GONADOLIBERIN DERIVATIVES, PROCESS FOR THE PREPARATION AND PHARMACEUTICAL AND VETERINARY COMPOSITIONS THEREOF

Abstract: New gonadoliberin derivatives of the formula Glp-His-Trp-Ser-Tyr-X-Lev-Arg-Pro-Y, wherein Y represents glycine-amide group or an -NH-alkyl group having 1 to 4 carbon atoms in the alkyl moiety, X stands for a D-tyroxyl, D-tyronyl or D-4-chlorophenyl-alanyl group, and acid addition salts and metal complexes thereof. The new nona- and decapeptide derivatives have an excellent luteinizing and folliculus stimulating hormone releasing activity, and they can therefore be used as active ingredients in pharmaceutical compositions for human or veterinary application.

Description: GOKADO ΣBER-Q. DERIVATIVES, PROCESS FOR THE PREPARATION AW PHARMACEUTICAL AUD VETERIHARY COMPOSITIONS THEREOP TECHNICAL PXE D This invention relates to new gonadoliberin derivatives 5. and process for their preparation. More partictilarly, the in¬ vention concerns Be gonadolifcerin derivatives of the for-πula I/ Glp-]=-is-T-- -Se-- * -!ζyτ^Σ-Iιeu-Arg-Pro-f /_/ wherein 0 X represents a D-4-chlorophenyl-alanyl, D-tyroxyl or D- -tyronyl group and Y is a glycine amide or -NH-al3-yl group, having 1 to 4 carbon atoms in the allcyl moiety, and acid addition salts and metal complexes thereof -, She in- vention also includes a process for the preparation of said nona- or decapeptide derivatives and pharmaceutical or vete¬ rinary compositions containing them. The abbreviations used in the above general formula and hereinbelow correspond to the generally accepted nomenclature of the chemistry of peptiάes see e.g. J. Biol. Cheπ 241. 527 /1966/_7 » In the specification the ollowing further abbrevia- tions .w ll be used: -Cpa - 4-chloropheπyl-alanine -Thy * tyronine -Thx » tyroxine BACKGROUND .ART Gonadoliberin /also referred to as gonadotropin releasing hormone, G-n-RH, luteinizing and folliculus stimulating hoxmone, releasing hormone, LH FSH-RH/ and its known derivatives are capable of releasing the luteinizing and folliculus stimulating hormones 1H and PSH/. It is known in the art βL Honahan et al., BLochemist-y 12. 4616-4620 /1973/'_/ that those gonadoliberin derivatives in which glycine in the 6-position is replaced by certain D-amino acids have a surplus effect related to gonadoliberin. This in¬ crease in the activity is most expressed in case of the compounds which contain D-tryptophane or D-tert.butyl-serine in the 6- -position /J. Sandow et al., Control of Ovulation, Butterworths, London, 1978, pp.49-70/. According to Nestor et al.^-J. Med. Chem * _ % 795-801 A982/, US-PS 4,234,5737 *? replacing the glycine in the 6- -posiϋ of gonadoliberin by D-amino acids having a highly lipophilic side-chain also effective gonadoliberin analogues can be prepared. It is further known that the biological activity of go¬ nadoliberin can further be increased by replacing the gy- cine-amide group in the 10-position with amide groups contain¬ ing aliphatic carbon chain J__. Pu^ino et al., J.ϊϊed.Chem. 16, PI According to Coy, D.H., Labz e, P., Eavary, VL. , Coy, E.J., Schally, A.V. Bioche . Biophys. Res. Commun. 