EP0777682A1

EP0777682A1 - New opioid peptide antagonists

Info

Publication number: EP0777682A1
Application number: EP95923629A
Authority: EP
Inventors: Peter-Appartement 1212 SCHILLER
Original assignee: Astra AB
Current assignee: AstraZeneca AB
Priority date: 1994-08-25
Filing date: 1995-06-14
Publication date: 1997-06-11
Also published as: SE9402838D0

Abstract

Compounds of formula (I) as well as methods for their preparation, their pharmaceutical preparations and their use. The compounds of formula (I) are useful in therapy, especially as analgesics and as immunosuppressive agents.

Description

New opioid peptide antagonists

The field of the Invention

This invention is related to a new class of opioid peptide analogs that are δ opioid receptor antagonists as well as to their synthesis and their use as analgesic and immunosuppressive compounds.

Background and prior art

A known nonpeptide δ opioid antagonist is naltrindole, which is described by P.S

Portoghese, et al J. Med. Chem. 31, 281-282 (1988). Naltrindole has similar δ antagonist, potency as the compounds according to this invention but is much less δ selective.

Furthermore, naltrindole has also quite high μ opioid receptor affinity (Kj^μ=12nM) in the receptor binding assay and potent μ antagonist properties (¥L_e=29nM) in the guinea pig ileum (GPI) assay, cf P.S. Portoghese, J. Med. Chem. 34, 1757-1762 (1991).

Another known δ-antagonist is the enkephaUn analog N,N-diaUyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) described by R. Cotton, et al. in Eur. J. Pharmacol. 97, 331-332 (1984). In comparison with the δ antagonists described in this patent application, ICI 174864 is much less δ-selective (10-300 times less) and has much lower antagonist potency in the MVD assay (40-1000 times less potent).

Peptides containing the H-Tyr-Tic-Aaa-sequence (Tic = 1 ,2,3,4-tetrahydroisoquinoline-3- carboxylic acid, Aaa = aromatic amino acid residue) at the N-terminus and which are very potent and highly selective δ antagonists have recently been disclosed by P.W. Schiller et al. in FASEB J, 6 (No.4), A 1575 (1992), at the International Narcotics Research (INRC) Meetings in Keystone, CO, June 24-29 (1992) and in Skδvde, Sweden, July 10-15 (1993), at the 2nd Japan Symposium on Peptide Chemistry Shi-zuoka, Japan, Nov. 9-13 (1992), at the 22nd European Peptide Symposium, Interlaken, Switzerland, Sept. 13-19(1992), in Proc. Natl. Acad.Sci. USA 89, 11871-11875 (1992), and in J. Med. Chem. 36, 3182-3187 (1993).

Thus, the problem underlying the present invention was to find δ opioid antagonists both with high δ antagonist potency and with high δ selectivity.

The Invention

It has now been found that peptides containing the H-Tyr-Tic-dipeptide segment at the N- terminus and a non-aromatic amino acid residue at the 3-position of the peptide sequence, as defined by the foUowing formula I, have

- extraordinary potency as δ antagonists

- high selectivity for the δ receptor

- total lack of μ antagonist properties.

The compounds according to the present invention have the general formula I

wherein

R-l is H; CH₃(CH2)rτ wherein n = 0-12; -CH2-CH2

— CH₂ <^j ^■ — CH₂— CH = CH₂ ; or arginine;

R2 is H; CH3(CH2)_n - wherein n = 0-12; CH3- ; — CH₂ <1 ; or ^

-CH₂-CH=CH₂;

R3, R4, R5 and R are all H; or

R4 and R5 are both H and R3 and Rg is each a Cj- C^ alkyl group; or R3, R₅ and R₆ are all H and R₄ is F, Cl, Br, OH, NH₂ or NO₂; R₇ is C=O or CH₂;

R_ is H or a Ci-Cβ alkyl group; R9 is selected from

C H₃ — (C H₂)_m-C H wherein m is 0- 12;

H₃C— CH-CH

OH

(CH₂)_p-CH wherein p is 0-4;

R_j0 is OH, NH₂ or

^R1 wherein

R_l i is H, NO₂, F, Cl, Br or I; q is 0-3;

R_l is COOH, CONH₂, CH₂OH, or any additional amino acid or peptide segment; or

Rio is wherein

R_l is as defined above.

