AP508A

AP508A - Novel opioid analogs that are 9 (delta) opioid receptor antagonists, their syntheis and their use as analgesic and immunosupressive compounds.

Info

Publication number: AP508A
Application number: APAP/P/1994/000605A
Authority: AP
Inventors: Peter Schiller
Original assignee: Ab Astra
Priority date: 1993-01-05
Filing date: 1994-01-05
Publication date: 1996-07-22
Also published as: NZ259597A; PL309778A1; IL108169A0; FI953302A0; LV10962A; KR960700269A; AU5844894A; AP9400605A0; WO1994015959A1; CN1096515A; SK83995A3; CZ171195A3; CA2152380A1; YU394A; ZA9455B; EE9400326A; NO952650L; LV10962B; HUT72597A; SI9400005A

Abstract

This invention is related to a new class of opiod peptide analogs that are 0 opioid receptor antagonists as well as to their synthesis and their use as analgesic and immunosuppressive compounds.

Description

The field of the Invention

C 10 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

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 δantagonist potency as the compounds according to this invention but is much less δ selective. Furthermore, naltrindole has also quite high μ opioid receptor affinity (1^= 12nM) in the receptor binding assay and potent antagonist properties (K_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 enkephalin analog N,N-diallyl-Tyr-Aib-Aib-Phe25 Leu-OH (ICI 174864) described by R. Cotton, et al. in Eur. J. Pharmacol. 97, 331332 (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).

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Prior art

Tetrapeptides, which are potent 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 Conference (INRC) Meeting, Keystone, CO, June 24-29, (1992) and at the 2nd Japan Symposium on Peptide Chemistry, Shizuoka, Japan, Nov. 9-13,

1992.

I

The Invention r io

It has now unexpectedly been found that the compounds of the following formula I have

- extraordinary selectivity for the δ receptor

- high potency as δ antagonists

- total lack of μ antagonist properties

- mixed μ agonist/δ antagonist properties in some cases (TIPP analogs with a Cterminal carboxamide group)

The compounds according to the present invention have the formula I

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AP.00508 r

r io

wherein

Rj is H, CHg(CH2)_n- wherein n = O-12-.CH2-C *-O

CH₂—<3 CH2*CH=CH2 or arginine;

AP/P/ 94/00605

R₂ is H, CH₃(CH₂)_n- wherein n = 0-12-, CH₂, CH₂-<] ,

CH₂-CH=CH₂;

Rg, R₄, Rg, Rg are all H or

R₄ and Rg are both H and Rg and Rg are both lower alky, groups or Rg, Rg, Rg are all H and R₄ is F, Cl, Br, OH, ΝΉ2 or NO2;

R7 is C=O or CH2;

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AP. 00508

Rg is H or a lower alkyl group R9 is

(CH₂)_m-CH wherein m is 0-2

/

N^NH

CH₂—CH or

AP . Ο Ο 5 Ο 8

AP/P/ 9 4 / 0 0 60 5 wherein

Rjq is H, F, Cl, Br or I and m is 0-2; Rj j is OH, NH2 ^or

Jr

^13 (CH₂)_m-CH — NH wherein R^ is H, NC^, F, Cl, Br or I, m is 0-2, Rj^ is COOH, CONH2, CH2OH, or any additional amino acid or peptide segment, or

wherein

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

with the exceptions of (i) the compounds wherein R,, R_:. R₃, R₄, R₅. R^, and R_s are all H, R_? is C = O and

R9 is

-CH and R,, is Phe-OH, Phe-NH_;, OH or NH_:, and (ii) the compound H-Tyr-Tic-Phe-PheLeu-Nle-Asp-NH·,.

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A lower alkyl group has according to this specification 1-6 carbon atoms.

Especially preferred compounds according to the invention are those wherein R? is

CH2 (as part of a reduced peptide bond). A reduced peptide bond gives the compound higher δ antagonist potency, increased δ-selectivity, better stability in organic solvents and resistance to enzymatic degradation.

AP.00508

Further preferred compounds according to the invention are those, wherein R4 and R^ are H and Rj and R^ are both methyl groups.

Synthesis

Most Boc-amino acid derivatives used in the peptide syntheses were 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) and 2-aminotetralin 2-carboxylic acid as described by P.W. Schiller et al in J. Med. Chem 34, 3125-3132 (1991).

