CA2089247A1 - Small peptide and pseudopeptide amides inhibiting the proliferation of small-cell and epithelial-cell lung cancer cells - Google Patents

Abstract

Abstract The invention relates to novel peptide and pseudopeptide amides of the formulae (1) to (6), wherein p-HOPA-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (1) D-MePhe-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (2) D-MePhe-D-Trp-Phe-D-Trp-Leu-MPA (3) D-Tyr-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (4) D-Tyr(Et)-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (5) D-MePhe-D-Trp-Tyr-D-Trp-Leu ?(CH2NH)Leu-NH2 (6) wherein p-HOPA means p-hydroxyphenylacetic acid, D-MePhe stands for D-N-methylphenylalanine, MPA represents 2-amino-3-methylpentane and ?(CH2NH) is a methyleneamino group being present instead of a peptide bond, as well as their pharmaceutically acceptable acid addition salts.
The invention further relates to pharmaceutical compositions containing these compounds as well as a process for preparing the compounds of formulae (1) to (6). The compounds according to the invention are useful for inhibiting the proliferation of cells of the small-cell and epithelial-cell lung cancer.

Description

n ~ r~ ?
. ? ~
2330~-1213 8MALL ~EPTIDE AND P8BUDOPEPTID~ ANIDE~ IN~IBI~ING
~KB PROLIFERATION OF 8MAL~-CBLL AND EPITHE~IAL-CELL LUNG

The invention relates to novel, therapeutically active peptide and pseudopeptide amides of the formulae (1) to (6), ~-HOPA-D-Trp-Phe-D-Trp-Leu ~(CH2NH)Leu-NH2 (1) 10 D-MePhe-D-Trp-Phe-D-Trp-Leu ~(CH2NH)Leu-NH2 (2) D-MePhe-D-Trp-Phe-D-Trp-Leu-MPA (3) D-Tyr-D-Trp-Phe-D-Trp-Leu r ~CH2NH)Leu-NH2 (4~
D-Tyr(Et)-D-Trp-Phe-~-Trp-Leu yr(cH2NH)Leu-NH2 (5) D-MePhe-D-Trp-Tyr-D-Trp-Leu ~(CH2NH)Leu-NH2 (6) wherein ~-HOPA means ~-hydroxyphenylacetic acid, D-MePhe stands for D-N-methylphenylalanine, MPA represents 2-amino-3-methylpentane and r(CH2NH) is a methyleneamino group bein~ present instead of a peptide bond, as well as their pharmaceutically acceptable acid addition salts and pharmaceutical compositions containing these compounds.
Furthermore, the invention relates to the preparation of the above compounds and compositions.
The compounds of formulae (1) - (6) according to the invention are new and possess valuable pharmacological activity. More particularly, they inhibit the proliferation of the cells of the small-cell and epithelial cell lung cancers.
It is known [Nature 316, 823 ~1985)~ that both bombesin (BN) occurring in the amphibia as well as the gastrin-releasing peptide (GRP), which is structurally closely related to bombesin and occurs in mammals, act as A4851-67-MRJKmO

autocrine growth factors in the small-cell lung cancer (SCLC). The formula of bombesin is Glp-Gln-Arg-Leu-Gly-A3~-Glr~-Trp-Ala-Val-Gly-llis-Leu-Met-NH2, whereas the fragment 14 to 27 of GRP is represented by the formula Met-Tyr-Pro-Arg-Gly-AQn-Hi~-Trp-Ala-Val-Gly-Hi~-Leu-Met-NH2.
It is also known [Am. J. Physiol. 252, 6439 (1987);
Regulatory Peptides 19, 10~ (1987); as well as Eur. J.
Pharmacol. 190, 31 (1990)] that structural analogues of BN or GRP (BN antagonists) inhibit the binding of [125I-Tyr4]-bombesin to BN receptors being present on Swiss 3T3 cells and prevent the proliferation of the small-cell lung cancer cell line; said proliferation can be measured by the incorporation of 3H-thymidine.
It has been observed [Proc. Natl. Acad. Sci. USA
82, 7616 (1985); Br. J. Cancer 57, 579 (1988)] that the peptide of the formula D-Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp--phe-n-Trp-Leu-Leu-NH2 (Spantide), i.e. an antagonist against "Substance P" (SP), acts as a BN antagonist on Swiss 3T3 mouse embryonal fibroblasts and inhibits the proliferation of the cells of the small-cell lung cancer.
Furthermore, it has been described [Biochem.
Biophys. Res. Commun. 156, 323 (1988)] that the N--acylated pentapeptide of the formula HOPA-D-Trp-Phe-D-Trp-Leu-Leu-NH2 (I) is a relatively strong SP antagonist (with a PA2 value of 5.73 on the guinea pig ileum) inhibiting the smooth muscle-contracting effect of SP.
Our investigations directed to the C-terminal pentapepti~e analogues of Spantide have shown the low molecular SP antagonist of the formula (I) to inhibit the binding of the labelled ~l25I-Tyr4]-bombesin to Swiss 3T3 cells and prevent the proliferation of NCI-H69 small-cell lung cancer cell line possessing no BN receptors.
Thus, the aim of the present invention is to 2 ~ t ! ~ ~ 7 prepare analogues not inhibiting the binding of [125I-Tyr4]-BN to Swiss 3T3 cells, i.e. analogues which are therefore not BN antagonists but exert a strong inhibitory action on the proliferation of the cells of the small-cell and epithelial-cell lung cancers. Such compounds might be very useful in the therapy of lung cancer.
It has been found that the analogues of formulae (1) to (6), containing a modified, mainly reduced peptide bond, show such an effect.
Abbreviations used in the description are as follows:
FCS foetal calf serum D-MEM Dulbecco's minimal essential medium 15 BSA bovine serum albumin HEPES N-(2-hydroxyethyl3piperazine-N'-(2-ethanesul-fonic acid) PBS phosphate-buffered saline SDS sodium dodecyl sulfate 20 TCA trichloroacetic acid Other abbreviations are in agreement with those described in "Nomenclature and Symbolism for Amino Acids and Peptides, Recommendations 1983" of the IUPAC-IUB
Joint Commission on Biochemical Nomenclature (JCBN) tBiochem. J. 219, 345 (1984)].
The novel compounds of formulae (1) to (6) and their acid addition salts can be prepared in such a way that, by using Leu r(CH2NH)Leu-NH2 or Leu-MPA as starting substances, the peptide of formula (1), or the derivatives of peptides of formulae (2) to (6) containing N-terminal benzyloxycarbonyl or tert-butoxycarbonyl protective group are built up, preferably by the successive use of the steps of reactive ester coupling and deprotection of the ~-NH2 group, then the protective group is removed by catalytic hydrogenation or acidolysis and, if desired, r ~ ~?, ~