Preferred compounds of the invention are those compounds wherein Rl is selected from H or CH3;

R is selected from H or CH3; R3 is selected from H or CH3; R₄ is H; R₅ is H;

Rg is selected from H or CH3; R7 is selected from CO or CH₂; Rg is selected from H or CH3;

R9 is selected from .

RlO is selected from R! , wterein Ri ! is H, NO₂, F, Cl, Br or I,

q is 1-3, and R_i2 is COOH. EspeciaUy preferred compounds of the invention are those compounds wherein Rl is selected from H or CH3;

R₂ is selected from H or CH3;

R3 is selected from CH3;

R4 is selected from H;

R5 is selected from H;

R6 is selected from CH3;

R7 is selected from CH₂;

R_ is selected from H or CH3;

R9 is selected from H , wherein p=0-4 ;

R₁₀ is selected from R_π , wherein Rπ is H, q is L and R_i2 is COOH.

Especially preferred compounds according to the invention are those

wherein R9 is — CH₂ — CH

(containing a cyclohexylalanine [Cha] residue in the 3-position of the peptide sequence). Substitution of Cha in the 3-position significantly enhances δ antagonist potency.

Further preferred compounds according to the invention are those, wherein R4 and R5 are hydrogen and R3 and Rβ are both methyl groups. Also preferred compounds according to the invention are compounds wherein R7 is a part of a reduced peptide bond.

The best mode of carrying out the invention known at present is to use the compounds of Examples 1, 2, , 11, 12 and 15.

Synthesis

Most Boc-amino acid derivatives used in the peptide syntheses are commercially available. 2,6-dimethyl-tyrosine (Dmt) was prepared as described by J.H Dygos et al. Synthesis, No 8 (August) pp. 741-743 (1992).

All peptides were prepared by solid-phase techniques. The usual polystyrene/- divinylbenzene resin was used for the solid-phase synthesis of peptides with a free C- terminal carboxyl group, whereas peptide amides were synthesized by using the p- methylbenzhydrylamine resin. Boc protection of the amino group was employed in the preparation of all peptides. The syntheses were performed according to protocols that have been extensively used in the inventor's laboratory (P.W SchiUer et al, Biochemisty 16, 1831-

1832 (1977)). Couplings were performed in CH₂C1₂, DMF or a mixture thereof, using

N,>T-dicyclohexylcarbodiimide/l-hydroxybenzotriazole (DCC/HOBt), N,N- diisopropylcarbodiimide/l-hydroxybenzotriazole, benzotriazolyloxytris-

(dimethylamino)phosphonium hexafluorophosphate, or any other suitable coupUng agent Completeness of coupling was carefully examined after each coupling step by means of the ninhydrin color test. The fully assembled peptide chain was cleaved from the resin and completely deprotected by treatment with liquid HF at 0°C and in the presence of anisole as scavenger (60-90 min).

Crude products obtained from soUd-phase peptide synthesis required extensive purification by various chromatographic techniques or by other methods. FoUowing HF cleavage and extraction of the resin, gel filtration on Sephadex (G-15 or G-25) was routinely performed. Various subsequent purification steps included partition chromatography on Sephadex G-25 (using various butanol-acetic acid-pyridine- water two phase systems), ion exchange chromatography (DEAE-Sephadex, SP-Sephadex and CM-cellulose) and reversed-phase chromatography on an octadecasilyl- silica column using linear gradients of methanol in 1 % trifluoroacetic acid (low pressure). If necessary, final purification to homogeneity was performed by semi-preparative HPLC. Semi-preparative μ-Bondapak C- 18 columns