67, 576-582 /1975/, potent gonadoliberin derivatives can be obtained also by re¬ placing the 6-glycine in gonadoliberin or in a corresponding amide by a D-phenylalanyl group. The invention relates to new gonadoliberin derivatives whic show a better biological activity than their known analogues. According to another aspect of the invention we provide a process or the preparation of the new nona- and decapeptide derivatives of the formula /I/ I and Y are as defined above/, by -which in place of the 6-glycine group other amino acids can be incorporated with a good yield, without racemization. DISCLOSURE OP THE INVENTION The invention is based on the surprisiag inding that the compounds which contain a D-tyroxyl, D-tyronyl or D-4-chloro- phenyl-alanyl group in place of the 6-glycine group of gonado¬ liberin have a better biological activity and can easily he. prepared. We have further found that the activity of these new go- nadoliberin derivatives can be increased further by replacing the glycine-amide group in the 10-position by an -NH-G. ,- -alkyl group. According to the invention the new nona- or decapeptide derivatives of the formula /_/ Glp-His-Trp-Ser-Tyr-X-Leu-Arg-?ro-Y /I/ wherein X and YThave the same meaning as defined above, and acid addition salts and coπφlexes thereof are prepared by.con¬ densing a pentapeptide azide of the formula /H/ GIp-Ξis-Trp-Ser-ϊζyT-H, /__/ -V OMP with a tetra- or pentapeptide of the formula IH/ X-Leu-Arg-Pro-Y /HI/ wherein X and Y are as defined above, and if desired, convert¬ ing the nona- or decapeptide amide obtained into an acid addi- . tion salt thereof by reacting it with a suitable acid, or if desired, deliberating the free base from an acid addition salt obtained with a base, and if desired, converting a nona- or decapeptide-amide obtained into a metal complex thereof, BEST WDS OR CARRYING OUT THE INVENTION The D-Thy -gonadoliberin derivatives are preferably pre¬ pared by reducing the D-Thx -gonadoliberin derivatives. The reduction is preferably carried out with hydrogen. If the reduc- t_ T5sperformed with tritium gas, in the presence of a ca¬ talyst, from D-rhx -gonadoliberia D-Thy -gonadoliberin specifi- cally labelled with tritium is obtained. These gonadoliberin derivatives which show a high specific radioactivity can ex¬ cellently be used is various tests and as diagnostics. The pentapeptide component of the fO-naula /__/ is pre¬ ferably prepared by the condensation of pyroglutaEyl-hysticlyl- -tryptophane-azide and seryltyrozine mβthylester. Hg- this method the undesired racemization can be avoided, -ξyroglutamyl- -hystidyl-tiyptophane used as a starting compound can be pre¬ pared by coupling pyroglutam±c acid with /N-imidazol-dinitro- pheryl/-hystidyl-txyptophane ethylester. The tetra- or pentapeptides of the fo-raula /HI/ are preferably prepared by acylating a tri- or tetrapeptide of the formula Leu-Arg-Pro-Y /Y is as defined above/ known in the art in* Yanaihara et al., J.Ifed.Chem. 16, 373 /1973 _7 OD i s a ino group with an ' X a ino acid supplied with a Boc or Z protecting group /X is as defined above/ by a method conven¬ tionally used in the peptide chemistxy. The LH-releasing activity of the new peptides according to the invention was tested by the following method, in which for comparison the known gonadoliberin was employed under the same conditions. The adenohypophysis of fe ale Sprague-Dawley rats weight¬ ing 150-200 g each was eliminated and pretrypsinized. Approxi¬ mately 40-50 pituitaries were placed, into separate flasks and allowed to stand in 25 ml of a 0.256 trypεin solution /Sigma, type 111/ at 0 to 4°C or 16 hours. The pituitaries were then stirred under 5/6 CO- atmosphere at 37°C or 20-25 minutes while they were decomposed to