Antitumor peptides

 

(57) Abstract:

Describes the new peptides of General formula (I) and (CH3)2-N-CHX-CO-A-B-D-E-K, where a represents the residue of valine; means the residue N-methylvaline; D means the residue of Proline; E means the residue of Proline; X is isopropyl; It means N(CH3)3, -NHCH(CH2CH2)CH(CH3)2, -N(CH3)C(CH3)3, -NHCH(C2H5)2,

-N(C2H5)CH(CH3)2, -N(CH3)CH(CH3)2, -N(CH3)2C2H5, -N(CH3)(C2H5)2,

-N(CH3)2CH(CH3)2, -N(C3H7)2, -N(C2H5)3H7, -N(CH3)2,

-N(CH3)2H5, -N(CH3)C(CH3)3, NH-cyclohexyl, NH-cycloheptyl, -N(CH3)OCH2CH3, -N(CH3)OS3H7, -N(CH3)OCH(CH3)2, -N(CH3)O(CH2)3CH3,

-N(CH3)OCH2WITH6H5, -N(CH3)2WITH6H5, -N(CH3)2CH2CH3, -N(CH3)(CH2CH3)2,

-N[CH(CH3)2]2, -N(CH32CH3)2CCH, -N(CH3)2CH2CH2HE N(CH3)2CH(CH3)2, -N(CH3)2CH2CH2CH3, -NHC(CH3)2CH2C6H5,

-N(OCH3)CH(CH3)2, -N(OCH3)CH2CH3, -N(OCH3)CH2CH2CH3, -N(OCH3)CH2WITH6H5,

-N(OCH3)6H5, -N(CH3)OS6H5, -N(OCH3)CH2CH2CH2CH3,

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and their salts with physiologically tolerated acids. The new compounds possess anti-tumor activity. In comparison with the known analogues, for example, dolastatin-10 or -15, these compounds exhibit high resistance to decomposition and can be used orally. 3 C.p. f-crystals, 2 tab.

This invention provides new peptides and their derivatives, which provide potentially improved therapeutic possibilities for the treatment of neoplastic diseases compared with dolastatin-10 and -15 (U.S. patent 4879276 and 4816444) and the compounds described in the international patent application 93/23424.

The compounds of this and is hydrogen, methyl or ethyl;

R2means methyl or ethyl, or

R1-N-R2together form pyrolidine loop;

A represents the residue was felled, isoleucyl, ALLO-isoleucyl, 2-tert-butylphenyl, 2-ethylglycine, norleucine or Norvaline;

In the mean residue N-methylvaline, N-methylmorpholine, N-methyl-laitila, N-methylisoleucine, N-methyl-2-tert-butylphenyl, N-methyl-2-ethylglycine or N-metallurigcal;

D means the remainder of the shed, homepolish, hydroxyproline or thiazolidin-4-carbonyl;

E means the remainder of the shed, homepolish, hydroxyproline, thiazolidine-4-carbonyl, TRANS-4-fluoro-L-prolyl, CIS-4-fluoro-L-prolyl TRANS-4-chloro-L-prolyl or CIS-4-chloro-L-prolyl;

X means ethyl, propyl, butyl, isopropyl, sec-butyl, tert-butyl, cyclopropyl or cyclopentyl;

G means the residue of L-2-tert-butylglycol, D-2-tert-butylglycol, D-cut, D-isoleucyl, D-laitila, D-Norvaline, 1-aminopentyl-1-carbonyl or 2,2-dimethylglycine;

s is 0 or 1;

It is alkylaminocarbonyl with 1-8 carbon atoms in the alkyl, alkynylamino with 3-8 carbon atoms in Alchemilla balance, alkynylamino with 3-8 carbon atoms in alchenilla balance, cycloalkylation with 6-8 carbon atoms in cycloalkyl, dialkylamino with 1-4 carbon atoms in one alkyl with 1-6 carbon atoms in each other, in which one methylene group may be replaced by oxygen or sulphur, one hydrogen atom by a phenyl or langroup or one, two or three hydrogen atoms may be replaced by fluorine, in addition to N-methoxy-N-methylaminopropyl, N-benzylamino or N-methyl-N-benzylamino, or By means

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and their salts with physiologically tolerated acids.

