GLYCYL-HYSTIDYLE-LYSINE (GHL) DERIVATIVES

Abstract: The invention refers to derivatives of the peptide Gly-His-Lys (GHL) having cytostimulant and cytoprotective activity, to their therapeutic use and to pharmaceutical compositions containing them. The derivatives of the invention are surprisingly resistant to hydrolytic enzymes in addition to increase therapeutic activity.

Description: GLYCYL-HISTIDYL-LYSINE (GHK) DERIVATIVES The present invention refers to Gly-His-Lys (GHL) peptide derivatives having cytostimulant an cytoprotective activity, to their therapeutic use as well as to pharmaceutical compositions containing them. The derivatives of the invention, together with improved therapeutic activities, are endowed with surprising resistance to hydrolytic enzymes. Many methods are known in medicine for woun management, including particular devices (from the anti-infective medicated plaster to the synthetic skin substitutes for burn patients) ointments, gels or other formulations for topical application containing for instance zinc derivatives. Recently, as a consequence of a deeper knowledge of the tissue repair mechanism and of the development of recombinant DNA techniques, different active principles have been prepared and studied, in order t promote and/or normalize the healing process and t heal tissue damages not otherwise curable (such as, fo instance, decubitus or diabetic ulcers) and to preven the formation of scars and cheloids: growth factors such as: bFGF, basic Fibroblas Growth Factor; EGF, epidermal growth factor; GH (also known as GHK), Plasma Copper-Binding Growt Factor, etc. ; components of the extracellular matrix such a fibronectin, collagens, etc.. However, a number of reasons make the practica use of these derivatives difficult. They comprise the availability of the substance, the risk of transmission of viral or bacterial diseases, due to their origin, and, above all, their instability to enzymatic systems (such as proteases and peptidases: both i-ri vitro and in vivo), characterising most proteins, glycoproteins or peptide substances and providing the major obstacle to their use as drugs, particularly in the pathologies in which the protease activity is increased, such as in inflammatory conditions, both systemic and local. There is therefore the need for more stable active principles in the most different therapeutic fields. In the specific case of healing, attempts to this aim on complex proteins such as EGF, bFGF and collagens - obtained by recombinant DNA or extraction - turn out to be difficult and the few known examples are based on the use of topical galenical forms containing both inhibitors of serine protease and, as for EGF, competitive substrates such as collagen (see for instance K. Okumura et Al. - Phar . Res., 1_, 1289, 1990). As far as simple peptide molecules, such as Arg- Gly-Asp (RGD: known as the cellular adhesion site of fibronectin, laminin and of other proteins of the extra-cellular matrix) and Plasma Copper-Binding Growt Factor (GHL or GHK: tripeptide Gly-His-Lys) are concerned, the stabilization strategy used until now is based on the modification of the N- or C-ends (or o equivalent groups of the amino acid side-chains): introduction, with ester- or amide- like bonds, o suitable residues on said ends, leaving the peptid backbone unchanged. These changes induce a certain resistance to protease degradation, but the half-life of the products is subjected to the local presence of different enzymatic systems such as esterases, more or less enhanced by the pathology of the interested tissue and able to cleave the introduced group (exposing therefore the resulting peptide to the natural demolition pathway). In this respect, Pierschbacher M. (WO 90/06767, La Jolla Cancer Res.Found.) proposes polypeptide polymers of Arg-Gly-Asp (RGD), conjugated with biodegradable polymeric matrices, such as jaluronic acid, chondroitin sulphate, heparan sulphate, etc.. Pickart L.R. (US Patent 4,665,054 of 12.05.1985, applicant Biohead Inc.) discloses the healing activity of the copper complexes of glycyl-histidyl-lysine (GHL) peptide derivatives having the general formula: glycyl- histidyl-lysyl-COOR (RO=residues of alkyl or aryl alcohols; -NH 2 group) and claims their utility in the facilitation of the healing process and in the inhibition of thromboxane production by platelets. In comparison to the natural peptide, the said derivatives have an higher stability to hydrolysis by carboxypeptidases, i.e. the enzymes hydrolyzing the molecule starting from the carboxy-terminus (C- terminal) . In fact, the following half-life times (approximate and in excess) with respect to these enzymes can be derived from the data reported in said US patent: natural GHL < 1'; GHL-C0NH 2 < 2*; GHLCOOMe < 1.5'; GHL-COOCH 2 C 6 H 5 > 8'-10'. It has now been surprisingly found that the derivatives of the GHL peptide (and of the corresponding copper complex) according to this invention impart a general stability increase