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 Schiller et al, Biochemisty 16, 1831-1832 (1977)). Couplings were performed in CH2CI2 or DMF, using dicyclohexylcarbodiimide/l-hydroxybenzotriazole (DCC/HOBt) as coupling agents. 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).

S 0 9 0 0 / » 6 /d/dV

BAD ORIGINAL '7 ,Ar

Analogs containing the C^NH peptide bond isostere were prepared by solid-phase synthesis according to a procedure developed by Sasaki and Coy (Y. Sasaki &

D.H. Coy, Peptides 8, 119-121 (1987)). With this method the C^NH peptide bond can be directly introduced by the reductive alkylation reaction between a Boc5 amino acid aldehyde and an amino group on the resin-bound peptide employing sodium cyanoborohydride in acidified DMF. No significant racemization was observed with this method. Boc-Tic aldehyde was synthesized via the corresponding Boc-Tic N-methoxy-N-methylamide by the reportedly racemizationfree L1AIH4 reduction method (J.A Fehrcntz & B. Castro, Synthesis, 676-678 (1983)). Peptides containing reduced peptide bonds were cleaved from the resin and deprotected by treatment with HF/anisole as described above.

Crude products obtained from solid-phase peptide synthesis required extensive purification by various chromatographic techniques or by other methods. Following

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-pyridinewater two phase systems), ion exchange chromatography (DEAE-Sephadex, SPSephadex 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 . ·. 1 . · ;

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I 1 nr acid analyses, peptides were hydrolyzed in 6N HCI 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 establish the correct molecular weights of the peptides.

EXAMPLES OF PARTICULAR ANALOGS EXAMPLE 1

H-Tyr-Tic-Hfe-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: CH2CI2 (3x 1 min), 50% (v/v) TFA in CH2CI2 (30 min), CH2CI2 (5x1 min), 10% (v/v) DIEA in CH2CI2 (2x5 min), CH2CI2 (5x1 min). Boc-Hfe-OH (425 mg, 1.52 mmol) was then coupled using HOBt (205 mg, 1.52 mmol) and DCC (313 mg, 1.52 mmol) in O^C^/DMF (3:1, v/v) for 17h. The resin was then washed with CH2CI2 (3x1 min), EtOH (1 min), CH2CI2 (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^Clj/DMF (3:1, v/v) (3x) and a recoupling step using the same amounts of Boc-Tic-OH, HOBt and DCC in CH2CI2/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 CH2CI2 (30 min), the resin was washed with CH2CI2 (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 first for 90 min at 0^eC and then for 15 min at room temperature. After evaporation of the HF, the resin was extracted three times with Et20 and, subsequently three

AP/P/ 94/00605

BAD ORIGINAL ίο 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 5 AcOH followed by re versed-phase chromatography on an octadecasilyl silica 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 solid form through lyophilization.

Yield: 45 mg

FAB-MS^H* = 648

TLC (silica) 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, Hfe 1.03, Phe 1.00 15

EXAMPLE 2

H-Tyr-TicT[CH₂-NH]Phe-Phe-OH

The synthesis of this peptide was performed as in the case of EXAMPLE 1 using 20 the same resin, except that the introduction of a reduced peptide bond between the

Tic³ and Phe³ residue required a reductive alkylation reaction between Boc-Tic aldehyde and the amino group of the resin-bound H-Phe-Phe dipeptide.

bad original

Preparation of N-t-butoxycarbonyl-L-1,2,3,4-tetrahydroisoquinoline-3-aldehyde (Boc-Tic aldehyde) via N-t-butoxycarbonyl-L-1,2,3,4-tetrahvdroisoquinoline-3-Nmethoxy, 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 CHjC^. After five minutes, Ndimethylhydroxylamine 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 HCI, a saturated aqueous solution of NaHCO₃ and a saturated aqueous solution of NaCl. The organic solution was dried over MgSC^ prior to evaporation of the solvent The resulting crude product of N-t-butoxycarbonyl-L-1,2,3,4tetrahydroisoquinoline-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 (silica) 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), 3.18 (3H, NCH₃),

3.8(3H, OCH₃), 4.42-4.90(3H, 2H-1 and 1H-3), 7.17(4H, ar)

To a stirred solution of N-t-butoxycarbonyl-L-1,2,3,4-tetrahydroisoquinoline-3-Nmethoxy, N-methyamide (1.2 g, 4mmol) in 30 ml ether 190 mg (5 mmol) of lithium 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 HCI, a saturated aqueous solution of NaHCO₃ and a

AP/P/ 9 4 / 0 0 6 0 5

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ΛΡ 00508 saturated aqueous solution of NaCl, and finally dried over MgSO^. After solvent evaporation the aldehyde was obtained in pure form as an oil.

