SK165497A3 - Peptides, preparation method thereof and pharmaceutical composition containing the same - Google Patents

Abstract

Peptides of general formula (I) R1R2N - CHX - CO - A - B - D - E - (F)t - K, wherein R1 is methyl, ethyl or isopropyl, R2 is H, methyl or ethyl, R1-N-R2 together pyrrolidine ring, A residues of Val, Ile, Leu, 2-terc-butylglycyl, 2-ethylglycyl, norleucyl or norvalyl, B residues of N-methyl-valyl, N-methylleucyl, N-methylisoleucyl, N-methylnorvalyl, N-methylnorleucyl, N-methyl-2-terc-butylglycyl, N-methyl-3-terc-butylalanyl or N-methyl- 2-ethylglycyl, D residues pro, 3,4-dehydroprolyl, 4-fluoroprolyl, 4,4-difluoropropyl, asetidine-2-carbonyl, homoprolyl, 3-methylprolyl, 4-methylprolyl, 5-methylprolyl or thiazolidine-4-carbonyl, E 3,4-dehydroprolyl, 4-fluoroprolyl, 3-methylprolyl, 4-methylprolyl, azetidine-2-carbonyl or 4,4-difluoroprolyl, F residues of Val, 2-tec-butylglycyl, Ile, Leu, 2-cyclohexylglycyl, norleucyl, norvalyl, neopentylglycyl, Ala, beta-Ala or amino isobutyroyl, X alkyl, cyclopropyl or cyclopentyl, T is 0 or 1, K alkoxy group, benzyloxy group or group R5-N-R6, or their salts with physiologically suitable acids, pharmaceutical preparation containing them and their use in the medicine, particularly for treatment of oncological diseases and mammals tumours.

Description

Technical field

The invention relates to peptides and to a pharmaceutical composition containing them and is suitable for the treatment of cancer.

BACKGROUND OF THE INVENTION

Peptides isolated from marine sources such as Dolastatin-10 (U.S. Pat. No. 4,816,444) and Dolastatin-15 (European Patent Specification No. EP-A-398558) are known to have great activity as cell growth inhibitors (Biochem Pharmacology 40; 8: 1859-1864 (1990); J. Natl.Cancer Inst. 85: 483-488 (1993 and references therein). Based on interesting results in experiments with tumor systems in vivo, further preclinical evaluations of these natural products are currently underway to initiate clinical trials for cancer patients.

However, natural products have the drawback that they are poorly soluble in aqueous solvents and the investment costs for their synthesis are high.

The invention relates to novel peptides and derivatives thereof which represent an improved therapeutic potential for the treatment of neoplastic diseases compared to Dolastatin-15. In addition, the novel peptides of the invention can be readily synthesized in the manner described in detail below.

SUMMARY OF THE INVENTION

The present invention provides peptides of formula I

- CHX - CO - ABDE - (F) _t ~ K (I)

R ¹ R ² N where is

R ^{1 is} methyl, ethyl or isopropyl,

R ² is hydrogen, methyl or ethyl,

R, -NR ² together pyroilidínový ring,

And the residues valyl, isoleucyl, leucyl, 2-tert-butylglycyl,

2-ethylglycyl, norleucyl or norvalyl,

B residues N-methylvalyl, N-methylleucyl, N-methylisoleucyl,

N-methylnorvalyl, N-methylnorleucyl, N-methyl-2-tert-butylglycyl, N-methyl-3-tert-butylalanyl or N-methyl-2-ethylglycyl,

D residues prolyl, 3,4-dehydroprolyl, 4-fluoroprolyl, 4,4-difluoroprolyl, azetidine-2-carbonyl, homoprolyl, 3-methylprolyl, 4-methylprolyl, 5-methylprolyl or thiazolidine-4-carbonyl,

E 3,4-dehydroprolyl, 4-fluoroprolyl, 3-methylprolyl, 4-methylprolyl, azetidine-2-carbonyl or 4,4-difluoropropyl,

F residues valyl, 2-tert-butylglycyl, isoleucyl, leucyl, 2-cyclohexylglycyl, norleucyl, norvalyl, neopentylglycyl, alanyl, β-alanyl or aminoisobutyroyl,

X alkyl preferably having 2 to 5 carbon atoms, cyclopropyl or cyclopentyl, t 0 or 1 a

K alkoxy preferably having 1 to 4 carbon atoms, benzyloxy or substituted or unsubstituted amino, or salts thereof with physiologically acceptable acids. The invention also relates to a process for the preparation of these peptides of general formula

I, pharmaceutical compositions comprising these peptides of the formula I together with pharmaceutically acceptable carriers and used in the treatment of cancer.

K is in particular C 1-6 alkoxy, benzyloxy or amino of the formula R ⁵ -NR ⁶ , where it is

R ⁵ is hydrogen or hydroxy or C ₁ _ ₄ alkoxy, or benzyloxy optionally substituted with up to two substituents each independently selected from the group consisting of CF _3, nitro, Item ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl, cyano, hydroxy, N (CH ₃ ) ₂ , COOMe, COOEt, COOiPr and COONH ₂ or R 6;

means phenyloxy optionally substituted until two substituents on himself independently elected from File including CF ₃ , heart C ₁ _ ₄ alkylsulfonyl, ^c 1-4 alkoxy, halogen, C ₁ _ ₄ alkyl, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr and COONH ₂ , of: R is hydrogen or C ₁ _ ₁₂ alkyl optionally substituted with at least one

or - (CH ₃ ) ₂ CN, -C (CH ₃ ) ₂ CCH, -C (CH ₃ ) ₂ C = CH ₂ , -C (CH ₃ ) ₂ CH ₂ OH, -C (CH ₃₎ ) ₂ CH ₂ CH ₂ OH or - (CH ₂₎ C ₃ _ ₇ cycloalkyl, wherein v is 0, 1 or 2 and which is optionally substituted by methyl, or represents norephedryl, norpseudoephedryl, quinolyl, pyrazolyl, adamantyl, _-CH2 -benzimidazoyl, -CH ₂ -adamantyl, α-methylbenzyl, α-dimethylbenzyl, - (CH ₂ ) _v phenyl, wherein v is 0, 1 or 2 and which is optionally substituted with one or two substituents independently selected from CF _3, nitro, C - ^ _ ₄ alkylsulfonyl, C - ^ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH ₂ , or - (CH ₂ ) _m naphthyl, wherein m is 0, 1 or 2, and which is optionally substituted with one or two substituents independently selected from CF ₃ , nitro, C 1-6 alkylsulfonyl, C 1-6 alkoxy , halogen, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is - (CH _{2) w} benzhydryl, wherein w is 0, 1 or and optionally bears one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2; or is biphenyl which is optionally substituted by one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halogen, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or represents pyridyl which is optionally substituted by one or two substituents each independently selected from the group consisting of CF _3, nitro, C ^ _ ₄ al alkylsulfonyl, C ^ _ ₄ alkoxy, halogen, Cj_ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is picolyl, which is optionally substituted by or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is -CH ₂ -CH ₂ -pyridyl, optionally substituted by one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halogen, C ₃ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is benzothiazolyl, which is optionally substituted by one or two substituents independently selected from CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is benzoisothiazolyl, which is optionally substituted by one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, , hydroxy, N (CH ₃ ) ₂ , and COONH ₂ , or is