to the peptide (and to the corresponding copper complex) both with respect to the carboxy- and amino- peptidases and to the esterases. The present invention provides a more favourable solution to the known problem in the stabilization of the GHL peptide. The derivatives of the invention provide an improvement to the applicative therapeutic fields (wound healing, ulcers and tissue damages of different etiology) and to cosmetic applications (increase of the subcutaneous fat; decrease of wrinkles and telangiectasia conditions; stimulation of hair growth) since, differently from the known derivatives disclosed by L.R. Pickart, they exhibit an higher and unexpected resistance to hydrolytic agents such as carboxypeptidases, aminopeptidases and esterases (the latter being able to hydrolyse the ester bond between GHL and an alcohol or amide residue, therefore exposing the resulting GHL to rapid subsequent degradation) . These products maintain the capacity of complexing the Cu ion and show biological activities higher then those of the natural derivative, both in terms of specific activity (defined as activity per weight unit) and in terms of prolonged action in time (both connected to the increased resistance to enzymatic hydrolysis) . The derivatives of the invention have the following general formula: Gly-His-Lys-OR (I) wherein: Gly is one of the fol lowing residues : - glycine ; sarcosine ; group of formula NH 2 -CH 2 NH- , [gem(Gly ) ] ; His is one of the following residues : L-his tidine ; - D-histidine ; H I N CH_ ϋ LN I - group of formula HN-CH-NH, [gem(His)]; - group of formula -CO-CH-CO m(His); I CH,—. N N I H Lys is one of the following residues: - L-lysine; D-lysine; group of formula -CO-CH-CO-(m-Lys) (CH 2 ) 4 NH 2 R is hydrogen, straight or branched Cm - , . alkyl, Cg-C, 4 aryl or aralkyl residue, with the proviso that Gly, His and Lys cannot be contemporaneously the natural amino acids glycine, L-histidine and L-lysine. The invention also comprises the pharmaceutically acceptable salts and the copper complexes of the compound I. Preferred compounds of formula I are thos wherein: 1 - His or Lys is a residue of the corresponding aminoacid and R is H; 2 - His or Lys is a residue of the corresponding aminoacid and R is different from H; 3 - His or Lys are both residue of the corresponding aminoacids and R is H; 4 - His or Lys are both residue of the corresponding aminoacids and R is different from H; 5 - Gly is the sarcosine residue whereas His, Lys an R are as defined in any one of the above point 1-4; 6 - Gly is a gem-diaminal residue as above define [gem(Gly) ] whereas one of the His and Lys residu is a residue m(His) or m(Lys) as above define whereas the other is a residue of the L or series and R is hydrogen; 7 - Gly is a gem-diaminal residue as above define [gem(Gly) ] whereas one of the His and Lys residu is a residue m(His) or m(Lys) as above define whereas the other is a residue of the L or series and R is different from hydrogen; 8 - Gly is glycine, His is gem-His and Lys is m(Lys as above defined and R is hydrogen; 9 - Gly is glycine, His is gem-His and Lys is m(Lys as above defined and R is different from hydrogen. With reference to the previously reporte biological properties of the derivatives of th invention, these may be used as therapeutic agents i pathological forms asking for a cytoprotective and/o cytostimulating activity. Particularly, the compounds of formula I ar conveniently used for the preparation of a drug usefu in treating ulcers, scars, tissue damages of differen kind and more generally of drugs for the treatment o autoimmune disease. The compounds of the invention are formulate in suitable pharmaceutical compositions alone o in combination or with other useful activ principles. The dosages will be determined by the physicia and will anyhow depend on the pathology to be treated age, weight and conditions of the patient. Examples o pharmaceutical compositions are topical forms such a creams, ointments, gels, aspersory powders, medicate plasters, controlled release topical forms, loca injection (e.g. intraarticular) ; systemic forms, suc as injectable, or oral forms such as tablets, capsule or other conventionally known forms. The composition of the invention may be prepared by usual methods, suc as those disclosed in Remington's Pharmaceutica Sciences Handbook, Mack Pub. Co., NY, USA. The following examples further illustrate th invention. The used reagents, chemicals and standards ar usually available from commercial sources. Th disclosed solid-phase synthetic methods for th derivatives containing either D or L aminoacids ar those usually used in the peptide synthesis but the should not be intended to limit the different syntheti possibilities which can be used according to th available knowledges, such as, for instance, the synthesis in homogeneous phase, used herein for the derivatives esterified at the C-terminus. The preparation of the retro-inverted derivatives containing also aminoacids of the L series relies on methods already known (inter alia: A.S.Verdini et al., J. Med. Chem. 1991, 34, 3372-3379). Unless otherwise specified, the analytical method used for the determination of the titer or the belonging to the steric series (L or D) of the single aminoacids contained in each tripeptide (excluded those retro-inverted) is based on the method of Noriyuki Nimura et al. in J. Chrom. 