NEW PEPTIDE DERIVATIVES WITH delta OPIOID RECEPTOR ANTAGONIST OR MIXED mu AGONIST/ delta ANTAGONIST EFFECTS

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

Description: New peptide derivatives with δ opioid receptor antagonist or mixed μ agonist/δ antagonist effects The field of the Invention This invention is related to a novel class of opioid peptide analogs that are δ opioid receptor antagonists or mixed μ agonist/δ antagonists as well as to their synthesis and their use as analgesics 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). However, naltrindole has also quite high μ opioid receptor affinity (K j =12nM) in the receptor binding assay and, unlike the compounds according to the present invention, has potent μ antagonist properties (K e =29 nM) 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- Phe-Leu-OH (ICI 174864) described by R. Cotton, et al. in Eur. J. Pharmacol. 97, 331-332 (1984). In comparison with some of the antagonists described in this patent application, ICI 174864 has much lower δ antagonist potency in the MVD assay (200 times less potent). Peptides containing the H-Tyr-Tic-Aaa sequence (Tic=l,2,3,4-tetra-hydro- isoquinoline-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), A1575 (1992), at the International Narcotics Research Conference (INRC) Meetings in Keystone, CO, June 24-29 (1992) and in Skovde, Sweden, July 10-15 (1993), at the 2nd Japan Symposium on Peptide Chemistry, Shizuoka, Japan, Nov. 9-13 (1992), at the 22nd European Peptide Symposium Interlaken, Switzerland, Sept. 9-13 (1992), in Proc. Nad. Acad. Sci. USA 89, 11871-11875 (1992), and in J. Med. Chem. 36, 3182-3187 (1993). Peptides structurally related to TIPP that are mixed μ agonist/δ antagonists have recendy been disclosed by P.W. Schiller et al. in Proc. Natl. Acad. Sci. USA 89, 11871-11875 (1992) and at the International Narcotics Research Conference (INRC) Meeting in North Falmouth, Mass., USA, July 16-21 (1994). Cyclic β-casomorphin analogs with mixed μ agonist/δ antagonist properties have recently been disclosed by R. Schmidt et al. in J. Med. Chem. 37, 1136-1144 (1994). On the basis of results obtained by E.E. Abdelhamid et al., J. Pharmacol. Exp. Ther. 258, 299- 303 (1991), mixed μ agonist/δ antagonists are of interest because they are expected to be analgesics with low propensity to produce tolerance and dependence. The problem underlying the present invention was to find new dipeptide derivatives with δ antagonist or mixed μ agonist/δ antagonist properties that have a low molecular weight and are highly lipophilic. These properties should facilitate passage across the blood-brain barrier. The Invention It has now been found that certain novel derivatives of the dipeptide H-Tyr-Tic- OH, as defined by the following formula I, have - high potency as δ antagonists - mixed μ agonist/ δ antagonist properties - total lack of μ antagonist properties The novel compounds according to the present invention have the general formula I wherein R. is H; CH 3 (CH 2 ) n - wherein n = 0-12; — C H 2 < ; - CH 2 -CH=CH 2 : or argininyl; R j is H; CH 3 (CH 2 ) n - wherein n = 0-12; — CH 2 -<] ; or -CH 2 -CH=CH 2 ; R 3 , R 4 , R 5 , R 6 are all H; or R 4 and R 5 are both H and R 3 and R 6 are both C 1 -C 6 alkyl; or R 3 , R 5 , R 6 are all H and R 4 is F, Cl, Br, OH, NH 2 or N0 2 ; R. is carbonyl or CH^ R 8 is H or C,-C 12 alkyl, or aralkyl wherein alkyl is C 7 -C ]2 alkyl; R,. is a linear or branched j -C 12 alkyl or aralkyl wherein alkvl is C.-C p alkyl, or C.