their cells* To the cell suspen¬ sion soya-bean trypsin inhibitor was added whereupon it was suspended and centrifuged in a Medium 199 solution /with -Sarie's salts, Plow Laboratories, UK/ three times /800 x g, 10 minutes/. The cell suspension was passed throug a sterile metal filter and the number of cells in one ml was counted. After counting, 3 ml of the cell suspension was dispensed into culture flasks each cozitaining 1.8 x 10 cells. The cells were cultured for 3 days at 37 C under S& C0„ atmosphere. On the 3rd day the medium was replaced by a fresh one and incubation was continued or one more day. On the 4th day the medium was —7 —12 changed again and a medium containing a 10 to 10 molar solution of the peptides to be tested was added to the cell cultures* The cells were incubated with the peptide solutions for 4 hours, the culture solutions were eliminated and the amount of the LH-hoxmone released from the cells was determined by radioiaunoassay. For the various peptides the following ED__- -" * υ -values and relative activities were obtained. GnHH 0.3433 ΏH 1.00 c D-Cpa -GnHH 0.02063 nH 16.6 D-Thx -GnHH 0.02892 nH 11.9 D-Thy -GnHH 0.02435 M 14.1 D-Cpa -GnEH-EA 0.01278 n 26.9 wherein Cpa: 4-chlorophenylalanine EAJ ethylamide According to a still further aspect of the invention there are provided human or veterinary pharmaceutical compositions, which are generally prepared by admixing at least one compound of the foπnula _/ or a pharmaceutically acceptable salt or complex thereof with, carriers conventionally used in the pre¬ paration of pharmaceutical compositions and formulating the com- positions obtained for example as tablets, dragees, capsules, suppositories, injection solutions, nasal spray, etc. In the veterinary therapy the peptides of the ormula /I/ can ef ectively be used for example as anoestronic or anovula- tory agents or for treating follicular cysts. They are generally administered is a single daily dose of 5-200,ug, preferably 50-100Λig, intramuscularly, subcutaneously or intravenously. When applying against follicular cysts a repeated treatment is also possible. They are effective in the treatment of various animals, e.g. catties. In the human therapy the sew compounds according to the invention proved to be effective contraceptives. For this pur¬ pose they are generally administered is a single 400 to €00 ,ug/day dose as a nasal spray * The sew compounds are further effective is treating mammasy carcinoma or cancer of the prostate. In this application their daily dose generally is 5 to 50 ,__ when administered subcutaneously, and 400 to 600 ,ug when a nasal spray- s employed. The pharmaceutical compositions con¬ taining the new peptides of the formula /_/ as an active in¬ gredient can be formulated and administered also as infection solutions or suppositories. The new peptides are effective also against cryptorchidism. The main advantages of the process according to the in¬ vention and the sew peptides obtained by this process are as followst a/ The new gonadoliberin derivatives according to the invention are more active than those knows in the art. b/ The pentapeptides of the formula /H/ are common intermediates of the preparation of all end products. c/ The X amino acids are incorporated isto the peptide* is the last step of the preparatios of the tetra- or penta¬ peptides of the formula /HI/, therefore the replacemest of the 6-amiso acid, which plays the