COOMe,

COOEt, COOiPr benzopyrazolyl, which is optionally substituted by one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system , cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is benzooxyzolyl which is optionally substituted by one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is - (CH _{2) m} - fluorenyl, wherein m is 0 or 1, optionally substituted with one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl, which can form a cyclic system, cyano , Hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is pyrimidyl, which is optionally substituted by one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is ~ ^(CH2 ^ m inúanyl wherein m is 0 or 1, optionally substituted with one or two of substituents are independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic a system, cyano, hydroxy, N (CH ₃ ) ₂ , COOMe, COOEt, COOiPr and COONH ₂ , or is - (CH ₂ CH ₂ O) y CH ₃ , wherein y is 0, 1, 2, 3, 4, or 5, or means - (CH ₂ CH ₂ O) y CH ₂ CH ₃ , wherein y is 0, 1, 2, 3, 4 or 5 or is -ΝΗ-ΟθΗ ^, which is optionally substituted by one or two m substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ^ _ ₄ alkoxy, halo, C ^ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2} , COOMe, COOEt, COOiPr, and C00NH _2, -NC H _3, or is C ₆ H _5, optionally substituted with one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is -NH-CH ₂ -CgH _5, which is optionally substituted with one or two substituents independently selected from the group consisting of

C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, halogen,

CF nitro,

C _1-4 alkyl which may form a cyclic system, cyano, hydroxy, N (CH ₃ ) ₂ ,

COOMe,

COOEt, COOiPr

COONH

Or 2 '

-NCH ₃ -CH ₂ -C ₆ H _5, optionally subtituted with one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C _L _ ₄ alkoxy, halo, C ₁ _ ₄ alkyl which may form a cyclic system, cyano, hydroxy, N (CH _{3) 2,} COOMe, COOEt, COOiPr, and COONH _2, or is a five heteroaryl, which is optionally substituted by one or two substituents each independently selected from: CF _3, nitro, C ₁ _ ₄ alkylsulfonyl, C ₁ _ ₄ alkoxy, thiomethyl, thioethyl, picolyl, acetyl, ^C 3 _'6 cycloalkyl, thiophenyl, CH ₂ COOEt, C ₃ _ ₄ alkylene forming a bicyclic system with the heterocycle, phenyl, optionally substituted with one or two substituents each independently selected from: nitro, _CF3, cyano, halo, and C ₁ _ ₄ alkyl, and further comprising benzyl which is optionally substituted by one or two substituents each inde dependently selected from: nitro, CF _3, halogen, C ₁ _ ₄ alkyl, C ₁ _ ₄ alkylsulfonyl, cyano, hydroxy, C ₄ dialkylamino, or is a five -CHR ⁷ -heteroaryl, optionally substituted by one or two substituents at the each independently selected halogen, from the group consisting of CF _3, nitro, cyano, COONH _a C ₁ _ ₄ alkyl,

COOMe, COOEt, COOiPr, and, phenyl, benzyl, naphthyl or C ₁ _ ₄ alkylsulfonyl, wherein R is hydrogen, C ₁ _ ₅ alkyl, straight or branched, or benzyl.

C ₁ _ ₄ alkoxy,> 7

2 '

For example, the five-membered -CHR ⁷ -heteroaryl moieties include:

For example, the following five-membered heteroaryl is mentioned:

CH ₃

N

CH

N

CH ₃

Ň λ «

CH

CH

I

N

CH

ch ₃

N

CH3

N

N

About ^CTi 0%

N '/ T

S -N /? zT

NN NN ch ₃

CK3

SCH 3

CH 2 CH 3 J, p

N - N? ^N? CH (CH 3) 2? NN

, NO ₂ r-CH ₃

N -N

N-N ';

Cl

As the group R ⁵ -NR ⁶ groups, for example, the following are mentioned

Preference is given to peptides of the general formula I, wherein

R ^{1 is} methyl or ethyl,

R ² is hydrogen, methyl or ethyl,

And the residues valyl, isoleucyl or 2-tert-butylglycyl,

B residues N-methylvalyl, N-methylisoleucyl or N-methyl-2-tert-butylglycyl,

D residues prolyl, 3,4-dehydroprolyl, 4-fluoroprolyl, 3-methylprolyl or azetidine-2-carbonyl,

E residues 3,4-dehydroprolyl, 4-fluoroprolyl, 3-methylprolyl or azetidine-2-carbonyl,

F residues D-valyl, 2-tert-butylglycyl, D-isoleucyl, D-leucyl or aminoisobutyroyl, x -ch (ch ₃ ) ₂ , -c (ch ₃ ) ₃ , -ch (ch ₃ ) ch ₂ ch ₃ ;

t 0 or 1 a

K is preferably

OC _{(CH3) 3,} -NH _2, -NH-C ₁ _ ₁₂ alkyl, -NH-C ₃ _g cycloalkyl,

-NH- [3,3, O] octyl, norephedryl, norpseudoephedryl, -NH-quinolyl, -NH-pyrazyl, _-NH-CH2 -benzimidazolyl, -NH-adamantyl, _{-NH-CH2-adamantyl,} -NH -CH _(CH3) -phenyl,

-NH-C _{(CH3) 2-phenyl,} -N (C ^ _ ~ ₄ alkoxy) C ^ _ ~ ₄ alkyl, "N (C ₁ _ ₄ -alkoxy) _-CH2 -phenyl, -N (C _{1 4-} alkoxy) phenyl,