the product obtained is tra~sformed into a pharma-ceutically acceptable acid addition salt or the free base is liberated from such a salt.
In the case of the compound of formula (1), the N--terminal ~-HOPA is incorporated without any protective group into the molecule, to obtain the final product by building up the peptide chain.
The methyleneamino (reduced peptide) bond of the pseudopeptide Leu- ~(CH2NH)Leu-NH2, used as starting substance, can be formed by reductive alkylation performed in a known manner [see e.g. Nature 299, 555 (1982)]. Thus, Leu-NH2 hydrochloride is alkylated with Z--Leu-H (Z-Leu-aldehyde) and the Schiff's base obtained is reduced in situ by sodium cyanoborohydride. The N--terminal benzyloxycarbonyl protective group of the protected pseudodipeptide Z-LeuYr~CH2NH~Leu-NH2 is removed by catalytical hydrogenation.
Leu-MPA may be prepared as follows. In the first step, 2-amino-3-methylpentane is acylated by a mixed anhydride formed from Z-Leu-OH and pivaloyl chloride, then the benzyloxycarbonyl protective group is removed by catalytical hydrogenation and the mixture of Leu-MPA
isomers is separated by chromatography on a silica gel column. Thereafter, the Leu-MPA isomer described in the experimental part is used.
After accomplishment of the synthesis, the obtained crude pentapeptide amides according to the invention are purified by chromatography on a silica gel column.
The biological investigation of the new ccmpounds of the invention was c~rried out in four tests. The BN--antagonizing effect of the compounds in question was determined in Test 1, based on measurin~ the displacement of the labelled t12~I-Tyr4]-BN from the BN receptors being present on the Swiss 3T3 mouse fibroblast cells.
Test ~ and Test 3 were used to study the inhibitory effect of the compounds of the invention on the proliferation and propagation of NCI-H69 small-cell lung cancer cells. The inhibitory effect of the compounds of the invention on non-small-cell lung cancer cells and epithelial-cell lung cancer cells was investigated in Test 4.
Test 1 Study of the binding of target compounds to BN
receptors on the Sviss 3T3 mouse fibroblast cell line Swiss 3T3 mouse fibroblast cells were grown on tissue-cultivating plates containing 24 holes in a D-MEM
culture medium containing FCS of 10 % until a confluent and restinq state (3x105 cell/hole). The cells were washed 3 times with 0.5 ml of D-MEM of pH 7.4 (containing 0.2 ~ of BSA and 24 mmol of HEPES, and cooled to 4 ~C), i.e. incubation mixture, each. Subsequently, the cells were incubated in a final volumP of 250 ~l at 4 C for 3 hours with an incubation mixture containing [125I-Tyr4~--BN in a constant concentration (1 nmol) and the compound to be tested in an amount of 1.13 to 75 ~mol. At the end of the reaction the cells were washed twice with the incubation mixture and then ~ times with PBS (pH = 7.4).
The washings were carried out with 750 ~1 volumes of the ice-cold liquids each. The cells were solubilized by adding 750 ~l of 0.5 M sodium hydroxide solution, carried over into test-tubes by pipet and the radio-activity bound to the cells was measured. The grade of aspecific binding was determined in the presence of a high concentration (1o~6 molJl) of cold ~Tyr4~-BN. The amount of the specifically bound, labelled hormone was expressed as the percentage of the maximum binding, i.e.
the specific binding in the absence of the compounds to be tested, as shown in Table l.