Specifically, It can be N3, -N2CH3, -NH(CH2)2CH3, -NH(CH2)3CH3, -NH(CH2)4CH3, -NH(CH2)5CH3, -NH(CH2)6CH3, -NH(CH2)7CH3, -NHCH(CH3)2, -N(CH3)CH2CH3, -N(CH2CH3)2, -NHCH(CH2CH2CH3)2, -N(CH3)3,

-N(CH2CH3)CH2CH2CH3, -NHCH(CH3)CH(CH3)2, -N(CH2CH3)CH(CH3)2,

-N(CH3)C(CH3)3, -NH-cyclohexyl, -NH-cycloheptyl, -NH-cyclooctyl, -N(CH3)OCH2CH3, -N(CH3)OCH2CH2With 2WITH6H5, -NH(CH2)2WITH6H5, -NH(CH2)3WITH6H5, -N(CH3)6H5,

-N(CH3)2WITH6H5, -N(CH3)2CH2CH3, -N(CH3)(CH2CH3)2, -NHCH[CH(CH3)2] 2, -NHC(CH3)2CN, -NHCH(CH3)CH(OH)C6H5, -NHCH2-cyclohexyl, -NHCH2C(CH3)3, -NHCH2CH(CH3)2, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2CH3)2, -NHCH2CF3,

-NHCH(CH2F)2, -NHCH2CH2F, -NHCH2-CH2OCH3, -NHCH2CH2SCH3, -NHCH2CH= CH2, -NHC(CH3)2CH=CH2, -NHC(CH3)2CCH, -NHC(CH2CH3)2CCH, -NHC(CH3)2CH2CH2OH, -NH(CH2CH2O)2CH2CH3, -N(CH3)2CH(CH3)2,

-NHC(CH3)2CH2CH2CH3, -NHC(CH3)2CH2C6H5, -N(OCH3)CH(CH3)2, -N(OCH3)CH2CH3, -N(OCH3)-CH2CH2CH3, -N(OCH3)CH2WITH6H5, -N(OCH3)C6H5, -N(CH3)OC6H5

Preferred compounds of formula I in which the substituents R1, R2A , b, D, E, X, G and s have the following meanings:

R1is hydrogen, methyl or ethyl, especially methyl;

R2is methyl or ethyl, especially methyl;

A - poured, isoleucyl, 2-tert-butylphenyl, 2-agillity, norlaili or Norwell, especially, was felled, isoleucyl, 2-tert-butylphenyl, 2-ethylglycol;

In - N-methylallyl, N-methylmorpholin, N-methylisoleucine, N-methyl-2-tert-butylphenyl, N-methyl-2-ethylglycol or N-mernorial, especially N-methylallyl, N-methyl-2-ethylglycine, N-mernorial, N-methylisoleucine or N-methyl-2-tert-butylglycol;

D - prolyl, homopolar or thiazolidin-4-carbonyl, especially, shed or thiazolidin-4-carbonyl;

E - shed, gemopolis, thiazolidin-4-carbonyl, TRANS-4-fluoro-L-prolyl, CIS-4-fluoro-L-prolyl TRANS-4-chloro-L-prolyl or CIS-4-chloro-L-prolyl, especially, shed, TRANS-4-FERROLI, CIS-4-FERROLI, TRANS-4-chlorpropyl or CIS-4-chlorpropyl;

X is ethyl, propyl, isopropyl, sec-butyl, tert-butyl or cyclopropyl, especially, ethyl, isopropyl, sec-butyl or tert-butyl;

G is a residue of L-2-tert-butylglycol, D-2-tert-butylglycol, D-cut, D-isoleucyl, D-laitila or 2,2-dimethylglycine;

s is 0 or 1.


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More preferably By means

-N3, -N2CH3, -NH(CH2)2CH3, -NH(CH2)3CH3, -NH(CH2)4CH3, -NH(CH2)5CH3, -NH(CH2)6CH3, -NH(CH2)7CH3, -NHCH(CH3)2, -N(CH3)CH2CH3,

-N(CH2CH3)2, -NHCH(CH2CH2CH3)2, -N(CH3)3, -N(CH2CH3)CH2CH2CH3, -NHCH(CH3)CH(CH3)2, -N(CH2CH3)CH(CH3)2, -N(CH3)C(CH3)3, -NH-cyclohexyl, -NH-cycloheptyl, -NH-cyclooctyl,

