DD299044A5 - METHOD FOR THE PRODUCTION OF NEW ANTICONVULSIVA / ANTIEPILEPTIKA - Google Patents

Abstract

The invention relates to the development and application of novel neuropsychopharmaceuticals which are suitable for the therapy of diseases of the epileptic type. The inventively developed substances contain as active ingredient peptides of des-tyrosine 1-b-casomorphine-5-type and their derivatives. They are distinguished by a novel spectrum of action, a long-term effect and good compatibility in comparison to the currently used standard injections. The invention makes it possible to therapeutically influence diseases of the epileptic type. {Neuropsychopharmaceuticals; anticonvulsants; antiepileptic drugs; Des-Tyrosin1-b-Casomorphin; derivatives; manufacture; Method; Effect profile; Side effects; Compatibility}

Description

The invention relates to novel neuropsychopharmaceuticals which contain linear or cyclic oligopeptides of the des-tyrosine-β-casomorphine type as active component and, in contrast to the known neuropsychopharmaceuticals, are distinguished by a novel activity profile. The invention is of importance for the pharmaceutical industry and for use in medicine for the therapeutic treatment of neuropsychiatric disorders.

Diseases of the epileptic type group are in the morbidity statistics among the diseases of the central nervous system and occupy a prominent because of their chronic health, social and economic problem dar. The lifelong necessary pharmacotherapy of the aforementioned disease picture is currently a purely symptomatic therapy The problem is that a considerable part of the patients is resistant to therapy. Furthermore, the antiepileptics used in therapy z.T. not inconsiderable side effects.

The discovery of highly effective, long-term effective neuropsychopharmaceuticals, which can also be used in therapy-resistant patients, is hardly to be expected on the basis of the bishei underlying effect profiles and mechanisms of action.

With regard to the chemical structure, the known antiepileptics are preferably heterocyclic compounds of different structural classes, which are accessible by chemosynthesis with sometimes considerable effort. Peptide-based psychotropic drugs were first reported by D. deWied et al. (See, Europ. J. Pharmacol. 49 [1978], 424; Arch. Gen. Psychiatry 36 (1979), 294; Life Sci 26 (1980), 1575; H.M. Grevon et al. see. Reel. trav. Chim. Pays-Bas 99 [1980], 284) as part of his studies on the behavioral physiology of des-tyrosine-v-endorphin described. By eliminating the N-terminal amino acid in γ-endorphin, an endogenous opiate in the mammalian organism? peptide, the activity spectrum of the molecule is changed. Des-tyrosine-y-endorphin, in contrast to γ-endorphin, has a psychotropic effect, without triggering analgetic effects. However, the synthetic effort for peptides of this chain length is still very high, despite advances made in recent years in the field of peptide synthesis. The resulting high economic outlay thus does not yet allow the use of these desyrate Tyr'-y endorphin peptides in medicine. In addition, the clinical findings are still very contradictory.

The aim of the invention is the development of novel neuropsychopharmaceuticals based on the basic simple oligopeptides and peptidic subtypes of the des-tyrosine ¹ -β-casomorphine type, which are superior to those currently used in the therapy of psychiatric disorders by a novel mode of action, a novel mechanism of action, and a wide spectrum of action, influenced by simple structural modification, distinguished and also have a high metabolic stability, long-term effect and good tolerability.

The invention serves to increase the quality and effectiveness of the medical care of neuropsychiatric patients and thus has a ho'ie health policy importance.

The invention has for its object to develop novel anticonvulsants / antiepileptics and to capture their application spectra.