(Waters; 0.7x25 cm), which, depending on the separation problem, permitted purification of 2-20 mg peptide material per run were used. Several highly sensitive and efficient analytical methods were used to demonstrate homogeneity of the prepared peptides and to verify their structures. Thin layer chromatography in at least two different solvent systems was used to establish purity. Furthermore, analytical HPLC in two or three different solvent systems was routinely used in the laboratory as a highly sensitive purity test Verification of peptide structures was mainly based on amino acid analysis and fast atom bombardment-mass spectrometry (FAB-MS). For amino acid analyses, peptides were hydrolyzed in 6N HC1 containing a small amount of phenol for 24 h at 110°C in deaerated tubes (in some case hydrolyses lasting for 12 and 48 h were also performed to take into account amino acid degradation). Hydrolysates were analyzed on a Beckman Model 121 C amino acid analyzer equipped with a system AA computing integrator. FAB mass spectrometry was used to estabUsh the correct molecular weights of the peptides.

Detailed description of the invention

The invention will now be described in more detail by the foUowing examples.

EXAMPLE 1

Preparation of H-Tyr-Tic-Cha-Phe-OH

Boc-Phe-O-resin (lg, 0.61 mmol Boc-Phe/g resin; Peninsula, Belmont, CA) was washed ^■ with reagents in the following sequence: CH₂C1₂ (3x 1 min), 50% (v/v) TFA in CH₂C1₂ (30

min), CH₂C1 (5x1 min), 10% (v/v) DIEA in CH₂C1₂ (2x5 min), CH₂C1₂ (5x1 min). Boc- Cha-OH (412 mg, 1.52 mmol) was then coupled using HOBt (205 mg, 1.52 mmol) and DCC (313 mg, 1.52 mmol) in C^Cl^MF (3:1, v v) for 17h. The resin was then washed

with CH C1₂ (3x1 min), EtOH (1 min), CH₂C1 (3x1 min). This sequence of washes and reactions was repeated for the addition of each of the residues with the following modifications. After coupling of Boc-Tic-OH the resin was washed with C^Cl^MF (3:1, v/v) (3x) and

a recoupling step using the same amounts of Boc-Tic-OH, HOBt and DCC in CH α₂/DMF (3: 1, v/v) was performed for another 17h. The same recoupling step was also carried out to couple Boc-Tyr(Boc)-OH. After final deprotection with 50% (v/v) TFA in CH₂C1₂ (30

min), the resin was washed with CH₂Q (3x1 min) and EtOH (3x1 min) and was dried in a desiccator. The dry resin was treated with 20 ml of HF plus 1 ml of anisole (per gram of resin) first for 90 min at 0^°C and then for 15 min at room temperature. After evaporation of the HF, the resin was extracted three times with Et₂O and, subsequently three times with

7% AcOH. The crude peptide was then obtained in solid form through lyophilization of the combined acetic acid extracts.

The peptide was purified by gel filtration on a Sephadex-G-25 column in 0.5 N AcOH foUowed by reversed-phase chromatography on an octadecasilyl siUca column with a linear gradient of 0-80% MeOH in 1% TFA. After solvent evaporation the pure peptide was dissolved in cone. AcOH and was obtained in soUd form through lyophilization.

Yield: 45 mg FAB-MS :MH⁺ = 640

TLC (siUca) Rf 0.75 n-BuOH/AcOH/H₂O (4/1/5, organic phase)

Rf 0.70 n-BuOH/Pyridine/AcOH/H₂O (15/10/3/12) Amino acid analysis: Tyr 0.96, Phe 1.00 EXAMPLE 2 Preparation of H-Tyr-TicψrCty-NHICha-Phe-OH

The synthesis of this peptide was performed as in the case of EXAMPLE 1 using the same resin except that the introduction of a reduced peptide bond between the Tic² and Cha residue required a reductive alkylation reaction between Boc-Ti*- aldehyde and the amino group of the resin-bound H-Cha-Phe dipeptide.

Preparation of N-t-butoxycarbonyl-L-1.2.3.4-tetrahydroisoquinoline-3-aldehyde (Boc-Tic aldehyde) via N-t-butoxycarbonyl-L-L2.3,4-tetrahydroisoquinoUne-3-N-methoxy. N- methylamide.