Yield: 635 mg (60%), oil

TLC (silica) 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 (1H, CHO)

Reductive alkylation reaction between Boc-Tic aldehyde and the H-Phe-Phe-0 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 lyophilized as described in EXAMPLE 1.

Yield: 180 mg

FAB-MS: MH⁺ = 633

TLC (silica) Rf 0.73 n-BuOH/AcOH/H₂O (4/1/5, organic phase)

Rf 0.69 n-BuOH/pyridine/AcOH/H₂O (15/10/3/12)

Amino acid analysis: Tyr 0.95, Phe 1.00

The following compounds according to the invention have been synthesized and tested as δ antagonists.

Ar . ν υ -· ν v r

C 10

Pharmacological testing in vitro of δ opioid antagonists

a) Biosassys based on inhibition of electrically evoked contractions of the mouse vas deferens (MVD) and of the guinea pig ileum (GPI). 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_e-values (H.W. Kosterlitz & A.J. Watt, Br. J. Pharmacol. 33, 266-276 (1968)). Agonist potencies are expressed as IC50 values (concentration of the agonist that produces 50% inhibition of the electrically induced contractions).

Bioassavs 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 determind with [Leu^]enkephalin as standard for each ileum and vas preparation, and IC50 values of the compounds being tested were normalized according to A.A Waterfield et al., Eur. J. Pharmacol. 58, 11-18 (1979). K_e values for the TIPP-related antagonists were determind 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. Kosterlitz &

A.J. Watt, Br. J. Pharmacol. 33, 266-276 (1968). These determinations were made with the MVD assay, using three different δ-selective agonists ([Leu^] enkephalin, DPDPE and (D-Ala²]deltorphin I].

0 9 0 0 /46 /d/dV

In the following Table 1 the results are given.

Table 1

K_-values of TIP(P) related peptides in the MVD assay (Antagonist potencies r~5-1 <

against the 6 agonists [Leu5] enkephalin, [D-Pen ,D-Pen ]enkephalin (DPDPE) 5 and [D-Ala²]deltorphin I) (Prior known compounds are marked (P)

K_e (nM)^a
Compound	(Leu⁵) Enkephalin	DPDPE	[D-Ala²] deltor- phin I
I H-Tyr-Tic-PhePhe-OH(P)(TIPP)	5.8610.33	4.8010.20	2.9610.02
H-Tyr-Tic-Phe- Phe-NH₂(P)(TIPP-NH₂)^b	15.712.4	18.012.0	14.412.2
H-Tyr-Tic-Phe- OH(P)(TIP)	11.7±1.8	16.111.9	12.611.8
H-Tyr-Tic-Phe- NH₂(P)(TIP-NH₂)	43.918.9	96.8114.1	58.917.7
Tyr(N^aMe)-Tic- Phe-Phe-OH	1.0110.15	1.2210.17	0.43610.071
Tyrib^CpmJ-Tic- Phe-Phe-OH	29.614.4	28.210.5	32.511.3
Tyr(N^aHex)-Tic- Phe-Phe-OH	9.3711.18	4.2810.56	10.611.9

f ι—/

1 ς'

K_e (nM)^a
Compound	[Leu⁵] Enkephalin	DPDPE	[D-Ala²] deltorphin I
Tyi-Cf^Et^-Tic- Phe-Phe-OH	3.3910.16	0.89310.112	2.3010.18
H-Dmt-Tic-Phe- Phe-OH	0.16910.015	0.19610.022	0.13010.017
H-Dmt-Tic-Phe- Phe-NH₂ ^b	0.22110.028	0.20910.037	0.26010.064
H-Tyr(3-F)-Tic- Phe-Phe-OH	5.8810.72	13.010.7	8.7311.21
H-Tyr(3-Cl)- Tic-Phe-Phe-OH	18.012.2	20.411.5	19.911.7
H-Tyr(3-Br)- Tic-Phe-Phe-OH	18.212.7	31.314.2	23.912.7
H-Tyr-Tic-v(CH₂- NH]Phe-OH	9.1211.57	9.0610.70	8.2411.12
H-Tyr-Tic-v(CH₂- NH]Phe-Phe-OH	2.4610.35	2.8910.23	2.8510.13
H-Dmt-Tic\g[CH₂- NH]Phe-Phe-OH	0.25910.043	0.19610.033	0.15710.028
H-Dmt-Ticy[CH₂- NH]Phe-Phe-NH₂ ^b	0.47010.078	0.42010.049	0.486+0.058