2 ? ~? ~ 7 T~bl~ 1 Displacement of tl25I-Tyr4]-BN on the Rwis~ 3T3 mou~e fibrobl~t c~ll line Displacement as percentage of control net binding Concentration Compound r~mol/l~ 1)(2~ (3) (4) (5) 1.18 108 lOS - 96 85 113 2.36 98 96 92 108 88 111 4.71 83 99109 92 79 106 9.42 55 10393 103 72 117 18.8 33 10699 108 61 116 37.6 18 10794 113 52 95 The data of Table 1 show that the BN-antagonizing effect being characteristic of SP antagonists is abolished by incorporating the methyleneamino kond into SP antagonists [compounds of formulae (1), (2), (4) and (5)] or by substituting an aliphatic amine for the C-ter-minal amino acid of the SP antagonists [compound (3)].
Test 2 ID~estigation of the inhibitory effect of target compoun~s on the prolifer~tion of NCI-~69 3mall-cell lunq oanc~r cell line po~essing no BN

3 0 recep~ors by measuring the incorporation of 3~-thymidine The cells gr~wn in suspension, i.e. in RPMI-1640 culture medium ~DIFC0, U.S.A.3 supplemented with FCS of 10 %, were collected by centrifuging at 4 C with 1200 rpm. After decanting the supernatant, the cells were 2'?`~

suspended in RPMI-1640 HITES medium (containing lx10-8 mol/l of hydrocortisone, 5 mg/l of insulin, 10 m~/l of transferrin, lxl0-8 mol/l of estradiol and 3x10-8 mol/l of sodium selenite). The cell number was adjusted to 5 5x105 cell/ml; 90 ,ul of this cell suspension were added into each hole of a tissue-cultivating plate containing 96 holes. 10 1~1 of solutions, each containing the target ~ompounds in various concentrations, were added to the cells. The cells were grown at 37 C under air containing 5 % by volume of carbon dioxide for 72 hours, then 37 kBq (1 ,uCi) of 3H-thymidine, dissolved in 10 ~l of culture medium, were added into the holes and the incubation was continued for additional 24 hours. Subsequently, the cells were solubilized in 10 ~l of 20 % SDS solution. 25 ,ul of solution from each hole were dropped onto Whatman 3 filter paper. After drying, the filter paper was washed 3 times with 5 % a~ueous cold trichloroacetic acid solution, and then with 96 % ethanol in order to de-hydrate it and to remove the traces of trichloroacetic acid.
After drying, the filter paper pieces were placed in scintillation tubes and 5 ml of scintillation cocktail [a mixture containing lO00 ml of toluene, 400 ml of abs.
ethanol, 10 ml of dioxane, 6 g of 2,5-diphenyloxazole and 0.15 g of 1,4-bis(5-phenyl-2-oxazolyl)benzene] were added to each. The activity of the samples was measured by using on I,KB Wallac 1211 beta-counter. The data were expressed as the percentage of control, where the 3H--thymidine uptake of NCI-H69 cells not treated with the compounds was considered to be 100 ~6 (control~ (see Table 2).

Table 2 Effect of the target compounds on the 3~-thymi~ine upta~e of NCI-~69 small-cell lu~g cancer cell line Uptake as percentage of control Concentration Compound [umol/l~ (I) (1) f2) (3) (4) (5) ~6) 0 100 100 100 10~ 100 100 100 0.75 92 - 75 115 101 105 1.56 77 - 48 104 76 155 82 3.125 104 77 51 49 88 137 65 6.25 66 34 2~ 34 59 67 63 12.5 63 44 8 18 51 17 58 200 1.7 1.3 1.7 - - --Te~t 3 Investigation of the i~hibitory effec~ of target compoun~s on the prop~gation of NCI-~69 small-cell lung c~ncer callq NCI-H69 cells grown in suspension, i.e. in RPMI-1640 culture medium supplemented with FCS of 10 ~, were collected by centrifuging at 4 C with 1500 rpm. After decanting the supernatant, the cells were suspended in a fresh medium and the cell number was adjusted to 1 x 105 cell/ml. 5 ml of cell suspension each were applied into tissue-culti~ating vessels of 25 cm2 size. The compounds to be tested w re used in a final concentration of lQ and 50 ~mol/l, respectively. The cell number was determined from 2x200 ~1 of cell suspension in each defined time 3~ interval. The volume of sample taken QUt was replaced with g a medium containing the target compound in a con-centration of 10 or 50 ~mol/l, respectively. The cell dilution resulted was corrected by calculation. The data were expressed as percentage of the starting cell number (lx105 cell/ml was considered to be 100 ~). The change in the number of cells not treated with the compounds was also indicated as control (see Table 3).
T~ble 3 Effect of the target compounds on the propagation of NCI-~69 qm.ll-cell lung ca~cer cell line Cell number as percentage of the starting cell number Compound Concent- (C) (I) (2) (3) (4) (5) ration t~mol/l] o 10 50 10 50 10 50 50 50 t days 0 lV0100 100 100 100 100 100 lO0 100 164- - 110 47 15~ 93 - 73 7 17~233 120 140 20 190 107 80 122 Note: C = control It can be seen from the data of Tables 2 and 3 that the target compounds retained the inhibitory effect on the maturation and propagation of NCI-H69 small-cell lung cancer cells, which is characteristic of SP antagonists;
the inhibitin~ effect of the compound of formula (2) is even stronger than that of the reference substance of the ~5 formula ~I).