most important role is the biological activity, takes plaoe easily, is a simple way and the usdesired racemisation can be avoided* d/ The D-Thy -gonadoliberin derivatives can be effective¬ ly labelled with tritium, and the derivatives obtained which have a high specific radioactivity can ery advantageously be used in pharmacological, phar akinetical, biochemical and phy- siological assays or as diagnostics * Further details of our invention are illustrated by the following son-limiting Examples, ∑n the Examples the melting points are indicated is C, the values are sot corrected. The thin layer chroma ographic R.-valu s were determined os Kieselgel DC, Alufolien, Merck/ plates, using the follow¬ ing solvent mixtures: 1 * Ethy^cetate/ϊ>yridise/acetic acid/water 30:20;6:11 2. et__y3.cetate/pyridise/acetic acid/water 60:20:6:11 3. ethyl acβtate/pyridise/acetic acid/water 120:20:6*11 4. ethyl acetate/pyridise/acetic acid/water 240:20:6ill 5. acetone/toluene 1:1 6. chloroform/methasol/acetic acid 85:10:5 7. methylethyl ketose/pyridise/water 65:15:20 8. j&-butasol/acetic acid/water/ethyl acetate 1:1:1:1 9. jn-butanol/acetic acid/water 4:1:1 10. n^butanol/acetic acid/water 4:1:5 /upper phase/ 11. n-butanol/pyridine/acetic acid/water 15:1:4:20 /upper phas 12. i-propanol/lM acetic acid 2:1 OMPI Example 1 Preparation of D-Cτa -gonadoliberin a/ BOC-His DEP/-Trp-OMs H*622.62 21.12 g /50 mmoles/ of BOC-His/DHP/-OH are dissolved is 100 __ of dimethyl foimamide and the solution is cooled to 0°C. Thereafter 10.32 g /50 m oles/ of dicyclohe2yl-carbodi- iπide and 7»66 g /50 mmoles/ of are added with stirring* The mixture is stirred at 0 C or 10 mi- sutes and the precipitated dicycloheχyl-urea is filtered off. 12 * 74 g /50 mmoies/ of H-Trp-OSe.BCl are dissolved is 70 ml of dimethyl fozxαa ide and the solution is cooled to 0 C. 6.93 ml /50 mmoles/ of triethyl gπi,d,e are added and the pre¬ cipitated triethylamine hydroc-hloride is filtered off after stirring or 5 minutes. The two solutions are combined and stirred at 0 C over¬ sight. Thereafter the precipitated DCU is filtered off and the solution is evaporated to dryness. The oily residue is dis¬ solved is 500 ml of ethyl acetate and shakes with three 100-ml portioss of ice cool 1 lϊ ΣΞSO, solstios, five 00-asl portioss of a saturated SaHCO- j solution asd finally with two 100-ml portioss of a 10$ HaCl solution * The organic phase is dried over magnesium sulfate, filtered and evaporated to dryness. The obtained .oily substance is powdered with petroleum ether, filtered and dried * She crystallise product obtained cas be recrystalli-Bed from ethyl acetate with petroleum ether. Yield: 27.70 _ /89g/ Sslting pointt 119 to 122°C H-Hi H:522.49 /free base/j 595.41 /.2B21/ 24.9 g /40 mmoles/ of B0C-Eis/ππ?/-0 e dipeptide are dissolved is 100 ml of methanol and 100 ml of a 4 n methanolic hydrochloric acid solution are added. The ϋ xture is allowed to stand at room temperature for 30 minutes, while the di- peptide ester 1-ydrochloridβ is crystallized. The crystals are filtered off, washed with ether and dried. Yield: 21.91 g /92$6/ Melting point. 