-N(CH3)OCH2CH3, -N(CH3)OCH2CH2CH3, -N(CH3)OCH(CH3)2, -N(OCH3)CH(CH3)2,

-N(CH3)OCH2WITH6H5,

-N(CH3)2WITH6H5, -N(CH3)2CH2CH3, -N(CH3)(CH2CH3)2, -NHCH(CH3)CH(OH)C6H5, -NHCH2-cyclohexyl, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2CH3)2, -NHCH(CH2F)2,

-NHC(CH3)2CH2CH2OH, -NH(CH2CH2O)2CH2CH3, -NHC(CH3)2CH(CH3)2, NHC(CH3)CH=CH2, -NHC(CH3)2CN, -N(CH3)2ONH2, -N(OCH3)C6H5, -N[CH(CH3)2]2, -N(OCH3)CH2WITH6H5, -N(OCH3)CH2CH3, -N(OCH3)CH2CH2CH3,

-N(OCH3)CH2CH2CH2CH3,

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Especially preferred compounds of formula I, where

R1and R2means methyl,

A represents the residue was felled, isoleucyl or 2-tert-butylglycol,

In the mean residue N-methylvaline, N-methylisoleucine or N-methyl-2-tert-butylglycol,

D means the remainder of the shed or thiazolidin-4-carbonyl, E means the remainder of the shed, CIS-4-fluoro-L-prolyl or CIS-4-chloro-L-prolyl,

X means isopropyl, sec-butyl and 3, -N(CH2CH3)2, -NHCH(CH2CH2CH3)2, -NHC(CH3)3, -NHCH(CH2CH3)CH2CH2CH3, -NHCH(CH3)CH(CH3)2, -N(CH2CH3)CH(CH3)2, -NHCH(CH3)C(CH3)3, -NH-cycloheptyl, -NH-cyclooctyl,

-N(CH3)OCH2CH3, -N(CH3)OCH2CH2CH3, -N(CH3)OCH(CH3)2, -N(OCH3)CH(CH3)2,

-N(CH3)OCH2WITH6H5, -NH(CH2)2WITH6H5, -NH(CH2)3WITH6H5, -N(CH3)6H5,

-NHC(CH3)2C6H5, -NHC(CH3)2CH2CH3, -NHC(CH3)(CH2CH3)2, -NHCH(CH3)CH(OH)C6H5, -NHCH(CH2F)2, -N(CH3)2CH2CH2HE, -NH(CH2CH2O)2CH2CH3, -NHC(CH3)2CH= CH2, -N(CH3)2CH(CH3)2, -N(OCH3)CH2CH3, -N(OCH3)CH2CH2CH3, -N(OCH3)CH2CH2CH2CH3, -NHC(CH3)2CN, -N[CH(CH3)2]2, -NHC(CH3)2CONH2, -N(CH3)2
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This invention also provides methods of making compounds of formula I, pharmaceutical compositions containing such compounds, together with a pharmaceutically acceptable carrier and methods of their use for the treatment of cancer in mammals.

New connections can be represented in the form of salts with physiologically tolerated acids such as: hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonate, acetic acid, formic acid, maleic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucus acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid and acetylglycine.

The new compounds may be obtained by known methods of peptide chemistry. Thus, peptides can be synthesized sequentially on the basis of amino acids, or by linking suitable small peptide fragments. A series connection, starting with the end of the peptide chain is extended gradually, each time for one amino acid. When whut to be received through the serial connection of the amino acids or from themselves when interacting fragments.

As a series connection and interaction of the fragments must be connected areas through the formation of amide bonds. Appropriate enzymatic and chemical methods.