-N (CH ₃ ) OBz 2, -NH- (CH ₂ ) _v -phenyl (v = 0,1,2,3), -NH- (CH ₂ ) _m -naphthyl (m = 0 or 1), -NH - (CH ₂ ) _n -benzhydryl (w = 0, 1 or 2), -NH-biphenyl, -NH-pyridyl, -NH-CH ₂ -pyridyl, -NH-CH ₂ -CH ₂ -pyridyl, -NH- benzothiazolyl, -NH-benzoisothiazolyl, -NH-benzopyrazolyl, ¹ -NH-benzoxazolyl, -NH- (CH2) m-fluorenyl (m = 0.1), -NH-pyrimidyl, -NH- (CH2) m-indanyl ( m = 0.1, -NH- (CH 2 CH 2 O) y -CH 3 (y = 0, 1,2,3,4,5), -NH- (CH 2 CH 2 O) y -CH 2 CH 3 (y = 0.1.2, 3,4,5), -NH-five-membered heteroaryl, as previously described, -NH-CHR ⁷ -five-membered heteroaryl, as described above, wherein R ⁷ is hydrogen, straight or branched C 1-5 alkyl, or K is a radical

Γ ~ \

-N S = o

N

In particular, K represents a residue

-nhch ₃ , -nhch ₂ ch ₃ , -nh (ch ₂ ) ₂ ch ₃ , -nh (ch ₂ ) ₃ ch ₃ , -nh (ch ₂ ) ₄ ch ₃ , -nh (ch ₂ ) ₅ ch ₃ , -nh (ch ₂ ) ₆ ch ₃ , -nh (ch ₂ ) ₇ ch ₃ , -nhch (ch ₃ ) ₂ , -nhch (ch ₃ ) ch ₂ ch ₃ , -nhch (ch ₃ ) ch ₂ ch ₂ ch ₃ , -nhch (ch ₂ ch ₃ ) ₂ , -nh (ch ₂ ch ₂ ch ₃ ) ₂ , -nhc (ch ₃ ) ₃ , -nhch (ch ₂ ch ₃ ) ch ₂ ch ₂ ch ₃ , -nhch ( ch ₃ ) ch (ch ₃ ) ₂ , -nhch (ch ₂ ch ₃ ) ch (ch ₃ ) ₂ , -nhch (ch ₃ ) c (ch ₃ ) ₃ , -NH-cyclopropyl, -NH-cyclobutyl, -NH -cyclopentyl, -NH-cyclohexyl, -NH-cycloheptyl, -NH-cyclooctyl,

-NH-bicyclo [3.3.0] octyl, -N (CH ₃ ) OCH ₃ , -N (CH ₃ ) OCH ₂ CH ₃ ,

-n (ch ₃ ) och ₂ ch ₂ ch ₃ , -n (ch ₃ ) och (ch ₃ ) ₂ , -n (ch ₂ ch ₃ ) och ₃ ,

-n (ch ₂ ch ₃ ) and ₂ ch ₃ , -n (ch (ch ₃ ) ₂ ) and ₃ , -n (ch ₃ ) and ₂ c ₆ h ₅ , -n (ch ₃ ) ch ₂ -c ₆ h ₅ , -n (ch ₃ ) oc ₆ h ₅ , -nh-ch ₂ -c ₆ h ₅ , -nh (ch ₂ ) ₂ c ₆ h ₅ , -nh (ch ₂ ) ₃ c ₆ h ₅ , - nhch (ch ₃ ) c ₆ h ₅ , -nhc (ch ₃ ) ₂ c ₆ h ₅ ,

-nhc (ch ₃ ) ₂ ch ₂ ch _{3 /} -nhc (ch ₃ ) (ch ₂ ch ₃ ) ₂ , -nhch (ch ₃ ) ch (oh) c ₆ h _{5 /} -NHCH ₂ -cyclohexyl, -NH- CH ₂ CF ₃ , -NHCH (CH ₂ F) ₂ ,

-NHC (CH ₃ ) ₂ CH ₂ CH ₂ OH, -NH (CH ₂ CH ₂ O) ₂ CH ₂ CH ₃ , -NHC (CH ₃ ) ₂ CH (CH ₃ ) 2 and -NHC (CH ₃ ) ₂ CN , -NHC (CH _{3) 2} CCH, -NHC (CH _{3) 2} CCH _2, norephedryl, norpseudoephedryl, -NH-quinolyl, -NH-pyrazyl,

-NH-adamantyl (1), '- NH-adamantyl (2), _{-NH-CH2-adamantyl,} -NH-CH ₂ -naphthyl, -NH-benzhydryl, -NH-biphenyl,

-NH-pyridyl, -NH-picolyl, _{-NH-CH2 -CH2} -pyridyl, -NH-benzothiazolyl, -NH-benzoisothiazolyl, -NH-benzopyrazolyl, -NH-benzoxazolyl, -NH-fluorenyl, -NH-pyrimidyl , -NH-thiazolyl (2), -NH-isoxazolyl (3), -NH-CH ₂ -furány1 (2),

-NH- (3-methyl) isoxazolyl (5), -NH- (3-methyl) isothiazolyl (5), -NH- (2-trifluoromethyl) thiadiazolyl (5), -OC (CH ₃ ) ₃ ,

-NH-CH ₂ - (4-methyl) thiazolyl (2), _{-NH-CH2-thienyl} (2),

_-NH-CH2 ~ (5-methyl) thienyl (2), -NH- (2-methyl) thiadiazolyl (5),

-NH- (2-cyclopropyl) thiadiazolyl (5), or K represents the radical of formula ' ^N. I -HS-11 o, - N

• N

H, C ^H and C HC H, C

AS) - «- K) * -« - k]

The examples given above merely illustrate the invention but do not limit it.

The peptides of formula I consist of L-amino acids, but F may also be D-amino acids.

The peptides of formula I may be in the form of salts with physiologically acceptable acids such as hydrochloric, citric, tartaric, lactic, phosphoric, methanesulfonic, acetic, formic, maleic, fumaric, malic, succinic, malonic, sulfuric, L-glutamic, L-aspartic, pyruvic, mucosal, benzoic, glucuronic, oxalic, ascorbic and acetylglycine.

The peptides of formula I may be prepared by methods known in the art from peptide chemistry. For example, they may sequentially consist of amino acids, or may be obtained by binding suitable small peptide fragments. Gradual folding starts from the C-terminus of the peptide chain, which is gradually extended by one amino acid each. In coupling the fragments, it is possible to bind fragments of varying length together, such fragments being obtained by sequential amino acid binding or also by coupling the fragments. In both of these methods, it is necessary to bind the units to each other by forming an amide bond. Both chemical and enzymatic methods are suitable for this purpose.