-C- 2 alkyl linked to a heterocyclic moiety. Illustrative examples of R^ are CH 3 -(CH 2 ) m -; C H 3 C H (C H 2 ) m - CH 3 < >— (CH 2 ) m — [>— (CH 2 ) m — adamantyl-(CH 2 ) -; wherein R. 0 is H, F, Cl, Br, I, OH, NH 2 , N0 2 , CH 3 or phenyl; wherein m is 1-20 in all given examples of the R g definition. Preferred compounds of the invention are those compounds wherein R, is selected from H or CH 3 ; R ^ is selected from H or CH 3 ; R 3 is selected from H or CH 3 ; R 4 is H; R 5 is H; R 6 is selected from H or CH 3 ; R. is selected from carbonyl or CH 2 ; R g is selected from H or C y and R9 is selected from V(CH 2 ) 2 ■ (CH 2 ) 2 — , - (CH 2 ) 3 or Espedally preferred compounds of the invention are those, wherein R 4 and R 5 are H and R 3 and R 6 are both methyl groups. The best mode of carrying out the invention known at present is to use the compounds of Examples 4, 14 and 18. Synthesis Most Boc-amino add derivatives used in the peptide syntheses are commercially available. 2',6'-dimethyltyτosine (Dmt) was prepared as described by J.H. Dygos et al. Synthesis, No. 8 (August) pp. 741-743 (1992). All dipeptide derivatives were prepared by solution synthesis by first coupling the C-terminal amine substituent to the carboxylic add function of Boc-Tic-OH (mixed anhydride method), subsequent deprotection with acid, preferably an inorganic acid, espedally preferred TFA, coupling of the Boc-protected N- terminal tyrosine or tyrosine analog (mixed anhydride method) and final deprotection with add. The preferred acid system for Boc-deprotection is aqueous 95% TFA containing anisole (3%). Compounds containing a reduced amide bond (ψfCH j -NH]) between the Tic residue and the C-terminal substituent were obtained by first preparing Boc-Tic-3- (N-methoxy-N-methylamide) which was then deprotected with add, TFA being especially preferred. The resulting product was coupled to the Boc-protected tyrosine or tyrosine analog (mixed anhydride method) and the dipeptide aldehyde was then obtained by reduction with lithium aluminium hydride. Subsequent reaction with the amine component, followed by treatment with sodium cyanoborohydride and final deprotection with TFA yielded the desired product. The HPLC system GOLD (Beckman) consisting of the programmable solvent module 126 and the diode array detector module 168 was used for the purification and the purity control of the peptides. Reversed-phase HPLC was performed using a gradient made from two solvents: (A) 0.1 % TFA in water and (B) 0.1% TFA in acetonitrile. For preparative runs a Vidac 218TP1022 column (250 x 22 mm) was used with a linear gradient of 15-40% B over a period of 45 min at a flow rate of 13 ml/min, absorptions being measured at both 216 nm and 280 run. The same gradient was used for analytical runs on a Vidac 218TP 0046 column (250 x 4.6 mm) over a period of 30 min at a flow rate of 1.0 ml/min. Purity of peptides was also established by TLC on precoated silica gel plates 60F-254 (E. Merck, Darmstadt, FRG) in the following solvent systems (all v/v): (A) CHCl 3 /MeOH/AcOH (85:10:5), (B) n-BuOH/ H,0/AcOH (4:1:1) and (C) EtOAc/ hexane (1:1). Peptides were visualized with UV and with the ninhydrin spray reagent. Molecular weights of the peptides were determined by FAB mass spectrometry on an MS-50 HMTCTA mass spectrometer interfaced with a DS-90 data system. Melting points were determined on an electrothermal melting point apparatus and are uncorrected. Detailed description of the invention The invention will now be described in more detail by the following examples. Peptide Synthesis - General methods 1) Mixed Anhydride Method NMM (1 equiv.) was added to a stirred solution of 1 mmol of the Boc-protected amino acid in THF. The mixture was cooled to -15°C, treated with IBCF (1 equiv.) and was allowed to react for 3-4 min. Subsequently, the amino component (1 equiv.) was added. The reaction mixture was stirred for 30 min at -15°C and was then allowed to reach room temperature. The solvent was then removed by vacuum evaporation and the residual oil was dissolved in 100 ml of EtOAc. The resulting solution was extracted consecutively with brine, 5% KHS0 4 , brine, saturated NaHC0 3 and brine. The organic phase was dried (MgS0 4 ), filtered and evaporated to dryness. The resulting crude products were used for deprotection without prior purification. 