Chemical methods for the formation of amide bonds described by Muller in the book. "Methods of organic chemistry", vol. XV/2, pp. 1-364, Thieme publishing house, Stuttgart, 1974; Stewart, young in the book. "Solid-phase peptide synthesis", pages 31-34, 71-82, publisher Pierce Chemical Company. Rockford, 1984; Bodansky, Klausner, Ondetti in the book. "Peptide synthesis", pp. 85-128, John Wiley & Sons, new York, 1976; "the Practice of peptide synthesis" by M. Bodansky, A. Bodansky, Springer, 1994, and other relevant studies on chemistry of peptides. Particular preference is given to the azide method, the method of symmetric and mixed anhydrides, generated in situ or pre-obtained activated esters, the use of urethane-protected N-carboxyanhydrides amino acids and the formation of amide linkages, using a combination of reagents, in particular, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-etoxycarbonyl-2-ethoxy - 1,2-dihydroquinoline (EEDQ), pivaloyloxy, the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide the C - pyrrolidinecarboxylic-phosphate (Rougher), diphenylphosphide (D), benzotriazol-1 yloxy-Tris(dimethylamino)-phosphonium-hexaphosphate (reagent Castro, THIEF, Rumor), O-benzotriazolyl-N, N, N', N'-tetramethyluronium salt (HBTU), O-isobenzofuranyl-N,N,N',N'-tetramethyluronium salt (HATU), diethylphosphoramidite (DEPCN), dioxide, 2,5-diphenyl-2,3-dihydro-3-oxo-4 - hydroxythiophene (reagent Steglich, HOTDO) and 1,1'-carbonyldiimidazole (CDI). Reagents for combination can be used separately or in combination with additives, such as N,N-dimethyl-4-aminopyridine (DMAP), N-hydroxy-benzotriazole (HOBt), N-hydroxybenzotriazole (HOOBt), isobenzofuran, N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine.

At that time, as it is usually possible to dispense with protective groups in enzymatic peptide synthesis, reversible protection of reactive groups not involved in the formation of amide linkages necessary for both reactant in chemical synthesis. The preferred three accepted methods with the use of protective groups for chemical peptide synthesis techniques using benzyloxycarbonyl(Z), tert-butoxycarbonyl (BOC) and 9-fluorenylmethoxycarbonyl (Fmoc) protective group.

Defined in each case is a protective g the Lehr in the book. "Methods of organic chemistry", volume XV/1, page 20-906, Thieme publishing house, Stuttgart, 1974. Areas to be used for binding in the peptide chain may react in solution, in suspension or in the manner similar to that described by Meregildo in J. Amer.Soc. 85(1963), 2149.

For carrying out peptide synthesis in solution fit all solvents which are inert under the reaction conditions, in particular, water, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile, dichloromethane (DHM), ethyl acetate, 1,4-dioxane, tetrahydrofuran (THF), N-methyl-2-pyrrolidone and mixtures of these solvents.

Peptide synthesis on polymeric carrier can be carried out in an inert organicheskikh solvents, which are used soluble amino acid derivatives. However, the preferred solvents additionally have cause swelling of the resin properties, for example, DMF, DHM, N-methyl-2-pyrrolidone, acetonitrile and DMSO and mixtures of these solvents. After completion of the synthesis, the peptide is separated from the polymer carrier. The conditions for removal from various types of resins are described in the literature. The most frequently used fission reactions are catalyzed by acids and palladium, baulenas or concentrated triperoxonane acid, catalyzed by palladium cleavage in THF or a mixture of THF-DHM in the presence of a weak base, such as morpholine, or splitting in mixtures of acetic acid/DHM/triptoreline. Depending on the selected protective groups, the latter may be held or in the same way be chipped off in terms of the splitting.

The partial withdrawal of the protection of the peptide may also be necessary when you have some reaction conversion.

Peptides, dialkylamino N-end may be obtained either during the interaction with the corresponding N,N-dialkylaminoalkyl in solution or on a polymeric carrier, the reductive alkylation associated with the resin peptide in TMPAL/% acetic acid using nutritionrelated and the corresponding aldehydes, with the hydrogenation of the peptide in solution in the presence of aldehyde or ketone and Pd/C.

Various naturally occurring amino acids, as well as various diaminotoluene plots presented here can be purchased or synthesized from commercially available materials using known ways to do this. For example, the amino acid building blocks for the desired connections with areas of R1and R2Evo Thieme, Stuttgart, 1974, hereinafter cited literature.

The compounds of this invention can be used for inhibition or treatment of solid tumors (e.g., tumors of lung, breast, colon, prostate, bladder, colon or endometrial) or hematologic malignancies (such as leukemias, lymphomas) when introducing the compound to the mammal.

A special advantage of the new compounds is their high resistance to enzymatic degradation and the fact that they can be administered orally.

The introduction can be carried out by any of the methods adopted for the pharmaceutical, preferably cancer, means, including oral and parenteral methods, such as the introduction subcutaneously, intravenously, intramuscularly and intraperitoneally.

Connections can be entered individually or in the form of pharmaceutical compositions containing a compound of formula I together with a pharmaceutically acceptable carrier suitable for the desired route of administration. Such pharmaceutical composition can be a combination of products, there may also contain other therapeutically active ingredients.