Chemical methods for amide bond formation have been described in detail by Mueller (Methoden der organischen Chemie, Vol. XV / 2, pp. 1 to 364, Thieme Verlag, Stuttgart, 1974; Stewart, Young, Solid Phase Peptide Synthesis, pp. 31-34, 71-71). 82, Pierce Chemical Company, Rockford, 1984; Bodanszky, Klausner, Ondetti, Peptide Synthesis, pp. 85-128, John Wiley & Sons, New York, 1976) and also described in other basic publications on peptide chemistry. Particularly preferred is the in situ azide process, symmetrical and mixed anhydride process

I generated and preformed active esters, · use of urethane protected with N-carboxyanhydrides of amino acids and amide bonding using coupling / activator system, mentioning in particular dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1-ethoxy , 2-dihydroquinoline (EEDQ), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), n-propanephosphonic anhydride (PPA), Ν, Ν-bis (2-oxo-3-oxazolidinyl) amidophosphoryl chloride (BOP) -Cl), bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBrop), diphenylphosphoryl azide (DPPA) of Castro reagent (BOP, PyBop),

O-benzotriazolyl-Ν, Ν, Ν ', Ν'-tetramethyluronium salt (HBTU), O-azabenzotriazolyl-N, N, N', Ν'-tetramethyluronium salt (HATU), diethylphosphoryl cyanide (DEPCN), 2,5-diphenyl -2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich reagent, HOTDO) and 1,1'-carbonyldiimidazole (GDI). The coupling agents can be used alone or in combination with additives such as N, N-dimethyl-4-aminopyridine (DMAP) N-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine.

While it is generally possible to dispense with protecting groups in the enzymatic synthesis of peptides, transient protection of reactive groups not involved in the amide bond is needed for the reactants in chemical synthesis. In the chemical synthesis of peptides, three conventional methods of protecting groups are preferred: the method using benzyloxycarbonyl (Z), t-butoxycarbonyl (Boe) and 9-fluorenylmethoxycarbonyl (Fmoc). In each case, a protecting group on the α-amino group of the chain extender is used. A detailed review of the amino protecting groups is given by Mueller (Methoden der organischen Chemie, Vol. XV / 1, pp. 20-906, Thieme Verlag, Stuttgart, 1974).

The units used to form the peptide chain can be reacted in solution, suspension or the like, as described by Murrifield (J. Amer. Chem. Soc. 85, 2149, 1963). Particularly suitable are methods in which the peptides are formed sequentially or by coupling the fragments using Z, Boe or Fmoc protecting groups. One of the reactants in the method described by Murrifield binds to an insoluble polymeric carrier (also referred to herein as resin). As a rule, the peptide is formed sequentially on a polymeric support using Boe or Fmoc protecting groups, the growing peptide chain is covalently linked by a C terminus to insoluble resin particles (Figs. 1 and 2). This process makes it possible to remove the reagents and by-products by filtration, and thus recrystallization of the intermediates is unnecessary.

The protected amino acids may bind to any suitable polymer, which must only be insoluble in the solvent used and must have a stable physical form which facilitates filtration. The polymer must contain a functional group to which the first protected amino acid can be firmly bound by a covalent bond. A variety of polymers are suitable for this purpose: for example, cellulose, polyvinyl alcohol, polymethacrylate, sulfonated polystyrene, a copolymer of chloromethylated styrene and divinylbenzene (Murrifield resin), 4-methylbenzhydrylamine resin (MBHA resin), phenylacetamidomethyl resin (Pamyl-benzamidamidyl resin), resin, benzhydrylamine resin (BHA-resin), 4- (hydroxymethyl) benzoyloxymethyl resin, resin described by Breipohl et al. (Tetrahedron Letters 28, p. 565, 1987, commercial product of BACHEM), 4- (2,4-dimethoxyphenylaminomethyl) phenoxy resin (commercial product of Novabiochem) or o-chlorotrityl resin (commercial product of Biohellas).

All solvents which are inert under the reaction conditions, especially water, Ν, Ν-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane, tetrahydrofuran (THF) are suitable for solution peptide synthesis. ), N-methyl-2-pyrrolidone (NMP) and mixtures of these solvents. The polymer-supported peptide synthesis can be carried out in all inert organic solvents in which the amino acid derivatives used are soluble. However, preferred solvents such as Ν, Ν-dimethylformamide, dichloromethane, N-methyl-2-pyrrolidone, acetonitrile and dimethylsulfoxide and mixtures thereof cause swelling of the resins. When the synthesis is complete, the peptide is cleaved from the polymeric support. The conditions under which cleavage from various resins is possible are described in the literature. The most commonly used cleavage reactions are catalyzed by acids and palladium, especially suitable cleavage in liquid anhydrous hydrogen fluoride, in anhydrous trifluoromethanesulfonic acid, in dilute or concentrated trifluoroacetic acid, palladium catalyzed cleavage in tetrahydrofuran or in a mixture of tetrahydrofuran and dichloromethane is present. or cleavage in mixtures of acetic acid and dichloromethane / trifluoromethanol. Depending on the protecting groups selected, these groups may be maintained or optionally cleaved under cleavage conditions. Partial deprotection of the peptide may also be valuable if certain derivatization reactions are to be performed. N-terminally dialkylated peptides can be prepared

(a) coupling the appropriate Ν, dial-dialkylamino acids in solution or on a polymeric support;

b) reductive alkylation of the resin-bound peptide in dimethylformamide / 1% acetic acid with NaCNBH 4 and the corresponding aldehyde or ketone,

c) hydrogenating the peptides in solution in the presence of an aldehyde or ketone and palladium on carbon.

The various non-natural amino acids of the invention are commercially available or can be synthesized from commercially available materials by methods known in the art. Commercially available starting materials are azetidine-2-carboxylic acid, 3-methyl-L-proline, 5-methyl-L-proline and a Boe or Fmoc protected 3,4-dehydroproline (ACROS, NOVABIOCHEM, BACHEM). Cis- and trans-4-fluoroproline can be prepared as described by Panasik et al. (N. Panasik, E. S. Eberhardt, A. S. Edison, D. R. Powell, R. T. Raines Int. J. Peptide Protein Res. 44, pp. 262-269, 1994) from hydroxyproline.

The compounds of the invention may serve to inhibit or otherwise treat solid tumors (e.g., lung, breast, colon, prostate, gallbladder, rectum or endometrial tumors) or hematological diseases (e.g., leukemia, lymphomas), by administering the compounds to mammals.

A particular advantage of the novel compounds of the invention is that they are more resistant to enzymatic degradation compared to Dolastatin-15.