The problem was solved by linear or cyclic oligopoptides of the basic structure Pro-Phe-Pro-Gly (Des-Tyr'-β-casomorphine-5), salts thereof or C- and / or N-terminally modified Derivatives can be synthesized, wherein proline in one or both positions by hydroxyproline, homoproline, dohydroproline, pipecolic acid, derived substituted compounds, alanine, phenylalanine, valine, serine, ornithine, lysine, 2,4-diaminobutyric acid, arginine, cystine and mothionine and Phenylalanine may also be replaced by homophenylalarine, N-alkyl tyrosine, (eg, N-methylphenylalanine), tyrosine, N-alkyl tyrosine), analogous ring-substituted compounds, tryptophan, tyrosine, valine, and alanine The amino acids may be in either the L or D configuration glycine may or may not be present, and if present, may be replaced by all amino acids in the L or D configuration, or as a racemate be changed. In addition, the above-mentioned basic structure can also be shortened to pro-Phe peptides, their salts and derivatives, preferably the corresponding dipeptide pyrrolidides and piperidides. Some preferred compounds are the oligopeptides Pro-D * Phe-Pro-Gly, Pro-Phe-D-Pro-Gly, Pro-Phe-D-Pip-Gly, Pro-D-Phe-N <Pro, Pro-Phe- N <to.

The oligopeptides used have as active components u.a. the following advantages:

1. Non-toxic structures

2. Effect potentiation and -rolongierung u.a. by incorporation of D-amino acids

3. High resistance to enzymatic degradation

4. Influencing psychotropic effects by structural modification

5. Influence on lipophilicity and other physicochemical parameters in terms of improving transport and penetration properties through targeted structural modification

The construction of the inventive peptide active ingredients is preferably carried out by stepwise extension of the peptide chain from the C-terminal end. As mentioned above, one of the preferred compounds is the peptide of the formula I.

H-Pro-D-Phe-Pro-Gly-OH (I)

The strategic variant of the step-by-step condensation for the preparation of the compound according to formula I, is characterized in that a glycine derivative H-Gly-Z stepwise with X-Pro-Y, XD-Phe-Y according to those customary in peptide chemistry Coupling methods (see E. Wünsch: Synthesis of peptides, in Houben-Weyl, Volume 15/11, Methods of Organ. Chemistry, Hgr. E. Müller, Georg Thieme Verlag, Stuttgart, 1974) to the protected tetrapeptide of the formula Il is extended,

X-Pro-D-Phe-Pro-Gly-Z (II)

X sonicated. Butyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxy-carbonyl, biphenylisopropyloxycarbonyl.a / i'-dimethyl

SB-dimethoxybenzyloxycarbonyl.o -nitrophenylsulfenyl, 9-fluorenylmethyloxycarbonyl Y for OH, pentafluorophenyl, pentachlorophenyl, trichlorophenyl, 4-nitrophanyl, N-hydroxysuccinimide, N-hydro>: y-5-norbornene-2,3- dicarboximide ester and hydrazide

Z is OH, methyl, ethyl, tert. Butyl, 4-nitrobeniyl and Phenacylester stands.

The prefabricated structure of the peptide of the formula II was preferably carried out by the mixed anhydride technique or the DCCI / Additiva method. As Schuizgruppenkombination is preferably tert. Butyloxycarbonyl / methyl ester used. The corresponding protected peptides according to formula II are purified by adsorption chromatography on a silica gel column by chloroform / methanol elution or by gel filtration on Sephadex LH 20 (eluent: methanol) or else by recrystallization from ethyl acetate / petroleum ether, ethyl acetate / diisopropyl ether or chloroform / Purified diisopropyl ether and then the respective amino and carboxyl protecting group simultaneously or sequentially with the customary in peptide deblocking (see Wünsch, supra), in particular by means of HCI / dioxane, HCl / glacial acetic acid, trifluoroacetic acid, HBr / glacial acetic acid, catalytic hydrogenation, transfer hydrogenolysis, zinc / Glacial acetic acid, piperidine / dimethylformamide or alkaline hydrolysis. The resulting peptide of the formula I is preferably isolated as the hydrochloride or as the free compound (release from the hydrochloride using triethylamine) and optionally purified by gel filtration on Sephadex G10 (eluent: methanol or water). In the case of the freer. Compound is a subsequent recrystallization on ethanol advantageous.