ΑΡ/Ρ/ 94/00605

K_e (nM)^a
Compound	[Leu⁵] Enkephalin	DPDPE	[D-Ala²] deltorphin I
H-Tyr-Ticy[CH₂- NCH₃]Phc-Phe-OH	6.2810.14	4.7610.48	2.8910.31
H-Tyr-Tic-T[CH₂- NH]Hfe-Phe-OH	2.2210.31	2.6410.47	1.9010.17
H-Tyr-Tic-Hfe-Phc-OH	1.2310.18	0.60910.043	0.40810.039
Tyr(NMe)-Ticv[CH₂- NH]Hfe-Phe-OH	0.78010.082	0.90210.135	0.41810.098
H-Tyr-Tic-Phg-Phc-OH	13.811.2	21.512.9	8.3111.75
H-Tyr-Tic-Trp-OH	10.612.1	6.2310.79	5.3610.77
H-Tyr-Tic-Trp-Phe- OH(P)	2.3710.54	2.5610.21	1.6510.18
H-Tyr-Tic-Trp-Phe- NH₂<^P>	3.2410.43	4.6510.92	2.3110.17
H-Tyr-Tic-His-Phe-OH	20.111.8	18.211.6	18.710.7
H-Tyr-Tic-2-Nal-Phe-OH	2.6410.17	4.4110.65	4.1710.58
H-Tyr-Tic-Atc-Phe-OH	1.6310.14	1.8510.16	0.92710.142
H-Tyr-Tic-Phe- Phe(pNO₂)-OH	3.6210.46	3.3010.35	2.79+0.46

♦ΛΙ

K_e (nM)^a
Compound	[Leu⁵] Enkephalin	DPDPE	[D-Ala²] deltorphin I
H-Tyr-Tic-TrpI Phe(pNO₂)-OH	1.8310.10	4.4010.55	2.2710.14
H-Tyr-Tic-Phe-Trp-NH₂	49.514.6	41.315.2	38.613.3
H-Tyr-Tic-Phe-Phe-Val- Val-Gly-NH₂	6.4810.59	6.3611.32	4.9610.77
H-Tyr-Tic-Phe-Phe-Tyr- Pro-Ser-NH₂	4.7810.80	4.6310.43	3.9010.63
H-Tyr-Tic-Trp-Phe-Tyr- Pro-Ser-NH₂	4.2011.13	3.6510.94	3.6510.14
H-Tyr-Tic-Trp- Phe(pNO₂)-Tyr-Pro-Ser- nh₂	3.6810.79	2.4810.34	3.9110.38 P 1,
Naltrindole(P)	0.85010.221	0.63210.161	0.63610,105 J

AP/P/ 9 4 / 0 060 5

Values are means of 3-8 determinations ± SEM ^bH-Tyr-Tic-Phe-Phe-NH₂, H-Dmt-Tic-Phe-Phe-NH₂ and H-Dmt-Ticy(CH₂NH]Phe-Phe-NH₂ are mixed μ agonist/δ antagonists showing IC50s of 17001220 ftp . -^508 nM, 18.2±1.8 nM and 7.71 ±0.32 nM, respectively, in the guinea pig ileum (GPI) assay.

Μ Γ'

Conclusion u Antagonist or μ Agonist behavior of ΤΊΡΡ-related δ antagonists

- All compounds show no μ antagonist activity in the GPI assay at concentrations as high as 10 μΜ.

- ΤΙΡΡ-related peptides with a free C-terminal carboxyl group have very weak μ f 10 agonist potency in the GPI assay (IC50 > ΙΟμΜ). On the other hand, TIPP-derived peptides with a C-terminal carboxamide function show full μ agonist potency in the GPI assay (e.g. H-Dmt-Tic-Phe-Phe-NF^) has an IC50 of 18.2±1.8 nM in the GPI assay.

Opioid receptor binding assays μ and δ opioid receptor binding constants (K^, Kj⁶) 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)).