BOP (benzotriazol-l-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate) (3.48 g, 10 mmol) was added to a stirred solution of Boc-Tic-OH (2.8 g, 10 mmol) and triethylamine (1.33 ml, 10 mmol) in CH₂C1₂. After five minutes, N-dimethylhydroxylamine hydrochloride (1.2 g, 12 mmol) and triethylamine (1.68 ml, 12 mmol) were added to the solution. The reaction was carried out for 17h. Subsequently, the reaction mixture was diluted with dichloromethane and washed with 3N HC1, a saturated aqueous solution of

NaHCO3 and a saturated aqueous solution of NaCl. The organic solution was dried over

MgSO4 prior to evaporation of the solvent. The resulting crude product of N-t- butoxycarbonyl-L- 1 ,2,3,4- tetrahydroisoquinoUne-3-N-methoxy, N-methylamide was purified by chromatography on a silica gel column in EtOAc/hexane(l:2, v/v).

Yield: 2.1 g (65%), oil TLC (sffica) Rf 0.57 EtOAc/hexane ( 1/1 ) Rf 0.30 EtOAc/hexane (1/2)

NMR (CDC1₃) δ 1.45 (9H,t-butyl), 3.00 (2H,H-4), 3J8 (3H, NCH3), 3.8(3H, OCH₃),

4.42-4.90(3H, 2H-1 and 1H-3), 7J7(4H, ar) To a stirred solution of N-t-butoxycarbonyl-L-l,2,3,4-tetrahydroisoquinoUne-3-N-methoxy, N-methylamide (1.2 g, 4mmol) in 30 ml ether 190 mg (5 mmol) of Uthium aluminium hydride were added. The reduction reaction was carried out for lh and the reaction mixture was then hydrolyzed with a solution of KHSO4 (954 mg, 7 mmol) in water (20 ml). Subsequently, the aqueous phase was separated and extracted with three 50 ml portions of ether. The four organic phases were combined, washed with 3 N HC1, a saturated aqueous solution of NaHCO3 and a saturated aqueous solution of NaCl, and finally dried over

MgSO4. After solvent evaporation the aldehyde was obtained in pure form as an oil.

Yield: 635 mg (60%), oil

TLC (siUca) Rf 0.84 EtOAc/hexane ( 1/1 ) Rf 0.57 EtOAc/hexane (1/2)

NMR(CDC1₃) δ 1.5 (9H, t-butyl), 3.0-3.27 (2H, H-4), 4.4-4.8 (3H, 1H-3 and 2H-1), 7.0 -

7.2 (4H, ar), 9,43 (IH, CHO)

Reductive alkylation reaction between Boc-Tic aldehyde and the H-Cha-Phe-O resin

The resin was washed with DMF (2x1 min) and then Boc-Tic aldehyde (392 mg, 1.52 mmol) in DMF containing 1% AcOH was added to the resin. Sodium cyanoborohydride (115 mg, 1.83 mmol) was then added portionwise over a period of 40 min and the reaction was allowed to continue for 3h.

After coupling of the N-terminal tyrosine residue and deprotection the peptide was cleaved from the resin, purified and lyophiUzed as described in EXAMPLE 1. Yield: 285 mg FAB-MS: MH⁺=627

TLC (silica) Rf 0.73 n-BuOH/AcOH/H₂O (4/1/5, organic phase) Rf 0.85 n-BuOH/pyridine/AcOH/H₂O (15/10/3/12)

The compounds of Examples 3-14 have been synthesized as described for Example 1 above, and the compound of Example 15 was synthesized as described for Example 2 above.

The compounds in Table 1 according to the invention have been synthesized and tested as δ antagonists.

TABLE 1

Pharmacological testing in vitro of δ opioid antagonists

Biosassys based on inhibition of electricaUy evoked contractions of the mouse vas deferens (MVD) and of the guinea pig ileum (GPI) were made. In the GPI assay the opioid effect is primarily mediated by μ opioid receptors, whereas in the MVD assay the inhibition of the . contractions is mostly due to interaction with δ opioid receptors. Antagonist potencies in these assays are expressed as so-called K^-values (H.W. KosterUtz & AJ. Watt, Br. J.

Pharmacol.21, 266-276 (1968)). Agonist potencies are expressed as IC50 values

(concentration of the agonist that produces 50% inhibition of the electrically induced contractions).