Analytical data for H-Pro-P-Phe-Pro-Gly-OH HCI

M.p .: 130-140 ⁰ C (decomposition)

[a \ h ² : -82.0 ° (c = 1, acetic acid) Chromatographisch uniformly (see Ausführungsbeispiei)

Analytical data for H-Pro-D-Phe-Pro-Gly-OH

Mp: 2? 8-230 ° C

[a] g ² : 105.0 "(c = 1, acetic acid)

Chromatographically uniform (see Ausführungsbeispiei)

The obtained oligopeptides of des-tyrosine-ß-casornorphin-B-type can in the form of anticonvulsive drugs effective as novel drugs for neuropsychiatric treatment of epileptic disorders in the dose range

from 0.2 to 5 pmol / kg are used. Their novel Wirkungsproiil and their novel mechanism of action against the standard preparations used currently in the therapy of convulsive diseases will be explained in more detail with reference to the embodiments.

Examples

List of abbreviations:

Amino acid symbols according to IUPAC-IUB Commission on Biochemical Nomenclature, J. Biol. Chom. 247 (1972) Pip = pipecolic acid

AcOH acetic acid

Boc tert. butyloxycarbonyl

CAIBE isobutyl chloroformate

TLC thin-layer chromatographic, chromatographic

ECS electroconvulsive shock

EE ethyl acetate

Mp melting point

h hours

vacuo. in a vacuum

LM solvent

MeOhI methanol

minutes

NEM N-ethylmorpholine

OMe methyl ester

PE petroleum ether

RT room temperature

se. subcutaneously

IHF tetrahydrofuran

Dec. decomposition

By the "usual work-up" one understands:

After completion of the coupling reaction, the respective crude product is taken up in ethyl acetate and the solution washed successively twice with water (NaCl-saturated), three times with 5% KHSO ^ solution, three times with water, three times with saturated sodium bicarbonate solution and three times with water. The organic phase is dried over Na ₂ SO ₄ and the crude product isolated by evaporation of the solvent in vacuo.

The following solvent systems (by volume) for thin layer chromatography on silica gel precast panels (Silufol UV 254, CSSR) were used:

BAE benzene-acetone-acetic acid

50 + 20 + 1 BAW 2-butanol-formic acid-water

75+ 13.5+ 11.5 BEWE 1-butanol-acetic acid-water-ethyl acetate

20 + 20 + 20 + BPEW 1-butanol-pyridine-acetic acid-water

30 + 20 + 6 + CM chloroform-methanol

90 + 10 EPEW ethyl acetate-pyridine-acetic acid-water

90+ 15 + 4.5 + 8.3

example 1

Synthesis of H-Pro-D-Phe-Pro-Gly-OH · HCl

1.1. Boc-Pro-Gly-OMe

5,38g Boc-Pro-OH were dissolved in 50 ml of THF and treated at -15 ⁰ C successively with 3.175 ml of NEM and 3.25 ml CAIBE. After 10 min, 3.14 g of H-Gly-OMe.HCl and 3.175 ml of NEM were added to the reaction mixture and the mixture was stirred at -15 ° C. for 1.5 h and stirred at RT for a further 12 h. The reaction was worked up as usual, and the crude product from EA-PE and recrystallized from diisopropyl ether, yield: 5.0 g (70% d.Th.) M.p .: 70.5 ⁰ C to 72 ⁰ C [a] g ^2: - 78.5 ° (c = 1, AcOH) DC: uniform in BAE, CM and EPEW

1.2. Boc-D-Phe-Pro-Gly-OMe

4.6 g Boc-D-Phe-OH, 2.2 ml of NEM and 2,25ml CAIBE were dissolved at -15 ⁰ C in 40 ml of THF, and after 10 minutes (with 3.85 g H-Pro-Gly-OMe · HCl obtained by deacylation of 1. with 1.1 N HCl / AcOH-oil: [a \ l ² -57.7 ° (c = 1 * AcOH) and 2.2 ml of NEM were reacted as described above, worked up as usual and crystallized the crude product from EE diisopropyl ether.

Yield: 5,25g (70% d.Th.) M.p .: 138 ° C Cbis139 ^c [a] g ^2: -102.5 ° (c = 1, AcOH) TLC: uniformly in BAE, CM and EPEW

1.3. Boc-Pro-D-Phe-Pro-Gly-OMe

1.64 g of Boc-Pro-OH were dissolved in 20 ml of THF at -15 ° C. and admixed with 0.97 ml of NEM ιιηΊ 0.99 ml of CAIBE. After 10 minutes they gave 2.02 g HD-Phe-Gly-Pro OMe · HCl (obtained by Entacyliei clothes of 1.2 with 1.1N HCl / AcOH -. Mp .: 128 ⁰ C 131 ⁰ C We [αψ -138.8 ° (c - 1, AcOH) and 0.97 ml of NEM to the reaction mixture and allowed to stir for 1 h at -15 ° C. and 4 h at RT The mixture was worked up as usual and the crude product from EE / PF reprecipitated.