In the foilwing Table 2 the results of opioid receptor binding assays are given. The ratio / Kj⁶ is a quantitative measure of the δ-selectivity.

The higher the ratio the better the δ-selectivity.

AP. 0 Ο 5 0 8

21)

Opioid receptor binding studies

The μ-, δ- and x-opioid receptor affinities of all new analogs were determined in binding assays based on displacement of μ-, δ-and κ-selective radioligands from rat brain membrane binding sites. In the case of κ-ligands guinea pig brain homogenates were used, since the relative proportion of x-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 cerebellum 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 die 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 pellet in the initial volume of fresh standard buffer yielded the final membrane suspension. Aliquots (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 following radioligands at the final concentration indicated: [³H]DAMGO, μ-selective, 0.7 nM; [³H]DSLET, [³H]DPDPE, or [³H]TIPP, δ-selective, 1.0 nM; and [³H]U69,563, κ-selective, 0.5 nM. The incubation was terminated by filtration through Whatman GF/B filters under vacuum at 4°C. Following 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 duplicates and repeated at least three times. Specific binding of each of the three radioligands 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 (1C50) of specific binding were obtained

AP.00508 graphically from semilogarithmic plots. From the measured IC50-vaIues, 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 κ-representative binding assays are a measure of the receptor c· selectively of the compound under investigation (e.g. K^/Kj indicates the selectivity for δ-receptors versus μ-receptors). None of the compounds according to the claimed invention had significant affinity for K-receptors.

Table 2

C' io

Receptor binding data of opioid peptide analogs ^a
Compound	Kj*¹ [nM]	Kj⁵ [nM]	Κ//Κΐ^δ
H-Tyr-Tic-Phe-Phe- OH(P)(TIPP)	1720150	1.2210.07	1410
H-Tyr-Tic-Phe-Phe- NH₂(P)(TIPP-NH2)	78.817.1	3.0010.15	26.3
H-Tyr-Tic-Phe- OH(P)(TEP)	12801140	9.0711.02	141
H-Tyr-Tic-Phe- NH₂(P)(TIP-NH2)	624±79	12.011.3	52.0
Tyr(N^aMe)-Tic-Phe- Phe-OH	134001600	1.2910.36	10400
TyriN^Hexj-Tic-Phe- Phe-OH	1080180	0.95110.123	1140
H-Dmt-Tic-Phe-Phe-OH	141±0.24	0.24810.046	569

AP/P/ 9 4 / 0 0 6 0 5

Receptor binding data of opioid peptide analogs ^a
Compound	KjM [nM]	Kj⁸ [nM]	κ,^μ/κ,^δ
H-Dmt-Tic-Phe-Phe- nh₂	l.l9±0.l l	0.11810.016	10.1
H-Arg-T yr-Tic-PhePhe-NH₂	10712	4.7910.15	22.3
H-Tyr-Ticy[CH₂- NH]Phe-OH	1O8OOH3OO	1.9410.14	5570
H-Tyr-Ticv[CH₂- NH]Phe-Phe-OH	32281439	0.30810.060	10500
H- Dmt-Tic\\|/(CH₂- NH]Phe-Phe-OH	95.5111.0	1.7010.40	56.2
H-Tyr-Ticv[CH₂- NCH₃]Phe-Phe-OH	134001700	0.84210.116	15900
H-Tyr-Tic-Hfe-Phe-OH	19901170	0.27710.001	7180
H-Tyr-Tic-Phg-Phe-OH	2900017200	3.0110.54	9630
H-Tyr-Tic-Trp-Phe- NH₂(^p)	176121	0.24810.009	709
H-Tyr-Tic-Trp-Phe- OH(P)	17901380	0.30110.042	5950
H-Tyr-Tic-His-Phe-OH	1700013700	1.4810.22	11500
H-Tyr-Tic-l- Nal-Phe-OH	11201130	1.1410.17	982

* »-A^r

Receptor binding data of opioid peptide analogs ^a
Compound	K/ [nMJ	ΐς^δ [nM]	κ,μ/ις⁵
H-Tyr-Tic-2-Nal-Phe- OH	63301130	1.3110.03	4830
H-Tyr-Tic-Phe- Phe(pNO₂)-OH(P)	28901660	0.70310.099	4110
H-Tyr-Tic-Trp- Phe(pNO₂)-OH(P)	1520142	0.33010.004	4610
H-Tyr-Tic-Phe-Trp- NH₂(P)	312175	1.2110.19	258
H-Tyr-Tic-Phe-Phe-Val- Val-Gly-NH₂	32901520	1.4310.25	2300
H-Tyr-Tic-Phe-Phe-Tyr- Pro-Ser-NH₂	6581136	0.90010.196	731
Naltrindole	12.211.9	0.68710.100	17.8

-end of table 2AP/P/ 9 4 / 0 0 6 0 5 ^aValues are means of 3 determinations ± SEM

AP Ο Π 5 P β

Potential use

The pure 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 ai., J. Parmacol.