Bioassays Using Isolated Organ Preparations

The GPI and MVD bioassays were carried out as reported in P.W. Schiller et al., Biochem. Biophys. Res. Commun 85, 1332-1338 (1978) and J. Di Maio et al., j. Med. Chem. 25, 1432-1438 (1982). A log dose-response curve was determined with [Leu⁵]enkephalin as standard for each ileum and vas preparation, and IC50 values of the compounds being tested were normaUzed according to A.A Waterfield et al., Eur. J. Pharmacol. 58, 11-18 (1979). Ke values for the δ opioid antagonists were determined from the ratio of IC50 values (DR)

obtained in the presence and absence of a fixed antagonist concentration (a) (K = a/(DR-l))

H.W. KosterUtz & AJ. Watt, Br. J. Pharmacol. 3 , 266-276 (1968). These determinations were made with the MVD assay, using two different δ-selective agonists DPDPE and [D- Ala²]deltorphin I.

Conclusion

All compounds show high δ antagonist properties.

Peptides containing a cyclohexylalanine (Cha) residue in the 3-position of the peptide sequence are more potent δ antagonists than corresponding peptides with an aromatic amino acid in position 3. - All compounds showed no μ antagonist activity in the GPI assay at concentrations as high as 10 μM.

In the GPI assay most compounds showed very weak partial μ agonist activity (maximal inhibition of electricaUy evoked contractions ranging from 20% to 53%)

Opioid receptor binding assays

μ and δ opioid receptor binding constants (Kj^μ, Kj^δ) of the compounds were determined by displacement of relatively selective μ and δ radioligands from binding sites in rat brain membrane preparations (calculated from the measured IC50 values on the basis of the equation by Cheng & Prusoff (Y.C. Cheng and W.H. Prusoff (Biochem. Pharmacol. 22, 3099-3102 (1973)). Opioid receptor binding studies

The μ-, δ- and -opioid receptor affinities of aU new analogs were determined in binding assays based on displacement of μ-, δ-and K-selective radioUgands from rat brain membrane binding sites. In the case of K-ligands guinea pig brain homogenates were used, since the relative proportion of K-binding sites is higher in guinea pig brain than in rat brain. The experimental procedure being used in our laboratory represents a modified version of the binding assay described by Pasternak et al. (Mol. Pharmacol. 11, 340-351, (1975)). Male Sprague-Dawley rats (300-350 g) from the Canadian Breeding Laboratories were decapitated and after removal of the cerebeUum the brains were homogenized in 30 volumes of ice-cold standard buffer (50 mM Tris-HCl, pH 7.7). After centrifugation at 30,000 x g for 30 min at 4°C the membranes were reconstituted in the original volume of standard buffer and incubated for 30 min at 37° C (to realease bound endogenous ligands). Subsequent centrifugation and resuspension of the peUet in the initial volume of fresh standard buffer yielded the final membrane suspension. AUquots (2 ml) of the membrane preparations were incubated for 1-2 h at 25°C with 1 ml standard buffer containing the peptide to be tested and one of the foUowing radioUgands at the final concentration indicated: [³H]DAMGO, μ-selective, 0.7 nM; [³H]DSLET, [1HJDPDPE, or [³H]TIPP, δ- selective, 1.0 nM; and [³H]U69,563, K-selective, 0.5 nM. The incubation was terminated by filtration through Whatman GF/B filters under vacuum at 4°C. FoUowing two washings with 5 ml portions of ice-cold standard buffer the filters were transferred to scintillation vials and treated with 1 ml Protosol (New England Nuclear) for 30 min prior to the addition of 0.5 ml acetic acid and 10 ml Aquasol (New England Nuclear). After shaking for 30 min the vials were counted at an efficiency of 40-45%. All experiments were performed in dupUcate and repeated at least three times. Specific binding of each of the three radioUgands was defined by performing incubations in the presence of cold DAMGO, DSLET and U69,563, respectively, at a concentration of 1 micromolar. Values of half -maximal inhibition (IC50) of specific binding were obtained graphicaUy from semilogarithmic plots.