Dozhnogo ingredient, that will depend on common factors, including biological activity of this used compounds, methods of administration, the age, health and weight of the recipient, the nature and prevalence of symptoms, frequency of treatment, the introduction of other therapeutic drugs and the desired effect. A typical daily dose will be from about 0.05 to 50 mg per kg of body weight for oral introduction and from about 0.01 to 20 mg per kg of body weight at parenteral administration.

The new compounds may be introduced in the form of a conventional solid or liquid pharmaceutical forms for administration, for example, coated or covered with a thin layer tablets, capsules, powders, granules, suppositories or solutions. These forms are prepared by a common way. The active substance may, for this purpose be processed with conventional pharmaceutical AIDS such as, for example, binding agents for making tablets, fillers, preservatives, means for disintegration of tablets, fluidity regulators, plasticizers, wetting agents, dispersing agents, emulsifiers, solvents, compositions for sustained release, antioxidant and/or dispersant gases (cf. N. Sucker etc.: "Farget usually 1-90% (by weight) of the active substance.

The invention is illustrated by the following examples. Included in the protein amino acids are reduced in the examples using a known three-letter code. Other applied abbreviations:

Me2Val=N,N-dimethylamine, MeVal=N-methylvaline.

A. General methods

I. Peptides, stated in paragraph 1, are synthesized either by classical synthesis in solution using standard techniques with Z - and BOC-protective groups as described above, or by using standard methods of solid-phase synthesis using BOC - and Fmoc-protective group.

In the case of solid-phase synthesis of N, N-dialkylphenol or Hexapeptide acids are released from the solid media, and then are connected with the corresponding C-terminal amines in solution. N, N-Bis(2-oxo-3-oxazolidinyl)aminophospholipid and bromo-Tris - pyrrolidinedithiocarbamate were used as reagents to attach the amino acid following the N-methyliminodiacetic. The reaction time is correspondingly increased. For reductive alkylation on N-end of the peptide-resin was removed protection at N-end, and then subjected to reaction with 3-fold molar excess of aldehyde or ketone in DMF/1% acetic acid was washed several times with water, isopropyl alcohol, DMF and dichloromethane.

During the synthesis in solution using either the internal N-carboxyanhydrides the BOC-protected amino acids (N-carboxyanhydride N-tert-butoxycarbonylamino), N-carboxyanhydrides Z-protected amino acids (N-carboxyanhydride N-benzyloxycarbonylamino), or the use of pualeilani as a condensing agent, respectively, is most advantageous to attach the amino acid following the N-methyliminodiacetic. Reductive alkylation on N-end may, for example, be carried out when the reaction is not protected by the N-end of the peptides or amino acids to the corresponding aldehydes or ketones using nutritionrelated or hydrogen in the presence of Pd/C.

II. Purification and characterization of peptides

Purification was performed by gel chromatography (Sephadex G-10, G-15/10% acetic acid, Sephadex LH20/methanol), medium pressure chromatography (stationary phase: HD-SIL C-18, 20-45 MK, 100 Angstrom; mobile phase: gradient with A= 0.1% of triperoxonane acid/methanol, B=0,1% triperoxonane acid/water) or preparative high performance liquid chromatography (stationary phase: Waters Delta-Pak C-18, 15 MK, 100 .

The purity of the resulting products was determined with analytical high performance liquid chromatography (stationary phase: 100 2. 1 mm VYDAC C-18, 51, 300 Angstrom; mobile phase: a gradient of acetonitrile-water, buffered with 0.1% triperoxonane acid, 40oC).

Okharakterizovali using amino acid analysis and mass spectroscopy with fast atom bombardment.

B. Specific techniques

Example 1 (sequence 1) Me2Val-Val-MeVal-Pro-Pro-NHCH(CH3)2< / BR>
a) Z-MeVal-Pro-OMe

Dissolved 66,25 g (250 mmol) of Z-MeVal-OH in 250 ml of anhydrous dichloromethane. After adding 36,41 ml (262,5 mmole) of triethylamine, the reaction mixture was cooled to -25oWith and added 32,27 ml (262,5 mmole) of pivaloate. After stirring for 2.5 h to the reaction mixture were added 41,89 g (250 mmol) H-Pro-OMe model HC1 x 250 ml dichloromethane, neutralized 36,41 ml (262,5 mmole) of triethylamine at 0oC. Stirring was continued for 2 h at -25oC and overnight at room temperature. The reaction mixture was diluted with dichloromethane and thoroughly washed with a saturated aqueous solution of sodium bicarbonate (three times), water (once), 5% citric acid (three times) and a saturated solution chlorestol Oleinik ether overnight and was filtered. Received of 62.3 g of the product.