Administration can be by conventional pharmaceutical, preferably oncologic means, including oral and parenteral formulations, subcutaneously, intravenously, intramuscularly and intraperitoneally.

The compounds may be administered alone or in the form of pharmaceutical compositions comprising a compound of Formula I together with a pharmaceutically acceptable carrier for a given route of administration. Such pharmaceutical compositions may be combination products and thus may contain other therapeutically active ingredients.

The dosage administered to a mammal comprises a tumor inhibiting active ingredient which depends on common factors such as the biological activity of the compound used, the route of administration, age, health and weight of the patient, nature and type of symptoms, frequency of treatment, administration.

other drugs and the desired effect is about 0.5 to 50 mg per kg of body administration and about 0.05 to 20 mg at

The novel compounds of the invention may be administered in the form of conventional solid or liquid pharmaceutical compositions, i.e., coated or uncoated tablets, capsules, powders, granules, suppositories or solutions. These are produced in the usual way. The active substances can be combined with conventional pharmaceutical auxiliaries, such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, emollients, solvents, delaying agents, antioxidants and / or carrier gases (H. Sucker and carrier gases). et al., Pharmazeutische Technologie, Thieme Verlag, Stuttgart, 1978). The drug forms thus administered contain normally from 1 to 90% by weight of active ingredient.

The following examples illustrate the invention without limiting it. In the examples, protein amino acids are abbreviated in the conventional 3-letter code. Other shortcuts are:

Me ₂ Val = Ν, Ν-dimethylvaline, MeVal = N-methylvaline.

DETAILED DESCRIPTION OF THE INVENTION

A. General procedures

I. The peptides of the invention of formula I are either synthesized by classical solution synthesis by standard methods described above using Z and Boe or by standard solid-state synthesis methods using Boe and Fmoc protecting groups.

a) Synthesis cycle for the Fmoc protecting group technique.

First washing with DMF 1 x 1 min Second 20% piperidine in DMF 1 x 4 min 3. 20% piperidine in DMF 1 x 15 min 4th washing with DMF 5 x 1 min 5th adding a pre - activated protection amino acid (activated with 1 equivalent) TBTU and 5 equivalents of DIPEA in DMF) peptide coupling 1 x 61 min 6th washing with DMF 3 x 1 min 7th if the conversion is incomplete, repetition of copulation (back to 5th) 8th washing with DMF 3 x 1 min

9. back to 2.

Reagents ΒΟΡ-Cl and PyBrop are used to coupling amino acids to N-amino acids. The reaction times are correspondingly increased by the inclusion of double bonds. In solution synthesis, either Boe-protected is most preferred for this coupling method. (N-carboxy anhydrides) of amino acids NCA, Z-protected (N-carboxyanhydrides) amino acids of NCA, or pivaloyl chloride as a condensing agent.

II. Reductive alkylation of the N-terminus

The Ala resin peptide is deprotected at the N-terminus (steps 2-4 in Ala) and then reacted with

3-molar excess of aldehyde or ketone in dimethylformamide / 1% acetic acid, with 3 equivalents of NaCNBH _{3 added} . After completion of the reaction (negative Kaiser test), the resin was washed several times with water, isopropanol, dimethylformamide and dichloromethane. For example, reductive alkylation in solution can be achieved by reacting N-terminally deprotected peptides, peptide fragments or amino acids with the corresponding aldehydes or ketones using NaCNBH ₃ or a hydrogen palladium on carbon system.

III1 Treatment of peptide resins obtained according to paragraphs 1b and II

The peptide resin is dried under reduced pressure and then treated with TFA / water (95: 5) for 1.5 hours (Wade, Tregear, Howard Florey Fmoc Workshop Manual, Melbourne, 1985). The resin was filtered off and washed with TFA and dichloromethane. The filtrate and washings were concentrated and the peptide was precipitated by the addition of diethyl ether. After cooling in an ice bath, the precipitate is filtered off, taken up in 30% acetic acid and lyophilized.

IV. When o-chlorotrityl resin (commercial product from Biohellas) is used, the peptide resin suspension in acetic acid / / trifluoroethanol / dichloromethane (1: 1: 3) is stirred at room temperature for 1 hour. The resin is filtered off under suction and washed thoroughly with cleavage solution. The combined filtrates were concentrated in vacuo and treated with ether. The precipitated solid is removed by filtration or centrifugation, washed with diethyl ether and dried under reduced pressure.

V. Purification and characterization of peptides

Purification is by gel chromatography (SEPHADEX G-10, G-15 (10% HOAc, SEPHADEX LH 2 O / MeOH) and / or medium pressure chromatography (stationary phase: HD-SIL C-8, 20-45 micron, 100 Angstrom; mobile phase) gradient with A = 0.1% TFA / water system, B = 0.1% TFA / MeOH system, or by preparative HPLC (stationary phase: Waters Delta-Pak C-18, 15 micrometer 100 Angstrom; mobile phase) gradient with A = 0.1% TFA / water system, B = 0.1% TFA / MeOH system).

The purity of the resulting products was determined by analytical HPLC (stationary phase: 100 2.1 mm VYDAC)

C-18 300 Angstrom; mobile phase: acetonitrile-water gradient, buffered with 0.1% TFA, 40 ° C). Characterization of fast atom bombardment by mass spectroscopy and NMR spectroscopy

B.- Specific procedures

Example 1 (SEQ ID NO: 1)

Me ₂ Val-Val-MeVal-Pro-L-azetidinyl-2-carboxamide

0.53 g of Fmoc-RINK resin (0.46 mmol / g substitution) was reacted, corresponding to a dose size of 0.25 mmol as in Ala with 0.4 mmol each.

Fmoc-azetidine-2-carboxylic acid,

Fmoc-Pro-OH,

Fmoc-NMe-Val-OH,

Fmoc-Val-OH,

Fmoc-Val-OH.

The amino acid following the N-methylamino acid is linked by a double bond with the ByProp coupling reagent. After iterative synthetic cycles have been completed, the N-terminal protecting groups (steps 2-4 in Ala) are removed from the peptide resin and further reacted with an aqueous formaldehyde solution as in AII and dried under reduced pressure. The resulting resin was cleaved by TFA as in AIII. The crude product was purified by preparative medium pressure chromatography to give 5 mg of the desired pure peptide (10 to 40% A for 10 '; 40 to 90% A for 200'). The compound is further characterized by fast atom bombardment mass spectroscopy and NMR spectroscopy. [M + H] < ^{+ &gt};.