From the measured IC50-values, binding inhibition constants (Kj)were then calculated based on Cheng and Prusoff s equation (Biochem, Pharmcol. 22, 3099-3102 (1973)). Ratios of the Kj-values in the μ-, δ- and K-representative binding assays are a measure of the receptor selectivety of the compound under investigation (e.g. Kj^μ/Kj^δ indicates the selectivity for δ- receptors versus μ-receptors). None of the compounds according to the claimed invention had significant affinity for K-receptors.

Potential use

The δ antagonists may be used in combination with analgesics of the μ agonist type (e.g. morphine) to prevent the development of tolerance and dependence, as suggested by the results of E.E. Abdelhamid et al., J. Parmacol. Exp. Ther. 258. 299-303 (1991).

The δ antagonists according to the invention may also be therapeutically useful as immunosuppressive agents. Immunosuppressive effects of the less δ-selective and less "pure" δ antagonist naltrindole have been described by K. Arakawa et al. Transplantation Proc. 24, 696-697 (1992); Transplantation 53, 951-953 (1992).

Abbreviations

Aib= α-aminoisobutyric acid Boc= tert-butoxycarbonyl Cha — cyclohexylalanine

DAMGO= H-Tyr-D-Ala-Gly-Phe(N^αMe)-Gly-ol DCC= dicyclohexylcarbodiimide DIEA= dϋsopropylethylamine Dmt= 2',6'-dimethyltyrosine

DPDPE= [D-Pen²,D-Pen⁵]enkephalin DSLET= H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH FAB-MS= fast atom bombardment mass spectrometry GPI= guinea pig Ueum

HOBt= 1-hydroxybenzotriazole

MVD= mouse vas deferens

Tic= l,2,3,4-tetrahydroisoquinoUne-3-carboxylic acid

TIPP= H-Tyr-Tic-Phe-Phe-OH U69,593= (5α, la, 8β)-(-)-N-methyl-[7-(l-pyιτolidinyl)-l-oxaspiro[4,5]dec-8- yl] benzeneacetamide

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Astra AB

(B) STREET: Patent Department

(C) CITY: Sodertalje

(E) COUNTRY: Sweden

(F) POSTAL CODE (ZIP): S-151 85

(G) TELEPHONE: 46-8 553 26 000 (H) TELEFAX: 46-8 553 28820

(I) TELEX: 19237 astra s

(ii) TITLE OF INVENTION: New opioid peptide antagonists (iii) NUMBER OF SEQUENCES: 15

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modiiϊed-site

(B) LOCATION: 2

(D) OTHER INFORMATION: label= X /note=

"X=Tic=l,2JJ-tetrahydroisoquinoline-3-caιboxylic acid"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= X /note= "X=Cha=cyclohexylalanine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Tyr Xaa Xaa Phe 1 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note= "X=Tic(psi)[CH2-NH]; Tic= 1,2,3 J-tetrahydroisoquinoline-3 -carboxylic acid"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= X /note= "X=Cha=cyclohexylalanine"

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 2:

Tyr Xaa Xaa Phe 1

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note=

"X=Tic= 1 ,2,3 ,4-tetrahydroisoquinoline-3-carboxylic acid"

(ix) FEATURE:

(A) NAME/KEY: Peptide

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= X /note= "X=Cha=cyclohexylalanine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Tyr Xaa Xaa Phe 1

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note=

"X=Tic= 1,2,3 ,4-tetrahydroisoquinoline-3 -carboxylic acid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Tyr Xaa Leu Phe 1

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note=

"X=Tic= 1 ,2,3 J-tetrahydroisoquinolϊne-3 -carboxylic acid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Tyr Xaa Val Phe 1 (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= x /note=

"X=Tic= 1,2,3 J-tetrahydroisoquinoline-3 -carboxylic acid"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= X /note= "X=Nva=norvaIine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Tyr Xaa Xaa Phe 1

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: label= x /note=

"X=l,2,3,4-tetrahydroisoquinoline-3-caιboxylic acid"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= X /note= "X=norleucine" (xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 7:

