 Hz, 3H, Thr); 0.67-0.87 (m, 18H, 12H of each VaI + 6H of Leu). HRMS (FAB) Calcd. For C₄₃H₆₃N₈O₁₀ : 851.4667. Observed m/z 851.4653 HPLC (t_R, min) system A: 21.2 system B: 23.7

(Preparation Example 7) Synthesis of Peptide 11 (LWYPAW) ¹H-NMR (DMSO- ₆, 300 MHz) δ 12.58 (s, IH, Thr); 9.14 (s, IH, Tyr); 8.47 (d, / = 8.4 Hz, IH, Tyr); 8.05-8.13 (app.m, 4H, 2H of Leu + IH of Ala + IH of Thr); 7.87 (d, / = 9.3 Hz, IH, VaI); 7.60 (d, / = 8.7 Hz, IH, VaI); 7.05 (d, / = 8.4 Hz, IH, Tyr); 6.62 (d, / = 8.4 Hz, IH, Tyr); 4.87-4.97 (m, IH, Thr); 4.53-4.64 (m, IH, Tyr); 4.30-4.40 (m, 2H, 2H of each VaI); 4.22-4.30 (m, IH, Leu); 4.10-4.21 (m, 3H, 2H of Thr + IH of Ala); 3.82-3.90 (m, IH, Pro); 3.55-3.65 (m, IH, Pro); 2.62-2.75 (m, 2H, Tyr); 1.80-2.00 (m, 5H, 2H of each VaI + 3H of Pro); 1.55-1.65 (m, IH, Leu); 1.45-1.55 (m, 2H, Leu); 1.23 (d, / = 7.2 Hz, 3H, Ala); 1.01 (d, / = 6.0 Hz, 3H, Thr); 0.70-0.90 (m, 18H, 6H of Leu + 12H of each VaI). HRMS (FAB) Calcd. For C₃₇H₆₀N₇Oi₀ : 762.4402. Observed m/z 762.4416 HPLC (t_R, min) system A: 14.9 system B: 17.6

(Preparation Example 8) Synthesis of Peptide 12 (LWYPWA)

¹H-NMR (DMSO-de, 300 MHz) δ 10.80 (s, IH, Trp); 9.15 (s, IH, Tyr); 8.46 (d, / = 8.4 Hz, IH, Tyr); 8.00-8.20 (m, 4H, 2H of Leu + 2H of each VaI); 7.88 (d, / = 9.6 Hz, IH, Thr); 7.60 (d, / = 7.8 Hz, IH, Trp); 7.55 (d, / = 8.1 Hz, IH, Trp); 7.27 (d, / = 8.1 Hz, IH, Trp); 7.11 (s, IH, Trp); 7.03 (d, / = 8.7 Hz, 2H, Tyr); 7.00-7.02 (m, IH, Trp); 6.95 (m, IH, Trp); 6.60 (d, / = 8.7 Hz, 2H, Tyr); 4.50-4.60 (m, 2H, IH of Trp + IH of Tyr); 4.20-4.30 (m, 3H, IH of Leu + IH of Ala + IH of VaI); 4.12-4.18 (dd, / = 9.6, 8.4 Hz, IH, VaI); 3.80-3.90 (m, IH, Pro); 3.52-3.62 (m, IH, Pro); 3.12-3.22 (m, IH, Trp); 2.97-3.05 (m, IH, Trp); 2.60-2.82 (m, 2H, Tyr); 1.81-1.93 (m, 3H, IH of Pro + 2H of each VaI); 1.65-1.75 (m, 3H, 2H of VaI + IH of other VaI); 1.53-1.61 (m, IH, Leu); 1.45-1.52 (m, 2H, Leu); 1.27 (d, / = 7.2 Hz, 3H, Ala); 0.70-0.90 (m, 18H, 6H of Leu + 12H of each VaI). HRMS (FAB) Calcd. For C₄₄H₆₃N₈O₉: 847.4718. Observed m/z 847.4716 HPLC (t_R, min) system A: 20.0 system B: 25.2 (Preparation Example 9) Synthesis of Peptide 13 (cyclic LWYPWT)

