HU207737B - Process for producing halogeno-methylketon-pentapeptide derivatives utilizable as opioide receptor specific affinity reagent - Google Patents

Abstract

Novel halogen-methyl-ketone-penta-peptide derivs. of formula H-TyR-D-ALA-Gly-Pme-X(CH2-Y)-0H where X = aspargic acid or glutamic acid and Y = Cl, Br or F. They are applicable as specific affinity reagents for opioid receptors, are prepd. by the removal of protective gps. of protected peptides of Gln (1) and by opt. conversion of unprotected peptides into acid adduct salts

Description

The present invention relates to a process for the preparation of halomethyl ketone pentapeptide derivatives (I) and their acid addition salts which are specific reagents for opioid receptors and exhibit selectivity for the delta receptor type.

Natural alkaloids known as opioids collectively, their semisynthetic and synthetic analogues, and natural or synthetic peptides with morphine-like properties share the common feature of so-called predominantly nerve tissue. they bind to the opioid receptor. The heterogeneity of these opioid receptors is confirmed by both pharmacological and biochemical data. A common property of opioid receptors is the development of tolerance and dependence (drug dependence) and antagonism with naloxone (naltrexone), so that the action of any compound can be called opioid when its action is antagonized by the morphantagonist naloxone within their specific range of action.

Opioid receptors are heterogeneous in their biochemical and pharmacological properties and their chemical structure. The relationship between chemical structure and functional diversity of binding sites is currently the subject of intensive research. At present, it is generally accepted that three pharmacologic and binding properties distinguish three opioid receptors: the mu or morphine receptor; delta or enkephalin type and kappa or benzomorphan type. The distinction is based on which type of molecule binds selectively or more selectively to the binding site.

The body's opioid receptors and their active substances are involved in many physiological regulatory functions. Most commonly, they play a role in regulating pain. However, they are involved in the regulation of, inter alia, mood, cardiovascular, respiratory, endocrine and immune functions. Most opioid receptor types are involved at different levels in each process, but the "dominance" of each receptor type may vary widely across processes and animals, and in some cases may be almost exclusive. This feature can be exploited by rational drug design, but requires an in-depth mapping of physiological processes and receptor types.

An important condition for an in-depth characterization of a receptor type is the availability of a selective, highly efficient ligand for that receptor type. Of particular importance for the study of the chemical structure of the receptor type is the selective ligand, which is able to interact irreversibly with the receptor molecule. This is because, during research to determine the chemical structure of the receptor, the receptor must be released from the cell membrane, subjected to a purification procedure, and the chemical structure of the purified composition determined. Because the object to be determined is present at very low concentrations in the operating media, monitoring and determining the success of each operation step can only be achieved if the receptor can be selectively reacted with a well-detectable ligand that remains in association with the receptor during operations.

Successful production and use of opioid receptor-selective irreversible ligand is known in opioid receptor research; such as the Tyr-D-Ala-Gly-MePhe-CH ₂ Cl peptide derivative (Hungarian Patent No. 197927, and Neuropeptides, 9, 225, 1987).

To date, efforts to produce a highly efficient irreversible ligand selective for the delta receptor type have been only partially successful (e.g., FEBS Lett. 179, 87, 1985; J. Med. Chem. 30, 1668, 1987).

SUMMARY OF THE INVENTION It is an object of the present invention to provide effective opioid peptide derivatives that are selective for the delta receptor and are capable of interacting irreversibly with the receptor.

Our principle was that an oligopeptide, preferably having an end-chain aminodicarboxylic acid (such as aspartic or glutamic acid) containing a halogenomethyl ketone at its omega position, would meet the covalent bond with the corresponding opioid structure at the receptor site. desired efficiency, selectivity and irreversibility.