352 (1986), 169-177, suitably modified. The IR spectra were- recorded in D-O or KBr on Jasco Mod. Ft/IR 5000 apparatus. The bonds at 3 1 404 cm-1, 3180 cm-1 (stretching N-H peptide and NH imidazole); 3100 cm " , 2995 cm " (stretching NH of NH, in NH 2 of lysine and/or salified terminal -NH 2 ) ; 1654 cm " (stretching CO amide: bond I of the amide); 1565 NH bond bending: band II of the amide); 1239 cm " (C-N-stretching + N-H bending: band III of the amide) agree with the peptide structure of the disclosed derivatives whereas the bands at 1469 cm " e 1442 cm " (ring stretching of the imidazole) agree with the presence of histidine. The FAB-MS (Fast Atom Bombardment Mass Spectroscopy) data were obtained with the VG-70-70 EQ- HF apparatus provided with a standard source, using Xe as gas, glycerol as matrix and temperature of 363°K. All the non retro-inverted samples not containing sarcosine showed a MH+ at 340 and those with sarcosine at 350, in full agreement with the proposed structure. The purity degree of the single derivatives and their behaviour with respect to different enzymes was determined by RP-HPLC on water 600E apparatus with Erbasil C18S (3μ), 4x250 mm apparatus, eluent 0.1 M NaClO. in phosphoric acid 0.1% v/v, or by capillary electrophoresis on Applied Biosystems 270 A-HT apparatus, H 2 P0 4 buffer, pH = 7.5, at 20 Kv and 30 β C. Abbreviations used: Z : benzyloxycarbonyl group Tos : tosyl group Bzl : benzyl group Boc : t-butyloxycarbonyl group Oct : n-octyl group D.C.C. : dicyclohexylcarbodiimide HOTB : 1-hydroxybenzotriazole PIP : piperidine DMF : dimethylformamide Pfp : pentafluorophenyl residue TFA : trifluoroacetic acid Sar : sarcosine (N-methy1-glycine) Bom : benzyloxymethyl group Example 1: Gly-His-D Lys (_1) Fmoc-D Lys(Boc)-R, (R: p-benzyloxybenzyl alcohol resin), after treatment with PIP/DMF to remove the protect the group from ^-amino group, added to the pentafluorophenyl activated ester Fmoc-His(Boc)-OPfp. The dimer immobilized on resin, Fmoc-His (Boc)-D Lys(Boc)-R, treated with PIP/DMF, yielded His(Boc)-D Lys(Boc)-R which was conjugated with Fmoc-Gly-OPfp. The immobilized and protected tripeptide, Fmoc- Gly-His(Boc)-D Lys(Boc)-R, after deprotection and separation from the resin to a 90% trifluoroacetic acid in water, was added to a stoichiometric amount (3:1) of HC1 and then lyophilized. The derivative Gly-His-D Lys.3HCl was obtained. A sample of the product was transformed in Gly-D His- Lys.AcOH (acetate) by adsorption on Nova Pack HR C18 (Water) HPCL column and elution with 0.2% acetic acid in water. The Gly-His-D Lys Cu (II) complex was prepared dissolving the peptide in water, adding an equimolar amount of monohydrate copper acetate and adjusting the pH with diluted sodium hydrate, under cooling. After centrifugation at low temperature, to make the solution clear, the product was lyophilized. Aminoacid content (three determinations) Gly: 1.02±0.01; His: 0.98±0.03; D Lys: l.OO±O.OO Example 2: Gly-D His-Lys (2 ^ ) Fmoc-Lys(Boc)-R (R: p-benzyloxybenzyl-alcohol resin), after treatment with PIP/DMF, was conjugated with Fmoc-DHis(Boc)-OH by means of dicyclo- hexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt). The reaction with Fmoc-Gly-OPfp and the subsequent deprotection with 90% TFA yielded the trifluoroacetate which, after addition of HC1 3:1 and lyophilization, yielded the tripeptide Gly-D His- Lys.3HC1. The corresponding acetate was obtained as disclosed in Example 1. The copper complex Gly-D His- Lys Cu (II) was prepared as disclosed in Example 1. Aminoacid content (three determination) Gly: 1.01±0.02; D His: 0.99±0.02; Lys: 9.01±0.03 Example 3: Gly-D His-D Lys (3_) Fmoc-D Lys(Boc)-R (R: p-bezyloxybenzyl-alcohol resin) was added, after treatment with PIP/DMF, to Fmoc-D His(Boc)-OH in the presence of DCC/HOBt and then to Fmoc-Gly-Opf. The recovery of the Gly-D His-D Lys.3HCl derivatives was carried out as in the previous examples. The Gly-D His-D Lys Cu(II) complex was obtained as disclosed in Example 1. Aminoacid content (three determinations) Gly: 0.99+0.02; D His: 1.04±0.03; D Lys: 1.00±0.03 Example 4: (N-methyl)Gly-D His-D Lys (Sar-D His-D Lys) (12) The synthesis of the Sar-D His-D Lys derivative was carried out according to the method disclosed for the derivative Gly-D His-D-Lys except for the conju¬ gation of the dipeptide H-D His (Boc)-D Lys (Boc)-R which was carried out with Fmoc-Sar-OPfp. The preparation of the derivatives Sar-D His-D Lys.3HCl, Sar-D His-D Lys.Ac (acetate) and of the complex Sar-D His-D Lys Cu (II) was carried out as described in the previous examples. Aminoacid