Tyr Xaa Xaa Phe 1

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note=

"X=Tic= 1 ,2,3,4-tetrahydroisoquinoline-3 -carboxylic acid"

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 8:

Tyr Xaa He Phe 1

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note=

"X=Tic=l,2,3,4-tetrahydroisoqmnoline-3-carboxylic acid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Tyr Xaa Thr Phe 1 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note=

"X=Tic= 1 ,2,3 ,4-tetrahydroisoquinoline-3 -carboxylic acid"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Tyr Xaa Met Phe 1

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label= X /note= "X=Dmt=2',6'-dimethyltyrosine"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label=- X /note=

"X=Tic=l,2JJ-tetrahydroisoquionoline-3-carboxyl ic acid"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: Λabel= X /note= "X=Cha*=cyclohexylalanine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Xaa Xaa Xaa Phe 1

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: label= X

/note= "X-=D-Dmt; Dmt=2',6^,-dimethyltyrosine"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note=

"X=Tic-=l,2J,4-tetrahydroisoquinoline-3-carboJ 'lic acid"

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: label= X /note= "X=Cha=cyclohexylalanine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Xaa Xaa Xaa Phe 1

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label= X /note= "X=Dmt=2',6'-dimethyltyrosine"

(ix) FEATURE:

(A) NAME KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note=

"X=Tic= 1 ,2,3 ,4-tetrahydroisoquinoline-3 -carboxylic acid"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= x /note= "X=Cha=cyclohexylalanine"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Xaa Xaa Xaa Phe 1

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: Λabel= X /note= "X=Tyr(3'-I)"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /label= X /note=

"X=Tic=l,2J,4-tetrahydroisoquinoline-3-carboxylic acid"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= X /note= "X=Cha=cyclohexylalanine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Xaa Xaa Xaa Phe 1

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: N-terminal

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /label= X /note= "X=Dmt=2\6'-dimethyltyrosine"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: label= X /note= "X=Tic(psi)[CH2-NH]; Tic=l,2,3,4-tetrahydroisoqiunoline-3-carboxylic acid"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3

(D) OTHER INFORMATION: /label= X /note= "X=Cha=cyclohexylalanine"

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 15:

Xaa Xaa Xaa Phe 1

Claims

1. A compound of the formula

wherein

R₁ is H;CH₃(CH₂)_n- wherein n= 0-12;-CH₂-CH₂

-CH₂-CH = CH₂ ;orargιnιne;

R₂ is H; CH₃(CH₂)_n - wherein n = 0-12; CH₃-;

-CH₂-CH=CH₂;

R₃, R₄, R₅ and R₆ are all H; or

R₄ and R₅ are both H and R₃ and R₆ is each a C₁-C₆ alkyl group; or R₃, R₅ and R₆ are all H and R₄ is F, Cl, Br, OH, NH₂ or NO₂; R₇ is C=O or CH₂;

R₈ is H or a C₁-C₆ alkyl group;

R₉ is selected from R₁₀ is OH, NH₂ or

wherein

R₁₁ is H, NO₂, F, Cl, Br or I; q is 0-3;

R₁₂ is COOH, CONH₂, CH₂OH, or any additional amino acid or peptide segment; or R₁₀ is

wherein

R₁₂ is as defined above.

2. A compound according to the formula I of claim 1, wherein

R₁ is selected from H or CH₃;

R₂ is selected from H or CH₃;

R₃ is selected from H or CH₃;

R₄ is H;

R₅ is H;

R₆ is selected from H or CH₃;

R₇ is selected from CO or CH₂;

R₈ is selected from H or CH₃; R₉ is selected from

R₁₀ is selected from R_{1 1} wherein R_{1 1} is H, NO₂, F, Cl, Br or I,

q is 1-3, and R₁₂ is COOH.

3. A compound according to the formula I of claim 1 , wherein

R₁ is selected from H or CH₃;

R₂ is selected from H or CH₃;

R₃ is selected from CH₃;

R₄ is selected from H;

R₅ is selected from H;

R₆ is selected from CH₃;

R₇ is selected from CH₂;

R₈ is selected from H or CH₃;

R₉ is selected from

R₁₀ is selected from R₁₁ wherein R_{1 1} is H, q is 1, and R₁₂ is COOH.