Exp. Ther. 258, 299-303 (1991). The latter study also suggested that compounds with mixed μ agonist/δ antagonist properties may be therapeutically useful as analgesics that do not produce tolerance and dependence. The TIPP-related peptides with a C-terminal carboxamide group described in this patent are the first mixed μ agonist/δ antagonists known.

The δ antagonists described in the patent 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).

AP . 0 0 5 0 8

Abbreviations

Aib= α-aminoisobutyric acid 5 Atc= 2-aminotetralin-2-carboxylic acid

Boc= tert-butoxycarbonyl Cpm=cyclopropylmethyl DAMGO= H-Tyr-D-Ala-Gly-PheO^MeJ-Gly-ol DCC= dicyclohexylcarbodiimide

DIEA= diisopropylethylamine Dmt= 2,6-dimethyItyrosine DPDPE= (D-Pen²,D-Pen⁵]enkephalin DSLET= H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH Et=ethyl

FAB-MS= fast atom bombardment mass spectrometry GPI= guinea pig ileum Hex= hexyl

Hfe= homophenylalanine HOBt= 1 -hydroxybenzotriazole

MVD= mouse vas deferens

1- Nal= 3-(l’-naphthyl)alanine

2- Nal= 3-(2’-naphthyl)alanine Phe(pNO₂)=4-nitrophenylalanine Phg= phenylglycine

Tic= l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid ΤΠ>= H-Tyr-Tic-Phe-OH TIP-NH₂= H-Tyr-Tic-Phe-NH₂ΤΙΡ(ψ)= H-Tyr-Ticv(CH₂-NH]Phe-OH TIPP= Η-Tyr-Tic-Phe-Phe-OH

TrPP-NH₂=H-Tyr-Tic-Phe-Phe-NH₂

AP/P/ 94/00605

AP . 00508

ΤΙΡΡ(ψ)= H-Tyr-Ticv(CH₂-NHlPhe-Phe-OH Tyr(3-Br)= 3-bromotyrosine Tyr(3-Cl)= 3-chlorotyrosine Tyr(3-F)= 3-fluorotyrosine

Tyr(N^<xMe)= N^mcthyltyrosine

U69,593= (5a, 7a, 8P)-(-)-N-methyl-[7-(l-pyrrolidinyl)-l-oxaspiro[4,5]dec-8 yljbenzeneacetamide

Claims

CLAIMS AP.00508

1. A compound of the formula I wherein

R| is H, CH₃(CH₂)_n- wherein n = 0-12-. CH

2-CH;

AP/P/ 9 4/ 0060 5 ^ch2—<3, CH2CH=CH2 or arginine;

20 R₂ is H, CH₃(CH₂)_n- wherein n = o-12-.CH₂, CH₂—<],

CH₂-CH=CH₂;

AH · 0 0 5 0 8

Rg, ^4’ ^5’ all Η or

R4 and Rg are both H and Rg and Rg are both lower alkyl groups or Rg, Rg, Rg are all H and R4 is F, Cl, Br, OH, NH2 or NO2;

R-j is C=O or CH₂:

5 Rg is H or a lower alkyl group R9 is r

p, 10 wherein m is 0-2 (CH )_m—CH or or

CH₂—CH / ² I or

V

AP. Ο Ο 5 Ο 8

AP/P/ 94/00605 ^Rio—ff—(CH₂)_m—CH wherein

Rjq is H, F, Cl, Br or I and m is 0-2; Rj j is OH, NH2 or

Αί . ύ ύ ί) 0 8 (CH₂)_m-CH — NH wherein Rj2 is H, NC^, F, Cl, Br or I, m is 0-2, Rj^ is COOH, CONH2, CH2OH, or any additional amino acid or peptide segment, or wherein

Rj4 is COOH, CONH2, CH2OH, or any additional amino acid or peptide segment;

with the exceptions of (i) the compounds wherein R,, R,, R₃, R₄, R₅, R^ and R₈ are all H, Rj is C = O and

R9 is and R,, is Phe-OH, Phe-NH,, OH or NH₂, and (ii) the compound H-Tyr-Tic-Phe-PheLeu-Nle-Asp-NH,.