¹H-NMR (OMSO-dβ, 300 MHz) 5 10.93 (s, IH, Trp); 9.24 (s, IH, Tyr); 8.91 (d, / = 4.5 Hz, IH, Tyr); 8.15 (d, / = 9.3 Hz, IH, Trp); 7.76 (d, / = 9.6 Hz, IH, Thr); 7.52 (d, / = 7.5 Hz, IH, VaI); 7.49 (d, / = 7.5 Hz, IH, VaI); 7.32 (d, / = 7.5 Hz, IH, Trp); 7.25 (s, IH, Trp); 7.07 (d, / = 7.8 Hz, IH, Trp); 6.92 (d, / = 8.4 Hz, 2H, Tyr); 6.90 (IH, Trp); 6.64 (d, / = 8.4 Hz, 2H, Tyr); 5.59 (s, IH, Leu), 4.64 (m, IH, Tyr); 4.55 (m, IH, Trp); 4.22-4.41 (m, 4H, IH of Leu + 2H of each VaI + IH of Thr); 4.06 (m, IH, Thr); 3.85 (m, IH, Pro); 2.62-2.91 (m, 4H, 2H of Trp + 2H of Tyr); 2.10-2.20 (m, 2H, each VaI); 1.98-2.10 (m, 2H, Pro); 1.82 (m, 2H, Pro); 1.63 (m, IH, Leu); 1.50 (m, 2H, Leu); 1.23 (d, / = 6.3 Hz, 3H, Thr); 0.78-1.02 (m, 12H of each VaI + 6H of Leu). MS (MALDI-TOF) Calcd. For C₄₅H₆₂N₈O₉: 859.0. Observed m/z 859.3 HPLC (t_R, min) system A: 26.3 system B: 30.2

(Preparation Example 10) Synthesis of Peptide 14 (cyclic AVVYPWT)

¹H-NMR (OMSO-dβ, 300 MHz) 5 10.95 (s, IH, Trp); 9.25 (s, IH, Tyr); 8.94 (d, / = 4.8 Hz, IH, Tyr); 8.41 (bs, IH, Thr); 8.14 (d, / = 8.1 Hz, IH, Trp); 7.78 (d, / = 7.2 Hz, IH, VaI); 7.49 (d, / = 7.8 Hz, IH, VaI); 7.29 (d, / = 8.1 Hz, IH, Trp); 7.13 (s, IH, Trp); 7.06 (d, / = 8.1 Hz, IH, Trp); 7.02 (d, / = 4.2 Hz, IH, Trp); 6.94 (d, / = 8.4 Hz, 2H, Tyr); 6.65 (d, / = 8.4 Hz, 2H, Tyr); 5.59 (s, IH, Ala); 4,61 (m, IH, Tyr); 4.50 (m, IH, Trp); 4.20-4.38 (m, 3H, IH of Ala + 2H of each VaI); 4.01 (m, IH, Thr); 3.85 (m, IH, Pro); 2.80 (m, 2H, Tyr); 2.01-2.21 (m, 2H, Pro); 1.50-1.59 (m, 2H, Pro); 1.31 (d. / = 7.2 Hz, 3H, Thr); 1.16 (d, / = 6.0 Hz, 3H, Ala); 0.78-1.02 (m, 12H, 12H of each Valine). MS (MALDI-TOF) Calcd. For C₄₂H₅₆N₈O₉: 816.9 Observed m/z 817.7 HPLC (t_R, min) system A: 22.2 system B: 25.7

(Preparation Example 11) Synthesis of Peptide 15 (Retroinverso-peptide; (D)-TWPYVVL)