Example 2 (SEQ ID NO: 1)

Me ₂ Val-Val-MeVal-Pro-3,4-dehydroprolylbenzylamide

a) Z-MeVal-Pro-OMe

66.25 g (250 mmol) of Z-Val-OH are dissolved in 250 ml of dry dichloromethane. After addition of 36.41 ml (262.5 mmol) of triethylamine, the reaction mixture is cooled to -25 ° C and 32.27 ml (262.5 mmol) of pivaloyl chloride are added. After stirring for 2.5 hours, 41.89 g (250 mmol) of H-Pro-OMe x HCl in 250 ml of dichloromethane are added to the reaction mixture and neutralized at 0 ° C with 36.41 ml (262.5 mmol) of triethylamine. . Stirring is continued for 2 hours at -25 ° C and overnight at room temperature. The reaction mixture was diluted with dichloromethane and washed thoroughly with saturated aqueous sodium bicarbonate solution (3x), water (1x), 5% citric acid (3x) and saturated sodium chloride solution. The organic phase is dried over sodium sulphate and evaporated to dryness. The residue (91.24 g) was stirred overnight with petroleum ether and filtered. 62.3 g of product are obtained.

b) H-MeVal-Pro-OMe

48.9 g (130 mmol) of Z-MeVal-Pro-OMe are dissolved in 490 ml of methanol. After addition of 10.9 ml (130 mmol) of concentrated hydrochloric acid and 2.43 g of 10% palladium on carbon, the reaction mixture is hydrogenated. Filtration and evaporation to dryness yielded 36.43 g of product.

!

c) Z-Val-MeVal-Pro-OMe

In 110 ml of dimethylformamide, 18.1 g of H-MeVal-Pro-OMe, 21.6 g (78 mmol) of Z-Val-N-carboxyanhydride and 22.8 ml (130 mmol) of diisopropylethylamine are stirred at 40 DEG C. for two days. for two days. After evaporating the dimethylformamide, dichloromethane is added and the organic phase is washed with saturated aqueous sodium bicarbonate solution (3x), water (1x), 5% citric acid (3x) and saturated sodium chloride solution. The organic phase is dried over sodium sulphate and evaporated to dryness. 29.3 g of product are obtained in the form of a viscous oil.

d) H-Val-MeVal-Pro-OMe

29.3 g (61.6 mmol) of Z-Val-MeVal-Pro-OMe are dissolved in 230 ml of methanol. After the addition of 1.15 g of 10% palladium on carbon, the reaction mixture is hydrogenated. Filtration and evaporation to dryness gave 21.96 g of product.

e) Z-Val-Val-MeVal-Pro-OMe

15.29 g (61 mmol) of Z-Val-OH and 21.96 g (61 mmol) of H-Val-MeVal-Pro-OMe are dissolved in 610 ml of dichloromethane and cooled to 0 ° C. After addition of 8.16 ml (73.2 mmol) of N-methylmorpholine, 2.77 g (20.3 mmol) of HOBt and 11.74 g (61 mmol) of EDCI, the reaction mixture is stirred overnight at room temperature, diluted with dichloromethane. and washed thoroughly with saturated aqueous sodium bicarbonate (3x), water (1x), 5% citric acid (3x) and saturated sodium chloride solution. The organic phase is dried over sodium sulphate and evaporated to dryness. 31.96 g of product are obtained.

f) Z-Val-Val-MeVal-Pro-OH

31.96 g (57 mmol) of Z-Val-Val-MeVal-Pro-OMe are dissolved in 250 ml of methanol and 102.6 ml (102.6 mmol) of a 1 M solution of lithium hydroxide in water are added. After stirring at room temperature for 24 hours, water was added and the methanol was evaporated under reduced pressure. The aqueous phase is extracted (3x) with ethyl acetate, adjusted to pH 2 at 4 ° C and extracted (3x) with ethyl acetate. The combined organic extracts were dried over sodium sulfate and the solvent was evaporated under reduced pressure. 30.6 g of product are obtained in the form of a white solid.

g) Me ₂ Val-Val-MeVal-Pro-OH

Prepare Ν, dim-dimethylated tetrapeptide acid from

Z-protected tetrapeptide acid by hydrogenation of the Z-protecting group and subsequent addition of aqueous formaldehyde solution to the hydrogenation mixture to give the desired product in almost quantitative yield.

h) Me ₂ Val-Val-MeVal-Pro-3,4-dehydroproline methyl ester

3.38 g of Me ₂ Val-Val-MeVal-Pro-OH (7.27 mmol) and 0.925 g of 3,4-dehydroproline methyl ester hydrochloride (7.27 mmol) are dissolved in 75 ml of dry dichloromethane. N-methylmorpholine (8.72 mmol), 0.332 HOBt (2.43 mmol) and 1.4 g EDCI (7.27 mmol) are added at 4 ° C. After stirring overnight at room temperature, dichloromethane is added and the organic phase is washed with saturated sodium bicarbonate solution (3x) and water (1x). The organic phase is extracted with 5% aqueous citric acid. The pH of the aqueous acid layer was adjusted to 8 with 1 N sodium hydroxide solution and extracted 4 times with dichloromethane. After drying over sodium sulfate and evaporation under reduced pressure, 2.82 g of pentapeptide methyl ester is obtained.

k) Me ₂ Val-Val-MeVal-Pro-3,4-dehydroproline-benzylamide

A solution of 2.0 g of dehydroproline containing pentapeptide methyl ester in 20 ml of methanol is treated at room temperature with 4.6 ml of 1N lithium hydroxide solution. After completion of the reaction (TLC), water was added, the methanol was evaporated and the compound freed from water by lyophilization. The white powder obtained is dissolved in 36 ml of dichloromethane and 0.388 * ml (3.55 mmol) of benzylamide is added. After addition of 0.476 ml of N-methylmorpholine (4.26 mmol), 0.163 g

0.684 g of EDCI (3.55 mmol) was stirred at room temperature overnight. Dichloromethane is added and the organic phase is washed 3 times with saturated sodium bicarbonate solution and water (1x). The organic layer was extracted twice with 5% citric acid. The acidic aqueous layer was adjusted to pH 9 with 5N sodium hydroxide solution and extracted with dichloromethane (4x). After drying over sodium sulfate and evaporation of the solvent under reduced pressure, 600 mg of crude pentaHOBt (1.19 mmol) is obtained and the reaction mixture of peptidebenzylamide is 4 ° C. 300 mg of this crude peptide was purified by liquid medium pressure chromatography (0-10% A in 5 ', 10-25% A in 10', 25-90% A in 450 ') to give analytically pure product. The compound is further characterized by fast atom bombardment mass spectrography ([M + H] ⁺ = 639.4).