2 ~ ~ t r TQ~t 4 ~xAmination of the effect of t~rget co~poun~s on the proliferation of 8R-M~B-1 epithelial-cell lung cancer cell line by determining the incorporation of 3H-thymidine The cells cultivated in a monolayer culture in D-MEM medium supplemented with FCS of 10 % were suspended by treatment with trypsin of 0.25 % and the cell concentration was adjusted to 5 x 105 cell/ml by adding fresh culture medium.
100 ~l of cell suspension were applied into each of 96 holes of a tissue-cultivating plate. After 48-hour incubation at 37 C under air containing 5 % by volume of carbon dioxide, the culture medium on the adhered cells was exchanged for 90 ~l of D-MEM-HITES-0.1 % BSA. 10 ~l of solutions each containing the target compounds in various concentrations were added to the cells and after 24 hours, 37 kBq (1 ~Ci) of 3H-thymidine were added into each hole, than the incubation was continued for additio-nal 24 hours.
The samples were taken and measured as describedabove for Test 2.
The data were expressed as percentage of the control. The 3H-thymidine uptake o SK-MES-l cells not treated with the compounds was considered to be 100 %
(control).
Table ~
~ffect on the 3H-t~ymidine uptake of ~K-M~S-l epithalial-cell lung cancer cell line Thymidi~e ~ptake n~ perce~tage of control Concentration Compound ~mollll (I) (2) (3) (4) (5) (6) ~ 100 lO0 lO0 lOQ 100 100 6.2~ 1~8 33 10~ 81 44 63 s Table ~ (cont~.) Thymi~ine upt~e as perce~tage of control Concentration Compound ~umolll] (I) (2) 13) (4) (5) (6~
12.~ 179 25 92 98 6 58 187 17 65 64 lO 49 The data of Table 4 indicate that the spectrum of inhibitory effects of the compounds according to the invention on the proliferation of lung cancer cells is broader than those of the known SP antagonists [see the reference substance of formula (I)~. The compounds of the invention prevent also the proliferation of SK-MES-l epithelial-cell lung cancer cell line.
According to a further aspect of the present invention there are provided pharmaceutical compositions comprising as aotive ingredient at least one peptide or pseudopeptide amide of the formulae (1) - (6) of the present invention or an acid addition salt thereof in admixture with suitable inert pharmaceutical carriers.
The pharmaceutical compositions of the present invention can be used in therapy for inhibiting the proliferation of cells of the small-cell and epithelial-cell lung cancer.
The peptide and pseudopeptide amides of the formulae (1) - (6) and salts thereof are formulated in forms generally used in therapy by methods of pharmaceutical industry known per se. The pharmaceutical compositions of the present invention may be formulated in solid, liquid or semiliquid forms and may contain one or more of generally used conventional carriers, diluents, fillers, auxiliary agents (e.g. stabilizing agents, salts for modifying the osmotic pressure), agents for adjusting the pH value and further additives.
The solid pharmaceutical compositions may be e.g.
tablets, dragées, capsules, wafers or powder ampouls useful in the preparation of injections. The liquid compositions may be e.g. injections, infusions, spoonfuls, wet packs and drops. The semiliquid compositions may be e.g. creams, ointments, balms, shaking mixtures or suppositories.
The pharmaceutical compositions of the present invention are administered in an amount which contains sufficient active ingredient to exhibit the desired effect.Thesaid dosedependsonthetypeand severenessofthe disease, the body weight of the patient and his (or her~
sensitivity against the active ingredient, the mode of application, the daily number of treatments, etc. The dose to be applied can be safely determined by the physician based on all circumstances of the given case.
In order to enable simple administration, the active ingredient is preferably finished im the form of dosage units which contain the active ingredient in the amount to be administered or a small multiple or part (e.g. half, one-third, one-fourth part) thereof. Such dosage units are e.g. the tablets which may be provided by groove(s) in order to simplify the division of a tablet into two or four parts.
To ensure the hypogastric absorption of the active ingredient Ihe tablets may be provided with a coating not soluble i~ acidic medium; i.e the tablets can be rendered enterosolvent. Similar effect can be achieved by encapsulating the active inqredient.
The pharmaceutical compositions of the present invention ~ay generally contain from about 1 mg to about 100 mg of the active ingredient per dosage unit. The above values are naturally of a mere illustrative character and the actual active ingredient content can be below or above the said limits as well.
The invention is illustrated in detail by the aid of the following non-lim-ting Examples.
Melting points were determined on a Tottoli's device (Buchi, Switzerland).
Thin layer chromatography (TLC) examinations were carried out on prefabricated silica gel adsorbent layers (DC-Fertigplatten, Merck) by using the following solvent systems:
1. Ethyl acetate:stock solution = l9:1 (sign1) 2. Ethyl acetate:stock solution = 4:1 (sign2) 3. Ethyl acetate:stock solution = 39:1 (sign3) 4. Ethyl acetate:methanol:n-hexane = 6:1:3 (sign4).
The stock solution was a 20:6:11 mixture of pyridine/acetic acid/water. The above quotients are volume ratios.
The chromatograms were detected by ninhydrin as well as by using KI/o-tolidine reagent after chlorina-tion.
High pressure liquid chromatography (HPLC) examina-tions were carried out on a BST Nucleosil 300 C18 5 ~m column. A Gilson 305 detector connected with a Gilson pump was used in these examinations. As eluent a 2:3 mix-ture of acetonitrile/water containing 0.1 ~ of trifluoro-acetic acid was used with a flow rate of 1.2 ml/min.
When investigated in this system, the purity of the compounds of the invention was found to be at least 95 %.
For amino acid analyses, the samples were parallel hydrolyzed in 6 mol/l hydrochloric acid an~ mercapto-sulfonic acid at 110 C under nitrogen for 2~ hours. The hydrolysates were examined in a Biotro~i~ LC 5000 amino acid analyzerO In addition to the common amino acids, MePhe and Leu ~ CH2NH)Leu pseudodipeptide were also detected in the hydrolysate.