198 to 202°C β_7^* 0.49° /c-1, DMF/ R f 3 - 0.461 H^ - 0.18| R f 5 - 0.06 c/ Glp-Eis-/DBP/-Trp-OMe M:633.60 4.85 g /36.8 mmoles/ of L-pyroglutamic acid, 7.58 g of dicyclohexyl-carbodiimide and 5,63 g of H-hydroxy-ben2tria- zolθ are dissolved is 100 ml of dimethyl foraamide. The mixture is stirred at 5 to lo°C for 10 minutes whereupon the preclpitat- ed DCU is filtered off. 20.84 g /35 mmoles/ of H-l-_is/DHP/-Trp-01-fe.2-__Cl are dis¬ solved in 100 ml of dimethyl foimamide and 9.72 ml /70 mmoles/ of triethyl amine are added to the solution. After stirring for 5 minutes the precipitated triethylamisβ hydrochloride is filtered off. The two solutions are combined ___ stirred at room temperature overnight* Thereafter 90 ml of acetone are- added to the mixture and the precipitated insoluble material is filtered off. Evaporation of the solutios affords as oily material, which is triturated with ethyl acetate, filtered asd dried. The dry crystalline material is washed with three 25-ml portions of water and dried * The product can be recryst l ized by dissolving is hot ethyl acetate and coolisg. Yield: 18.42 g /83.W Melting point: 148 to 151°C Z? ξ 3 " +5 - 2 ° /cβl » DMP/ R j 3 " 0.53} R f 4 » 0.38 d/ Glp-His-Trp-OHθ M.466.50 15.84 g /25 mmoles/ of Glp-His DKP/-Trρ-0Me protected tripeptide are dissolved is a mixture of 100 ml of dimethyl O ? fo_mand.de and 40 ml of water. 4 ml of mercapto-ethasol are added and the pH of the solution is adjusted to 8 with triethyl amise. It is allowed to stasd at room temperature for 30 mi¬ nutes, asd is evaporated to drysess is vacuo. The oily material is triturated with ether, filtered asd dried. The product ob¬ tained is dissolved is a snail portion.. of methasol asd is re- crystallized by adding ether. The precipitated crystals are filtered off and dried. Yield, 10.92 g /93.6$6/ Melting point: 228 to 232°C £g^- * +4.04° /c-0.42, DMF/ R f 2 » 0.30 9.33 g /20 mmoles/ of Glp-His-Trp-OMe tripeptide are dissolved is 250 ml of methasol. To the solutios 20 ml of a 98$ hydrazise hydrate are added. The reaction mixture is stirred at 40 C for 3 hours and subsequently at room temperature over¬ night* The precipitated material is. then filtered off, washed with cold methasol and dried* Yield: 7.58 g /81.2S6/ Melting point: 166 to 169°C f/ Glp-His-Trp-Ser-Tyr-OMe M:716.8 7.0 g /15 mmoles/ of Glp-His-Trp--_r 2 _-L obtaised is Step g are dissolved is 60 ml of dimethyl formamide. The solutios is cooled to 0 C asd 7.5 ml /45 mmoles/ of a 6 s EC1 solutios are added with stirrisg. Thereafter 1.035 g /15 mmole. of NaNO- are added to the mixture as a coscestrated aqueous solution,-drop- wise, and the mixture is stirred at 0°C for a further 15 minutes. To the reaction mixture a solution of 4,78 g /_5 mmoles/ of H-Ser-Tyr-OHe-HCl is 15 ml of dimethyl forπa idβ are added^ The pH is adjusted to neutral with 6.25 ml /45 mmoles/ of triethyl amine and the mixture is stirred at 0 to 4°C oversight. Os the following day it is evaporated to dryness in vacuo and the oily product is triturated with ether. 12.1 g θO%/ of the title compound are obtained, contain¬ ing a small amount of impurity* This product cas be eosverted isto iiydrazide without purificatios asd the hydrazide obtaised is readily crystallizable. The physical properties of a costrol sample recrystallized from methasol: Melting point: 188 to 190°C ^ m 7_ 2 " -3.48° /c-1, DMF/ R j 1 - 0.58; R j 2 - 0.26 g/ Glp-His-Trp-Ser-Tyr-HgH- M:716.8 12 g of crude, powdered δlp-His-Trp-Ser-Tyr-OMe penta¬ peptide ester are dissolved is 200 ml of methasol, 10 ml of a 98% hydrazine hydrate are added and the mixture is stirred at 40 C for 3 hours. The mixture is stirred at room temperature oversight, whereupon the precipitated crystals are filtered off and dried is as exsiccator, over concentrated sulfuric acid. The dry crystallise material is dissolved is 250 ml of a 0.5 s hydrochloric acid solutios, its pH is adjusted to 8 with saturate sodium carbonate solution, it is allowed to stand at 0°C for 2 hours asd the precipitated crystals are filtered, washed with ice cold water asd dried. Yield: 6.12 g /56.9S6 calculated for the two steps/ Meltisg poist: 205 to 206°C £?