b) H-MeVal-Pro-OMe

Dissolved 48,9 g (130 mmol) of Z-MeVal-Pro-OMe in 490 ml of methanol. After addition of 10.9 ml (130 mmol) of concentrated hydrochloric acid and 2.43 g of 10% Pd/C, the reaction mixture was first made. After filtration and evaporation to dryness received 36,43 g of the product.

in), Z-Val-MeVal-Pro-OMe

18,1 g (65 mmol) of H-MeVal-Pro-OMe, of 21.6 g (78 mmol) Z-Val-N-carboxyanhydride and 22.8 ml (130 mmol) of diisopropylethylamine was stirred in 110 ml of DMF at 40oWith in 2 days. After evaporation of DMF was added dichloromethane and the organic phase is washed with saturated aqueous sodium bicarbonate (three times), once with water, 5% citric acid (three times) and a saturated solution of sodium chloride. The organic phase was dried over sodium sulfate and evaporated to dryness. Product (29.3 g) was obtained as a viscous oil.

g) H-Val-MeVal-Pro-OMe

Dissolved 29,3 g (61.6 mmole) of Z-Val-MeVal-Pro-OMe in 230 ml of methanol. After addition of 1.15 g of 10% Pd/C, the reaction mixture was first made. After filtration and evaporation to dryness received 21,96 g of the product.

d) Z-Val-Val-MeVal-Pro-OMe

Dissolved 15,29 g (61 mmol) Z-Val-OH and 21,96 g (61 mmol) of H-Val-MeVal-Pro-OMe in 610 ml of dichloromethane and cooled to 0oC.

After adding 8,16 ml (73.2 mmole) of N-metalmark the reaction mixture was stirred over night at room temperature, was diluted with dichloromethane and thoroughly washed with aqueous saturated solution of sodium bicarbonate (three times), water (once), 5% citric acid (3 times) and a saturated solution of sodium chloride. The organic phase was dried over sodium sulfate and was evaporated to dryness, got 31,96 g of the product.

e) Z-Val-Val-MeVal-Pro-OH

Dissolved 31,96 g (57 mmol) Z-Val-Val-MeVal-Pro-OMe in 250 ml of methanol. Added 102,6 ml of 1 n solution of lithium hydroxide and the mixture was stirred over night at room temperature. After addition of 500 ml water, the aqueous phase was washed three times with ethyl acetate, was established pH 2 at 0oWith and was extracted three times with ethyl acetate. The organic phase was dried over sodium sulfate and was evaporated to dryness, got 30,62 g of the desired product as a white solid.

W) Z-Val-Val-MeVal-Pro-Pro-NHCH(CH3)2< / BR>
Was dissolved 2 g (3,35 mmole) of Z-Val-Val-MeVal-Pro-OH and 0,664 g (3,35 mmole) N-RHS-MNSN(CH3)2in 34 ml of anhydrous dichloromethane. After cooling to 0oWith added 1.35 ml (12,1 mmole) N-methylmorpholine, 0,114 g (from 0.84 mmole) of N-hydroxybenzotriazole and to 0.645 g (3.35 mmole) of the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and the reaction mixture was stirred over night at room temperature. Added 80 is GDI), water (once), 5% citric acid (three times) and once with a saturated solution of sodium chloride. The organic phase was dried over sodium sulfate and was evaporated to dryness, received a 1.96 g of product which was used without further purification in the next reaction.