¹H-NMR (OMSO-dβ, 300 MHz) δ 10.88 (s, IH, Trp); 9.11 (s, IH, Tyr); 8.77 (d, / = 7.2 Hz, IH, Tyr); 8.13 (d, / = 7.2 Hz, IH, Trp); 7.87 (d, / = 9.0 Hz, IH, Leu); 7.82 (d, / = 9.0 Hz, IH, VaI); 7.70 (d, / = 7.8 Hz, IH, VaI); 7.58 (d, / = 7.8 Hz, IH, Trp); 7.33 (d, / = 7.8 Hz, IH, Trp); 7.27 (s, IH, Trp); 7.04-7.12 (m, IH, Trp); 7.00 (d, / = 8.4 Hz, 2H, Tyr); 6.90-7.00 (m, IH, Trp); 6.60 (d, / = 8.4 Hz, 2H, Tyr); 5.40-5.52 (m, IH, Thr); 4.72-4.80 (m, IH, Tyr); 4.46-4.55 (m, IH, Trp); 4.32-4.38 (m, IH, Leu); 4.16-4.30 (m, 3H, IH of Thr + 2H of each VaI); 3.76-3.84 (m, IH, Pro); 3.48-3.50 (m, 2H, Pro); 3.08-3.17 (m, IH, Trp); 2.91-3.00 (m, IH, Trp); 2.82-3.00 (m, IH, Tyr); 2.67-2.75 (m, IH, Tyr); 1.88-2.00 (m, 3H, IH of Pro + 2H of each VaI); 1.70-1.80 (m, 3H, Pro); 1.57-1.66 (m, IH, Leu); 1.45-1.53 (m, 2H, Leu); 1.18 (d, / = 6.0 Hz, 3H, Thr); 0.74-0.90 (m, 18H, 6H of Leu + 12H of each VaI). HRMS (FAB) Calcd. For C₄₅H₆₅N₈Oi₀ : 877.4824. Observed m/z 877.4805 HPLC (t_R, min) system A: 23.1 system B: 26.1

(Preparation Example 12) Synthesis of Peptide 16 (VYP)

¹H-NMR

300 MHz) 5 12.51 (s, IH, Pro); 9.23 (s, IH, Tyr); 8.66 (d, / = 7.8 Hz, IH, Tyr); 7.98 (bs, 2H, VaI); 7.12 (d, / = 8.4 Hz, 2H, Tyr); 6.67 (d, / = 8.4 Hz, 2H, Tyr); 4.62 (m, IH, Tyr); 4.21 (d, / = 4.5 Hz, IH, Pro); 3.72 (m, IH, VaI); 3.58 (m, 2H, Pro); 3.44 (m, IH, VaI); 2.73 (d, / = 5.7 Hz, 2H, Tyr); 2.09 (m, 3H_x IH of Pro + IH of VaI); 1.80-1.92 (m, 3H, Pro); 0.89 (m, 6H, VaI). MS (MALDI-TOF) Calcd. For Ci₉H₂₇N₃O₅ : 377.4 Observed m/z 377.7 HPLC (t_R/ min) system A: 11.9

(Preparation Example 13) Synthesis of Peptide 17 (VYPW)

¹H-NMR (DMSO-de, 300 MHz) (512.61 (s, IH, Trp); 10.85 (s, IH, Trp); 9.28 (s, IH, Tyr); 8.66 (d, / = 7.5 Hz, IH, Tyr); 7.99 (d, / = 7.5 Hz, IH, Trp); 7.56 (d, / = 7.8 Hz, IH, Trp); 7.33 (d, / = 8.4 Hz, IH, Trp); 7.18 (s, IH, Trp); 7.11 (d, / = 8.4 Hz, 2H, Tyr); 7.03 (m, IH, Trp); 6.98 (m, IH, Trp); 6.64 (d, / = 8.4 Hz, 2H, Tyr); 4.61 (m, IH, Tyr); 4.52 (m, 2H, IH of VaI + IH of Trp); 4.41 (m, IH, Pro); 3.71 (m, 2H, Pro); 3.13 (m, 2H, Trp); 2.60-2.81 (m, 2H, Tyr); 2.07 (m, 2H, Pro); 1.78-1.82 (m, 3H, IH of VaI + 2H of Pro); 0.94 (dd, / = 6.6, 7.2 Hz, 6H, VaI). MS (MALDI-TOF) Calcd. For C₃₀H₃₇N₅O₆ : 563.7 Observed m/z 562.8 HPLC (t_R, min) system A: 18.2