NMR spectra of the new compounds ~ere taken on a Varian VXR-300 type NMR spectrometer at room temperature in deuterated dimethylsulfoxide (DMSO-d6) solution, by using tetramethylsilane (TMS~ as internal standard. The signal of the CH2 moiety of the methyleneamino group appeared in the lH-NMR spectrum as a multiplet close to 2.4 ppm, whereas the signal of the CH2 moiety showing a triplet multiplicity was found close to 52 ppm in the 13C-NMR spectrum and simultaneously, the signal of the carbon atom in the C=O moiety of the peptide bond was absent.
kxample 1 Preparation of Z-NePhe-D-Trp-Phe-D-Trp-Leu ~(C~2N~)Leu-(1) (a) Z-~eu r(CH2N~)Leu-N~2 After suspending 3.6 g (100 mmol) of lithium alumi-num hydride in 30 ml of tetrahydrofuran (THF) cooled to O C, the solution of 14.6 g (200 mmol) of diethylamine in 50 ml of a-hexane is dropwise added thereto at O C.
After stirring the suspension at O C for 10 minutes, 21.3 g (76 mmol~ of Z-Leu-OCH3 dissolved in 120 ml of n--hexane are portionwise added. After stirring at O C for 90 minutes, the reaction mixture is diluted with 70 ml of ethyl acetate and acidified to pH 2 by adding 6 mol/l hydrochloric acid solution under strong cooling. After separation of the two phases, the aqueous layer is extracted with ethyl acetate, then the combined organio phase is washed with cold water. The ethyl acet~te solution is dried over anhydrous sodium sulfate and evaporated.
The Z-Leu-H aldehyde obtained (18 g, 69 mmol) is dissolved in 300 ml of a 99:1 mixture of methanol/acetic acid cooled to O C and 8 g (50 mmol) of Leu-NH2.HCl are added. To the solution obtained 4.8 g (80 mmol) of sodium cyanoborohydride dissolved in 100 ml of anhydrous ~ ~ . t tetrahydrofuran are dropwise added at O C. The reaction mixture is stirred at o C for 30 minutes and then at room temperature for 2 hours.
After evaporating the solvent, the residue is dis-solved in 300 ml of ethyl acetate, washed twice with 100ml of 5 % sodium hydrogen carbonate solution each and twice with water. After drying, the solution is evapo-rated and the residue is solidified by adding 50 ml of n--hexane. The crude product is recrystallized from 20 ml of ethyl acetate to obtain 7 g (38 % yield calculated for Leu-NH2 HCl) of Z-Leu ~(CH2NH)Leu-NH2, m.p.: 108-110 C.

(1)(b) H-Leu ~(CH2N~)Leu-NH2~2HCl To a solution containing 5.0 9 (13.7 mmol) of Z-Leu r(CH2NH)Leu-NH2 in 100 ml of methanol, 5 ml of 5.3 mol/l hydrogen chloride solution in dioxane and then 0.5 g of 10 % palladium-on-carbon catalyst are added, then hydrogen is bubbled through the suspension.
The reaction becomes complete within 2 hours. After filtering off the catalyst, the filtrate is evaporated under reduced pressure, the residue is solidified by adding 50 ml of ether and the crude product obtained is recrystallized from an 1:5 mixture of ethanol and ether to give the aimed product in a yield of 3.4 g (82 S);
t~]D = +7-3 (c = 2, methanol), m.p.: 110 C ( decomposi-tion).

c) 5~-MeP~e-D-Trp-Phe-D-'rrp-Leu ~( CH2N~{)Leu-N~2 1.12 ml (8 mmol) of triethylamine and 4.0 g (8.5 mmol) of Boc-D-Trp-OPfp (OPfp means pentafluorophenoxy group) are added to a solution of 2.4 g (8 mmol) of Leu (C~2NH)Leu-NH22HCl in 30 ml of dimethylformamide at O C. After stirring the reaction mixture at room tem-perature ~or 90 minutes, meanwhile portionwise adding 1.5 ml of triethylamine, the reaction mixture is evaporated 4 ~