2) Deprotection The Boc-protected peptides were deprotected using aqueous 95% TFA containing thioanisole (3%) under stirring and cooling with ice. After evaporation in vacuo, the pure TFA salts of the peptides were obtained by crystallization from EtOH/ether or by HPLC purification. EXAMPLE 1 A) Preparation of H-Tic-NH-(CH 3 -Ph (Compound 1) Boc-Tic-OH (1.5 mmol) was coupled with H 2 N-(CH 2 ) 3 -C 6 H 5 (3-phenyl-l- propylamine, 1.5 mmol) according to method 1. After deprotection compound \ was obtained by crystallization from EtOH/ether in 95% yield. FAB-MS: MH + =295 TLC (silica): Rf=0.40 (A), Rf=0.68 (B) mp=148-150°C. B) Preparation of H-Tyr-Tic-NH-(CH .,-Ph Using the mixed anhydride method, Boc-Tyr(Boc)-OH (1 mmol) was coupled with the TFA salt of compound 1_(1 mmol) in the presence of NMM (1 equiv.). After deprotection the crude product was purified by HPLC. Compound 2 in pure form was obtained in 80% yield. FAB-MS: MH + =458 TLC (silica): Rf=0.29 (A), Rf=0.65 (B) HPLC: K'=6.8. The compounds of Examples 2-19 have been synthesized as described for Example 1 above. EXAMPLE 20 A) Preparation of Boc-Tyτ(Boc)-Tic-3-(N-methoxy-N-methylamide) (Compound 2) Boc-Tic-3-(N-methoxy-N-methylamide), prepared as described by P.W. Schiller et al., in J. Med. Chem. 36, 3182-3186 (1993), was deprotected using method 2) described above (95 % yield; TLC (silica): Rf =0.31 (A)). Coupling of Boc-Tyr(Boc)- OH was performed according to method 1) described above and the resulting crude product was purified by flash chromatography on silica gel with hexane/EtOAc (2:1) as eluent (48 % yield). FAB-MS:MH + =584 TLC (silica): Rf=0.85 (A); Rf=0.28 (C). B) Preparation of Boc-Tyr(Boc)-Tic-3-aldehyde (Compound 3) 580 mg (1 mmol) of compound 2 was dissolved in 10 ml of ice-cold EtOH and 100 mg (2 mmol) of lithium aluminium hydride was added under stirring. The reaction was carried out for 1 h at 0 °C. 50 ml of ether followed by an ice-cold solution (50 ml) of 20 % citric acid were then added and the mixture was vigorously stirred for 30 min. The organic layer was collected and the aqueous phase was extracted again with ether (50 ml). The combined ether extracts were washed with saturated NaHC0 3 , brine, 10 % citric acid and H 2 0, dried over MgS0 4 , and evaporated in vacuo to yield a clear oil (50 % yield). TLC (silica): Rf=0.49 (C). C) Preparation of H-Tyr-Tiαi/fCH.-NHKCH ^-Ph The aldehyde (Compound 3) (270 mg, 0.5 mmol) was dissolved in 10 ml of MeOH/ AcOH (99:1) containing 3-phenylpropylamine (97 μl, 0.5 mmol). Sodium cyanoborohydride (38.2 mg, 0.6 mmol) was then added portionwise over a period of 45 min and the reaction was carried out for 15 h. At the end of the reaction saturated NaHC0 3 (50 ml) was added under stirring, followed by an addition of 100 ml EtOAc. The organic phase was collected, washed with water and dried over MgS0 4 . After evaporation of the solvent, the obtained product was deprotected by using method 1) described above, and the crude target compound was purified by HPLC (yield=42 %). FAB-MS: MH * =444 TLC (silica): Rf=0.24 (A); Rf=0.18 (B) Examples of compounds prepared according to the invention are given below in Table 1. Table 1 Pharmacological testing in vitro of δ opioid antagonists with a u agonist component Biosassys based on inhibition of electrically evoked contractions of the mouse vas deferens (MVD) and of the guinea pig ileum (GPI) were performed. 