4. A compound according to formula I of claim 1, wherein R₇ is a part of a reduced peptide bond.

5. A compound according to formula I of claim 1, wherein R₄ and R₅ are hydrogen and R₃ and R₆ are both methyl groups.

6. A compound according to formula I of claim 1, wherein

7. A compound according to claim 1, being

H-Tyr-Tic-Cha-Phe-OH;

H-Tyr-Ticψ[CH₂-NH]Cha-Phe-OH;

H-Tyr-Tic-Cha-Phe-NH₂;

H-Tyr-Tic-Leu-Phe-OH;

H-Tyr-Tic-Val-Phe-OH;

H-Tyr-Tic-Nva-Phe-OH;

H-Tyr-Tic-Nle-Phe-OH;

H-Tyr-Tic-lle-Phe-OH;

H-Tyr-Tic-Thr-Phe-OH;

H-Tyr-Tic-Met-Phe-OH;

H-Dmt-Tic-Cha-Phe-OH;

H-D-Dmt-Tic-Cha-Phe-OH;

H-Dmt-Tic-Cha-Phe-NH₂;

H-Tyr(3 -I)-Tic-Cha-Phe-OH; or H-Dmt-Ticψ[CH₂-NH]Cha-Phe-OH.

8. A compound according to claim 7, being

H-Tyr-Tic-Cha-Phe-OH;

H-Tyr-Ticψ[CH₂-NH]Cha-Phe-OH;

H-Tyr-Tic-Val-Phe-OH;

H-Dmt-Tic-Cha-Phe-OH;

H-D-Dmt-Tic-Cha-Phe-OH; and H-Dmt-Ticψ[CH₂-NH]Cha-Phe-OH.

9. A compound according to claim 1 for use in therapy.

10. A compound according to claim 1 for use as an analgesic.

11. A compound according to claim 1 for use as an immunosuppressive agent.

12. A method for preparing peptides of the formula I of claim 1 by means of soUd-phase synthesis, whereby the coupling step is performed in an inert solvent, using an appropriate coupling agent.

13. The method according to claim 12 wherein the inert solvent is CH₂Cl₂, DMF or a mixture of CH₂Cl₂/DMF(3:1 v/v) and the coupling agents are N, N'-dicyclohexyl- carbodiimide/1-hydroxybenzotriazole, N,N'-dusopropylcarbod iimide/1-hydroxy- benzotriazole or benzotria-zolyloxytris-(dimethylamino)phosphonium hexafluoro- phosphate.

14. A method according to claim 13 whereby an additional step for attachement of the Boc-Tic-OH residue to the dipeptide resin (recoupling step) and an additional step for the attachement of the Boc-Tyr(Boc)-OH residue to the peptide resin (recoupling step) is performed.

15. The method for preparing peptides of the formula I according to claim 1 containing a reduced peptide bond (-CH₂-NH-) between the Tic² residue and the 3-position residue, whereby the reaction between Boc-Tic aldehyde and the amino group of the resin-bound peptide is based on a reductive alkylation employing sodium cyanoborohydride in acidified DMF.

16. A method for preparing N-t-butoxycarbonyl-L-1, 2, 3, 4-tetrahydroisoquinoline-3- aldehyde (Boc-Tic aldehyde) used for preparing peptides with a reduced peptide bond (-

CH₂-NH-) between the Tic² residue and the 3-position residue according to claim 12.

17. A pharmaceutical preparation comprising an effecive amount of a compound according to claim 1 together with one or more pharmaceutical carriers.

18. Use of a compound according to the formula I of claim 1 in the manufacture of a medicament for use as an analgesic.

19. Use of a compound according to the formula I of claim 1 in the manufacture of a medicament for use as an immunosuppressive agent.

20. A method for the treatment of pain whereby an effective amount of a compound of the formula I according to claim 1 is administered to a patient in the need of such treatment.

21. A method for producing immunosuppressive effects whereby an effective amount of a compound of the formula I according to claim 1 is administered to a patient in the need of such treatment.