The following compounds were prepared and can be prepared according to Examples 1 and 2:

2. Xaa Val Xab Pro Xac

4. Xaa Val Xab Pro Xad

6. Xaa Val Xab Pro Xae

6. Xaa Val Xab Pro Xaf

7. Xaa Val Xab Pro Xa g * Xaa Val Xab Pro Xaa

g. Xaa Val Xab Pro Xai

10. Xaa Val Xab Pro Xak

11. Xaa Val Xab Pro Xal

12. Xaa Val Xab Pro Xam

13. Xaa Val Xab Pro Xan

14. Xaa Val Xab Pro Xao

15. Xaa Val Xab Pro Xap

16. Xaa Val Xab Pro Xaq

17. Xaa Val Xab Pro Xar

18. Xaa Val Xab Pro Xas

Xaa Val Xab Pro Xat

20. Xaa Val Xab Pro Xau

21. Xaa Val Xab Pro Xav

22. Xaa Val Xab Pro Xaw

23 Xaa Val Xab Pro Xax

Xaa Val Xab Pro Xay

25. Xaa Val Xab Pro Xaz

26. Xaa Val Xab Pro Xba

27. Xaa Val Xab Pro Xbb

28. Xaa Val Xab Pro XBC

Xbd Val Xab Pro Xar

30. Xbe Val Xab Pro Xar

31. Xaa Val Xab Pro Xcx

32. Xaa Val Xab Pro Xcy

33. Xaa White Xab Pro Xar

34. Xaa Xbh Xab Pro Xar

35th Xaa wall XBF for XAR 36th Xaa wall xbg for XAR 37th Xaa wall xab for XBI 38th Xaa wall xab for XBK 39th Xaa wall xab for XBL 40th Xaa wall xab for Xbra 41st Xaa wall xab for xbn 42nd Xaa wall xab for xbo 43rd Xaa wall xab for XBP 44th Xaa wall xab for XBQ 45th Xaa wall xab for XBR 46th Xaa wall xab for XBS 47th Xaa wall xab for XBT 48th Xaa wall xab for XBU 49th Xaa wall xab for XBV 50th Xaa wall xab for XBW 51st Xaa wall xab for xbx 52nd Xaa wall xab for XBY 53rd Xaa wall xab for xbZ 54th Xaa wall xab for xca 55th Xaa wall xab for xcb 56th Xaa wall xab for xcc

57th Xaa wall xab for XCD 58th Xaa wall xab for XCE 59th Xaa wall xab for xcf 60th Xaa wall xab for XCG 61st Xaa wall xab for XCH 62nd Xaa wall xab for XCI 63rd Xaa wall xab for XCK 64th Xaa wall xab XCN XCO 65th Xaa wall xab XCN Xak 66th Xaa wall xab XCN Xaq 67th Xaa wall xab XCN XAR 68th Xaa wall xab XCN Xay 69th Xaa wall xab XCP Xak 70th Xaa wall xab XCP Xaq 71st XCW wall xab XCP XAR 72nd xCX wall xab XCP Xay 73rd Xaa wall xab for XCN XCR 74th Xaa wall xab for XCN XCS 75th Xaa wall xab for XCN XCT 76th Xaa wall xab for XCN x Cu 77th Xaa wall xab for XCN XCV 78th Xaa wall xab for XCQ XCR 79th Xaa wall xab for XCQ XCS 80th Xaa wall xab for XCQ XCT 81st Xaa wall xab for XCQ x Cu 82. · Xaa wall xab for XCQ XCV 83rd Xaa wall xab for XCW XCR 84th Xaa wall xab for XCW XCS 85th Xaa wall xab for XCW XCT 86th Xaa wall xab for XCW x Cu 87th Xaa wall xab for XCW XCV 88th Xaa wall xab for XCW OCH ₃ 89th Xaa wall xab for XCL 90th Xaa wall xab for Xcra 91st Xaa wall xab for XCZ 92nd Xaa wall xab Pró Xda

Examples of MS-characterization of synthesized compounds are shown in the following table.

new

Example Analysis of MS in fast atom bombardment

no. [Mol. weight (measured)]

61 627 64 546 88 552 89 655

Table I - Sequence identification of compounds prepared according to Examples 1 and 2

Compound No.

Sequence ID number

1-32, 35-63, 88-92

2

3

64-72 4

74-87 5

The symbols Xaa in sum have the following meanings:

Xaa:

xab:

N, N-Dimethylvaline N-Methylvaline

XAC:

xad:

xae:

XAF:

XAG:

XAH:

N '

H ₃ x

Xai:

oh ₃ c

Xak:

XAL:

Xam:

Xan:

Xao:

XAP:

Xaq:

XAR:

XAU

Xav:

xaw:

XAX:

Xay:

x and z:

Xba:

xbb:

XBD: Ν, Ν-dimetylizoleucín XBE: N, N-dimety1-2-tert-butyl-glycine XBF: N-methyl isoleucine xbg: N-methyl-2-tert-butyl-glycine XBH: 2-tert-butylglycine

XBL:

./NO h ₃ g

F

N

CH.

CH.

XBK;

O h ₃ c

XBL:

XBM:

xbo:

XBP:

XBQ:

XBR:

XBS:

XBT:

XBU:

XBV.

XBW.

Xbx *.

XBY:

xbZ:

A

O, C

xca

xcb:

xcc:

XCD:

XCG:

'1

XCH:

XCI

XCK:

3,4-dehydroproline-adamantyl (1) amide

XCN:

XCO:

XCP:

XCQ:

3,4-dehydroproline

3,4-dehydroproline amide

L-thiazolidine-4-carbonyl

4-fluoro-proline

XCR:

XCS:

XCT:

^ </ γ ^Ν ^ € Η ₂

x Cu:

XCV:

XCW:

CH 2 ^N 2 CH ₃

ABOUT

CH, _N- yY ^N , -cH ₃

H ₃ C ⁰

L-azetidine i-2-carbonyl

xCX:

xcy:

XCZ:

Xda

The compounds of the invention can be tested for anti-cancer activity by conventional methods including, but not limited to:

A. In vitro methodology

Cytotoxicity is measured by standard methodology for adherent cell lines such as microculture tetrazolium assay (MTT). Details of this assay are described in the literature (Alley, MC et al. Cancer Research 48: 589-501, 1988). Exponentially growing cultures of tumor cells, such as HT-29 colon-carcinoma or LX-lung tumor cells, are used to obtain microtiter plate cultures. Cells are seeded at 5,000 to 20,000 per well in 96-well plates (in 150 μΐ medium) and cultured overnight at 37 ° C. Compounds are added at a 10-fold dilution of 10 ^-4 M to 10 ^-10 M. The cells are then incubated for 48 hours. To determine the number of viable cells in each well, add MTT dye (50 μΐ of a 3 mg / ml solution of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide in brine). The mixture is incubated for 5 hours at 50 µl of 25% SDS, pH 2, added to the temperature. The absorbance of each well is then read at 550 nm by ELISA. Mean +/- SD data from replicated wells are calculated using the% T / C formula (% of treated live cells / control cells).