and the residue is dissolved in 100 ml of ethyl acetate.
The solution is twi~e extracted with 30 ml of 1 mol/l hydrochloric acid each and then with 30 ml of water. The combined aqueous phase is neutralized by adding sodium carbonate and extracted twice with 30 ml of ethyl acetate each. After drying over anhydrous sodium sulfate, the ethyl acetate solution is evaporated. The protected tripeptide is solidified by adding ether to obtain a yield of 3.2 g (77 %), m.p.: 180 - 182 C.
Rfl = 0 3 (1 means solvent system 1, 2 means solvent system 2, etc.) After dissolving 3.0 g (5.8 mmol) of Boc-D-Trp-Leu-~(CH2NH)Leu-NH2 in 15 ml of an 5.3 mol~l hydrogen chloride solution in dioxane, the solution is diluted with 100 ml of ether after 20 minutes. The precipitate is filtered off and washed with ether to give 2.8 g (98 %) of product, m.p.: 170 C (decomposition); Rf2 = 0.1.
After adding 1.4 ml (10 mmol) of triethylamine and 2.3 g (5.3 mmol) of Boc-Phe-OPfp to t~e solution of 2.45 g (5 mmol) of H-D-Trp-Leu r(CH2NH)Leu-NH2-2HCl in 30 ml of dimethylformamide, the reaction mixture is stirred at room temperature for 1 hour, then the dimethylformamide is distilled off and the residue is solidified by adding 50 ml of water. The precipitate obtained is filtered, washed with 50 ml of water and 30 ml of ethanol to obtain the pseudotetrapeptide Boc-Phe-D-Trp-Leu ~(CH2NH)Leu-NH2 in a yield of 2.73 g (82 ~), m.p.: 212-214 C (decompo-sition); Rf2 - 0 7 2.5 g (3.7 mmol) of the pseudotetrapeptide Boc-Phe-D-Trp-Leu ~ CH2NH3Leu-NH2 are dissolved in 30 ml of 5.3 molJl hydrogen chloride solution in dioxane and after 30 minutes, the mixture is diluted with 100 ml of eth~r. After filtering and washing with 25 ml of ether, the precipitate is dissolved in 25 ml of dimethyl-formamide. To this solution, 1.3 ml of triethylamine and 1.9 ~ (4 mmol) of Boc-D-Trp-OPfp are added, the reaction mixture is stirred at room temperature overnight, then dimethylformamide is distilled off. The residue is dis-solved in 50 ml of ethyl acetate and the solution is twice shaken with 20 ml of 1 moltl hydrochloric acid solution each and then with 20 ml of water. The aqueous phases are extracted with 20 ml of ethyl acetate each.
The combined organic solution is dried over anhydrous sodium sulfate and evaporated. The residue is solidified by addin~ ether to obtain the pseudopentapeptide in a yield of 2.83 g (90 %~, m.p.: 133 C (decomposition);
Rf2 = 0 7 2.6 g (3 mmol) of Boc-D-Trp-Phe-D -Trp-Leu ~--(CH2NH)Leu-NH2 are treated with 20 ml of 5.3 mol/l hydrogen chloride solution in dioxane, and after 30 minutes, the solution is diluted with 100 ml of ether.
After filtering the precipitate and washing with ether, the free pseudopentapeptide dihydrochloride is obtained in a yield of 2.4 g (92 %), m.p.: 167 C (with decomposition); Rf2 = 0.15.
After dissolving 1.23 g (1.48 mmol) of H-D-Trp-Phe--D-Trp-Leu ~(CH2NH)Leu-NH2 2HCl in 20 ml of dimethylform-amide, 0.96 g (2 mmol) of Z-D-MePhe-OPfp and 0.7 ml (5 mmol) of triethylamine are added to the above solution.
After stirring the reaction mixture at room temperature for 1 hour and evaporating, the residue is dissolved in 30 ml of chloroform. The chloroform solution is twice washed with 10 ml of water each. The organic solution is dried over anhydrous sodium sulfate and then evaporated.
The final product is solidified by adding 50 ml of ether to obtain Z-NePhe-D-Trp-Phe-D-Trp-Leu~r-(CH2NH)Leu-NH2 in a yield of 1.32 g (~5 %), m.p.: 88 C (decomposition);
t~D20 = +5.7~ (c = 1, methanol).

?