content (three determinations): Gly: 1.03±0.03; D His: 1.01±0.02 ; D Lys: 0.97±0.04 Example 5: (N-methyl)Gly-His-D Lys (Sar-His-D Lys: 11) and (N-methyl)Gly-D His-Lys (Sar-D His-Lys: 10) The two derivatives were synthesized as in the previous examples, using the intermediates H-His(Boc)-D Lys(Boc)-R and H-D His(Boc)-Lys(Boc)-R in the conjugation with Fmoc-Sar-OPfp. The copper complexes were obtained as in Example 1. Chemico-physical characteristics: Aruninoacid content (three determinations) lit Sar: 1.01±0.03; His: 0.98±0.02; D Lys: 0.99+ 0.04 _10_: Sar: 0.97± 0.02; D His: 1.01±0.03; Lys: 0.98+0.02 Example 6: Gly-D His-D Lys-O-benzylester (j?) In a suitable solvent (hexane/ethyl acetate) H-D Lys(Z)-0-Bzl and Boc-His(Tos) were conjugated in the presence of D.C.C.. After treatment with NaHC0_, extraction and washing with water, the organic phase was evaporated and the residue crystallized from hexane-ethyl acetate. The crystallized product was dissolved in 50% hydrofluoric acid in dichloromethane and the solvent was then removed. The residue dissolved in hexane/ethyl acetate and in the presence of D.C.C. , was conjugated with Boc-Gly. After drying, dissolution in glacial acetic acid and hydrogenation in the presence of Pd/C, the tripeptide was purified by means of cation and anion exchange resins. The eluates were neutralized with acetic acid or HCl. Gly-D His-D Lys-O- Bzl was transformed into the Cu(II) complex as disclosed in Example 1. Aminoacid content (three determinations) : Gly: 1.01±0.02; D His: 0.97±0.05; D Lys: 1.00±0.03 Example 7: Gly-D His-Lys-O-octylester (4_) Preparation of the derivative (D or L) Lys (Z)-O-R (R: alkyl residue from 1 to 14 straight or branched carbon) according to the patent by L. Pickart (cited patent): H- L (or D) Lys(Z)-OH was treated with an excess of the desired alcohol in anhydrous benzene and traces of p-toluensulfonic acid chloride (or anhydrous HC1) . After refluxing for about 2 hours, th benzene/water azeotrope was continuously distilled fo about 12 hours. After this period the mixture was kep at 0°C and the precipitated product was treated wit potassium bicarbonate in water and methylene chlorid and the organic phase, was evaporated after washin with water and drying on anhydrous magnesium sulphate The residue was purified by chromatography. The derivative L Lys (Z)-O-Oct, prepared a described above starting from n-octyl alcohol, wa dissolved in anhydrous solvent and reacted with Boc- His (Tos)-OH in the presence of D.C.C. and HOBT. Th subsequent treatment with 50% HF in methylene chloride followed by coupling with Boc-Gly as disclosed i Example 6, optionally in the presence of HOBT, yield Gly-D His-Lys-O-oct, purified by chromatography. The preparation of corresponding Cu (II) comple was carried out as in Example 1. Aminoacid content (three determinations) Gly: 0.97±0.02; D His: 0.98±0.05; D Lys: 0.96±0.05 Example 8: Sar-D His-D Lys-O-benzylester (18) The derivative D Lys (Z)-O-Oct, prepared a disclosed in Example 7, is reacted with Boc- His(Tos)-OH in the presence of D.C.C. and HOBT in th same conditions above reported; the obtained derivativ was condensed with Boc-Sar with D.C.C./HOBT and th crude product, dissolved in glacial acetic acid, wa hydrogenated with Pd/C and the final product purifie by chromatography. The complex Sar-D His-D Lys-O-benzylester Cu (II was obtained as disclosed in Example 1. Aminoacid content (three determinations) Sar: 0.96±0.04; D His: 0.98±0.02; D Lys: 0.97±0.05 Example 9: Gly-gem His-(R,S)m-Lys (19) a) Bσc-Gly-g-His(Bom)-H 1.2 g (3.05 mmol) of Boc-His(Bom)-OH (Bachem) we dissolved in 20 ml of dichloromethane containi 3.05 mmol pyridine and 3.05 mol pentafluoropheno 7.625 mmol of dicyclohexylcarbodiimide (D.C.C were added to this solution, cooled at 0 β C. Aft 3 h the reaction mixture was filtered, dried a the residue, repeatedly washed with petrole ether, was suspended in 10 ml of tetrahydrofura 2.6 ml of 25% ammonia were added, under stirrin to this suspension, obtaining a yellow solutio this was evaporated and the residue dissolved 100 ml of chloroform. The chloroform solution w washed with 10 ml of water, 20 ml of 5% NaHCO then again with NaCl saturated water unt neutrality of the washing liquid. The resid obtained from the chloroform solution w suspended in 12 ml of HCl 3.2 M and stirred 60 β C for 20 min; the solution obtained, w adjusted to pH 7.5 with NaHCO- (3.6 g) aft cooling. A residue was obtained from th evaporated solution, which was thoroug dehydrated by means of repeated additions benzene-methanol 6:4 azeotropic mixture, and th the inorganic salts were removed by repeat chloroform extractions. The obtained amide (1.787 mmol) was dissolved 13 ml of dichlorometane together with equivalent amount of triethylamine. After the addition of 1.787 mmol of Boc-Gly-OPfp (obtained starting from Boc-Gly, Bachem, by activating the carboxy group with pentafluorophenol according to the method above disclosed for histidine) the solution was left under stirring at room temperature for 2 h. The reaction mixture was evaporated, dissolved in acetonitrile and evaporated again, repeating this operation many times. The residue was finally dissolved with the minimum amount of CHC1, and purified on 70-230 mesh Merck silica gel column, 20 g, using CHCl,-MeOH 9:1 as eluent. 