There were two simple natural peptide structure samples with delta receptor selectivity. One of these is Met ⁵ / Leu ⁵ encephalin (Tyr-Gly-Gly-Phe-Met (Leu), the other mammalian deltorphin [(Tyr-DAla-Phe-Asp (Glu) -Val-Val-Gly) -NH _2)]. Knowing the enkephalins previously elucidated impact structure relationships (eg., Br. Med. Bull., 39, 5, 1983), D-Ala favorable position 2 of enkephalin structure substituents, whereas aminopeptidase enzyme confers resistance to impact, potency enhancer and maintains or enhances delta receptor selectivity based on the biological activity assay of Tyr-D-Ala-Phe-Asp, Tyr-Gly-GlyPhe-Asp and Tyr-D-Ala-Gly-Phe-Asp (Table I, their methodological description 1 later), the Tyr-D-Ala-Gly-Phe-Asp basic structure was chosen because it is known that at position 5 of the enkephalin structure, there is a hydrophobic, aliphatic side chain and a negative charge for delta receptor selectivity necessary, at least for impact analysis applied in one biological system, mouse iron deferens (Biochem. Biophys. Gap. Commun. 91, 1239, 1979; Eur J. Pharmacol. 177, 99, 1990), the position for the formation of the halomethyl ketone was selected by the biological activity assay of the alpha and beta methyl esters of Tyr-DAla-Gly-Phe-Asp. Studies have shown that the beta position is satisfactory (Table II).

Based on the above principle, it has been found that the pentapeptide of formula I is irreversibly inhibited by delta ligand binding according to the biological assays detailed below, although its effect on the mu receptor is not negligible.

The invention thus relates to a process; pen1 of formula (I)

EN 207 737 Β tapeptide and, if desired, its acid addition salts (hereinafter DAACK).

According to the invention, the compound of formula (I) protected with a protecting group (s) commonly used in peptide chemistry is deprotected. The acid addition salt formation is carried out in a manner known to those skilled in the art, i.e. the free compound (I) is reacted with a molar equivalent of the desired acid.

The biological activity study of the compound of the invention of the present invention was performed on two isolates containing opioid receptors, electrically stimulated guinea pig ileum longitudinal smooth muscle (GPI) and mouse (CFLP strain) iron deferens (MVBS short) (74). , 182, 1977, and Biochem. Biophys. Rés. Commun.

In GPI, opioid receptors regulate neuromuscular transmission by mu and kappa, whereas in MVD, delta, mu and kappa opioid receptors are regulated (Pharmacol. Rev. 39, 1977, 1987). In these systems, the potency of an agonist-type opioid agent is characterized by a 50% inhibitory concentration (IC ₅₀ ), which is given everywhere in nmol / l (nM). According to the measurement constants of our own system, mu receptor agonists have approximately the same potency on GPI and MVD, delta agonists are 15-300 times more potent on MVD than GPI, and kappa agonists are 15-30 times more potent on GPI, as on MVD. The type of receptor on which agonist substances act is characterized by determining in our systems the affinity of the opioid antagonist naltrexone for the receptors at which the substances in question act. Affinity was characterized by the Ke / equilibrium dissociation constant, which gives the concentration of the antagonist (expressed in nM) at which the antagonist binds to 50% of the receptors. For mu receptors, this value is in the range of 0.2-0.8 nM, while in the case of delta and kappa, it is in the range of 4.0-14.0 nM.

Receptor binding assays {Neurochem. Gap. 10, 627, 1985 and J. Neurochem. 43, 957,984 (rat) / PVG / C strain}, prepared according to Molar Pharm. 11, 340 (1975). For receptor labeling, ³ H-naloxone (antagonist, 73 Ci / mmol, J. Labell. Comp. Radiopharm. 19, 1021, 1982) and ³ H-Tyr ¹ , D-Ala ² , D-Leu ⁵ -encephelin (short. ³ H-DADLE delta agonist, 37 Ci / mmol, Neurochem. Res. 10: 627, 1985) and ³ H-dihydromorphinone (abbr. ³ HDHM, Mu agonist, 68 Ci / mmol, Radio Chem. Radio Anal. Lett., 56.209, 1982).