_7^» 21.51° /c»l, DMF/ R j 1 - 0.39I 2 j 2 « 0.14 -Ξty-drazise sitroges: found t 3.7956, 3.76J6 theoretical: 3.91 * 6 h/ BOC-D-Cpa-OFFP M:465.8 A solutios of 6.0 g /20 mmoles/ of BOC-D-Cpa-OH is 50 ml of ethyl acetate is cooled to 0°C asd 4.0 g /22 mmoles/ of peπtafluorophenol and subsequently 4.52 g /22 -aaoles/ of dic>yclohe-^l-carbodiimide are added. The precipitation of DCU starts ismediately. The mixture is stirred at 0 C for one hour and at 20°C for a further ose hour, whereupon the precipitated DCU is iltered off * She solutios is evaporated is vacuo asd the residue is crystallized from a mixture of ethyl acetate asd petroleum ether * The substance is allowed to stand is a refrigerator whereupon it is filtered, washed with a 1:1 mix¬ ture of ethyl acetate asd petroleum ether asd dried* Yields 7.8 g /84U/ °C Asalysis for C p -E ^ O.KClF c calculated: C 51.57*, H 3.67#, H 3.005*, CI 7.6156 ound: C 51.5256, H 3.6656, H .05S6 f CI . 8* / H-D-Cpa-Leu-Arg-Pro-Gly-NI- .ΣHF M:662.15/2HF 300 mg /0.39 mmoles/ of the pestapeptide prepared is Step i/ are suspended is 0.9 ml of asisole asd 30 ml of hydrogen fluoride are condensed on the suspension at -80°C in a teflon reaction vessel* It is stirred at 0°C for 45 minutes and hydrogen fluoride is distilled off. To the residual asisole solution ab¬ solute ether is added and the precipitated substance is filtered after standing or one hour, washed with more portions of ether and d±±ed over sodium hydroxide. Yield: 220 mg /85?6/ Melting poist: 169 to 181°C β j^ « -0.37 /c-0.5, E^O/ R j 1 - 0.5} R j 2 - 0.1; R f δ - 0; R j 8 0.45; R 10 «0.35 OMPI Amino acid analysis: Cpa 1.0, Arg 1.0, Pro 1.01, Gly 0.95, Leu 1.0 k Glp-His-Trp-Ser-Tyr-D-Cpa-Leu-Arg-Pro-Gly-EHg M:1324.9 36 mg /0.05 mmoles/ of Glp-His-Trp-Ser-Tyr-HgH- penta- peptide-hydrazide are dissolved in 1 ml of dimethyl formaτπi,de, the solutios is cooled to -10°C and 3 l /0.2 mmoles/ of a 6 n hydrochloric acid solutios are added with stirring. The tempe¬ rature of the mixture is kept at -10°C while a coscentrated aqueous solution of 3.8 mg /0.055 mmoles/ of sodium sitrite is added with stirrisg and the mixture is stirred or further 10 minutes between -5 asd -10 C. Thereafter to the azlde solutios prepared as described above a cooled solutios of 35.3 mg /0.055 mmoles/ of D-4-chlorophenyl-alanine-leucyl-arginyl-prolyl- ±Ώ. -gl cineamide hydrogen fluoride p. ml of dimethyl forma ide is added and the pH of the reaction mixture is adjusted to 7-8 with triethyl amine. It is stirred at a temperature between -5 and -10 C for one hour and at 0 C for one further hour and allowed to stand in a refrigerator overnight. The reaction mixture is evaporated in vacuo and the crude decapeptide obtained is puri¬ fied on a Sephadex G-25- column /3 x 100 cm/ /Pharmacia, Uppsala, Swedes/ by partitios chromatography, is a 15:1:4:20 mixture of butasol, propasol, acetic acid and water. 