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 potendes 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 potendes 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 [Leu°Jenkephalin 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 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 e = 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 two different δ-selective agonists (DPDPE and [D- 2 Ala Jdeltorphin I). Conclusion Based on the results from the tests performed, the following condusions could be made: - All compounds showed δ antagonist properties All compounds showed no μ antagonist activity in the GPI assay at concentrations as high as 10 μM. - All compounds were either partial or full μ agonists in the GPI assay. Compounds 4, 14 and 18 were a potent mixed μ agonist/δ antagonists. Opioid receptor binding assays μ and δ opioid receptor binding constants (K ; , K 4 ) 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)). 2 The ratio K. μ / K ( was a quantitative measure of the δ-versus μ receptor selectivity. Opioid receptor binding studies The μ-, δ- and K-opioid receptor affinities of all new analogs were determined in binding assays based on displacement of μ-, δ-and -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 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 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 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 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: fΗjDAMGO, μ-selective, 0.7 nM; [ 3 H]DSLET, følDPDPE, or [ 3 H]TIPP, δ-selective, 1.0 nM; and [ 3 H]U69,563, K-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 (IC50) of spedfic binding were obtained graphically from semilogarithmic plots. From the measured IC50- values, binding inhibition constants (K,)were then calculated based on Cheng and Prusoff 's equation (Biochem, Pharmcol. 22, 3099-3102 (1973)). Ratios of the K -values in the μ-, δ- and K-representative binding assays are a measure of the receptor selectivety of the compound under investigation (e.g. , /K t 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 with a weak partial μ agonist component may be used in combination with analgesics of the μ agonist type (e.g. morphine) to prevent the development of tolerance and dependence, refer to the results of E.E. Abdelhamid et al., J. Pharmacol. Exp. Ther. 258, 299-303 (1991). The latter study also supports the conclusion that compounds with mixed μ agonist/δ antagonist properties are therapeutically useful as analgesics that do not produce tolerance and dependence. The dipeptide derivatives containing 2 ,6'-dimethyltyrosine (Dmt) in place of Tyr described in the present patent application are potent mixed μ agonist/δ antagonists of relatively low molecular weight. The δ antagonists with a weak partial μ agonist component described in the present patent application may also be therapeutically useful as immunosuppressive agents. Immunosuppressive effects of the 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 DAMGO = H-Tyr-D-Ala-Gly-Phe(N α Me)-Gly-ol Dmt = 2',6'-dimethyltyrosine DPDPE = [D-Pen 2 ,D-Pen 5 ]enkephalin DSLET = H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH FAB-MS = fast atom bombardment mass spectrometry GPI = guinea pig ileum HPLC = high performance liquid chromatography IBCF = isobutylchloroformate MVD = mouse vas deferens NMM = N-methylmorpholine TFA = trifluoroacetic acid THF = tetrahv/ drofuran Tic = l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid TIPP = H-Tyτ-Tic-Phe-Phe-OH TLC = thin layer chromatography U69,593 = (5α, 7a, 8β)-(-)-N-methyl-[7-(l-pyrrolidinyl)-l-oxaspiro[4,5]dec-8- yljbenzeneacetamide