C) Me2Val-Val-MeVal-Pro-Pro-NHCH(CH3)2< / BR>
Dissolved 1,96 g of Z-Val-Val-MeVal-Pro-Pro-NHCH(CH3)2in 11 ml of methanol, was added 0,054 g 10% Pd/C in nitrogen atmosphere and the reaction mixture was first made at room temperature for 4 h After addition of 0,86 ml (11,24 mmole) of 37% aqueous formaldehyde and 0,281 g 10% Pd/C hydrogenation was continued for 5 hours After filtration and removal of solvent received 2,77 g of the crude product. Further purification was carried out by dissolving the peptide in water, bringing the pH to 2 and triple extraction of the aqueous phase with ethyl acetate. Then the pH of the aqueous phase was brought to 8-9 and was extracted four times with dichloromethane. The organic phase was dried over sodium sulfate and got to 1.37 g of the pure product as a white foam. Then the substance was purified using liquid chromatography medium pressure (10-50% in 10 min; 50-90% And 320 min). Containing the product fractions were combined, subjected to freeze-drying, re-dissolved in water and octanal the blockhead sodium and evaporated to dryness. Lyophilization resulted in 500 mg of pure product, which okharakterizovali using mass spectrometry with fast atom bombardment ([M+H]+=593).

Example 2 (sequence 1)

Me2Val-Val-MeVal-Pro-Pro-NHC(CH3)3< / BR>
and Z-Val-Val-MeVal-Pro-Pro-NHC(CH3)3< / BR>
Was dissolved 2 g (3,35 mmole) of Z-Val-Val-MeVal-Pro-OH and 0,692 g (3,35 mmole) N-RHS-N(CH3)3in 34 ml of anhydrous dichloromethane. After cooling to 0oWith added 1.35 ml (12,1 mmole) of N-methyl-research, 0,114 g (from 0.84 mmole) of N-hydroxybenzotriazole and to 0.645 g (3,35 mmole) of the hydrochloride of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and the reaction mixture was stirred over night at room temperature. Was added 80 ml of dichloromethane and the organic phase is thoroughly washed three times with saturated aqueous sodium bicarbonate solution, once with water, three times with 5% citric acid and once with saturated sodium chloride solution. The organic phase was dried over sodium sulfate and was evaporated to dryness, received 1.8 g of product which was used in the next reaction without further purification.

K) IU2Vl-Vl-Vl-RHS-RHS-NH(CH3)3< / BR>
Was dissolved 1.8 g of Z-Val-Val-MeVal-Pro-Pro-NHC(CH3)3in 10 ml of methanol, was added in atmosphere ml (11,24 mmole) of 37% aqueous formaldehyde and 0,252 g 10% Pd/C hydrogenation was continued for 5 hours After filtration and evaporation was received 1,82 g of the crude product. Next, the compound was purified using liquid chromatography medium pressure (10-50% in 10 min; 50-90% And 320 min). Containing the product fractions were combined, subjected to freeze-drying, re-dissolved in water and with 1 n lithium hydroxide was established pH 9. After extraction with dichloromethane the organic phase was dried over sodium sulfate and evaporated to dryness. Lyophilization resulted in 547 mg of pure product, which was characterized using mass spectrometry with fast atom bombardment ([M+H]+= 607).

According to the examples 1 and 2 were obtained or can be obtained the following compounds (see below).

Compounds of the present invention can be tested for anticancer activity using conventional methods, including, for example, the methods described below.

A. In vitro methodology

Cytotoxicity was measured using a standard methodology for fused cell lines, for example, the analysis of micro cultures using bromide 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (thiazolinones blue (MTT). Details of this analysis have been published (M. C. Alley, etc., Cancer Research 48: 589-601, 1988). the Pujol easy LX-1 for preparing crops for tablets titration. Cells seeded at 3000 cells in a cell in 96-cell plates (150 μl medium) and grown overnight at 37oC. Compounds subjected to the test, added in 10-fold dilutions ranging from 10-4M to 10-10M. the cells are Then incubated for 72 h to determine the number of viable cells in each cell is added to the dye MTT /50 μl solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in saline at a concentration of 3 mg/ml/. This mixture is incubated at 37oC for 5 h, then in each cell add 50 µl of 25% sodium dodecyl sulfate, pH 2. After incubation overnight, the absorption at 550 nm in each cell is determined using a reader device used in enzyme immunosorbent analysis (ELISA). Calculated values for the average +/- standard deviation of the data from the replicated cells, using the formula % O/C (% treated viable cells/control)

< / BR>
The concentration of the tested compound, which gives About/To 50% growth inhibition, was designated as IR50.

B. In vivo methodology

Compounds of the present invention is further subjected to preclinical testing of the who were transplanted (xenotransplantation) tumor tissue, preferably of human origin, as is well known in this field. Evaluated the effectiveness of the studied compounds as antitumor subsequent administration to mice with xenografts.