° C and then after Easter

OD treated cells

x 100 =% T / C

OD control cells

The concentration of test compound that gives a T / C growth inhibition value of 50% is referred to as an IC 50 value.

Example compound IC ₅₀ [M]

1 1 X 10 ~ ⁶ 2 1 X 10 ' ⁹ 61 1 X 10 ⁷ 64 1 X 10 ^-8 88 1 x 10 ^-8 89 1 x 10 ^-8

B. In vivo methodology

The compounds of the invention were further tested in preclinical assays for in vivo activity indicative of clinical use. Such assays are performed in nude mice transplanted with tumor tissues, preferably of human origin, as is well known in the art. Test compounds are evaluated for anti-tumor efficacy after administration to xenograft mice.

Human breast tumors (MX-1) that grew in an athymic nude mouse are transplanted into a new host mouse using tumor fragments of about 50 nm in size. The transplant day is day 0. Six to ten days later, mice are treated with test compounds administered as intravenous injections in groups of 5 to 10 mice at each dose. The compounds were administered every other day for three weeks at doses of 1 to 100 mg / kg body weight. Tumor diameters and body weight were measured twice a week. Tumor volumes were calculated from calliper diameters and tumor volumes were calculated for each treatment group according to the formula * O 0 (length x width) / 2 = mm tumor volume

Mean tumor volumes are calculated for each group and T / C values for each group versus untreated control tumors are determined.

I

The novel compounds show good tumor inhibition properties.

Industrial usability

A peptide for the manufacture of pharmaceutical compositions for the treatment of oncological diseases in mammals.

Sequence Listing (1) General Information

(i) Applicant

A. Addressee: BASF Aktiengesellschaft

B. Street: Carl-Bosch-Strasse 38

C. City: Ludwigshafen

E. Country: Federal Republic of Germany

F. ZIP: D-67056

G: Telephone: 0621/6048526 H: Telephone: 0621/6043123

I. Telex: 1762175170 ii) Title of the invention: Peptide and pharmaceutical composition containing it iii) Number of sequences: 5 iv) Form for computer

A. Media Type: 3.5 inch floppy disk, 2 DD

B. Computer: IBM AT-compatible, 80286 processor

C. Operating System: MS-DOS version 5.0

D. Software: WordPerfect

2. SEQ ID NO: 1 information:

i) Sequence characteristics

A. Length: 5 amino acids

B. Type: amino acid

D. Topology: linear ii) Type of molecule: peptide xi) Sequence description: SEQ ID. NO: 1:

Xaa Val Xaa For Xaa

2. SEQ ID. NO: 2:

i) Sequence characteristics

A. Length: 5 amino acids

B. Type: amino acid D. Topology: linear ii) Type of molecule: peptide xi) Sequence description: SEQ ID. NO: 2:

Xaa White Xaa For Xaa

2. SEQ ID. NO: 3:

i) Sequence characteristics

A. Length: 5 amino acids

B. Type: amino acid D. Topology: linear ii) Type of molecule: peptide xi) Sequence description: SEQ ID. NO: 3: Xaa Xaa Xaa For Xaa

2. SEQ ID. NO: 4:

i) Sequence characteristics

A. Length: 5 amino acids

B. Type: amino acid D. Topology: linear ii) Type of molecule: peptide xi) Sequence description: SEQ ID. NO: 4: Xaa Val Xaa Xaa Xaa

2. SEQ ID. NO: 5:

i) Sequence characteristics

A. Length: 6 amino acids

B. Type: amino acid D. Topology: linear ii) Type of molecule: peptide xi) Sequence description: SEQ ID. NO: 5: Xaa Xaa Xaa Xaa f / ¢ ££ 7-97-

Claims (5)

PATENT CLAIMS 1 O> R-N-R together pyroilidine ring, And the residues valyl, isoleucyl, leucyl, 2-tert-butylglycyl, 2-ethylglycyl, norleucyl or norvalyl, B residues N-methylvalyl, N-methylleucyl, N-methylisoleucyl, N-methylnorvalyl, N-methylnorleucyl, N-methyl-2-tert-butylglycyl, N-methyl-3-tert-butylalanyl or N-methyl-2-ethylgiycyl, D residues prolyl, 3,4-dehydroprolyl, 4-fluoropropyl, 4, Peptides of the general formula I R ^L R ² N - CHX - CO-ABDE - (F) _t-C (I) wherein R ^{1 is} methyl, ethyl or isopropyl, R ² is hydrogen, methyl or ethyl, Peptides of formula I according to claim 1, or salts thereof, for use in medicine, in particular for the treatment of cancer. A pharmaceutical composition comprising a therapeutically effective amount of a peptide of formula I according to claim 1, and a pharmaceutically acceptable support. 4. Method treatment tumor of mammals j ú c i sa t that is mammals with cancer submitted tumors inhibitory number peptide of general formula of formula I according to claim 1. 4-difluoropropyl, azetidine-2-carbonyl, homoprolyl, 3-methylprolyl, 4-methylprolyl, 5-methylprolyl or thiazolidine-4-carbonyl, E 3,4-dehydroprolyl, 4-fluoroprolyl, 3-methylprolyl, 4-methylprolyl, azetidine-2-carbonyl or 4,4-difluoropropyl, F residues valyl, 2-tert-butylglycyl, isoleucyl, leucyl, 2-cyclohexylglycyl, norleucyl, norvalyl, neopentylglycyl, alanyl, β-alanyl or aminoisobutyroyl, X alkyl preferably having 2 to 5 carbon atoms, cyclopropyl or cyclopentyl, 10 or 1 and K alkoxy preferably having 1 to 4 carbon atoms, benzyloxy or substituted or unsubstituted amino, or salts thereof with physiologically acceptable acids. A process for the preparation of a peptide of formula I according to claim 1, characterized in that the peptide is prepared by methods known per se from peptide chemistry.