600 mg of pure Boc-Gly-His(Bom)-NH 2 were thus obtained. The dipeptide obtained in form of amide, as above disclosed (1.386 mol) has been dissolved in 6 ml of CH,CN and 1.5 ml of H-,0 were then added; the bis-trifluoroacetoxy-iodobenzene reagent has been added thereto (820 mg; 2.087 mmol) and then 220 μl of pyridine. The reaction mixture was then stirred at room temperature for 1 h and 526 mg of solid NaHCO, were then added and the mixture was stirred for 10 additional minutes. The mixture was finally evaporated and, after drying by means of repeated evaporations with benzene-methanol azeotropic mixture, the residue was purified on Merck 70-230 mesh silica gel column, 55 g and mutiple-step elution. The used mobile phases were respectively: 1, CHCl 3 -MeOH 7:3, 2), CHCl- j -MeOH 10:1, 3), CHC1 3 - MeOH 7:1, 4), CHCl 3 ~MeOH 6:1. 505 mg of Boc-Gly-g-His(Bom)H were thus obtained, b) 2,2-Dimethyl-5-(N-trifluoroacetyl-butyl)-l,3- dioxan-4, 6-dione 4-NH 2 -butyrraldheyde diethylacetal (3.7 ml, 21 5 mmol) was dissolved, together with 2.62 g diethylaminopyridine in 40 ml of CHC1, and t solution cooled at 0 β C; 3 ml of trifluoroacet anhydride were added dropwise, under stirrin Stirring was continued for 30 minutes, maintaini 10 the temperature between 5 and 10 β C, and then aga for 2 additional h at room temperature. T suspension was filtered, the filtrate washed wi 10 ml of NaHCO-. After one night at ro temperature, this solution was saturated with Na 15 and extracted with dichloromethane (1x30 ml, 1x ml and 4x10 ml). The residue of the pooled a concentrated organic phases was a yellow oi about 3 g, which was directly used in t subsequent step. 20 8.7 ml of a cyanoborohydride solution (lM in TH were evaporated under vacuum and the resid dissolved in 5 ml of DMF. The Meldrum acid (2, dimethyl-l,3-dioxan-4,6-dione) 1.79 g, 12.4 mo was also added to this solution. This solution w 25 then added to the oil obtained in the first ste cooling at 0-5 β C to avoid an exothermic reacti and the mixture was left at room temperature f 2 h. The reaction mixture was then diluted with 40 30 of cold H 2 0: a white precipitate was formed a the pH was adjusted to 4 by careful adding con HCl . This suspension was stirred, always at 0-5°C for 1 h and then filtered; the product, washed o the filter with H 2 0 and then with ether, an thereafter thorougly dried on P 2 ° S ' hac ^ m -P- 129 130°C, with final yield of 40%. c) 2 ,2-Dimethy1-5-(N-Boc-butyl)-1,3-dioxan-4, 6-dione 1.17 g (3.77 mol) of N-trifluoroacetylbuty derivative (prepared in b) were suspended in 8 m dioxane. 1.9 ml of 5 N NaOH were added and th resulting clear solution was left under stirrin at room temperature for 4 h. After cooling to 5 β C di-tert-butyldicarbonate (1.6 ml, 7.54 mmol) wa added dropwise. The reaction mixture was kept a room temperature for 12 h adjusting the pH to 8 if necessary. At the end, the solution was dilute with 10 ml water and filtered. The filtrate wa evaporated under vacuum to halve its volume diluted again, and the same process was repeate until elimination of dioxane. The pH was adjusted to 5 with 1M KHSO. and th solution extracted with CHC1- (1x50 and 2x20 ml) After evaporation of solvent, the oily residue wa purified on a silica gel column (50 g) wit CHCl 3 EtOAC 5:1. The pure product was obtained with 74% yield. d) Boc-Gly-g-His(Bom)-m-Lys(Tfa)-OH 1.012 g (3.25 mol) of 2,2-dimethyl-5-(N trifluoroacetylbutyl)-1,3-dioxan-4 ,6-dione wer mixed with 598 mg of pentafluorophenol and heate to the melting temperature of the mixture (abou 110 β C). The fusion mixture was stirred for 3 h a this temperature and then subjected to vacuum b water pump for 15 more minutes. The residue wa dissolved in 10 ml of dichloromethane and 1.311 of Boc-Gly-g-His(Bom)H and 850 μl of triethylamin were added. After stirring overnight at roo temperature, the reaction mixture was purified b preparative HPLC on Deltapack column C18-100 Waters, 15 μ, eluted with a gradient from solutio A (H 2 0 + 0.1% Tfa) + Sol. B (CH 3 CN-H 2 0, 80:20 0.09% Tfa) 50:50, up to 0-100 in 20 minutes. Flo 12 ml/min. e) Boc-Gly-g-His(Bom)-m-Lys(Tfa)-OPh 528.2 mg (1.7 mmol) of 2,2-dimethyl-5-(N trifluoroacetylbutyl)-l,3-dioxan-4,6-dione wer mixed with 159.7 mg of phenol and heated to th melting temperature of the mixture (110 β C), unde stirring for 25 h and then 15 minutes unde vacuum. After cooling the oil was dissolved in ml of NaHCO- saturated solution which was washe with CHC1 3 (1x10 and 2x5 ml). The organic phases washed again with 5 ml of H 2 0 and 5 ml of NaC saturated solution, dried on Na-SO. an evaporated, gave a white solid residue consistin of pure product with 74% yield. 