All reagents were of analytical purity. The volume of the incubation mixture was 1 ml. The reaction mixture contained 100 microliters of radioligand, 100 microliters of water or unlabeled ligand to be tested and 800 microliters (0.3-0.5 mg / ml protein) of membrane protein in pH 7.4 Tris buffer. The binding reaction was initiated by the addition of membrane protein and stopped at the end of the incubation by rapid filtration under vacuum (Whatman GF / B and GF / C glass fiber filters). Unbound radioligand was removed by buffer wash. The radioactivity of the filters was measured in a toluene cocktail using a LKB Minibeta liquid scintillation counter. Non-specific binding was determined in the presence of 10 micro-M unlabeled (non-radioactive) ligands. Specific (receptor) binding was defined as the difference between total (measured in the presence of radioligand only) and non-specific (excess radioligand + unlabeled ligand) binding.

The compound of structure (I) of the present invention is a highly potent, agonist-type opioid peptide with high potency, high delta receptor selectivity, and biological assays. This property was confirmed in the receptor binding assay. Further biological assays confirm the irreversibility of receptor binding.

The process of the invention is described in detail below without limiting the scope of the invention to any of the examples.

The compound of formula (I) of the present invention was prepared by classical liquid phase synthesis followed by analysis of the unit product after an HPLC purification phase. Conditions for HPLC purification were as follows:

Stationary phase: LICHROPREPRP 18

Mobile phase: MeOH: 0.02 M NH ₄ OAc buffer pH ₄ = 40:60

Temperature: room temperature

Pressure / Fluid velocity: 4 bar / 2 ml / min.

t _R = 18.6 min. to = 5.2 min

Purity Control: Analytical HPLC, ODS-Hypersil column, MeOH: NaOAc 0.02 M, pH4 = 60: 40 1.1 mL / min, t _R = 4.0 min.

The main method of structural verification was amino acid analysis and elemental analysis.

Amino acid analysis:

Tyr: 0.81 (D) Ala: 1.06

Gly: 1.11

Phe: 1.03

According to Schöniger, the covalent chlorine content was determined by the reaction of 4-p-nitrobenzyl) pyridine (Anal.

Chem. 27, 1435, 1955). The incorporation rate of chloromethyl ketone is estimated to be approximately 88% based on the Asp content of the product prior to the HPLC purification step.

RESULTS OF BIOLOGICAL TESTS

1. The results of the isolated organ examinations performed during the design phase are shown in Figures I and Π. table.

Table I

Material IC ₅₀ (nM) Effect- strength ratio (MV D / GPI) MVD GPI Tyr-D-Ala-Phe-Asp 100000 100000 - Tyr-Gly-Gly-Phe-Asp 2150 32400 15 Tyr-D-Ala-Gly-Phe-Asp 87 206 2.4

The results were obtained in 3-8 independent experiments.

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II. spreadsheet

Material ICso (nM) Effectiveness ratio (MVD / GPI) MVD GPI Tyr-D-Ala-Gly-Phe-Asp-OH 87 206 2.4 Tyr-D-Ala-Gly-Asp-Phe-OMe 1 OH (Alpha) 32 106 3.3 Tyr-D-Ala-Gly-Phe-Asp-OH 1 OMe (Beta) 4 159 40

The results were obtained in 5 to 8 independent experiments.

2. Analysis of the acute effect of the DAACK halogenomethylketone peptide derivative of formula (I).

2.1 Isolation tests 20

As reference normorphomt (agonist), Tyr-DAla-Gly-MePhe-Gly-ol (cf. DAGO, agonist), morphiceptin (agonist), Met ^5- encephalin (delta agonist), DADLE (delta agonist) and PD 117 302 (kappa agonist, Br. J. Pharmacol. 93,618, 1988) was used. 25

III. spreadsheet

Comparative study of opioid agonist potency

DAAC 1.9 300 158 Normorfm 237 166 0.7 DAGO 44 48 1.1 Morficeptin 969 1034 1.1 Met ⁵ encephalic 7.2 232 32 DADLE 0.3 71 237 PD 117 302 71 4.4 0.06

The results were obtained in 3-220 independent experiments.

ARC. spreadsheet

Determination of the affinity of Naltrexon

Material Naltrexon K _c (nM) MVD GPI DAAC 12 0.3 normorphine 0.6 0.6 DAGO 0.3 0.3 Morficeptin 0.2 0.5 Met ⁵ encephalic 6.8 0.8 DADLE 7.9 0.3 PD 117 302 4.6 8.1

The results were obtained in 3-16 independent experiments.