Yield: 3.2 g (79% of theory)

Mp: 65 ^° C. to 71 ^° C. (decomposition)

[a] § ² : -117.9 ° (c = 1, AcOH)

DC: uniform in BAE, CM and EPEW

1.4. Boc-Pro-D-Phe-Pro-Gly-OH

3.18 g of Boc-Pro-D-Phe-Pro-Gly-OMe were dissolved in 40 mL of acetone and reacted with 6 mL of 1N NaOH. After a reaction time of 5 h at RT, the organic LM was i.Vak. evaporated and the residue dissolved in 40 ml of saturated NaHCOj solution. The aqueous phase was extracted twice with EA and then acidified to pH 3 with solid citric acid. After repeated extraction with EA, the combined EA phases were washed neutral with water, dried over Na ₂ SO ₄ and concentrated by evaporation in vacuo. evaporated. The product was reprecipitated from EE / PE and CHCl ₃ / diisopropyl ether.

Yield: 2.76 g (89% of theory)

Fp .: abaO ° C replacement

[a) l ² : -113.5 ° (c = 1, AcOH)

DC: uniform in BAE, CM and EPEW

1.5. H-Pro-D-Phe-Pro-Gly-OH HCI

2.7 g of Boc-Pro-D-Phe-Pro-Gly-OH were dissolved in 16 ml of 1.1 N HCl / AcOH. After 30 min at RT, the product was precipitated with ether, filtered off and washed with ether. The BAW, BEWE and BPEW DC-uniform product yielded precipitation from MeOH / ether.

Yield: 2.14 g (90% of theory)

Mp: from 13O ⁰ C decomposition

[a] g ² : -82.1 ° (c = 1, AcOH)

Pharmacological effects

The pharmacological effect of the peptides characterized by the claim has been determined in numerous animal experiments, which are customary for the characterization of anticonvulsive properties with particular attention to influencing behavioral regulation.

At the same time there is a study on him »Resistance to enzymatic degradation in vitro and in vivo.

To speak for novel possibilities of a clinical application:

- the profile of action deviating from comparable anticonvulsants / antiepileptic drugs already in use in the absence of toxic side effects in higher doses;

- The deviating from previous standard drugs mechanism of action;

the achievable high stability against enzymatic degradation;

- the long-lasting effect and

- the effectiveness of systematic and oral administration.

1. Study of the Anticonvulsant Activity Profile of Des-Tyr-β-Casomorphine Derivatives The anticonvulsant effect of β-casomorphine derivatives was tested in the models pentotrazole spasm, picrotoxin spasm and electroconvulsive shock in the mouse.

The Des-Tyr-ß-Casomorphinderivate were injected at a dose of 1 μηηοΙ / kg se. And after a Vdrbehandlungzeit of 20min, the Konvulsiva or ECS were applied.

The experimental results show that of the selected derivatives Des-Tyr-D-Phe ³ in the model of the electroconvulsive shock, on the other hand Dos-Tyr-D-Pro ⁴ in the pentetrazole spasm and picrotoxin-reaming models have anticonvulsant effects.

Peptide spasm model

Pentetrazole Picrotoxin ECS

Des-Tyr-D-Phe ³ 0 0 +

Des-Tyr-D-Pro ⁴ + + 0

Effect of des-Tyr-β-casomorphin derivative in different spasmodules -l- significant anticonvulsivo effect ( ~>< 0.05)

0 without clear effect

2. Duration of Protektlven effect of Des-Tyr-D-Phe ³ against electrically evoked spasms in mice

The duration of the protective effect of Des-Tyr-D-Phe ³ against electrically induced convulsions was after se. Application of

1 μιηοΙ / kg tested. A significant anticonvulsant effect (p <0.05) was found 0.3-5 hours post applicationem; the tendency of an effect is recognizable until 12 hours post applicationem.

