IE66529B1 - Novel CNS-active hexapeptides with an antiamnesic effect - Google Patents

Abstract

The present invention relates to hexapeptides of the general formula I R<1>-A<1>-A<2>-A<3>-A<4>-A<5>-A<6>-NH(CH2)n-NH2 (I> where R<1> denotes Z, formyl or (subst.) alkanoyl; A<1> denotes Met, Met(O) or Met(O2), all of which can be in the L or D form; A<2> denotes Glu, D-Glu, Ala or pyroglutamyl; A<3> denotes His, Ala, Phe or D-Lys; A<4> denotes the radical of a neutral aliphatic or aromatic alpha -amino acid; A<5> denotes Lys or D-Lys; A<6> denotes (subst.) Phe and n denotes an integer from 4 to 10. Processes for the preparation thereof, the use thereof as medicines, and pharmaceutical compositions containing these are described.

Description

CMS-active peptides with aa antiamnesic e££ect or effect on the cholinergic system have been disclosed ia EP-A 179 332- However,, the hexapeptides described therein have the disadvantage that they are easily transformed or decomposed,, that is to say are relatively unstable. With a view to use as pharmaceuticals „ this is a particular problem with regard to the production of pharmaceutical formulations and the shelf life thereof.

The object of the present invention is therefore to provide compounds which have an antiamnesic effect? are stable and thus can be handled without difficulty and can be used to produce pharmaceuticals with a long shelf life.

This object is surprisingly achieved by the compound of th® formula Ac-Met (O2) -Glu-His-Phe-D-bys-Phe-MH- (CH2) 8-NH2. and the physiologically tolerated salts thereof.

Examples of physiologically tolerated salts of the above compound are those which form basic groups? that is to say the amino groups in particular? with Inorganic or organic acids. Examples of such acids are hydrochloric acid? sulfuric acid? phosphoric acid? acetic acid? citric acid? benaoic acid, maleic acid? fumaric acid? tartaric acid and p-toluenasulfoaie acid. A preferred acid is acetic acid.

The invention furthermore relates to a process for the preparation of the abovementioned compound? which comprises condensing a fragment with an M-terminal free amino group with a fragment with a C-terminal free carboxyl group? where in these fragments primary and secondary amino groups which are not invovled in the reaction are protected with urethane-type protective groups which can be eliminated with acid (such as? for example? Soc) and carboxyl groups are protected with ester-type protective groups which can he eliminated with acid (such as, fox· example, Buc) s eliminating with acid the protective groups introduced to protect ami»© and carbostyl groups in the compound obtained in this way, and * converting the compound where appropriate into its physiologically tolerated salts. ·* Protective groups of the urethane type and ester type are described in Schroder» I«ubke, Academic Press, The Peptides, vol. I, Mew York, London 1965«, page 22 et sag», and page 52 et. seq„» and in A- Bubbuch, Kontakte Merck 3/79, pages 14-23.

The process is advantageously carried out In such a way that a) a compound of the formula Ila Is condensed with a compound of the formula Ilia, Ac-Met (Q2) -OB a-Glu-His-Phe-D-Lys-Phe-Z (Ila) (Ilia) b) a compound of the formula Xlb I® condensed with a compound of the formula IZIb, Ac-Met (02) -Glu-ΟΞ H-Bis-Phe-D-Lys-Phe-Z (lib) (IZIb) c) & compound of the formula He is condensed with a compound of the formula XHc, Ac-Met (O2)-Glu-His-OH B-Phe-D-Lys-Phe-X (He) (IIIc) & d) a compound of the formula lid is condensed with a compound of the formula Hid, Ac-Met (02) -Glu-Bis-Phe-OH (lid) H-D-Lys-Phe-Z (Hid) IS ®) a compound. ©£ the formula He is condensed with a compound of the formula XXXe? Ac-Het (02) -Glu-His-Phe~D~Lys-OS (He) S-Phe-X (Ills) where X is protected -SOH(CHa) -SH.,? but free primary and secondary amino groups? excepting the N-terminal groups of the compounds of the formulae XXXa-e? are protected with urethane-type protective groups which can be eliminated with acid? and free carboxyl groups? excepting the C-terminal groups of the compounds of the formulae Ila-e? are protected with ester-type protective groups which can be eliminated with acid? subsequently protective croups introduced to protect amino and carboxyl groups in the compound obtained, according to a J - e) are eliminated with acid? aad the resulting cowound is converted where appropriate into its physiologically tolerated salts. Reaction of the compound of the formula Ila with the compound of the formula XXXa is preferred.