(Preparation Example 14) Synthesis of Peptide 18 (VYPWT)

¹H-NMR {OMSO-de, 300 MHz) £ 10.79 (s, IH, Trp); 9.24 (s, IH, Tyr); 8.64 (d, / = 7.8 Hz, IH, Tyr); 7.84 (d, / = 7.8 Hz, IH, Trp); 7.76 (d, / = 8.4 Hz, IH, Thr); 7.59 (d, / = 7.8 Hz, IH, Trp); 7.28 (d, / = 8.4 Hz, IH, Trp); 7.15 (s, IH, Trp); 7.09 (d, / = 8.4 Hz, 2H, Tyr); 7.01 (t, / = 6.9 Hz, IH, Trp); 6.92 (m, IH, Trp); 6.65 (d, / = 8.4 Hz, 2H, Tyr); 4.68 (m, IH, Tyr); 4.57 (m, IH, Pro); 4.49 (m, IH, VaI); 4.33 (m, IH, Trp); 4.24 (dd, / = 3.3 Hz, IH, Thr); 4.13 (m, IH, Thr); 3.51 (m, 2H, pro); 3.17 (d, / = 5.4 Hz, 2H₇ Trp); 3.05 (d, / = 7.8 Hz, 2H, Tyr); 2.08 (m, IH, VaI); 1.76-1.91 (m, 4H, Pro); 1.05 (d, / = 6.6 Hz, 3H, Thr); 0.83-1.02 (m, 6H, VaI). MS (MALDI-TOF) Calcd. For C₃₄H₄₄N₆O₈ : 664.8 Observed m/z 664.2 HPLC (t_R/ min) system A: 18.9

(Preparation Example 15) Synthesis of Peptide 19 (YPW)

¹H-NMR (OMSO-dβ, 300 MHz) 5 12.70 (s, IH, Trp); 10.86 (s, IH, Trp); 9.35 (s, IH, Tyr); 8.18 (d, / = 7.5 Hz, IH, Trp); 8.01 (s, 2H, Tyr); 7.58 (d, / = 8.7 Hz, IH, Trp); 7.34 (d, / = 8.1 Hz, IH, Trp); 7.21 (s, IH, Trp); 7.14 (d, / = 8.4 Hz, 2H, Tyr); 7.06 (dd, / = 6.9, 7.2 Hz, IH, Trp); 6.95 (m, IH, Trp); 6.68 (d, / = 8.4 Hz, 2H, Tyr); 4.70-4.53 (m, 2H, IH of Tyr + IH of Trp); 4.22 (m, IH, Pro); 3.57-3.65 (m, 2H, Pro); 3.09 (m, 2H, Trp); 2.76-2.99 (m, 2H, Tyr); 2.06 (m, IH, Pro); 1.73-1.85 (m, 3H, 2H of Pro + IH of Pro). MS (MALDI-TOF) Calcd. For C₂₅H₂₈N₄O₅ : 464.5 Observed m/z 464.7 HPLC (t_R, min) system A: 18.8

(Preparation Example 16) Synthesis of Peptide 20 (YPWT)