Exampl~ 2 Preparation of H-Leu-MPA
After suspending 26.7 g (60 mmol) of Z-Leu-OH di-cyclohexylamine salt in 300 ml of ethyl acetate, the salt is decomposed by 150 ml of 1 mol/l sulfuric acid solution. The ethyl acetate solution is dried over anhydrous sodium sulfate and evaporated~ Z-Leu-OH ob-tained as a residue is dissolved in 160 ml of anhydrous tetrahydrofuran. After cooling the solution to -10 C, lo 9.4 ml (67 mmol) of triethylamine are added, and then 9.O
ml (73 mmol) of pivaloyl chloride are dropwise added while maintaining the temperature at -10 C. After stirring the reaction mixture at -lQ C for 15 minutes, a solution containing 6.7 g (74 mmol) of 2-amino-3--methylpentane in 20 ml of tetrahydrofuran are portion-wise added. The reaction mixture is stirred at 0 C for 30 minutes and at room temperature for 3 hours and then evaporated. The residue is dissolved in 300 ml of ethyl acetate and the solution is successively washed twice with 120 ml of 1 mol/l hydrochloric acid solution each, twice with 5 % sodium hydrogen carbonate solution and, finally, once with 120 m' of water. After drying and evaporation, Z-Leu-methylpentylamide is obtained as an oil in a yield of 20.5 g (98 ~); Rf3 -- 0.8.
Th~ obtained oily product is dissolved in 200 ml of methanol and hydrogenated in the presence of 2.5 g of 10 ~ palladium-on-carbon catalyst. The reaction becomes complete within 90 minutes. Subsequently, after filtering off the catalyst and evaporating methanol, the oily residue is separated to two components on a silica gel column (80x2.5 cm size; 250 g of silica gel; eluent:
6:1:3 mixtuxe of ethyl acetate/methanol/n-hexane; flow rate: 40 ml/hour) and the fractions collected are evaporated. The yield of the thus obtained isomer of H-Leu-MPA is 4r2 g ~19.5 mmol); Rf4 = 0-3, r~D24 = +11.6 (c = 1, methanol); [~]D24 = -20.4 (c = 1, ethyl acetate).
In the following, this isomer is used as described in Example 1. The characteristics of the protected peptide amides and pseudopeptide amides obtained in this synthesis are indicated in Table 5.
~xnmpl~ 3 Preparation of D-MePhe-~-Trp-Phe-D-Trp-~eu ~CH_NH)Leu-Na A solution containing 1.0 g (0.96 mmol) of Z-D--MePhe-D-Trp-Phe-D-Trp-Leu r(CH2NH)Leu-NH2 in 25 ml of methanol is hydrogenated in the presence of 0.3 g of palladium-on-carbon catalyst for 3.5 hours. After filter-ing off the catalyst and evaporating the solution, the residue is solidified by adding ether to obtain the crude title product ln a yield of 0.73 g.
The crude final product is purified on a silica gel column by using a 4:1 mixture of ethyl acetate with a 20:6:11 mixture of pyridine/acetic acid/water as eluent with a flow rate of 20 ml/hour. Fractions of 10 ml each are collected; the purity of the fractions is observed by thin layer chromatography. After evaporating the frac-tions containing the pure final product, repeatedly twice 10 ml of water each and 10 ml of ethanol each are added to the evaporation residue in such a way that before adding an additional amount of liquid, the amount of liquid previously added is distilled off. The final evap-oration residue is solidified by adding ether and after filtering, the precipitate is washed with ether to obtain the amorphous title product in a yield of 0.32 g;
~]D20 = -22.8 (c = 0.5, 10 % acetic acid).
Amino acid analysis: Phe 1.0 (1), Trp 1.7 (2), MePhe, Leu ~(CH2NH)Leu.
The characteristics of the new compounds are summarized in Table 6.

? ~`t I V . ' Table 5 Compound Yield M.p. [~]D2 ( c=l, methanol ) 1%) (C) ( ) Z-D-Me-Phe-D-Trp-Phe-D-Trp--Leu ~(CH2NH)Leu-NH2 75 120 (d~ +1.7 Z-D-Me-Phe-D-Trp-Phe-D-Trp--Leu-MPA 56 109 (d) -22.9 Boc-D-Tyr-D-Trp-Phe-D-Trp--Leu r(cH2NH)Leu-NH2 71 136 ~d) -19.2 Boc-D-Tyr(Et)-D-Trp-Phe-D--Trp-Leu ~(CH2NH)Leu-NH2 58 114 (d) -23.4 Z-D-Me-Phe-D-Trp-D-Tyr(Bzl)-15 -D-Trp-Leu r(CH2NH)Leu-NH2 74 88 (d) +5.7 Note: (d) means: decomposition.
Table C

20 Compound M.p.[~]D2 ( C)( ~
p-HOPA-D-Trp-Phe-D-Trp--Leu ~r(CH2NH)Leu-NH2 132-137-29.1 (c=l, 50% AcOH) D-Me-Phe-D-Trp-Phe-D-Trp-25 -Leu r(CH2NH)LeU-NH2 90 (d) -22.8 (c=0.5, 10~ AcOH) D-Me-Phe-D-Trp-Phe-D-Trp--Leu MPA 153-155 -41.0 (c=l, EtOH) D-Tyr-D-Trp-Phe-D-Trp--Leu ~(CH2NH)Leu-NH2 las (d~ -17.1 (c=l, MeOH) D-Tyr~Et)-D-Trp-Phe-D-Trp--Leu ~ (cH2NH)Leu-NH2 166 (d) -35.2 ~c=1, MeOH) D-Me-Phe-D-Trp-Tyr -D -Trp--Leu ~(CH2NH)Leu-NH2 110 (d) -18.5 (c=l, 10~ AcOH) Note: (d) means: decomposition.

Ex~mple ~
Powder ampoule for injection purposes 500 mg. of active ingredient and 9.5 g. of lactose are dissolved in 80 ml. of distilled water suitable for injection purposes. 0.1 g. of methyl ~-hydroxybenzoate are added to the solution, then the volume of this solution is adjusted to 100 ml. by using distilled water suitable for injection purposes. The homogenic solution is filtered to sterile, filled in a volume of 1 ml. each into vials fitted with gum cap, subjected to freeze-drying and finally the vials are provided with a gum plug. Powder ampoules containing 5 mg. of active ingredient each are obtained.
If one intends to obtain ampoules with a different active ingredient content, the amount of lactose is selected in such a manner that, based on 100 ml. of the solution, the common weight of the active ingredient and lactose be about 10 g. One can use mannitol in the same amount instead of lactose.
When administering an injection the powder of these ampoules is dissolved in an aqueous sodium chloride solution of such concentration which allows to obtain an isotonic solution.