3. Investigation of the Antiepileptic Effect Profile In the Model Pentfcrazole Kindling on Mice

The Des-Tyr-β-casomorphine derivatives Des-Tyr-D-Phe ³ and Des-Tyr-D-Pro ⁴ were tested at doses of 20,200 and 1,000 nmol / kg after subcutaneous injection for their efficacy against the developed Kindling phenomenon. It could be shown that the peptides in doses of 200 and 1000 nmol / kg significantly (p <0.05) the developed Kindling phenomenon

suppress. Compared to standard antiepileptic drugs (diazepam, phenytoin, phenobarbital), the β-casormorphine derivatives used were superior in their efficiency.

4. Effect of Dos-Tyr-U-Phe ³ in Combination with Tested Antieplleptlka The De-Tyr-β-Casomorphine derivative Des-Tyr-D-Phe ³ was administered in doses of 100.500 and 1000 nmol / kg in combination with diazepam ( 1,4,8,16 pmol / kg) phenobarbital (110, 220, 330 l / kg) phenytoin (100,200,300μιηοΙ /! ^) · ..) Model elktrctonvulsiver check and m »t diazepam (0,2, 2, 4 , θμηιοΙ ^ ο.) r'honoberbital (110, 220, 330 μmol / kg) phenytoin (100, 100, 300 μmol / kg) tested in the model pentetrazole spasm. In the case of diazepam and phenobarbital, a significant increase in the effect of Pentetrazolkrfampf was found. In the electroconvulsive shock model, the peptide potentiated the effects of all antiepileptic drugs used.

Claims (6)

Ί. Process for the preparation of novel anticonvulsants / antiepileptics, characterized in that peptides of the des-tyrosine ¹ -β-casomorphine-5-type and their derivatives with customary carriers and excipients are brought into a parenteral application form as active component. 2. The method according to claim 1, characterized in that as effective components linear or cyclic ü'igopeptide des-tyrosine ¹ -β-casomorphine-5-type, their salts or C- and / or N-terminal modified derivatives are added wherein in the basic structure Pro-PhePro-Gly Proline in one or both positions also by hydroxyproline, homoproline, dehydroproline, pipecolic acid, derived substituted compounds, alanine, phenylalanine, valine, serine, ornithine, lysine, 2,4-diaminobutyric acid, arginine , Cysteine and methionine, and phenylalanine can also be replaced by homophenylalanine, N-alkylphenylalanine, tyrosine, N-alkyl tyrosine, analogous ring-substituted compounds, tryptophan, tyrosine, valine and alanine both in the L or D configuration and as the racemate, glycine may be present or absent, and if present, it may also be replaced by all amino acids in the L or D configuration or as a racemate and moreover, the above-mentioned basic structure can also be shortened to pro-Phe peptides, their salts and derivatives, preferably the corresponding dipeptide pyrrolidides and piperidides. 3. The method according to claim 1 and 2, characterized in that as active components preferably the oligopeptides Pro-D-Phe-Pro-Gly, Pro-Phe-D-Pro-Gly, Pro-Phe-D-Pip-Gly, Pro D-Phe-N <Z3, Pro-Phe-N <C3 . 4. The method according to claim 1, 2 and 3, characterized in that the oligopeptides used as effective components of Des-tyrosine ¹ -β-casomorphine-5-type are prepared by segment condensation, but preferably by stepwise construction of the C-terminal end. 5. The method according to claim 1,2,3 and 4, characterized in that the oligopeptides used as active components of Des-tyrosine ^1- ß-Ca? Omorphin-5-type in the form of anticonvulsive / antiepileptic drugs effective with novel mechanism of action, broad Spectrum of action, high metabolic stability, pronounced La.igzeitwirkung, minor side effects and good tolerability for the treatment of diseases of the epileptic form circle are used. 6. The method according to claim 1,2,3,4 and 5, characterized in that on the basis of known anticonvulsive / antiepileptic drugs acting in combination with peptides of des-Tyr ¹ -β-casomorphine-5-type and its derivatives improved therapeutics produced with a novel profile of action.