In conclusion, DAACK is a high potency, high delta receptor selectivity agonist opioid peptide. There is no evidence of agonist activity at the Kappa receptor.

2.2. Receptor binding assays

To characterize the receptor binding specificity, DAACK suppressor activity against ³ H-DHM (1 nM) and ³ H-DADLE (2 nM) was investigated. From the dose-response curve, the concentration of ligand (IC ₅₀ in nmol / L (nM)) required to inhibit 50% of the specific binding was determined, which is characteristic of the affinity of the particular ligand. Morphine, DAGO, and unlabeled DADLE were used as reference (Table V).

Table V.

Material 1C _{5 O} (nM) delta / art selectivity ³ H-DADLE (delta) ³ h-dhm (artwork) DAAC 1 50 50 Morphine 70 1 0.01 DAGO 80 2 0.03 DADLE 4 20 5

It can be stated that DAACK is a high affinity and high delta receptor selective ligand in the receptor binding assay.

3. Irreversibility of DAACK effect / binding

3.1. isolated organ examinations

The baseline phenomenon was investigated with MVD. The formulations were incubated for 20 minutes with 10 ⁵ M DAACK (at 31 ° C, routine operating temperature of the preparation), and after washing, the material was washed for an additional 50-24 times (20-25 per wash). double volume excess). For comparison, under the same experimental conditions, the pentapeptide Tyr-D-Ala-Gly-Phe-Asp beta (aka DAA-OMe), which is highly reversible, but very close in structure, potency and delta receptor selectivity, was used. The results are shown in Figures 1 and 2.

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Figure 1, Part A shows that after a single wash after incubation with 10 ~ ⁵ M DAAOMe for 20 minutes, the preparation returned to a fast, almost complete 2 minutes; the same is shown as a summary of 3 independent experiments on the left of Figure 2 (blank circle symbols). In contrast, after 1025 washes after DAW incubation, the inhibition was practically complete (Figure 1, Part B and Figure 2, right, dark circles, respectively), and further return was slow. This phenomenon is indicative of DAACK's persistent binding tendency. A similar phenomenon does not occur when DAACK is used under the same conditions on a GPI without a delta receptor type; this shows the receptor-type specificity of the phenomenon.

3.2. Receptor binding assays

Irreversible binding of DAACK to rat brain membrane preparation was determined by the preincubation-wash method (Life Sci. 32, 2777, 1983). During the test, the membranes were incubated with 10 ^-5 M DAAC at room temperature for 90 minutes, then the incubation mixture was diluted 10-fold with ice-cold buffer, and pelleted 25,000 xg after disszociáltatás. The sediment is approx. It was resuspended in 30 volumes of fresh buffer and centrifuged again after 15 minutes of incubation. The leaching step, whereby the free and reversibly bound ligands were released, was repeated 4 times. The final pellet, after resuspension, was incubated with various labeled opioid ligands and the receptor binding (residual opioid activity) was determined as usual and corrected for protein content in the samples. As a control, membranes were preincubated with buffer or with a reversible opioid ligand. Reversible ligands can be completely washed out of the system by the above method, and control-specific binding can be well reproduced.

After the above experiment, the "residual" ³ H-DADLE (2 nM, delta-specific ligand) specific binding was 30% of the control, while the "residual" ³ H-naloxone (1 nM, mu-specific ligand) specific binding was the control. 47%. Non-opioid chloromethyl ketones (TPCK, TLCK) do not induce irreversible inhibition at high concentrations, so the chloromethyl ketone group is not an active substance alone.

Thus, DAACK inhibits binding of both delta and artificial ligands in a manner considered irreversible: the effect on delta receptors is greater but not negligible on the artificial receptor.

Claims (1)

Patent Claim Point Process for the pentapeptides of formula (I) H-Tyr-D-Ala-Gly-Phe-X-OH CH ² -Y (I) where X = aspartic acid or glutamic acid, And Y is chlorine, bromine or fluorine, and the acid addition salts thereof, characterized in that the compounds of formula (I) protected by the protecting group (s) commonly used in peptide chemistry are cleaved in a manner known per se in peptide chemistry; compounds are also converted to the desired acid addition salts in known manner.