1.677 g (4.83 mol of the previously obtained phenylester, 1.95 (4.83 mol) of Boc-Gly-g-His(Bom)-H, 0.390 μl o pyridine, 783 mg (5.705 mol) o hydroxybenzotriazole were dissolved in 40 ml o dichloromethane and the solution cooled to 0 β C under stirring, 1.315 of DCC were then added an the reaction was continued for 3 h at roo temperature. At the end the suspension was filtered, the filtrate diluted with 400 ml of dichloromethane, washed with 50 ml of 10% Na 2 C0 3 solution and then with different portions of H_0 and NaCl saturated solution. The solvent, dried and concentrated, gave 2 g of the completely protected tripeptide as yellow solid, 57% yield. f) Boc-Gly-g-His(Bom)-m-Lys (Boc)-OH 770 mg (1.91 mmol) of Boc-Gly-g-His(Bom)-H were suspended in 15 ml of CH_C1 2 under stirring in Argon atmosphere and 1.2 g (5.91 mol) of N,0-bis- silylacetamide (BSA) were added to the suspension. A clear solution was obtained which was refluxed for 6 h and thereafter cooled to room temperature. 542 mg (1.72 mmol) of 2,2-dimethyl-5-(N-Boc- butyl)-l,3-dioxan-4,6-dione were then added and stirred overnight (about 16 h). The reaction mixture was finally evaporated and the ter-butyloxycarbonyl group was directly removed as disclosed in Example 7. g) Removal of Boc groups from Gly and Lys The residue from the previous reaction (see Example 6) was treated with 29 ml of trifluoroacetic acid and stirred at room temperature for 20 minutes, then evaporated, dissolved again in 10 ml of CH 3 CN and purified on preparative HPLC column: Column: Deltapack 19x300 mm, 15 micron, Waters. Mobile phase: A, 0.1% Tfa in H 2 0 B, 20% CH 3 CN in H 2 0 + 0.09 Tfa, linear gradient of B from 0 to 20% in 20 min. Flow: 19 ml/min; injection volume: 1 ml The recovered residue consisted of 586 mg of Gly- g-His(Bom)-m-Lys-OH in trifluoroacetate form, 95% pure. Yield 42.5%. h) Removal of the benzyloxymethyl group from the gem- histidine residue 586 mg (0.73 mmol) of Gly-g-His(Bom)-m-Lys-OH and 4.74 g of thioanisole (38.16 mmol) were dissolved in 24 ml of TFA in Argon atmosphere. After cooling to l-2 β C 8.48 g (38.16 mmol) of triflate (trimethyl-silyl-trifluoromethansulfonate) were added and the mixture was stirred for 30 min. 36 ml of ethyl ether were then added: a white precipitate, separated by centrifugation, was obtained. The residue was washed by decantation with other 20 ml of ether three times. The residue was finally dissolved in 21 ml of 5% NH.OH containing 1.18 g of NH.F, stirred for 1 h and the pH adjusted to 5 with AcOH 5N. Chromatographic conditions: Column: Novopack 19x300 mm, HRC18, A°, 6 micron, Waters Mobile phase: 0.2% AcOH H 2 0 Flow: 12 ml/min; 200 μl injection Each fraction containing the product was pooled and concentrated, the residue dissolved with 1 ml of H-0 and evaporated again, repeating this operation many times, so as to remove the excess acetic acid from the residue. At the end, after lyophilization, 86 mg of pure peptide were obtained corresponding to the following structure: H-Gly-g-His-m-Lys-OH.AcOH.2.5.H 2 0. Elemental analysis: Calc. %: C 43.14, H 7.41, N 18.87 Trov. %: C 42.82, H 7.18, N 18.83 1 H-NMR, 10% in D 2 0, ppm: 8.38, 7.31 (s, CH imid.); 5.95 (t, CH g-His); 3.90 (s, CH 2 Gly); 3.38-2.98 (m; CH 2 g-His; CH 2 NH 2 m-Lys; CH m-Lys); 2.04 (s, CH 3 COO~); 1.78 (m, o\ and β CH 2 m-Lys); 1.38 (q, V CH 2 m-Lys) . i) Removal of all the protective groups of the tripeptide Boc-Gly-g-His(Boc)-m-Lys(Tfa)OPh 125 mg (0.17 mmol) of the fully protected peptide, obtained as described in e), were dissolved in 0.5 ml of MeOH + 0.17 ml of NaOH 5N and stirred for 4 h at room temperature. Sodium hydroxide was then neutralized with 3N HC1 and methanol was evaporated. The residue was diluted in 2 ml of H 2 0 and washed with CHC1 3 (2x0.5 ml). The aqueous phase was evaporated under vacuum and H-0 traces were removed by repeated evaporations with azeotropic mixture. The residue (232 mg) was directly used in the subsequent step. The previous residue was suspended with 200 mg of Nal in 2 ml of CH..CN, under stirring and Argon atmosphere. 0.17 ml of trimethylsilyl chloride were added to the suspension which was kept at 80 e C for 3 h. 130 mg of Nal and 0.11 ml of TMSiCl were then added and left in the same conditions for three hours. After cooling to room temperature, 270 mg of sodium thiosulfate in 4 ml of H 2 0 were added to the mixture; a clear solution was obtained having pH 4 which was then adjusted to 7 with NaOH. The formed precipitat was removed by filtration and the dissolve product exactly purified as in Example 8. The pure peptide H-Gly-g-His-m-Lys-OH monoacetat had the same characteristics as those obtaine with a different synthetic method as disclosed i h). 