The starting compounds of the formulae IXa-e and XXXa~@ are known or can be obtained in a manner known per se, for example by fragment condensation.

Suitable as M® protective group is preferably the benzyloxycarbonyl or 9-fluoreaylmethoxycarbonyl radical? and as carboxyl protective group Is the tert,-butyl radical? Ia the synthesis of the peptides according to the invention» The condensation in the process according to the invention is carried out by the general methods or peptide chemistry? for example by the nixed anhydride method? via active esters? azides or by the carbodiimide method? especially with the addition of substances which Increase the reaction rate and prevent racemization? such as? for example? 1-hydroxybensotriazole? N-hydroxysuccinimide? 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-beazotriasiae, SJhydroiy-5-aorbomeae-2,3-dicarboximida, and wish she use of activated derivatives of l-hydroxybenaotriazole or anhydrides of phosphoric, phosphonic and phosphinic * acids.

I Solvents are diaethylfiormsuaide, dimethylacefcamide, hexane fchylphosphoric triamide, N-methylpyrrolidon© ox dimethyl sulfoxide. It is also possible to employ solvents such as methylene chloride or chloroform where the solubility of the components permits it. The said methods are described, for example, in Heienhofier-Gross: The Peptides, Academic Press, vol. I, (1979).

The compound according to the invention is, by reason of its pharmacological properties,, suitable for the treatment of cognitive dysfunctions of a variety of etiologies, as occur, for example, in Alzheimer's disease or senile dementia. The improvement in cognitive functions by the compounds according to the invention (nootropic effect) was tested in animal experiments on mice using the inhibitory (passive) avoidance test (step-through model) and on rats using the radial mass test (8-arm radial maze). These are standard tests described in the literature for testing learning and memory functions in animal experiments. (Inhibitory avoidance; J. Bures, 0. Buresova and J. Huston In Techniques and Basic Experiments for the Study of Brain and Behavior", Elsevier Scientific Publishers, Amsterdam 1983, Radial Base: U. Staubli, M. Baudry and G. Lynch, Behavioral and Neural Biology 40, 58-69 (1984).) J In accordance with the statements in the said literature, a substance is said to have nootropic activity when it t counteracts or is even able to abolish the increase In the rat© of errors induced in the experimental animals by scopolamine, electric shock or inhibitors of protein synthesis in these tests.

The experiments were carried out by the test methods described above. The known nootropic 2-oxo-l-pyrrolidinylacetas&ide (piracetam) was used as comparison compound. t The distinct superiority of the compounds according to the invention was evident from 'the fact that the ’i scopolamine-induced amnesia is abolished, ia. the inhibitory-avoidance test with an HED (minimum effective dose) of 0.3 - 1 jzg/kg s.e. and ia the radial maze test with an MED of 10 jtg/kg s.c., while the comparison compound has an MED of 500 - 1000 mg/kg orally in these tests., This! peptide according to the invention has moodlightening, antidepressant and anxiolytic effects in humans. It increases awareness of the surroundings, , improves learning .and memory performance, has a beneficial effect In resocialization processes and can. be used for all diseases, post-traumatic and degenerative brain damage associated with reduced function of central acetylcholine metabolism,, for example mild dementia as well as early forms of Alzheimer's disease etc. It also reduces hvpereaaotivity.