5-ml fractions are collected and the elution is monitored by u.v. absorption or thin layer chromatography.. The fractions containing the desired material are lyoph£lized. The lyophilization is repeated from dilute acetic acid. Yield: 33 mg / <W j 1 - 0.5. E j 8 - 0.55, j 100 - 0.10, R j 12 - 0.66 Amino acid analysis: Glp 0.155, His 0.140, Ser 0.148, Tyr 0,105, - Leu 0.200, Arg 0.135, Gly 0.170, Pro could not be calculated. OMPI Example 2 The φreτaratios of D-Thx -gosadoliberis a/ BOC-D-T-_α:-Iιeu-Arg-Pro-G_-y-I--- M:1300.5 87.7 mg /0.1 mmoles/ of BGC-D-Thx are dissolved in 1 ml of dimethyl for-x * amide, 50 mg /O.ll mmoles/ of leu-Arg-Pro- -G-ty-H-__ 2* -3F asd 15yUl /O.ll mmoles/ of triethyl amine are added, the reactios mixture is cooled to 0°C asd a solution of 84 * 5 mg /O.ll mmoles/ of F-complex is dimethyl forrnamide is added. It is stirred at 0°C or one hour and at * room temperature overnight * It is then evaporated is vacuo, triturated with ethyl acetate, filtered asd chromat©graphed on a silica gel plate /I cm. x 15 cm . The column is equilibrated with a I20?6:lls20 mixture of ethyl acetate, pyridine, acetic acid and water and the desired material is eluted with this solvent mixture * About 2-ml fractions are collected in every 15 minute, The fractions having the same R j -value are combined, evaporated is vacuo. triturated with ether, iltered asd dried * Yields 32 mg Melting points 208°C /decomp./ ^Xj 20 - -6 * 8 /c=0.5, DSF/ S j 2 - 0.35, R j 8 * 0.7, H j 9 - 0.6 b/ H-D-TI-X-Leu-Arg-Pro-(--ly-BH 2 »ECl MJ1236.9 50 mg of a protected pentapeptide according to Step a/ are dissolved is 5 ml of glacial acetic acid asd 1 ol of a 1 M solution of hydrochloric acid in glacial acetic acid is added* It is stirred at room temperature. After 4 hours the reaction is complete. The title material is precipitated with ether, filtered asd dried in as exsiccator, over sodium hydroxide * Yield: 40 mg /90g/ j 2 - 0.16, R j 8 * 0,67- c/ Glp-His-Trp-Ser-Tyr-D-Thx-Leu-Arg-Pro-cay-EHg M:1860.2 28,7 mg /0.04 mmoles/ of Glp-His-Trp-Ser-Tyr-K^ pesta- peptide-hydrazide are dissolved is 1 ml or dimethyl forma ide asd the solutios is cooled to -10°C and 26 ,ul of a 6 JB hydrochloric acid solutios are added with stirrisg. The temperature of the mixture is kept at -10°C and a coscestrated aqueous solution of 3 mg of sodium sitrite is added with stirrisg, asd the ndi- ture is stirred at a temperature betwees -5 asd -10 C or further 10 misutes* To the aside solution obtained a cooled solutios of 49*5 mg /0.044 mmoles/ of E-Φ-Thx-Leu-Arg-Pro-Gly-S3=^.B31 is 1 ml of dimethyl formamide is added, asd the pH of the reaction mixture is adjusted to 7-8 * with triethyl amine* The mixture is stirred at -5 to -10°C for one hour and at 0°C or a further one hour and is then allowed to stasd in a refrigerator overnight* The mixture is evaporated in vacuo. The residual crude deca¬ peptide is purified on a Sephadex S-25 column /3 * 100 cm/ by partition chromatography, in a 15:l:4s20 mixture of butanol, propanol, acetic acid and water* 5-ml fractions are collected asd the elution is monitored by tuv* absorption or thin layer chromatography. The fractions containing the title product are lyophilized, Iyophilization is repeated from dilute acetic acid * Yields 18.7 Hg /255έ/ R j 1 - 0.65, R j 8 -0.68, j U « 0.30, R j 12 - 0.81 Example 3 Preparation of injections for istra-miscular, subcutane¬ ous or intravenous administrations a/ The gonadoliberin derivatire of the fort-tula /I/ is dissolved in distilled water, a physiological saline solutios or