1) Preparation of copper complex of the tripeptide G gH-mL 32.3 mg (0.095 mmol) of G-gH-mL were dissolved i 0.3 ml of H 2 0 and 0.7 ml of an aqueous solutio containing 22.6 mg of Cu(CH 3 COO) 2 .H 2 0 (0.113 mmol were added to this solution. The solution wa neutralized with 0.1N (2.4 ml) NaOH, lef overnight in the refrigerator (4 β C) and the centrifuged and decanted. The surnatant was eluted through a Sephadex G- column, eluted with distilled water a lyophilized, to give 34.7 mg of blue powde consisting of the G-gH-mL-Cu. Example 10: H-gem Gly-D His-(R,S) m Lys OH (22) The product was obtained starting from t monophenyl ester of the 2-N-trifluoroacetylbuty malonic acid (described in Example 9) and H-D His(Bom) O-tBu in the presence of HOBT/DCC. After ac treatment, tBu-O-D His(Bom)-(R,S) mLys(TFA)-O-Ph obtained was transformed in HO- D His(Bom) -(R, mLys (TFA)-0- Ph which was reacted with glycinamide the presence of HOBT and DCC. The amide residue of the derivative H 2 N-C0-CH--N CO-D His (Bom)- (R, S) mLys(TFA)-OPh was transformed amino group with TIB in DMF. The removal of the protective groups, carried out with alkali first and then with trimethylsilyliodide, yielded the derivative H-gem Gly-D His-(R,S) mLys-OH. The NMR spectrum confirms the expected structure. Similarly, H-gem Gly-(R,S) m-His-L Lys-OH and H- gem Gly-(r,s) His-D Lys-OH were prepared. Example 11: biological activities a) wound healing First method: Dunkin-Hartley guinea pigs were anesthetized and five square wounds (7x7 mm) were excised on the mid- back, completely removing the dermal surface, after shaving and cleaning the dorsal skin (F. Buffoni et al. - Pharmacol. Res. 2J5, suppl. 2, 332, 1992). The wounds, both in control and treated animals, were allowed to heal. Four, eight and eleven days after surgery, five animals/group were sacrificed and newly formed tissue was dissected and analyzed by histological and biochemical methods. The following biochemical parameters were determined: hydroxyproline production (index of collagen formation) (J.F. Woessner, Arch. Biochem. Biophys. 9_3, 440, 1961); protein content (O.H. Lowry et al., J. Biol. Chem. 193, 265, 1951); DNA content (C. Labarca et al., Anal. Biochem. 102, 344, 1980). Histology was performed after staining wit hematoxylin/eosin. Experimental groups (15 animals/group) : 1) controls, receiving 20 μl of distilled water; 2) animals treated with 10 μg of the Cu (II) complexes described in Table 1 dissolved in 20 μl of distilled water; 3) animals treated with 10 μg of GHL-Cu(II) complex in 20 μl of distilled water. Administration route: water solutions were applied onto the wound. TABLE (1 ) mg/g of fresh tissue The pieces of tissue removed from the wounds on the 4th day contain both the regenerated tissue and the scab (mainly cluster of dead cells) and thus the three analyzed parameters presented higher values than those at the 8th and the 11th day. However, the results obtained from the animals treated with the products hereinbefore described show higher chemotaxis and/or increased production of extracellular matrix in comparison to those obtained from animals untreated or treated with GHL-Cu(II). The comparison of the three parameter values obtained at the 8th and 11th day clearly shows that in the animals treated with the compounds described in the present patent application the skin was already well restored at the 8th day whereas in the animals treated with GHL-Cu(II) the same degree of healing was reached at the 11th day. This indicates that the compounds reduced the time of wound healing with respect to the controls and the GHL-Cu(II) treated. At the histological analysis, the tissues from the animals treated with the compounds hereinabove described turned out well organized. Second method: Hairs were removed from the dorsal skin of Wistar rats (approximate weight 220 g) and two round wounds (0 10 mm) were induced by punch under anesthesia. Ten min after the excision, the wounds were treated with 20 μl of distilled water alone (control group) or containing 10 μg of the products to be tested (treated groups), then covered with a surgical gauze. The gauze was kept distant from the wound bed by a teflon ring with appropriate diameter fixed with an adhesive bandage. The treatment was repeated for three consecutive days. On the 5th, 8th, and 12th day the percentage rate of wound healing was evaluated assuming: 100% : total healing 0% : absence of healing The experimental groups consisted of 6 animals/group. Administration route: water solution applied onto the wound. The results, shown in Table 2, indicate that the compounds described in the present application reduced the time of wound healing with respect to the native product. Therefore the histological analysis shows a complete restitutio ad integrum of the tissues and an angiogenetic effect similar to those obtained with physiological timing. TABLE 00 b) Superoxide dismutase activity The superoxide dismutase activity of the products hereinafter described was determined according to C. Beauchamp et al. (Anal. Biochem. 