5 4.

5.

r6.

AP. Ο Ο 5 Ο 8

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

3. A compound according to formula I of claim 1, wherein R< and R_s are hydrogen and R₃ and R<, are both methyl groups.

A compound according to any one of claims 1 to 3 for use in therapy.

A compound according to any one of claims 1 to 3 for use as analgesic.

A compound according to any one of claims 1 to 3 for use as in immunosuppressive agent.

7. A method for preparing a compound according to claim 1 which 10 method employs solid-phase synthesis.

8. A method according to claim 7, comprising an additional step of attaching the Boc-Tic-OH residue to the dipeptide resin (recoupling step) and an additional step of attaching the Boc-Tyr(Boc)-OH to the peptide resin (recoupling step).

9. A method according to claim 7 or 8 wherein there is employed, as inert 15 solvent, CH₂C1₂ or a mixture of CH₂C1₂/DMF(3:1 v/v) and the coupling agents are

Ν,Ν’-dicyclohexylcarbodiimide. 1-hydroxy-benzotriazole.

10. A method for preparing a compound according to claim 1 containing a reduced peptide bond (-CH₂-NH-) between the Tic² residue and the 3-position residue, which method comprises a reductive alkylation reaction between Boc-Tic aldehyde and

20 the amino group of the resin-bound peptide employing sodium cyanoborohydride in acidified DMF.

11. The method for preparing N-t-butoxycarbonyl-L-1,2,3,

4-tetrahydroAPIPl 9 4/ 0 0 6 0 5

BAD ORIGINAL isoqu;noline-3-aldehyde (Boc-Tic aldehyde) for preparing peptides containing a reduced peptide bond (-CH₂-NH-) between the Tic² residue and the 3-position residue according to claim 10 which method comprises reduction of N-t-butoxycarbcnyl-L-1.2,3.4tetrahydroisoquinoline-3-N-methoxy, N-methylamide, hydrolysis of the product obtained

5 and subsequently separating the aqueous phase.

12. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1 to 3 together with one or more pharmaceutically acceptable carriers.

13. Use of a compound according to any one of claims 1 to 3 in the 10 manufacture of a medicament for the treatment of pain.

14. Use of a compound according to any one of claims 1 to 3 in the manufacture of a medicament for producing immunosuppressive effects.

BAD ORIGINAL 2

AP.00508

Abstract

Compounds of the formula I as well as methods for their preparation, their pharmaceutical preparations and their use wherein

AP/P/ 9 4/ 0 0 60 5

R| is H, CH^Cl^^- wherein n = 0-12-. CHj-CHg—f \

20 cHg——<^] , CH2*CH=CH2 or arginine;

R₂ is H, CH₃(CH₂)_n- wherein n = 0-12-. CH₂, CH₂—<ζ] CH₂-CH=CH₂;

BAD ORIGINAL &

Rj, R^, Rj, R^ are all H or

R4 and R^ are both H and Rj and R^ are both lower alkyl groups or Rj, Rj, Rg are all H and R4 is F, Cl, Br, OH, NH2 or NO2;

R7 is C=O or CH2;

Rg is H or a lower alkyl group

R9 is

C >-(CH₂)_m-CH wherein m is 0-2 or or r\

N^NH

CH₂—CH / ² I or

Mr

AP/F/ 9 4 / 0 0 6 0 5 ^Rio—4 (C^R2)m QH

25 wherein

R₁₀ is H, F, Cl, Br or I and m is 0-2; R| j is OH, NH2 or

5 9 wherein Rj2 is H, NC^, F, Cl, Br or I, m is 0-2, Rjj is COOH, CONH2, CH2OH, or any additional amino acid or peptide segment, or ^R11 ^is

NH wherein

Rj4 is COOH, CONH2, CH2OH, or any additional amino acid or peptide segment;

with the exceptions of the compounds wherein Rj, R2, Rj, R4, R5, R^ and Rg are all H, R7 is C = O and

R^ is

CH,

CH and R j j is Phe-OH, Phe-NH₂, OH or NH₂- /