Claims: CLAIMS 1. A compound of the formula wherein R. is H; CH 3 (CH 2 ) n - wherein n = 0-12; — H 2 <_^ ; - CH 2 -CH=CH ; or argininyl; R j is H; CH 3 (CH 2 ) n - wherein n = 0-12; — CH 2 <] ; or -CH 2 -CH=CH 2 ; R g , R 4 , R 5 , R 6 are all H; or R 4 and R. are both H and R g and R 6 are both C 1 -C 6 alkyl; or R g , R 5 , R 6 are all H and R 4 is F, Cl, Br, OH, NH 2 or N0 2 ; R~ is carbonyl or CH 2 ; R g is H or C^-C^ alkyl, or aralkyl wherein alkyl is C.-C 12 alkyl; R„ is a linear or branched C,-C 12 alkyl or aralkyl wherein alkyl is C.-C 12 alkyl, or a C.-C 12 alkyl linked to a heterocyclic moiety. 2. A compound of the formula I according to claim 1, wherein R j is selected from H or CH 3 ; R 2 is selected from H or CH 3 ; R g is selected from H or CH 3 ; R 4 is H; R 5 is H; R 6 is selected from H or C y R-, is selected from carbonyl or CH 2 ; R g is selected from H or CH 3 ; and is selected from — (CH 2 ) 3 — (CH 2 ) 2 — Y — (CH 2 ) 3 — or 3. A compound according to formula I of claim 1, wherein R 4 and R. are H and R 3 and R 6 are both methyl groups. 4. A compound of the formula I according to claim 1, being H— Tyr— Tic— NH— (CH 2 ) 3 -^TS H-Tyr-Tic-NH-(CH 2 ) 3 -CH 3 ; H-Tyr-Tic-NH-(CH 2 ) 5 -CH 3 ; H-Tyr-Tic-NH-(CH 2 ) 9 -CH 3 ; H-Tyr-Tic-NH-(CH 2 ) 11 -CH 3 ; H— Dmt— Tic— Ψ 5. A compound of the formula I of daim 1, being 6. A compound according to formula I of claim 1 for use in therapy. 7. A compound according to formula I of claim 1 for use as an analgesic. 8. A compound according to formula I of claim 1 for use as an immunosuppressive agent. 9. A method for preparing compounds of formula I according to claim 1, which method is solution synthesis comprising the steps of - coupling the C-terminal amine substituent to the carboxylic add function of Boc-Tic-OH; - subsequent deprotection with add; - coupling of the Boc-protected N-terminal tyrosine or tyrosine analog to the amino group of the Tic amide derivative; - final deprotection with add. 10. A method according to claim 9, wherein couplings are performed by the mixed anhydride method. 11. A method for the preparation of the compound comprising the steps: (i) deprotection of Boc-Tic-3-(N-methoxy-N-methylamide) with acid; (ii) coupling of Boc-Tyr(Boc)-OH to H-Tic-3-(N-methoxy-N-methylamide); (iii) reduction with lithium aluminium hydride to obtain the dipeptide aldehyde; (iv) reaction of the dipeptide aldehyde with the desired amine component, followed by treatment with sodium cyanoborohydride and final deprotection with acid. 12. A pharmaceutical preparation comprising effective amounts of a compound of the formula I according to claim 1 as an active ingredient, optionally together with one or more pharmaceutically acceptable carriers. 13. Use of a compound of the formula I of claim 1 in the manufacture of a medicament for use as an analgesic. 14. Use of a compound of the formula I of claim 1 in the manufacture of a medicament for use as an immunosuppressive agent. 15. 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 host in need of pain relief. 16. A method for produdng immunosuppressive effects, whereby an effective amount of a compound of the formula I according to claim 1 is administered to a host in need of achieving immunosuppressive effects.

Inventors: SCHILLER PETER, SCHILLER, PETER
Applicants: ASTRA AB, SCHILLER PETER, ASTRA AKTIEBOLAG, SCHILLER, PETER
Application year: 1995
Main classification: C07K5
IPC codes: A61K31, A61K38, A61P25, A61P37, C07D217, C07K1, C07K5