¹H-NMR (OMSO-dβ, 300 MHz) 3 12.63 (s, IH, Thr); 10.80 (s, IH, Trp); 9.36 (s, IH, Tyr); 8.26 (d, / = 8.4 Hz, IH, Trp); 8.01 (s, 2H, Tyr); 7.81 (d, / = 8.4 Hz, IH, Thr); 7.64 (d, / = 7.2 Hz, IH, Trp); 7.29 (d, / = 7.8 Hz, IH, Trp); 7.20 (s, IH, Trp); 7.12 (d, / = 8.4 Hz, 2H, Tyr); 7.02 (dd, / = 7.2, 8.4 Hz, IH, Trp); 6.90 (m, IH, Trp); 6.68 (d, / = 8.4 Hz, 2H, Tyr); 4.92 (m, IH, Pro); 4.70 (m, IH, Tyr); 4.42 (m, IH, Trp); 4.25 (m, IH, Thr); 4.15 (m, IH, Thr); 3.57 (m, 2H, Pro); 3.11-3.28 (m, 2H, Trp); 2.73-3.11 (m, 2H, Tyr); 2.01 (m, IH, Pro); 1.64-1.80 (m, 3H, 2H of Pro + IH of Pro); 1.02 (d, / = 6.3 Hz, 3H, Thr). MS (MALDI-TOF) Calcd. For C₂₉H₃₅N₅O₇ : 565.6 Observed m/z 566.1 HPLC (t_R, min) system A: 16.6

(Preparation Example 17) Synthesis of Peptide 21 (PWT) ¹H-NMR (DMSCW₆, 300 MHz) δ 10.84 (s, IH, Trp); 8.73 (d, / = 8.1 Hz, IH, Trp); 8.09 (d, / = 8.7 Hz, IH, Trp); 7.71 (d, / = 8.1 Hz, IH, Thr); 7.34 (d, / = 8.1 Hz, IH, Trp); 7.20 (s, IH, Trp); 7.07 (dd, / = 7.2, 8.7 Hz, IH, Trp); 6.95 (dd, / = 7.2, 8.1 Hz, IH, Trp); 5.02 (m, IH, Pro); 4.79(m, IH, Trp); 4.28 (dd, / = 3.0, 3.3 Hz, IH, Thr); 4.19 (m, IH, Thr); 4.07 (m, IH, Pro); 3.16-3.24 (m, 2H, Trp); 2.98 (m, IH, Pro); 2.27 (m, IH, Pro); 1.81 (m, 2H, Pro); 1.07 (d, / = 6.3 Hz, 3H, Thr). MS (MALDI-TOF) Calcd. For C₂OH₂₆N₄O₅ : 402.5 Observed 402.9 HPLC (t_R, min) system A: 13.4

(Preparation Example 18) Synthesis of Peptide 22 (WYPWT)

¹H-NMR (DMSO-J₆, 300 MHz) δ 12.57 (s, IH, Thr); 10.78 (s, IH, Trp); 9.13 (s, IH, Tyr); 8.30 (d, / = 7.8 Hz, IH, VaI); 8.18 (d, / = 9.0 Hz, IH, Tyr); 7.99 (s, 2H, VaI); 7.83 (d, / = 7.8 Hz, IH, Trp); 7.74 (d, / = 8.4 Hz, Ih, Thr); 7.58 (d, / = 7.8 Hz, IH, Trp); 7.31 (d, / = 8.4 Hz, IH, Trp); 7.15 (s, IH, Trp); 7.02-7.07 (m, IH, Trp); 7.01 (d, / = 8.4 Hz, 2H, Tyr); ;6.93 (dd, / = 7.8, 8.4 Hz, IH, Trp); 6.61 (d, / = 8.4 Hz, 2H, Tyr); 4.88 (m, IH, VaI); 4.65 (m, IH, Tyr); 4.58 (m, IH, VaI); 4.32 (m, IH, Pro); 4.25 (m, IH, Trp); 4.20 (m, IH, Thr); 4.11 (m, IH, Thr); 3.53-3.67 (m, 2H, Pro); 3.17 (m, 2H, Trp); 3.02 (m, IH, Pro); 2.61-2.82 (m, 2H, Tyr); 1.90-2.11 (m, 3H, 2H of each VaI + IH of Pro); 1.74 (m, 3H, IH of Pro + 2H of Pro); 1.06 (d, / = 6.3 Hz, 3H, Thr); 0.80-0.92 (m, 12H, two VaI). MS (MALDI-TOF) Calcd. For C₃₉H₅₃N₇O₉ : 763.9 Observed 763.9 HPLC (t_R, min) system A: 19.4

(Example 1) Antagonist activity at recombinant mouse P2Xi and human P2X₃ receptors

Xenopus oocytes were harvested and prepared as previously described. Defolliculated oocytes were injected cytosolically with mouse P2Xi and human P2X₃ receptor cRNA (40 nL, 1 μg/mL), respectively, incubated for 24 h at 18 °C in Barth's solution and kept for up to 12 days at 4°C until used in electrophysiological experiments.