buffered aqueous solution, in a concentration of 1 to Λ mg/ml. The solution is filtered to sterile, 1 - small portions , _. which. containing 50-500 ,ug of active ingredient/are filled into aa- OMPI poules and lyophilized, asd the ampoules are sealed* The active ingredient content of the ampoules is freshly dissolved before treat-ient b adding 1-10 ml of distilled water asd a volume, corresponding to the desired dose Is administered. V 20-500 Ug/ml of a gonadoliberin derivative of the formula /I/ are dissolved in as aqueous solution containing 0*9* of EaCl asd 0.9* of benzyl alcohol* The solutios is steril¬ ized by filtration, 50-500-,ug active ingrediest-containing portions are filled into ampoules which are then sealed * The solutions obtained cas directly be injected*

Claims: CLAIMS 1. Ho_-βpeptid J θ-C- ,-alkyl amides and decapeptide amides of the formula /_ Glp-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y /_/ wherein X represents a D-tyroxyl, D-tyronyl or D-4-chlorophesyl- alanyl group, Y stands for a glycine-amide group or an -EΞ-alkyl group containing 1 to 4 carbon atoms in the alkyl moiety, acid addition salts and metal complexes thereof * 2. Human and veterinary pharmaceutical compositions which comprise as an active ingredient at least one gonadolibe¬ rin derivative of the formula /I/, whereis X and Y are as de— fised is claim 1, or a pharmaceutically acceptable acid addi- tion salt or metal complexthereof, is admixture with solid and/or liquid carriers or additives conventionally used is the preparation of pharmaceutical compositions * 3 * Pharmaceutical compositions as claimed is claim 2 in the form of tablets, dragees, capsules, suppositories, is- jec ion solutions, sasal sprays* 4. A process for the preparatios of gonadoliberin deri- vatives of the formula /I Glp-Eis-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y /I/ whereis X represents a D-tyroxyl, D-tyronyl or D-4-chlorophenyl— alasyl group, Y is glycine-amide or an —EH-aliyl group containing 1-4 carbon atoms in the alkyl moiety, acid addition salts and metal complexes thereof, which process comprises condensing a pentapeptide-azide of the formula /H/ Glp-Ξis-Trp-Ser-Tyr-Iϊ- /__/ - IS - with a tetra- or pentapeptide of the ormula /HI/ X-Leu-Arg-Pro-Y ' /___/ whereis X asd Y are as defined above, asd if desired, convert¬ ing a nona- or decapeptide-amide obtained into an acid additios salt thereof, or if desired, deliberating the free base from an acid addition salt obtained by reacting with a base, and/or if desired, converting a nona- or decapeptide-amide obtained into a metal complex. 5 * A process for the preparation of human or veterinary pharmaceutical compositions, which process comprises admixing at least one gonadoliberin derivative of the formula f_I% where¬ in X and Y are as defined in claim 4, or a pharmaceutically acceptable acid addition salt osp metal complex thereof with solid and or liquid car&iers or additives * OMPI

Inventors: ANTONI FERENC, ERCHEGYI JUDIT, HORVATH ANIKO, KERI GYOERGY, MEZOE IMRE, NIKOLICS KAROLY, SEPROEDI JANOS, SZELL ANDRAS, SZOEKE BALAZS, TEPLAN ISTVAN, ANTONI, FERENC, ERCHEGYI, JUDIT, HORVATH, ANIKO, KERI, GYOERGY, MEZOE, IMRE, NIKOLICS, KAROLY, SEPROEDI, JANOS, SZELL, ANDRAS, SZOEKE, BALAZS, TEPLAN, ISTVAN
Applicants: RICHTER GEDEON VEGYESZET, RICHTER GEDEON VEGYESZETI GYAR R.T.
Application year: 1983
Main classification: C07C103
IPC codes: A61K38, C07K7, C07K14