More specifically, human breast tumor (MX-1), which grew in hairless mice without thymus gland, was transferred to a new recipient mice, using fragments of the tumor mass of about 50 mg a Day of transplantation was defined as day 0. After 6-10 days, the mice were treated with the studied compounds, which were introduced in the form of intravenous injections or orally, in groups of 5-10 mice per dose. Compounds were injected every other day for 3 weeks at doses of from 1 to 200 mg/kg of body weight.

The diameters of the tumor and body weight were measured twice a week. The volume of tumor was calculated using the diameters measured by Vernier calipers, according to the formula

(length x width2)/2=tumor volume in mm3.

The average volume of tumors are calculated for each treated group, and the values Of/K are determined for each group relative to the untreated control tumors.

The new compounds possess hut resistance to splitting due to exposure to enzymes, as evidenced by the results of the next experiment, summarized in table.2.

This resistance is much higher than the resistance exhibited by the compounds described in the next similar, the application WO 93/23434, as evidenced by the comparison of data is also summarized in the following table.

Experience

100 μl of 1.35 μm solution trading enzyme proletarisation was added to 1 ml of 0.1 M phosphate buffer (pH 7) and 250 ál 810 μm solution of tested compound at a temperature of 37oC. After 1, 2 and 4 h, a sample was taken, after which add a 0.3% aqueous solution of triperoxonane acid was subjected to high performance liquid chromatography. It was determined metabolism in %. The compounds and the results of the experiment are summarized in the following table.

Compounds according to the invention belong to the category of srednetonnazhnyh substances.

The sequence listing, see the end of the description.

1. The peptides of formula (I)

(CH3)2-N-CHX-CO-A-B-D-E-K I,

where a represents the residue was felled;

In the mean residue N-methylvaline;

D means the remainder of the shed;

E means the remainder of the shed;

X means isopropyl;

It means N(CH3)3<, N(C2H5)CH(CH3)2, -N(CH3)CH(CH3)2, -N(CH3)2C2H5,

N(CH3)(C2H5)2, -N(CH3)2CH(CH3)2, -N(C3H7)2, -N(C2H5)3H7, -N(CH3)2, -N(CH3)2H5, -N(CH3)C(CH3)3, NH-cyclohexyl, NH-cycloheptyl, -N(CH3)OCH2CH3, -N(CH3)OS3H7, -N(CH3)OCH(CH3)2, -N(CH3)O(CH2)3CH3,

-N(CH3)OCH2WITH6H5, -N(CH3)2WITH6H5, -N(CH3)2CH2CH3, -N(CH3)(CH2CH3)2,

-N[CH(CH3)2]2, -N(CH3)2JV-N(CH3)CH(OH)6H5, -NH-C(CH3)2CH= CH2, -NHC(CH3)2CCH, -NHC(CH2CH3)2CCH, -N(CH3)2CH2CH2HE N(CH3)2CH(CH3)2, -N(CH3)2CH2CH2CH3, -NHC(CH3)2CH2C6H5,

-N(OCH3)CH(CH3)2, -N(OCH3)CH2CH3, -N(OCH3)CH2SUB>)OS6H5, -N(OCH3)CH2CH2CH2CH3,

< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
and their salts with physiologically tolerated acids.

2. The peptides of formula (I) under item 1, where a represents the residue was felled; In denotes the N-methylvaline; D means the remainder of the shed; E means the remainder of the shed; X is isopropyl; It means N(CH3)3, NHCH(CH3)C2H5, NHCH(C2H5)2, N(C2H5)CH(CH3)2, N(CH3)CH(CH3)2, N(CH3)C(CH3)3,

N(CH3)2C2H5, N[CH(CH3)2]2, N(CH3)2CH(CH3)2, N(C3H7)2, N(C2H5)3H7, NH-cyclohexyl, NH-cycloheptyl, N(CH3)OS3H7, N(CH3)2C6H5, N(CH3)(C2H5)2, NHC(CH3)2CCH, N(CH3)2CH2CH2HE NHCH(CH3)2N(OCH3)CH2CH2WITH6H5,

< / BR>
< / BR>
< / BR>
and their salts with physiologically tolerated acids.

3. The peptides of formula (I) under item 1, where a represents the residue was felled; means the residue N- 3
)3and their salts with physiologically tolerated acids.

4. The compounds of formula (I) under item 1 or their salts with physiologically tolerated acids with antitumor activity.

 

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