ATP-activated membrane currents (Vh = -90 mV) were recorded from cRNA-injected oocytes using the twin-electrode voltage-clamp technique (Axoclamp 2B amplifier). Voltage recording (1-2 MΩ tip resistance) and current-recording microelectrodes (5 MΩ tip resistance) were filled with 3.0 M KCl. Oocytes were held in an electrophysiological chamber and superfused with Ringer's solution (5 mL/min, at 18 "C) containing (mM) NaCl, 110; KCl, 2.5; HEPES

(N-[2-hydroxyethyl]piperazine-N' -[3-propanesulfonic acid]), 5; BaCl₂, 1.8, adjusted to pH 7.5.

ATP was superfused over the oocytes for 60-12Os then washed out for a period of 20 min. For inhibition curves, data were normalized to the current evoked by ATP, at pH 7.5. Test substances were added for 20 min prior to ATP exposure; all peptides were tested for reversibility of their effects. The concentration required to inhibit the ATP response by 50% (IC50) was taken from Hill plots constructed using the formula: log(J/I_maχ - T), where I is the current evoked by ATP in the presence of an antagonist. Data are presented as mean ± SEM (n=4) for the data from different batches of oocytes.

(Example 2) Ethidium⁺ accumulation assay at human P2X7 receptors

Human P2X7-expressing HEK293 cells were grown as monolayer culture at 37 °C in a humidified atmosphere of 5% CO₂ DMEM supplemented with 10% fetal bovine serum. Cells were harvested with treatment of Trypsin/ EDTA solution, collected by centrifugation (200 g for 5 min). The cells were resuspended at 2.5 X 10⁶ cells/ mL in assay buffers, consisting of (in mM) HEPES 10, KCl 140, glucose 5, EDTA 1 (pH 7.4), and then ethidium bromide (100 μ M) was added. Cell suspensions were added to 96 well plates containing the P2X7 receptor agonist, ATP or BzATP, at 2 X 10⁵ cells/wells. Plates were incubated at 37 °C for 120 min and cellular accumulation of ethidium+ was determined by measuring fluorescence with Bio-Tek instrument FL600 fluorescent plate reader (excitation wavelength of 530 nm and emission wavelength of 590 nm). When the effects of antagonists, such as PPADS, KN62, and test peptides were studied, antagonists were treated together with agonist without pre-incubation.

Claims

WHAT IS CLAIMED IS :

1. A spinorphin derivative as human P2X₃ receptor antagonist selected from the group consisting of (a) peptide AWYPWT (SEQ ID NO: I)₇ (b) peptide LAVYPWT (SEQ ID NO: 2), (c) peptide LVAYPWT (SEQ ID NO: 3), (d) peptide LWAPWT (SEQ ID NO: 4) and (e) cyclic peptide LWYPWT (SEQ ID NO: 5).

2. A P2X₃ receptor antagonist pharmaceutical composition comprising spinorphin derivative of claim 1 as active ingredient and pharmaceutically acceptable carriers.

3. A process for preparing spinorphin derivatives using a stepwise fluoren-9-yl-methoxycarbony (Fmoc) solid phase synthesis method comprising the steps of: i) storing Fmoc amino acids as 0.5 M of N-methyl-2-pyrrolidone

(NMP) solution; ii) pre-dissolving the coupling reagents in 0.5 M of NMP solution; iii) reacting 10 equivalents of amino acids with coupling agent; iv) performing Fmoc deprotection with 20% of piperidine in NMP solution; v) cleavaging peptide from the resin of amino acid side chains with

TFA/H₂O (95:5=v/v); and vi) precipitating peptide product with diethylether and lyophilizing the obtained peptide product after centrifuging it.