Hence the invention also relates to pharmaceutical products which contain this abovementioned compound.

For an adult of norma,1 weight, Intranasal administration Is employed, preferably la a dosage of 0.1 tug to 1 mg per dose, and 1 to 500, especially 5 to 200, pg per dose are particularly preferred. The pharmaceuticals according to the invention can be administered, for example, up to 6 } times, preferably up to 3 times, per day. In many cases administration of one dose per day also suffices. The compound according to the invention can also be administered subcutaneously with 0.001 to 10 tug/kg, preferably 0.01 to 5 tug/kg, especially 0.05 to 2 /xg/kg. Its oral absorbability varies with the constitution. The dose range on oral administration is comparatively large and Is between 0.1 and 50 mg a day, divided into several administrations.

The preferred single dose for the abovementioned compound is 0.1 to 10 aagPharmaceutical products contain an effective amount of aa 5 active compound of the abovementioned compound together with an inorganic or organic pharmaceutically utilizable excipient. Intraaasal, Intravenous, subcutaneous or oral us® is possible.

For a form for oral use, the active compound Is mixed with the additives customary for 'this purpose,, such as excipients, stabilisers or inert diluents, and converted by customary methods into suitable dosage forms such as tablets, coated tablets, hard gelatin capsules, aqueous, alcoholic or oily suspensions or aqueous or alcoholic solutions. Examples of Inert vehicles which can be used are gum arable, magnesium carbonate, potassium phosphate, lactose, glucose or starch, especially com starch. This formulation can be carried out both as dry or wet granules. Examples of suitable oily excipients or solvents are vegetable and animal oils, such as sunflower oil or fish liver oil.

For subcutaneous or intravenous administration, the active ccsmouad or the physiologically tolerated salts thereof is converted, if desired with the substances customary for this purpose, such as solubilisers, esaulsiflers or other auxiliaries. Into a solution, suspension or emulsion. Examples of suitable solvents for the novel active compound and . the corresponding, physiologically tolerated salts are: water, physiological saline solu30 tions or alcohols, for example ethanol, propanediol or glycerol, as well as sugar solutions such as glucose or mannitol solutions, or else a mixture of the various solvents mentioned.

Example 1 Ac-Met (O2) -Glu-Kis-Phe-D-Lys-Phe-KH- (CH2) 8-NS2 . 2 CHjCOOS a) Ac a tvl - L -me thi on.ia._e sul f one (Ac -Met (Q, ) _tlQH) .0 g of acetyl-h-methionine (Pluka) are dissolved la 50 ml of trifluoroacetic acid and? while stirring at -20°C? reacted with a mixture of 12.5 ml of 30 % strength hydrogen peroxide and 12.5 al of water. After removal of the cooling? the mixture is stirred at room temperature? 1 g of palladium/carbon catalyst is added? and the mixture is stirred for 5 minutes and filtered with suction to remove catalysts. The filtrate is evaporated in vacuo? the oily residue Is treated with 11 of diisopropyl ether? and the resulting solid substance is filtered off. The product Is washed with diisopropyl ether and dried In vacuo.

Yield 10.31 g (88 % of theory) Melting point 141 - 146°C [e;3D = 4-17.6° (C s 1 In methanol) 4-5.1° (C = 1 ia water) b) Ac-Met (02) -Glu (OtBu) -nis^Phe-D-Lys _(Bpc.)__-Phe-MH_~·. (CH,,) g-NH-Boc 32.1 g of Ξ-Glu(OtBu)-His-Phe-D-Lys(Boc)-Phe-SB(CH2)s-MH-Boc - HTbsOH (Example Ila of SP-A 179 332) are dissolved in 250 ml of dimethylformamide? and 5.86 ml of SJ-ethylmorpholine? 3.44 g of 1-hydroxybenzotriazole and 5.7 g of Ac-Met (02)-OH are added and? after cooling to 0°C? 5.25 g of dicycloheseyicarbodiimide are added. The reaction mixture is stirred at room temperature for IS hours? filtered to remove the precipitated dicyclohexylurea and, washed with dimethylformamide, and the filtrate is evaporated in vacuo. The oily residue is dissolved in water-saturated n-butanol? and the solution is extracted successively with aqueous potassium bisulfate and sodium bicarbonate solution and water. The n-bufcsnol phase is separated off and evaporated in vacuo, and the oily residue is dissolved In hot methanol. The solution is poured into 1 % strength aqueous sodium bicarbonate solution and, after stirring, the precipitated product is filtered off, washed with water and dried in vacuo.