Claims (13)

1. A novel peptide amide or pseudopeptide amide of any one of formula (1) to (6), p-HOPA-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (1) D-MePhe-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (2) D-MePhe-D-Trp-Phe-D-Trp-Leu-MPA (3) D-Tyr-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (3) D-Tyr(Et)-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (5) D-MePhe-D-Trp-Tyr-D-Trp-Leu ?(CH2NH)Leu-NH2 (6) wherein p-HOPA means p-hydroxyphenylacetic acid, D-MePhe stands for D-N-methylphenylalanine, MPA represents 2-amino-3-methylpentane and ?(CH2NH) is a methyleneamino group being present instead of a peptide bond, or a pharmaceutically acceptable acid addition salt thereof.

2. A pharmaceutical composition, which comprises as active ingredient a therapeutically effective amount of a peptide amide or pseudopeptide amide of any one of the formulae (1) to (6) as defined in claim 1, or a pharmaceutically acceptable acid addition salt thereof in association with a pharmaceutically acceptable diluent or carrier.

3. A process for preparing a peptide amide or pseudopeptide amide of any one of formulae (1) to (6), p-HOPA-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (1) D-MePhe-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (2) D-MePhe-D-Trp-Phe-D-Trp-Leu-MPA (3) D-Tyr-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (4) D-Tyr(Et)-D-Trp-Phe-D-Trp-Leu ?(CH2NH)Leu-NH2 (5) D-MePhe-D-Trp-Tyr-D-Trp-Leu ?(CH2NH)Leu-NH2 (6) wherein p-HOPA means p-hydroxyphenylacetic acid, D-MePhe stands for D-N-methylphenylalanine, MPA represents 2-amino-3-methylpentane and ?(CH2NH) is a methyleneamino group being present instead of a peptide bond, or a pharmaceutically acceptable acid addition salt thereof, which process comprises, using Leu (CH2NH)Leu-NH2 or Leu-MPA as a starting substance building a peptide of formula (1), or a derivative of a peptide of any one of formulae (2) to (6) additionally containing a N-terminal benzyloxycarbonyl or tert-butoxycarbonyl protective group by successive reactive ester coupling and deprotection of the .alpha.-NH2 group, then removing the protective group by catalytic hydrogenation or acidolysis and, if required, transforming the product obtained into a pharmaceutically acceptable acid addition salt or liberating a free base form of said peptide from an acid addition salt.

4. A process as claimed in claim 3, which comprises carrying out the step of reactive ester coupling by using the pentafluorophenyl ester of the protective amino acid to be incorporated.

5. A process as claimed in claim 3 or claim 4, which comprises carrying out the step of reactive ester coupling by using a reactive ester derivative of the amino acid protected by a tert-butoxycarbonyl protective group.

6. A process as claimed in claim 3 or claim 4, which comprises carrying out the step of reactive ester coupling by using a reactive ester derivative of the amino acid protected by a benzyloxycarbonyl protective group.

7. A process as claimed in any of the claims 3 to 5, which comprises removing the protective group by acidolysis.

8. A process as claimed in any of the claims 3, 4 or 6, which comprises removing the protective group by catalytic hydrogenation.

9. A process for the preparation of a pharmaceutical composition, which comprises mixing as active ingredient a therapeutically effective amount of a novel peptide amide or pseudopetide amide of any of the formulae (1) to (6), wherein p-HOPA, D-MePhe, MPA and ?(CH2NH) are as defined in claim 1, or a pharmaceutically acceptable acid addition salt thereof with one or more auxiliaries commonly used in the pharmaceutical industry and transforming the mixture into a pharmaceutical composition.

10. Method for inhibiting the proliferation of cells of the small-cell and epithelial-cell lung cancer of mammals including man, charaterized by administering the mammal to be treated a therapeutically effective amount of a peptide amide or pseudopeptide amide of any of the formulae (1) to (6), wherein p-HOPA, D-MePhe, MPA and ?(CH2NH) are as defined in claim 1, or a pharmaceutically acceptable acid addition salt thereof alone or in the form of a pharmaceutical composition.

11. Use of a compound according to claim 1 to combat small-cell or epithelial-cell lung cancer in a mammal.

12. A commercial package comprising a pharmaceutically effective amount of a compound according to claim 1 together with instructions for use thereof to combat small-cell or epithelial-cell lung cancer in a mammal.

13. A process for preparing a compound of any one of formula (1) to (6), according to claim 1 which process comprises condensing a first reagent A comprising Leu(CH2NH)Leu-NH2 or Leu-MPA and which from then on comprises an incomplete balance of any one of the compound of formula (1) to (6) with a second reagent (B) comprising, when compared with reagent A, the balance of said any one of the compound of formula (1) to (6), the reagents (A) and (B) being optionally protected and, if required removing any protecting group and then, if required forming a pharmaceutically acceptable acid addition salt thereof or liberating a free base form of said peptide from an acid addition salt.