44_, 276, 1971). The method is based on the appearance of coloured species with a maximum of absorption at 560 nm by NBT (nitroblue tetrazolium) in the presence of superoxide ion. The reduction in the intensity of absorption at 560 nm in the presence of the product under investigation determines its SOD-like activity. This activity is expressed in units defined as the concentration of product, in nmoles/ml, able to induce 50% of the maximum inhibition. The results, reported in Table 3, show that the tested derivatives presented an activity equal to GHL-Cu(II) activity. TABLE 3 Product Units of activity (nmol/ml) Gly-His-Lys (Cu) 3.3 ± 0.2 2 (Cu) 3.1 ± 0.2 3 (Cu) 2.8 ± 0.1 9 (Cu) 2.9 ± 0.2 12 (Cu) 3.3 ± 0.2 12 (Cu) 3.3 ± 0.1 19 (Cu) 3.4 ± 0.1 22 (CU) 3.1 ± 0.2 c) Anti-thromboxane activity The activity of the products under examination was determined in rabbit PRP (platelet rich plasma), according to N. Lad et al. (Br. J. Pharmacol. 6>9_, 3, 1980). TxB2 final titration was performed with the Thromboxane B2 125-1 Assay System (Amersham Life Science, U.K. ) . The results, reported in Table 4, show that the investigated products have an activity equivalent or superior to GHL-Cu(II) one. TABLE 4 Product % of inhibition of the (1 μg/ml) B 2 production Gly-His-Lys (Cu) 83 ± 3 2 (Cu) 86 ± 5 4 (Cu) 89 ± 4 12 (Cu) 92 ± 5 17 (Cu) 88 ± 2 19 (Cu) 91 ± 3 22. (CU) 90 ± 3 Example 12: resistance to peptidases The stability of the derivatives described i Table 5 was tested in presence of mitochondrial leucineaminopeptidase (aminopeptidase M) , carboxypeptidase and human plasma from health volounteers. The residual quantity of each one of th tested products was determined at differen experimental times by HPLC using the above describe conditions. The results obtained after 5 and 35 min o exposure to the enzymatic action (phosphate buffer pH 7.4 at 37°C for both pure enzymes and undiluted plasma) are reported in Table 5. The data show a much highe resistance of the tested products to the enzyme, i comparison to native GHL. TABLE (1) No longer analitically detectable at t ^ 20' (2) No longer analitically detectable at t ^ 150' (3) This value remains constant up to t > 120' This increased resistance accounts for the greater biological activities shown in vivo by these compounds. No significant difference between free peptides and peptide copper complexes has been detected as far as their biological activity.

Claims: CLAIMS 1. Compounds of general formula (I) Gly-His-Lys-OR (I) wherein: Gly is one of the following residues: glycine; sarcosine; group of formula NH 2 -CH 2 NH-, [gem(Gly)]; His is one of the following residues: L-histidine; D-histidine; group of formula ]; group of formula Lys is one of the following residues: L-lysine; D-lysine; group of formula -CO-CH-CO- (m-Lys) (CH 2 ) 4 NH 2 R is hydrogen, straight or branched C.-C,. alkyl, Cg-C 14 aryl or aralkyl residue, with the proviso that Gly, His and Lys cannot be contemporaneously the natural amino acids glycine, L-histidine and L-lysine, and pharmaceutically acceptable salts and the copper complexes thereof. 2. Compounds according to claim 1 wherein His or Lys is a residue of the corresponding D aminoacid and R is H. 3. Compounds according to claim 1 wherein His or Lys is a residue of the corresponding D aminoacid and R is different from H. 4. Compounds according to claim 1 wherein His or Lys are both residue of the corresponding D aminoacids and R is H. 5. Compounds according to claim 1 wherein His or Lys are both residue of the corresponding D aminoacids and R is different from H. 6. Compounds according to claim 1 wherein Gly is the sarcosine residue whereas His, Lys and R are as defined in any one of the above points 1-4. 7. Compounds according to claim 1 wherein Gly is a gem-diaminal residue as above defined [gem(Gly)] whereas one of the His and Lys residue is a residue m(His) or m(Lys) as above defined whereas the other is a residue of the L or D series and R is hydrogen. 8. Compounds according to claim 1 wherein Gly is a gem-diaminal residue as above defined [ge (Gly) ] whereas one of the His and Lys residue is a residue m(His) or m(Lys) as above defined whereas the other is a residue of the L or D series and R is different from hydrogen. 9. Compounds according to claim 1 wherein Gly is glycine, His is gem-His and Lys is m(Lys) as above defined and R is hydrogen. 10. Compounds according to claim 1 wherein Gly is glycine, His is gem-His and Lys is m(Lys) as above defined and R is different from hydrogen. 11. Use of the compounds of claims 1-10 as therapeutic agents. 12. Use of the compounds of claims 1-10 for the preparation of drugs useful in the treatment of ulcers, tissue damages of different etiology, autoimmune degenerative pathological conditions. 13. Pharmaceutical compositions containing a therapeutically effective amount of a compound of claims 1-10 as the active principle.

Inventors: DAL POZZO ALMA, SPORTOLETTI GIANCARLO, DAL POZZO, ALMA, SPORTOLETTI, GIANCARLO
Applicants: DAL POZZO ALMA, ELLEM IND FARMACEUTICA, SPORTOLETTI GIANCARLO, ELLEM INDUSTRIA FARMACEUTICA S.R.L.
Application year: 1993
Main classification: C07K5
IPC codes: A61K31, A61K38, A61P43, C07D233, C07K5