The dried product is dissolved and allowed to crystallize five times in succession In a 416:45:413 mixture of n-heptane, n-butanol and methanol using (R)Dicalite as filtration aid. After the last filtration, methanol is added to the residue and, after stirring, the filtration aid is filtered off, the filtrate is distilled in vacuo, and the residue is stirred with water. The product is filtered off, washed with wafer and dried in vacuo.

Yield 14.5 g (44 % of theory). c) Ac - Me t (O2) - Glu -His- Phe -D - Lvs - Phe - NB (CB2) a - MH2. ch?coqh .2 g of Ac-Met (O2)-Glu (OtSu)-Bis-Phe-D-Lys (Boc)Phe-ΜΠ- (CS2) θ-Boc are dissolved in a mixture of 40.8 ml of trif luoroacetic acid and 4.5 ml of water. The solution Is stirred for 30 minutes and then distilled In vacuo. The oily residue is dissolved in water, applied to a column packed with (R,A»berlite IRA 93 (acetate form) and eluted with water. The eluate Is freeze-dried .Yield 9.12 g (99.9 % of theory).

Comparison. Example a) 200 mg of Ac-Met (02) -Glu-His-Phe-D-I«ys-Phe-NH(CB2) 8~ NS2 · 2 CH3CGOB were dissolved In 20 ml of water and - 9 ~ heated at 80°C for 8 days. Cooling was followed by freeze-drying. HPLC investigation revealed that no decomposition had taken place.

HPLC conditions: Column?; Flow rate: Temperature s Detection: Mobile phase: Mucleosil 120 5μ C18 1.5 ml /sain RT Absorption at 220 nm 800 water/100 acetonitrile/3H3PO4 TBA pH 3.0 b) Repetition of a) with 200 mg of H-Met(O2)"Glu-HisPhe-D~Lys-Phe-NH-(CE2)8-SE2 (Bxasople IS of 3P~& 179 332) reveals serious decomposition of the substrate.

Claims (7)

1. CLAIMS;

1. A hexapepfcide of the Somls Ac-^ 2 )-Glu-His-PheD-Lys-^hss-Na- (CH 2 ) @ -SE 2 , and the physiologically tolerated salts thereof.

2. A process fox the preparation of a hexapeptide of the formula as claimed in claim 1, which comprises condensing a fragment with an ST-terminal free amino group with a fragment with a C-terminal free carboxyl group, where in these fragments primary and secondary amino groups which are not involved in the reaction are protected, with urethane-type protective groups which can be eliminated with acid (such as, for example, 3ocJ and carboxyl groups are protected with ester-type protective groups which can be eliminated with acid (such as, for exssaple, 3u c ) , eliminating with acid the protective groups introduced to protect amino and, carboxyl groups in the compounds obtained in this way, and converting the compounds where appropriate into their physiologically tolerated salts.

3. . A pharmaceutical composition containing a hexapeptide of the formula as claimed in claim 1.

4. A compound according to claim 1, substantially as hereinbefore described and exemplified.

5. A process for the preparation of a compound according to claim 1, substantially as hereinbefore described and exemplified.

6. A compound according to claim 1, whenever prepared by a process claimed in a preceding claim.

7. A pharmaceutical composition according to claim 3, substantially as hereinbefore described.